Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodies

Seishiro Hirano; Osamu Udagawa

doi:10.1371/journal.pone.0268835

. 2022 May 20;17(5):e0268835. doi: 10.1371/journal.pone.0268835

Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodies

Seishiro Hirano ^1,^*, Osamu Udagawa ¹

Editor: Claude Prigent²

PMCID: PMC9122205 PMID: 35594310

Abstract

Promyelocytic leukemia (PML) proteins are involved in the pathogenesis of acute promyelocytic leukemia (APL). Trivalent arsenic (As³⁺) is known to cure APL by binding to cysteine residues of PML and enhance the degradation of PML-retinoic acid receptor α (RARα), a t(15;17) gene translocation product in APL cells, and restore PML-nuclear bodies (NBs). The size, number, and shape of PML-NBs vary among cell types and during cell division. However, topological changes of PML-NBs in As³⁺-exposed cells have not been well-documented. We report that As³⁺-induced solubility shift underlies rapid SUMOylation of PML and late agglomeration of PML-NBs. Most PML-NBs were toroidal and granular dot-like in GFPPML-transduced CHO-K1 and HEK293 cells, respectively. Exposure to As³⁺ and antimony (Sb³⁺) greatly reduced the solubility of PML and enhanced SUMOylation within 2 h in the absence of changes in the number and size of PML-NBs. However, the prolonged exposure to As³⁺ and Sb³⁺ resulted in agglomeration of PML-NBs. Exposure to bismuth (Bi³⁺), another Group 15 element, did not induce any of these changes. ML792, a SUMO activation inhibitor, reduced the number of PML-NBs and increased the size of the NBs, but had little effect on the As³⁺-induced solubility change of PML. These results warrant the importance of As³⁺- or Sb³⁺-induced solubility shift of PML for the regulation intranuclear dynamics of PML-NBs.

Introduction

Promyelocytic leukemia proteins (PMLs) are proapoptotic molecules involved in cell proliferation, senescence, and tumor suppression [1, 2]. The human PML family comprises six nuclear isotypes (I-VI) and one cytosolic isotype (VII) [3–5]. The residence-time of PML in PML nuclear bodies (PML-NBs) ranges from several minutes to one hour, whereas that of other PML-NB client proteins such as Sp100 and Daxx varies from several seconds to one minute [6], which led to the notion that PML is a scaffold protein for PML-NBs. Sp100 is not essential for the maintenance of PML-NBs in human embryonic NT2 cells and the C-terminal region of Daxx is sufficient for interaction with PML [7]. The mutation of three small ubiquitin-like modifier (SUMO) conjugation sites of PML (K65, K160, and K490) did not affect the formation of PML-NBs, although this mutant could recruit neither Sp100 nor Daxx [8]. The SUMOylation of PML is thought to stabilize and promote the assembly of PML-NBs, since free PML tends to disperse throughout the nucleoplasm and SUMO1-conjugated PML localizes to PML-NBs [9]. PML-NBs are structurally and positionally stable in interphase and appear to associate with euchromatin via SUMO molecules [10].

PML is a member of RBCC/tripartite motif (TRIM) family. Cysteines in both B-box zinc finger domains (B₁ and B₂) are required for PML-NB formation [11]. Trivalent arsenic (As³⁺) binds to cysteine residues of the RING and B₂ domains [12] and the core region of RING is requisite for the solubility change and SUMOylation of PML in response to exposure to As³⁺ [13, 14]. C₂₁₂ in the B₂ domain is required for As³⁺-induced degradation of PML-retinoic acid receptor α (PML-RARα), a t(15;17) gene translocation product [15]. The subdomain comprising F₅₂Q₅₃F₅₄, and L₇₃ in the RING motif (aa 55–99) are unique to PML and are absent in other TRIM family members. The unique subdomain appears to play a role in PML tetramerization and the subsequent PML-NB formation, and also in As³⁺-induced SUMOylation of PML [16]. Together, intact zinc finger domains of RBCC and the unique RING motif of PML are all necessary for functional PML-NBs and biochemical responses of PML to As³⁺. PML-RARα functions as a dominant negative form and causes acute promyelocytic leukemia (APL) [17, 18]. Both As³⁺ and all-trans retinoic acid (ATRA) have been used to treat APL [19, 20]. As³⁺-binding to PML renders PML-RARα susceptible to ubiquitin-mediated degradation by RNF4 which non-covalently binds to polySUMOylated PML via SUMO-interacting motifs (SIMs) [4, 21].

Canonical PML-NBs are present in the nucleus as dense granular bodies or hollow toroid-like oblate (doughnut shaped) spheres [22], and the shape and size of PML-NBs vary depending on the cell type [23]. Non-spherical forms of PML-NBs have also been reported. Fiber-like PML-NBs are formed along with doughnut-like PML-NBs in the nuclei of IDH4 cells, human fibroblasts immortalized with the dexamethasone-inducible SV40 [24]. Nuclear envelope-associated linear and rod-like PML bodies reportedly arise transiently during the very early transitions of human ES cells towards cell-type commitment [25]. Progerin, a truncated form of lamin A, has been shown to colocalize with aberrant toroid-like and thread-like PML-NBs in fibroblasts obtained from Hutchinson-Gilford progeria syndrome patients [26], suggesting that the topology of PML-NBs may be linked with some pathological consequences.

Although PML-NBs are stable in interphase nuclei, they aggregate to form mitotic accumulation of PML proteins (MAPPs) after nuclear membrane breakdown. MAPPs associate with nuclear pore components in daughter cells after mitosis. At this stage the aggregated PML bodies are called cytoplasmic assemblies of PML and nucleoporins (CyPNs) and reside in peri-nuclear regions [27, 28]. MAPPs are inherited asymmetrically in daughter HaCaT cells after mitosis [29], and daughter cells that receive the majority of PML-NBs exhibit increased stemness in primary human keratinocytes [28]. Since PML-NBs feature phase-separated protein condensates in the nucleus [30, 31], it is of interest to investigate how their biochemical/biophysical properties change upon cell division and exposure to As³⁺.

In the present work, we performed a topological characterization of PML-NBs using CHO-K1 and HEK293 cells stably transduced with GFP-conjugated PML-VI. PML-VI is the only nuclear PML isoform that lacks a SIM motif, and therefore the SUMO-SIM interaction between PML and the other proteins, which is implicated in liquid-liquid phase separation (LLPS) [32], occurs only when PML-VI is SUMOylated. PML-NBs were toroidal in CHO cells and granular dot-like in HEK cells. Two-hour exposure of these cells to As³⁺ induced a robust SUMOylation of PML-VI although the size and number of PML-NBs were not changed. The solubility shift of PML-VI occurred prior to SUMOylation of this protein. We also show that inhibition of SUMOylation rather than the As³⁺-induced robust SUMOylation regulated the number and size of PML-NBs.

