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. 2013 Sep 19;14(12):1158–1166. doi: 10.4161/cbt.26525

The DeISGylase USP18 limits TRAIL-induced apoptosis through the regulation of TRAIL levels

Cellular levels of TRAIL influences responsiveness to TRAIL-induced apoptosis

Ivana Manini 1,†, Andrea Sgorbissa 1,†, Harish Potu 1, Andrea Tomasella 1, Claudio Brancolini 1,*
PMCID: PMC3912039  PMID: 24153058

Abstract

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising molecule for anti-cancer therapies. Unfortunately, cancer cells frequently acquire resistance to rhTRAIL. Various co-treatments have been proposed to overcome apoptosis resistance to TRAIL. Here we show that downregulation of the deISGylase USP18 sensitizes cancer cells to rhTRAIL, whereas, elevate levels of USP18 inhibit TRAIL-induced apoptosis, in a deISGylase-independent manner. USP18 influences TRAIL signaling through the control of the IFN autocrine loop. In fact, cells with downregulated USP18 expression augment the expression of cellular TRAIL. Downregulation of cellular TRAIL abrogates the synergism between TRAIL and USP18 siRNA and also limits cell death induced by rhTRAIL. By comparing the apoptotic responsiveness to TRAIL in a panel of cancer cell lines, we have discovered a correlation between TRAIL levels and the apoptotic susceptibility to rhTRAIL, In cells expressing high levels of TRAIL-R2 susceptibility to rhTRAIL correlates with TRAIL expression. In conclusion, we propose that cellular TRAIL is an additional factor that can influence the apoptotic response to rhTRAIL.

Keywords: IFN-α, IFNAR, TRAIL, TRAIL-R2, USP18, autocrine loop

Introduction

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) belongs to the TNF superfamily of extracellular signals. TRAIL has been subjected of intense research because of its specific pro-apoptotic activity against transformed cells.1 TRAIL is exposed at the surface of many cell types and its expression is stimulated by IFNs.2,3 Five TRAIL receptors have been identified, of which only two, DR4/TRAIL-R1 and DR5/TRAIL-R2, can engage the extrinsic apoptotic pathway.4-6

The resistance to TRAIL induced apoptosis, frequently acquired by cancer cells has limited the therapeutic possibilities of recombinant human TRAIL (rhTRAIL), or agonistic antibodies against its receptors.1,7 Neoplastic cells adapt different strategies to limit the apoptotic influence of TRAIL including increased expression of: decoy receptors,8 cFLIP,9 X-linked inhibitor of apoptosis (XIAP), and of anti-apoptotic Bcl-2 family members.10-12 Also the engagement of different signaling pathways such as the NFκB and the PI3K/Akt can influence the responsiveness to TRAIL.13,14

Several research groups have explored the use of different compounds or treatments to overcome resistance to TRAIL-induced apoptosis. A combined approach could represent an ideal therapy to elicit apoptosis in resistant cells.1,7 Histone deacetylase inhibitors, the proteasome inhibitor bortezomib, or compounds that trigger ER-stress can synergize with TRAIL to kill resistant cells.15-17 Bortezomib, for example can sustain TRAIL induced apoptosis through multiple mechanisms. In several cancer cells bortezomib can elicit ER-stress, Noxa and DR5 accumulation,18,19 in glioblastoma cells bortezomib stabilizes the cleaved form of Bid (tBid)20 and inhibits the NFκB pathway.21 In malignant B cells it can suppress Bax degradation.22

Also interferons can sensitize neoplastic cells to the pro-apoptotic efforts of TRAIL.23 Moreover, TRAIL is an ISG (interferon stimulated gene), which plays a key role in the apoptotic response to IFNs.24,25 Hence cells treated with IFN express higher amounts of TRAIL and are more prone to die in response to TRAIL. However, it is unclear whether the boost of TRAIL production sensitizes cells to treatments with exogenous TRAIL.

ISGs production, including TRAIL, in response to IFNs is a timely regulated process that operates through negative feedback loops. The ISG USP18 is an important element of these negative feedback loops. USP18 is a component of the molecular machinery that modifies protein through the addition of the Ubl protein ISG15.26 Viral infection and the interferon response elicit protein ISGylation through an enzymatic cascade similar to the system for poly-ubiquitination, with dedicated E1, E2, and E3 enzymes.27 This response is an important piece of the antiviral defense system. USP18 reverse protein ISGylation by removing the conjugated ISG15 from target proteins.26 In addition to the deISGylase function, USP18 acts as negative regulator of IFN-signaling by suppressing STAT activation.28

We have recently identified in USP18 an important regulator of the apoptotic response to interferons and to other pro-death stimuli such as proteasome inhibitors and genotoxic stresses.29 The pro-survival function of USP18 depends on its ability to buffer the spontaneous interferon signaling. In this manuscript we have investigated the ability of USP18 to modulate TRAIL-induced apoptosis in the glioblastoma cell line T98G. Our data indicate that USP18 limits the apoptotic influence of rhTRAIL.

Results

INF-α pretreatment sensitizes T98G glioblastoma cells to TRAIL-induced apoptosis

T98G glioblastoma cells are susceptible to TRAIL-induced apoptosis and in this cell line is active the spontaneous interferon response.30 Hence, they represent a good model to investigate the synergisms between TRAIL ad the IFN signaling. To evaluate the effect of the IFN-α treatment on apoptosis responsiveness to TRAIL, T98G cells were treated with IFN-α and next incubated with increasing amounts of rhTRAIL. Cell death was scored by trypan blue staining 24 h later. Figure 1A illustrates that pre-treatment with IFN-α sensitizes T98G cells to cell death induced by rhTRAIL. To prove that cell death under the described experimental conditions assumed the characteristic of apoptosis, we also evaluated caspase-3/-7 activation. Also in this case caspase activity in response to TRAIL was augmented in cells pre-incubated with IFN-α (Fig. 1B).