Materials and methods

Chemicals

TAK243 (Ub-activating E1 enzyme inhibitor, Selleckchem, Houston, TX) and ML792 (SUMO E1 inhibitor, MedKoo Bioscience, Morrisville, NC) were dissolved in DMSO and used at a final concentration of 0.1% DMSO. A 20S proteasome assay kit was purchased from (Enzo, Famingdale, NY). Bismuth (III) nitrate pentahydrate and antimony (III) chloride were purchased from Wako (Osaka, Japan). They were dissolved in 0.1 M citrate buffer (pH 7.4) at 10 mM and used as stock solutions. Sodium m-arsenite was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in PBS solution. These solutions were sterilized using a syringe filter (0.45 μm pore size). Unless otherwise specified, common chemicals of analytical grade including cadmium sulfate were obtained from Sigma-Aldrich or Wako.

Cells and GFP monitoring

The human PML-VI gene engineered into the pcDNA6.2N-EmGFP DEST vector was transduced into HEK293 and CHO-K1 cells [33]. Stable transfectants were selected using blasticidine S and designated as HEKGFPPML and CHOGFPPML cells, respectively. The cells were cultured in glass-bottom culture dishes and GFP fluorescence was monitored by confocal laser scanning microscopy (TCS-SP5, Leica Microsystems, Solms, Germany) or fluorescence microscopy (Eclipse TS100, Nikon, Kanagawa, Japan).

Protein extraction and western blotting

The cell monolayers were rinsed with Hank’s balanced salt solution (HBSS) and lysed with ice-cold RIPA buffer (Santa Cruz, Dallas, TX) containing protease (Santa Cruz) and phosphatase inhibitor cocktails (Calbiochem/Merk Millipore, San Diego, CA) on ice for 10 min. The lysate was centrifuged at 9,000 g for 5 min at 4°C. The supernatant was collected and labelled as the RIPA-soluble fraction (Sol). The pellet, which contained nucleic acids, was rinsed with PBS and treated with Benzonase^® nuclease (250 U/mL in Tris buffer, Santa Cruz) of the same volume as the RIPA buffer at 25°C for 2 h with intermittent mixing (Thermomixer comfort, Eppendorf, Wesseling-Berzdorf, Germany). The digested sample was labelled as the RIPA-insoluble fraction (Ins). The following antibodies were used for immunoblot analyses, with dilution rates of 1:750 for primary antibodies and 1:2500 for secondary antibodies in Can-Get-Signal^TM solution (TOYOBO, Osaka, Japan): anti-PML antibody (Bethyl, A301-167A, rabbit polyclonal); anti-SUMO2/3 (MBL, 1E7, mouse, monoclonal); anti-multi ubiquitin (MBL, FK2, mouse, monoclonal); anti-SUMO1 (CST, C9H1, rabbit, monoclonal); anti-UBA2 (CST, D15C11, rabbit, monoclonal); anti-GFP (Abcam, goat, polyclonal); HRP-conjugated anti-histone H3 (CST, #12648, rabbit, monoclonal); HRP-conjugated anti-α-tubulin (MBL, #PM054-7, rabbit, polyclonal); HRP-conjugated goat anti-rabbit IgG (Santa Cruz, sc-2054); HRP-conjugated goat anti-mouse IgG (Santa Cruz, sc-2055); HRP-conjugated mouse anti-goat IgG (Santa Cruz, sc-2354). Aliquots of the RIPA-soluble fraction and the nuclease-treated pellet suspension (RIPA-insoluble fraction) were mixed with LDS sample buffer (1x TBS, 10% glycerol, 0.015% EDTA, 50 mM DTT, and 2% LDS) and heated 95°C for 5 min. Proteins in the samples were resolved by LDS (SDS)-PAGE (4–12%) and electroblotted onto PVDF membranes. The membranes were blocked with PVDF Blocking Reagent (TOYOBO) before probing with antibodies. Signals on the ECL (Prime, GE Healthcare, Buckinghamshire, UK)-treated PVDF membrane were then captured with CCD cameras (Lumino Imaging Analyzer, FAS-1100, TOYOBO; Amersham ImageQuant 800, GE Healthcare, Uppsala, Sweden).

Immunofluorescent staining

CHOGFPPML cells were grown in an 8-well chamber slide (Millicell EX slide, Merck Millipore, Burlington, MA) to early confluency. The cells were washed with warmed (37°C) HBSS and fixed with 3.7% formaldehyde solution for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and treated with Image-iT™ FX Signal Enhancer (Invitrogen-ThremoFischer, Carlsbad, CA) for another 10 min. The cells were immunostained with Alexa Fluor® 594-labeled anti-SUMO-2/3 for 45 min. Anti-SUMO2/3 antibody (MBL) was labelled with Alexa Fluor^® 594 using an Alexa Fluor™ 594 Antibody Labeling Kit (Invitrogen-ThermoFischer) and diluted to 1/200 in Can-Get-Signal^TM Immunostain solutions (TOYOBO). DAPI was used to counter-stain the nuclei. The fluorescent images were captured using a fluorescence microscope (Eclipse 80i, Nikon, Tokyo) and the digital images were assembled using Adobe PHOTOSHOP® software.

Immunoprecipitation

The cells were lysed with cold RIPA solution and clear supernatants were obtained by centrifugation (9000 g, 5 min). The protein concentration of the supernatant was adjusted to 2 mg/mL with RIPA. The sample was diluted to one fourth the initial concentration with 150 mM Tris-HCl solution (pH 7.2) and reacted with anti-GFP mAb-magnetic beads (MBL) or control IgG2a-magenitic beads (MBL) at 4–8°C for 2 h with intermittent vortexing. The magnetic beads were washed 3 times with 150 mM Tris-HCl solution (pH 7.2) containing 0.1% NP40 and the pellet was boiled with 2X LDS(SDS)-PAGE sample buffer for 5 min. The sample was centrifuged, then the supernatant was subjected to LDS (SDS)-PAGE and subsequent western blot analysis as described above.

Cell viability

The cells were pre-cultured to early confluency in a 96-well culture dish, then further cultured in the presence or absence of As³⁺, Sb³⁺, Bi³⁺, or ML792 and/or TAK792. The cells were washed twice with HBSS and the viable cell numbers were assayed colorimetrically by WST-8 (Wako) using a microplate reader (POLARstar OPTIMA, BMG Labtech, Offenburg, Germany).

Data analyses

Data were presented as means ± SEM. Statistical analyses were performed by ANOVA followed by Tukey’s post-hoc test. The number and size analyses of PML-NBs were performed using ImageJ (NIH, https://imagej.nih.gov/ij/) or NIS-Elements software (Nikon). A PML-NB agglomerate was defined as an intranuclear assembly of five or more than five PML-NBs in an aggregated form. A chi-squared (χ²) test was applied to compare frequencies of cells with PML-NB agglomerates among groups. A probability value of less than 0.05 was accepted as indicative of statistical significance.

Results

Topology and dynamics of PML-NBs

Most interphase PML-NBs occurred as toroidal and granular dot-like bodies in CHOGFPPML (Fig 1A) and HEKGFPPML cells (Fig 1B), respectively. The number and size of PML-NBs vary among cells from 4 to 27 in the number and ca. 0.3–1.2 μm in diameter (Fig 1C). Neither the number nor the size was significantly changed by exposure to As³⁺ for 2 h. We next studied time-course changes in the shapes of PML-NBs by confocal laser scanning microscopy using the z-stack mode to further interrogate the dynamic nature of PML-NBs. Fig 2A shows that aggregated PML toroids were found after nuclear membrane breakdown in dividing CHOGFPPML cells (0:00:00). Another large toroidal PML aggregate was freshly formed in the peri-nuclear region via condensation of vague small GFP speckles (arrows) in 87 sec (from 0:11:55 to 0:13:22) as cytokinesis proceeded (See also S1 Fig for a freshly formed peri-nuclear PML toroid). Small nascent PML-NBs (arrowheads) were formed as the daughter cells spread in both untreated (Figs 2A and S1) and As³⁺-exposed CHOGFPPML cells (S2 Fig). Although most PML-NBs of HEKGFPPML cells were granular dot-like, a small percentage (0.5%) of HEKGFPPML cells accommodated unstable toroidal PML-NBs (Fig 2B). The sizes of these toroidal PML-NBs were comparable to those of CHOGFPPML cells. The toroidal PML-NBs in HEKGFPPML cells were, however, gradually dissipated and transformed into small dot-like PML-NBs in 1.5 h (boxed areas), suggesting that the toroidal form of PML-NBs in HEKGFPPML cells are less stable than those in CHOGFPPML cells and eventually transformed into the granular dot-like PML-NBs.

Fig 2 — Topological changes of PML–NBs and peri–nuclear PML aggregates in CHOGFPPML (A) and HEKGFPPML cells (B). GFP live images were obtained by confocal laser scanning microscopy in z–stacking mode. (A) Time–lapsed images of CHOGFPPML cells were captured during cell division and spreading. The upper 8 panels show GFP images, and the lower 8 panels show overlaid images with the corresponding bright field (BF). The time counter is shown in the right upper corner of each GFP panel. Arrows show *de novo* appearance of a large peri–nuclear PML aggregate. The accretion of small GFP speckles began at 0:11:55 and the large toroidal PML aggregate was formed clearly after 1 min and 27 sec (at 0:13:22). Arrowheads show small nascent PML–NBs that appeared in daughter cells after cell division. (B) Time–lapsed images of HEKGFPPML cells were captured. The time counter is shown in the right upper corner of each panel. GFP fluorescence images in the boxed areas were enlarged and are shown in the bottom 4 panels. The toroidal PML–NBs were dissipated or dissolved (1:25:57), then were transformed into small granular dot–like PML–NBs (1:52:14) in the non–dividing cell.

Effects of As3+, Sb3+, and Bi3+ on PML and intranuclear distribution of PML-NBs

This experiment was performed to study the specificity of As³⁺ as a modifier of intranuclear dynamics of PML-NBs. Both As³⁺ and Sb³⁺ are metalloids, while Bi³⁺ is a heavy metal belonging to Group 15 of the elements. Bi³⁺ was slightly less cytotoxic than either As³⁺ or Sb³⁺ in CHOGFPPML cells (Fig 3A). Exposure to 3 μM As³⁺ or Sb³⁺ for 2 h induced an overt protein solubility change and converted cold RIPA-soluble GFPPML into the RIPA-insoluble form, and SUMOylated GFPPML. However, these biochemical changes of GFPPML were not observed in Bi³⁺-exposed cells (Fig 3B). We next examined a possibility that As³⁺ and Sb³⁺ inhibited the proteasome activity and SUMOylated PML proteins were not eliminated efficiently by ubiquitin-proteasome system (UPS). The 20S proteasome activity was inhibited slightly by 30 μM As³⁺, Sb³⁺, Bi³⁺, and Cd²⁺. However, Bi³⁺ inhibited more efficiently than As³⁺ and Sb³⁺ (S3 Fig), suggesting that the degradation of SUMOylated PML by UPS was not involved in the different SUMOylation level of PML between Bi³⁺-exposed and As³⁺- or Sb³⁺-exposed cells, although we do not deny a possibility that As³⁺ and Sb³⁺ affected the 26S moiety of proteasomes and decreased the UPS activity

Fig 3 — Changes in cell viability (A), and solubility shift and SUMOylation of PML (B) in response to trivalent arsenic (As), antimony (Sb), or bismuth (Bi) ions in CHOGFPPML cells. (A) The cells were exposed to various concentrations of As³⁺, Sb³⁺, or Bi³⁺ for 24 h. As³⁺ was significantly more cytotoxic than Sb³⁺ and Bi³⁺ in CHOGFPPML cells as analyzed by two–way ANOVA followed by Tukey’s multiple comparison. Data are presented as means ± SEM of quadruplicated wells. (B) The solubility of PML in cold RIPA buffer decreased in response to 2 h–exposure to 1–3 μM As³⁺ or Sb³⁺. GFPPML was SUMOylated (SUMO1) following exposure to either 3 μM As³⁺ or Sb³⁺. These biochemical changes were not observed in Bi³⁺–exposed cells. The major cold RIPA–insoluble proteins observed by Ponseau S–staining are histones. ‘Sol’ and ‘Ins’ denote the RIPA–soluble and–insoluble fractions, respectively.

Contrary to the explicit biochemical changes in GFPPML, the microscopically observed size, number, and intranuclear distribution of PML-NBs did not remarkably change by the 2-h treatment with either As³⁺ or Sb³⁺. However, after 24-h exposure to 1–3 μM As³⁺ or Sb³⁺, PML-NBs agglomerated in the nucleus of CHOGFPPML cells. Exposure to Bi³⁺ did not cause these microscopic changes (Fig 4). The agglomeration was reduced after removal of As³⁺ or Sb³⁺ from the culture medium (Fig 5A), suggesting that the agglomeration of PML-NBs was not an irreversible process. Neither As³⁺-induced solubility shift nor SUMOylation of PML was irreversible phenomena, because removal of As³⁺ from the culture medium reverted those As³⁺-induced biochemical changes in CHOGFPPML cells (Fig 5B) and in HEKGFPPML cells (S4 Fig).

Fig 4 — The cells were exposed to 1 or 3 μM As³⁺, Sb³⁺, or Bi³⁺ for up to 24 h. Contrary to the dramatic solubility change and SUMOylation of PML in response to As³⁺ and Sb³⁺, the intranuclear distribution of PML–NBs was not explicitly changed by 2 h–exposure to As³⁺ or Sb³⁺. After exposure to As³⁺ or Sb³⁺ (1 and 3 μM) for 24 h, however, agglomeration of PML–NBs was observed, while most PML–NBs were evenly distributed in Bi³⁺–exposed and untreated cells. GFP images within the white boxes were enlarged and are shown in the right panels.

Fig 5 — Changes in the percentage of cells with agglomerated PML–NBs (A), and recovery of PML in the RIPA–soluble fraction (Sol) from a 24 h–exposure to As³⁺ in CHOGFPPML cells (B). (A) The cells were exposed to 3 μM As³⁺, Sb³⁺, or Bi³⁺ for 24 h, and further cultured in fresh medium for 16 h. The numbers of cells with the agglomerated PML–NBs were counted at 2, 4, 8, 16, 24, 32 (8 h in recovery), and 40 h (16 h in recovery). At least 300 cells of each group were examined for the presence of agglomerated PML–NBs. The agglomeration was defined as five or more than five PML–NBs in an aggregated form. *, Significantly different among the groups as examined by χ² test. (B) The cells were first exposed to 3 μM As³⁺ for 24 h. The cells were lysed with the RIPA buffer immediately or washed and further cultured in As³⁺–free culture medium for 8 or 24 h before lysis. Lane 1 and 5, untreated; Lane 2 and 6, 24–h exposure to As³⁺; Lane 3 and 7, 24–h exposure to As³⁺and 8–h recovery in As³⁺–free culture medium; Lane 4 and 8, 24–h exposure to As³⁺ and 24–h recovery in As³⁺–free culture medium. The unconjugated GFPPML and GFPPML conjugated with SUMO2/3 in the RIPA–insoluble fraction (Ins) decreased during the culture in As³⁺–free culture medium. An open arrowhead indicates SUMO2/3 monomers.

SUMOylation and ubiquitination of PML

PML proteins are known to be modified by SUMOylation and subsequent ubiquitination by RNF4 E3 ubiquitin ligase in As³⁺-exposed cells [21, 34]. We investigated whether post-translational modification of GFPPML with SUMO and ubiquitin molecules occurs while GFPPMLs are still soluble in cold RIPA. Fig 6 shows that exposure to As³⁺ converted GFPPML from the RIPA-soluble to the RIPA-insoluble form, and SUMOylated GFPPML remained in the RIPA-soluble fraction at 1 h post-exposure in both CHOGFPPML (Fig 6A) and HEKGFPPML cells (Fig 6B). Accordingly, immunoprecipitation was performed using the RIPA-soluble fraction with magnetic bead-tagged anti-GFP antibody using the clear supernatants of RIPA lysates obtained from untreated and As³⁺-exposed (1 h) CHOGFPPML cells (Fig 7A) and HEKGFPPML cells (Fig 7B). Modification on GFPPML with SUMO2/3 and SUMO1, and ubiquitination, were clearly observed in these cells after 1 h-exposure to As³⁺. It should be noted that the amount of SUMOylated GFPPML immunoprecipitated in the untreated cells was less than that of As³⁺-exposed cells.

Fig 6 — Time–course changes in the solubility and SUMOylation of PML following exposure to 3 μM As³⁺ in CHOGFPPML (A) and HEKGFPPML cells (B). The cells were exposed to 3 μM As³⁺ for 0 (untreated), 0.5, 1, and 2 h. SUMOylation of GFPPML was observed in the RIPA–soluble fraction (Sol) after 0.5 h–exposure. The solubility of unmodified GFPPML decreased after 1 h, and most of the GFPPML and SUMOylated GFPPML was recovered in the RIPA–insoluble fraction (Ins) after 2 h–exposure to As³⁺. An open arrowhead indicates SUMO2/3 monomers.

Fig 7 — Immunoprecipitation analyses to detect the SUMOylation and ubiquitination of PML in CHOGFPPML (A) and HEKGFPPML cells (B). The cells were exposed to 3 μM As³⁺ (As) or left untreated (C) for 1 h and the RIPA–soluble fractions were collected. The samples were centrifuged again to obtained clear supernatants and subjected to immunoprecipitation with anti–GFP antibody–magnetic beads. The immunoprecipitates were resolved by SDS–PAGE and subsequent western blotting. The membranes were sequentially probed with anti–multi–Ub, anti–SUMO1, and anti–GFP antibodies (membrane 1) or with anti–SUMO2/3, anti–PML, and anti–GFP antibodies (membrane 2).

Effects of SUMOylation and ubiquitination inhibitors on PML-NBs

Recently, TAK243 (an inhibitor of UBA1-Ub thioester bond formation) and ML792 (an inhibitor of UBA2-SUMO thioester bond formation) were shown to effectively inhibit the ubiquitination and SUMOylation of PML, respectively [35, 36]. Exposure to 20 μM ML792 for up to 8 h did not significantly change the viability of HEKGFPPML cells, although longer exposure slightly reduced the number of viable cells compared to untreated cells (S5A Fig). TAK243 was cytotoxic at a concentration of 10 μM and cells started to shrink after 5 h of culture with TAK243. It is noteworthy that ML792 reduced the cytotoxicity of TAK243 (S5B Fig), suggesting that SUMOylation may enhance the adverse effects caused by the accumulation of ubiquitin-free and improperly folded proteins.

Inhibiting SUMOylation by ML792 reduced the number of PML-NBs and reciprocally increased their sizes in both CHOGFPPML (Fig 8A) and HEKGFPPML cells (Fig 8B), although the size difference was not statistically significant in CHOGFPPML cells. The number of PML-NBs was slightly reduced by TAK243 in CHOGFPPML cells but not in HEKGFPPML cells. These results indicated that the SUMOylation of PML is important to maintain the number and size of PML-NBs.

Fig 9 shows that TAK243 completely inhibited the ubiquitination of RIPA-soluble proteins in both CHOGFPPML (Fig 9A) and HEKGFPPML cells (Fig 9B). In the RIPA-insoluble fraction, the major anti-multi-ubiquitin reactive protein (24 kD) that disappeared upon TAK243 treatment is probably mono-ubiquitinated H2A and/or H2B [37]. SUMOylation of GFPPML with either SUMO1 or SUMO2/3 was inhibited by ML792. SUMO2/3 monomers, present in the RIPA-soluble fraction, essentially disappeared in CHOGFPPML (Fig 9A) and were reduced in HEKGFPPML cells (Fig 9B) after 2 h-exposure to As³⁺. However, the amount of SUMO2/3 monomers was not changed by As³⁺ in ML792-pre-treated cells. Exposure to As³⁺ caused the solubility shift and SUMOylation of GFPPML. Although SUMOylation was inhibited by ML792, a substantial amount of non-SUMOylated GFPPML was recovered in the RIPA-insoluble fraction after exposure to As³⁺, suggesting that SUMOylation is not the cause but rather the consequence of the solubility change of GFPPML. Alternatively, the solubility shift and SUMOylation of GFPPML may occur independently in response to As³⁺. UBA2, a SUMO-activating enzyme, was not detected in the RIPA-insoluble fraction, regardless of exposure to As³⁺.

Finally, we investigated the intracellular distribution of SUMO2/3 when SUMOylation was inhibited. CHOGFPPML cells were pre-cultured in the presence or absence of ML792 and exposed to As³⁺ or left untreated for 2 h before immunostaining with anti-SUMO2/3. It is of interest to note that the ratio of SUMO2/3 to PML of the peri-nuclear PML aggregates was lower than that of PML-NBs regardless of As³⁺-exposure in the absence of ML792 as judged by the relative fluorescence intensities (arrows, Fig 10). In contrast, the SUMO/PML ratio was almost the same between peri-nuclear PML aggregates and PML-NBs when the cells were pre-treated with ML792, suggesting that ML792 disturbs the intranuclear restoration of SUMO molecules.

Fig 10 — The cells were treated with 0.1% DMSO or 20 μM ML792 for 3 h, then further treated with 3 μM As³⁺ or left untreated for 2 h. Note that SUMO2/3 in peri–nuclear PML aggregates (arrow) was decreased in the DMSO–treated cells, whereas a substantial amount of SUMO2/3 retained with GFPPML in the ML792–treated cells. Scale bar = 10 μm.

Discussion

PML-NBs and peri-nuclear PML aggregates

Most PML-NBs occurred in the toroidal shape in CHOGFPPML cells and in the granular dot-like shape in HEKGFPPML cells (Figs 1 and 2). In addition to PML-NBs, peri-nuclear PML aggregates appeared to be accretions of several toroidal PML bodies in CHOGFPPML cells. These observations suggest that the CHO cells have a propensity for toroidal PML-NBs, although it is not clear why the annular condensation of GFPPML occurs in CHO cells. Given that SUMOylation of GFPPML proceeded normally in both cell types, PML-NBs may need some other scaffold or client molecules to form stable toroidal shapes. The disassembly of large toroidal PML-NBs into small granular dot-like PML-NBs in the HEKGFPPML cell nucleus (Fig 2B) indicates that PML-NBs are not rigid aggregates, and that toroidal PML-NBs in HEK cells are more labile than those in CHO cells. Non-spherical PML-NBs are prone to forming hydrogels with less fluidity in the sol-gel balance of non-membrane bodies because a liquid-like nature would make these bodies spherical due to viscous relaxation [38]. Our current finding that toroidal PML-NBs were converted into small granular dot-like PML-NBs in the HEKGFPPML cell nucleus may shed light on the rheological nature of PML-NBs.

A systematic regulatory network deciphering study indicated that most cells with increased PML-NBs contain S phase DNA and low 5-ethynyl-2’-deoxyuridine (EdU) incorporation, suggesting that increased PML-NBs correlate with decreased DNA replication rates [39]. It has been reported that the number of PML-NBs increases in early S phase in SK-N-SH and GFP-PML-IV transduced U-2OS cells due to fission of the parent NBs and not to increased PML protein levels [10]. Although the transformation of large toroidal PML-NBs into small granular dot-like ones in HEKGFPPML cells (Fig 2B) appeared to begin with dissolution or dissipation rather than fission in the present study, the intranuclear dynamics of PML-NBs may be fine-tuned with DNA synthesis.

Effects of As3+, Sb3+, and Bi3+on PML

One of the most notable biochemical changes in As³⁺-exposed cells was the solubility shift from the cold RIPA-soluble to the RIPA-insoluble form, and SUMOylation of PML [40, 41]. These changes were also observed in Sb³⁺-exposed but not in Bi³⁺-exposed GFPPML-expressing cells (Fig 3), which is consistent with findings using other cell types [13, 42]. Bi³⁺ forms a stable complex with metallothionein in the cytosol and might not efficiently reach the nucleus [43]. The solubility shift and SUMOylation of PML appear to be induced by TGF-β, as well as by As³⁺ or Sb³⁺ [44]. The PML protein level, and the number and size of PML-NBs, are increased by IFNα in BL41 cells; however, the solubility change of PML does not occur in IFNα-treated cells. In contrast, TGF-β disrupts PML-NBs and changes PML from the RIPA-soluble (nucleoplasm) to the RIPA-insoluble form (nuclear matrix), and further enhances the SUMOylation of PML. Accordingly, a large amount of SUMOylated PML was found in the RIPA-insoluble fraction in TGF-β-treated and IFNα-pre-treated cells [44]. However, the involvement of cytokines in As³⁺-exposed cells remains to be elucidated. Intriguingly, prolonged exposure to As³⁺ caused agglomeration of PML-NBs which was reversible after removal of As³⁺ from the culture medium (Figs 4 and 5). To our best knowledge, this is the first quantitative study to report the agglomeration of PML-NBs. Since the agglomeration of PML-NBs was observed 4 h after exposure to As³⁺, either solubility change or SUMOylation of PML or both may lead to the agglomeration.

A role of SUMOylation in PML-NBs

MAPPs are aggregated forms of PML that appear after nuclear membrane breakdown, and CyPNs are formed in the peri-nuclear regions of daughter cells after mitosis [45]. MAPPs and CyPNs appear to be devoid of SUMO, Sp100, and Daxx, which are well-known PML-NB client proteins [46, 47]. We did not differentiate MAPPs and CyPNs, referring to these PML extranuclear PML bodies as peri-nuclear PML aggregates in the present study. Although the biological functions of these peri-nuclear PML aggregates have not been elucidated [48], the aggregates can be used as markers of laminopathies [49]. Our findings that PML colocalized with SUMO2/3 in PML-NBs and peri-nuclear PML aggregates contained less SUMO2/3 are consistent with these previous studies. SUMO2/3 distributed almost evenly between PML-NBs and peri-nuclear PML aggregates in the presence of ML792, an inhibitor of SUMO-activating enzymes, irrespective of As³⁺ exposure (Fig 10). Since SUMOylation of GFPPML was almost completely inhibited by ML792 (Fig 9), it is plausible that SUMO monomers non-covalently associate with peri-nuclear PML aggregates as well as with PML-NBs in the presence of ML792.

The 69-kDa isoform of PML mutated at the B-box (B1C17C20AΔAla, B1C25C28AΔAla, and B2C21C24ΔAla) does not form PML-NBs and is instead present diffusely over the nucleus [11]. The three main SUMOylation sites (K65, 160, and 490) and the SIM are not essential for formation of the PML multimer lattice or the outer shell of PML-NBs. SUMO2/3 appears to be more closely associated with PML compared to other partner proteins such as Sp100, Daxx, SUMO1, and RNF4 [50]. In addition, depletion of SUMO3 reduces the number of PML-NBs [51]. Recently, PML-specific sequences (F52QF54 and L73) were shown to play essential roles in PML-NB assembly, recruitment of Ubc9, and SUMOylation [16]. The depletion of SUMO-specific protease 1 (SUSP1/SENP6), which dismantles highly conjugated SUMO2/3 proteins, caused marked accumulation of SUMO2/3 in PML-NBs [52] and increased the average number of PML-NBs per nucleus from 3.7 to 5.9 in HeLa cells [53]. Thus, the balance of SUMOylation and deSUMOylation of PML is critical for the appropriate maintenance of PML-NBs. In addition to SUMOylation and deSUMOylation of PML, PML-NBs might be regulated by non-PML-NB-associated proteins. The expression of either Ki-1/57 or CGI-55 proteins, which have three SUMOylation sites, reduces the number of PML-NBs in both untreated and As³⁺-treated HeLa cells, whereas the expression of Ki-1/57 mutant (K/3R) reduces the number of PML-NBs in untreated cells but not in As³⁺-treated cells [54]. The inhibition of SUMOylation by ML792 increased the size of PML-NBs in the present study (Fig 8), suggesting that the SUMOylation indeed regulates the scaffold structure of PML-NBs.

Blocking ubiquitination using TAK243 resulted in the build-up of SUMOylated proteins in HeLa and U-2OS cells, with PML-NBs apparently functioning as primary accumulation sites for newly synthesized and SUMOylated proteins [36]. Ubiquitination by RNF4 E3 ligase via the reciprocal SUMO-SIM interaction is proposed to be the main cascade for the proteasomal degradation of SUMOylated PML-RARα [8, 19, 55]. PML-NBs may be transiently employed to store harmful SUMOylated proteins by phase separation before proteolysis by the ubiquitin-proteasome system [56]. However, functional roles of SUMOylated PML remain elusive, because modification with SUMO protects IκBα [57] and Glis2/NPHP7, a Cys2/His2 zinc finger transcription factor [58], from ubiquitin-proteasome degradation by competing the same lysine [3]. An ancillary but interesting finding in the present study is that the cytotoxic effect of TAK243 was antagonized by ML792 (S5 Fig), suggesting that SUMOylation may be harmful to the cells when ubiquitination is inhibited.

Conclusions

In summary, topological features of PML-NBs were different between CHO and HEK cells, although biochemical responses of PML to As³⁺ such as solubility shift and SUMOylation were almost the same between these two different cells. The solubility shift of PML occurred prior to SUMOylation. The number, size, and shape of PML-NBs were not changed by 2-h exposure to As³⁺. However, the prolonged exposure to As³⁺ or Sb³⁺ caused agglomeration of PML-NBs which is reversed after removal of these metalloids from the culture medium. The present results warrant the importance of SUMO-dependent and -independent dynamic nature of PML-NBs which are implicated in phase separation cell biology and human diseases.

Supporting information

S1 Fig. Emergence and uneven partitioning of peri-nuclear PML aggregates in dividing untreated CHOGFPPML cells.

Live GFP images were captured during cell division by confocal laser scanning microscopy in z-stacking mode. The upper and middle 8 panels show GFP alone and the corresponding GFP-bright field overlaid images, respectively. The lower 8 panels show enlarged and intensified GFP images of a peri-nuclear PML aggregate indicated with arrows on the top four panels with four additional time-lapsed GFP images. The time counter is shown in the right upper corner of each panel. The peri-nuclear PML aggregates appear to be comprised of 2–4 toroids. The nascent small PML-NBs (arrowheads) appeared as the daughter cells spread.

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S2 Fig. Uneven partitioning of peri-nuclear PML aggregates in As³⁺-exposed CHOGFPPML cells.

Image capture started 30 min after the addition of As³⁺. The upper and lower 10 panels show GFP images alone and the corresponding GFP-bright field overlaid images, respectively. The nascent small PML-NBs (arrowheads) appeared as the daughter cells spread. See also the legend to S1 Fig.

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Click here for additional data file.^{(407.1KB, pdf)}

S3 Fig. Inhibition of proteasomal activity by As³⁺, Sb³⁺, Bi³⁺, and Cd²⁺.

The proteasomal inhibitory effect was assayed using a 20S proteasome assay kit. Briefly, the fluorescence (Ex/Em, 360/460 nm) of the reaction mixture (proteasome, Suc-LLVY-AMC, and SDS) was monitored every 1 min in the presence or absence of As³⁺, Sb³⁺, Bi³⁺, Cd²⁺, or epoxomicin (a positive control) at 30°C for 20 min using a microplate reader (Infinite M Plex, TECAN, Männedorf, Switzerland). As³⁺, Sb³⁺, Bi³⁺, and Cd²⁺ were added to the reaction mixture at a final concentration of 30 μM. The fluorescence was increased linearly and the increment of fluorescence in 20 min was used for evaluation of relative inhibitory activity of proteasome. Data are presented as means ± SEM of 4 replicate measurements.

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S4 Fig. Recovery of SUMO2/3 and GFPPML proteins in the RIPA-soluble fraction (Sol) from 24 h-exposure to As³⁺ in HEKGFPPML cells.

The cells were exposed to 3 μM As³⁺ for 24 h or left untreated. The As³⁺-exposed cells were lysed with the RIPA buffer immediately or washed and further cultured in As³⁺-free culture medium further for 8 or 24 h. 1, untreated; 2, 24 h exposure to As³⁺; 3, 24 h exposure to As³⁺and 8 h recovery in As³⁺-free culture medium; 4, 24 h exposure to As³⁺and 24 h recovery in As³⁺-free culture medium. An open arrowhead indicates SUMO2/3 monomers. The unconjugated GFPPML and GFPPML conjugated with SUMO2/3 in the RIPA-insoluble fraction (Ins) decreased during the culture in As³⁺-free culture medium.

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S5 Fig

Time-course changes in the number of viable ML792-exposed cells (A) and the inhibitory effects of ML792 on the cytotoxicity of TAK243 (B) in HEKGFPPML cells. (A) The cells were exposed to 0.1% DMSO (open columns) or 20 μM ML792 (hatched columns) for 2, 4, 8, 15 h. The number of viable cells was assayed using WST-8 agent. Data are presented as means ± SEM of five wells. (B) The cells were exposed to 0, 2.5, 5, and 10 μM TAK243 for 24 h in the presence or absence of 20 μM ML792. Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparison. The viability of ML792-treated cells was significantly elevated compared to 0.1% DMSO-treated cells in the presence of 10 μM TAK243.

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S1 Raw images

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S1 Data

(XLSX)

Click here for additional data file.^{(45KB, xlsx)}

Acknowledgments

The authors would like to thank Dr. Ayaka Kato-Udagawa and Ms. Mihoko Tadano for their technical assistance.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was partially supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (16K15386 given to SH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0268835.r001

Decision Letter 0

Claude Prigent

30 Dec 2021

PONE-D-21-36000Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodiesPLOS ONE

Dear Dr. Hirano,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised during the review process.

The experiments are well conducted but the originality and significance of the data can be better presented and exploited.

I invite you to rewrite the manuscript to better highlight the biological questions addressed and the original results obtained. The manuscript also needs English proofreading to make the style clearer

Please improve the quality of microscopy images.

Please define more precisely the different types of bodies they mention (toroid bodies, annular bodies, amorphous bodies, small dots, …) and provide metrics to characterize these bodies and their dynamics.

Please extend the discussion to better explain the significance of your findings on the understanding of PML biology

Please submit your revised manuscript by Feb 09 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Claude Prigent

Academic Editor

PLOS ONE

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The authors would like to thank Dr. Ayaka Kato-Udagawa and Ms. Mihoko Tadano for their technical assistance. This work was partially supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (16K15386).

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors analyze in the manuscript the effect of Arsenic on the solubility of the PML protein (isoform VI), using 2 different cell lines stably expressing exogenous, GFP-tagged PML-VI. They document that PML-nuclear bodies (PML-NBs) have different shapes and stability depending on the cells. They show that treatment of the cells for 2hrs with 3µM Arsenic (As3+) and also to as lesser extent antimony (Sb3+), but not bismuth (Bi3+), induces PML SUMOylation together with its relocalization from a soluble to an insoluble compartment, without affecting size, number and intranuclear distribution of PML NBs. However, upon prolonged (24 hrs) treatment, PML NBs agglomerated in the nucleus, in a reversible manner. The authors further show that upon As3+ treatment PML SUMOylation precedes its relocalization, but is not necessary for this relocalization. Rather, PML SUMOylation appears important to maintain the number and size of PML NBs.

General comments:

Overall, I find the manuscript technically sound, with data that support the conclusions presented by the authors. What remains unclear to me is the originality of the issues addressed in the manuscript, and consequently the novelty of the conclusions, as to my knowledge most of the information found in the manuscript has already been published previously. It seems to me that the interest of the manuscript could be greatly improved if the authors extensively rewrite it to better highlight the biological questions they want to address and the original results they obtained, while reducing the length given to what is already known.

Specific concerns:

- the authors analyze in the manuscript the isoform VI of PML, which is lacking a SUMO-interacting motif present in the other isoforms. Isn't this choice a concern when studying the solubility and the SUMOylation of the protein? I think this point should be discussed.

- the quality and resolution of the microscopy images is low (as judged from the pdf available for reviewing) and should be increased. Actually, it would be useful for many readers to display the exact contour of the nuclei, either by outlining them in the images of by adding photos of a DAPI (or equivalent) staining.

- I am not sure of the pertinence of testing the possible inhibition of the proteasome by As3+ and Sb3+ on 20S proteasome only. Indeed, one could imagine that these compounds inhibit more the 26S than the 20S proteasome, by interfering not directly with the proteolytic activities but with other activities or properties of the 26S proteasome. To strengthen this point, one possibility would be to better document that there is no accumulation of ubiquitylated proteins in cells upon these treatments, as indicated by the input fractions in Fig.7.

- if overall the quality of the English is adequate, the reading is sometimes difficult and it seems to me that the manuscript would benefit from editing by a native English speaker to make the style clearer. As it is, the meaning and therefore the conveyed message of some sentences is sometimes obscure. For example, may be I missed the point but I could not understand the sentence "suggesting that the topology was retained in the extranuclear region" in the first paragraph of the results.

- the use of the term 'life-time' in the introduction is not correct. The authors probably meant 'residence-time'?

- Fig 10 legend: SUMO2/3 is more (not less) abundant in peri-nuclear PML aggregates in ML792-treated cells

Reviewer #2: The promyelocytic leukemia (PML) protein is regulating multiple cellular activities (including DNA repair, apoptosis and senescence) and plays a critical role in oncogenesis and tumor progression. PML is sensitive to arsenic and regulated by SUMOylation and ubiquitylation. It is also well known to accumulate in nuclear bodies which are important for its activity. In this manuscript, the authors investigate the effect of arsenic and other metalloids on the accumulation of PML in nuclear bodies, its solubility and its SUMOylation and ubiquitylation. The results that are presented are solid are will certainly contribute to a better understanding of the interplay between arsenic treatment, PML posttranslational modifications, and PML bodies formation, although their novelty and significance is not obvious for non-specialists.

Major comments:

1) The authors have studied the topology of PML nuclear bodies and their dynamics in two cell lines. They have quantified the size and number of PML bodies, but most of their other observations on the shape of the bodies, remain qualitative, poorly documented and difficult to visualize in the figures that are provided. They should more precisely define the different types of bodies they mention (toroid bodies, annular bodies, amorphous bodies, small dots, …) and provide metrics to characterize these bodies and their dynamics.

2) The authors should complete their discussion and conclusion to better explain the significance of their findings on the understanding of PML biology.

Other comments:

3) Figure 1 and Figure S1: The quantification provided in Figure S1 is important and should be included in the main Figure rather than in the supplementary data.

4) The PLOS data policy requires authors to make all data underlying the findings described in their manuscript fully available. Thus, the individual data points used for the plots of Figures 3A, 4A, 8A, 8B, S1 S4 and S6 should be provided, either as scatter plots or in supplementary tables.

5) The legend of Figure 8 should state whether the indicated variations of PML nuclear bodies number and size are SD or SEM. Actually, I encourage the authors to use SD rather than SEM as summary statistics for this and all other figures as this enables the reader to evaluate data dispersion.

6) Figure annotations: The annotations of many figures could be improved to facilitate their understanding and interpretation. For instance, in Figure 1 the panel A and B represent similar experiments performed with two different cell lines (CHOGFPPML and EKGFPPML). The authors should include the names of these cell lines in the corresponding panels of this (and other) Figure(s). Similarly, in Figure 5B, the lanes 1,2,3 and 4 correspond to different time points of As3+ treatment. This could be explicitly notated in the Figure panel rather in the legend. These two examples are not exhaustive and I encourage the authors to think for each Figure panel whether more information could be provided to improve their legibility.

7) The authors should state the mechanism of action of TAK243 and ML792 in the main text.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Olivier Coux

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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PLoS One. 2022 May 20;17(5):e0268835. doi: 10.1371/journal.pone.0268835.r002

Author response to Decision Letter 0

24 Jan 2022

Very helpful. Thanks all.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(24.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0268835.r003

Decision Letter 1

Claude Prigent

14 Mar 2022

PONE-D-21-36000R1Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodiesPLOS ONE

Dear Dr. Hirano,

As you will see from their comments, both reviewers consider the revisions to the manuscript, while they improve the manuscript, to be insufficient to allow publication.

I am willing to propose a second round of minor revisions, but you will need to address all of their requests (see reviewer #1's list).

For reviewer #2, the definition and quantification of bodies still needs improvement. The text also needs to be improved as suggested.

Translated with www.DeepL.com/Translator (free version)

Please submit your revised manuscript by Apr 28 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Claude Prigent

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have addressed the concerns made by the 2 reviewers, but unfortunately in the minimal way: the changes mostly concern the text of the manuscript, and fail to really clarify the biological significance of the presented findings and to make the manuscript easier to read. Overall, I still find the manuscript weak, both in the solidity and in the originality of the conclusions that can be drawn from the experiments.

Having said that, the presented data globally support the conclusions proposed by the authors, and I think the manuscript could be published, depending of the opinion of the other reviewer and of the editor.

However, I thing that the following modifications, noted as I read the revised manuscript, should be made before acceptance for publication, as they will help to improve the readability:

> first paragraph: the residence-time of PML ranges --> the residence-time of PML in PML bodies ranges...

> beginning of second paragraph: define B1 and B2 domains

> second paragraph: The unique subdomain appears to play a role in PML tetramer --> tetramerization? Same sentence: the following subsequent PML-NB formation --> remove either following or subsequent as both terms are redundant

-Material and methods: the real term is confoncal laser scanning microscopy and not confoncal laser microscopy

- Results: the definition of what the authors consider toroidal or irregular-shaped punctates is still unclear: this makes it difficult to visualize in the images the structures referred to in the text. Typically, I don't understand how the authors can conclude that in HEKGFPPML cells, the "occasionally found" toroidal PML-NB are those that dissipate in small PML-NBs.

> page 9, last sentence before Fig. 3 legend: ..: a possibility that that...

> Fig 5A: the m of agglomerates is missing in the vertical legend of the graph

>Fig 7: the authors must comment why there is less immunoprecipitated GFPPML from the soluble fraction of untreated cells than from that of As-treated cells. Shouldn't the opposite be expected according to the results shown in Fig.3?

> Fig 8: the meaning of the superscript symbols used in the tables should be explained

- Sup data:

> legend of S2 refers to the legend of S2

Reviewer #2: In their revised manuscript, the authors answered many questions of the reviewers. The implemented changes improved the overall quality of the manuscript. In particular, the authors now provide in a supplementary file the original data underlying most of the plots presented in the different figures of the manuscript. This is a significant improvement, not only because it is a requirement from the PLOS data policy, but also because it enables to assess data variability, which is not represented in the plots (the error bars represent SEM, not SD). The authors also improved the annotations of several figure so that they are now easier to understand. Other small qualitative improvements have also been properly implemented, as listed by the authors in their point-to-point answer.

Yet, in my opinion, two of the most critical concerns that I and the other reviewer had raised were not properly addressed:

1) I had asked the authors to precisely define and quantify the different types of bodies they observe. In response to this request, the authors simplified the naming of the different structures. Still, I have difficulties with the qualitative distinction that is made between the toroidal structures vs the “punctates” (actually, although I am not a native English speaker, I am not sure that using the word “punctate” as a noun is appropriate: I think it is mainly used as an adjective). These two types of structures are very difficult to distinguish on the Figure images, which makes this distinction difficult to understand and evaluate. Wouldn’t it be better (and more meaningful?) to categorize the different types of structures by their apparent size on 2D images (or volume when 3D information is available) rather than by their qualitative shape (toroidal vs punctate)? Furthermore, the authors could then use this metrics all along their manuscript to more quantitatively describe the transitions that they observe during the cell cycle and after heavy metal treatment. I believe this would be more rigorous and informative than the current classification.

2) Reviewer 1 asked the authors to “extensively rewrite” the manuscript and stressed that “the reading is sometimes difficult”. Similarly, I asked the authors to “better explain the significance of their findings”. Although some text changes have been made, the overall manuscript remains difficult to read and the conveyed messages are not always clear. In my opinion, more efforts could and should be made to more clearly explain the context of the study, the rationale of the experiments, the main findings and their biological implications. Actually, in some cases the implemented text changes did not improve clarity. For instance, I do not understand the meaning of the new sentence “the SUMO-SIM interaction, which is implicated in liquid-liquid phase separation [32], occurs only when PML-VI is SUMOylated” (page 4) which the authors added in response to a question of reviewer 1. How can there be a “SUMO-SIM” interaction if PML-VI has no SIM ? Do the authors refer to interactions with the SIM of other proteins? As another example, the authors replaced in the introduction the term “life-time” by “residence-time” (page 2). Indeed, as noted by reviewer 1, the term “life-time” was not correct. But the authors omitted to rephrase the rest of the sentence, which in my opinion is still unclear. To clarify this sentence, the authors simply need to explicitly write that what they describe is the “residence-time of PML in nuclear bodies”. Thus, I really encourage the authors to make further efforts to improve the clarity of their manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Olivier Coux

Reviewer #2: No

PLoS One. 2022 May 20;17(5):e0268835. doi: 10.1371/journal.pone.0268835.r004

Author response to Decision Letter 1

14 Apr 2022

Valuable comments.

Attachment

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.^{(249.6KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0268835.r005

Decision Letter 2

Claude Prigent

10 May 2022

Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodies

PONE-D-21-36000R2

Dear Dr. Hirano,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Claude Prigent

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0268835.r006

Acceptance letter

Claude Prigent

13 May 2022

PONE-D-21-36000R2

Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodies

Dear Dr. Hirano:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Claude Prigent

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Emergence and uneven partitioning of peri-nuclear PML aggregates in dividing untreated CHOGFPPML cells.

(PDF)

Click here for additional data file.^{(505.4KB, pdf)}

S2 Fig. Uneven partitioning of peri-nuclear PML aggregates in As³⁺-exposed CHOGFPPML cells.

(PDF)

Click here for additional data file.^{(407.1KB, pdf)}

S3 Fig. Inhibition of proteasomal activity by As³⁺, Sb³⁺, Bi³⁺, and Cd²⁺.

(PDF)

Click here for additional data file.^{(147.3KB, pdf)}

S4 Fig. Recovery of SUMO2/3 and GFPPML proteins in the RIPA-soluble fraction (Sol) from 24 h-exposure to As³⁺ in HEKGFPPML cells.

(PDF)

Click here for additional data file.^{(246.2KB, pdf)}

S5 Fig

(PDF)

Click here for additional data file.^{(148.5KB, pdf)}

S1 Raw images

(PDF)

Click here for additional data file.^{(2.9MB, pdf)}

S1 Data

(XLSX)

Click here for additional data file.^{(45KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(24.7KB, docx)}

Attachment

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.^{(249.6KB, pdf)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodies

Seishiro Hirano

Osamu Udagawa

Roles

Abstract

Introduction

Materials and methods

Chemicals

Cells and GFP monitoring

Protein extraction and western blotting

Immunofluorescent staining

Immunoprecipitation

Cell viability

Data analyses

Results

Topology and dynamics of PML-NBs

Fig 1.

Fig 2.

Effects of As3+, Sb3+, and Bi3+ on PML and intranuclear distribution of PML-NBs

Fig 3.

Fig 4. Agglomeration of PML–NBs in response to As3+ or Sb3+ in CHOGFPPML cells.

Fig 5.

SUMOylation and ubiquitination of PML

Fig 6.

Fig 7.

Effects of SUMOylation and ubiquitination inhibitors on PML-NBs

Fig 8.

Fig 9.

Fig 10. Fluorescent immunostaining of ML792–pretrreated CHOGFPPML cells to detect PML and SUMO2/3.

Discussion

PML-NBs and peri-nuclear PML aggregates

Effects of As3+, Sb3+, and Bi3+on PML

A role of SUMOylation in PML-NBs

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Claude Prigent

Roles

Author response to Decision Letter 0

Decision Letter 1

Claude Prigent

Roles

Author response to Decision Letter 1

Decision Letter 2

Claude Prigent

Roles

Acceptance letter

Claude Prigent

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Fig 4. Agglomeration of PML–NBs in response to As³⁺ or Sb³⁺ in CHOGFPPML cells.