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Figure 1. IFN-α pretreatment sensitizes T98G glioblastoma cells to TRAIL-induced apoptosis. (A) T98G cells were pre-treated with IFN-α (1000 units/ml). Twelve hours later cells were treated or not, for further 24 h with increasing doses of rhTRAIL as indicated. Cell death was evaluated by trypan blue staining. Columns, mean (n = 3); bars, ± SD. (B) T98G cells were pre-treated with IFN-α. Twelve hours later cells were treated or not, for further 24 h with increasing doses of rhTRAIL as indicated. Apoptosis was evaluated by scoring caspase activity (DEVDase activity).

USP18 downregulation increases the responsiveness to TRAIL-induced apoptosis

USP18 is a negative regulator of the IFN signaling. Downregulation of USP18 increases apoptosis in response to different stimuli including DNA damage, inhibition of protein degradation, and ER-stress. The pro-survival effect of USP18 is correlated with its function as inhibitor of the spontaneous interferon signaling.29

To evaluate whether USP18 can influence TRAIL induced apoptosis we generated T98G cells stably expressing a shRNA specific for USP18. Clones were isolated and treated with IFN-α to evaluate the robustness of silencing. In the isolated clones sh#3 and sh#12, upregulation of USP18 was impaired compared with a clone expressing the control shRNA (Fig. 2A). Next we evaluated the apoptotic response to IFN-α treatment. T98G cells with impaired USP18 expression entered cell death after IFN-α treatment, whereas the control cell line was resistant (Fig. 2B). Finally we explored whether impaired USP18 expression could augment apoptosis in response to TRAIL treatment. The different cell lines were incubated with increasing amounts of rhTRAIL and cell death was scored 24 h later. Figure 2C shows that TRAIL-induced cell death was increased in cells with reduced levels of USP18. The observed cell death assumed the characteristic of apoptosis, as testified by the caspase assay in Figure 2D.

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Figure 2. USP18 downregulation increases the responsiveness to TRAIL-induced apoptosis. (A) Analysis of the expression levels of USP18 in T98G cells expressing a shRNA against USP18 or a control shRNA (shPuro). Lysates from T98G clones silenced as indicated and treated for 24 h with IFN-α were prepared and subjected to immunoblot analysis using the anti-USP18 antibody. Nucleoporin p62 was used as loading control. (B) T98G clones expressing a shRNA against USP18 or a control shRNA (shPuro) were treated or not, for 24 h with IFN-α. Cell death was evaluated by trypan blue staining. (C) T98G clones expressing a shRNA against USP18 or a control shRNA (shPuro) were treated or not for 24 h with increasing doses of rhTRAIL. Cell death was evaluated by trypan blue staining. (D) T98G clones expressing a shRNA against USP18 or a control shRNA (shPuro) were treated or not for 24 h with increasing doses of rhTRAIL. Apoptosis was evaluated by scoring caspase activity (DEVDase activity). Columns, mean (n = 3); bars, ± SD. (E) Analysis of the expression levels of USP18 in T98G cells expressing USP18-wt, its catalytic inactive mutant USP18-C64S or the resistance gene. Lysates from T98G clones were prepared and subjected to immunoblot analysis using the anti-USP18 antibody. Nucleoporin p62 was used as loading control. (F) T98G cells expressing USP18-wt, its catalytic inactive mutant USP18-C64S or the resistance gene were treated or not, for 24 h with increasing doses of rhTRAIL. Cell death was evaluated by trypan blue staining. (G) T98G cells expressing USP18-wt, its catalytic inactive mutant USP18-C64S or the resistance gene were treated or not, for 24 h with increasing doses of rhTRAIL. Apoptosis was evaluated by scoring caspase activity (DEVDase activity).

USP18 suppresses TRAIL induced apoptosis with a catalysis-independent mechanism

USP18 is a cysteine-protease that removes the Ubl protein ISG15, from target proteins. To confirm the influence of USP18 to TRAIL-induced apoptosis and to clarify whether the enzymatic activity of USP18 is required for this outcome, we generated T98G cells stably expressing USP18-wt or the catalytic inactive mutant C64S. After retroviral infection T98G cell lines expressing USP18-wt, its catalytic mutant C64S or the hygromicin resistance gene were isolated (Fig. 2E). To evaluate the apoptotic response to rhTRAIL cells were treated with increasing doses of the death ligand (Fig. 2F). USP18 was able to limit the apoptotic response to rhTRAIL. Furthermore T98G cells overexpressing USP18 or the C64S mutant were equally resistant to TRAIL induced apoptosis, both in terms of trypan blue assay and of DEVDase activity (Fig. 2G). Hence these results confirm that USP18 influences TRAIL induced apoptosis and evidence a deISGylase independent activity.

Cellular TRAIL is required to augment the apoptotic response to rhTRAIL in USP18 silenced cells

In fibroblasts transformed with the oncogene E1A, USP18 can increase the apoptotic susceptibility to different apoptotic stimuli in a TRAIL-dependent manner, by augmenting the spontaneous interferon response.29 Hence, we decided to explore whether also the improved apoptotic response to rhTRAIL, observed in cells with reduced USP18 expression was dependent on cellular TRAIL. E1A-transformed fibroblasts were co-transfected with two different RNAi specific for USP18 and for TRAIL, or the relative controls (Fig. 3).

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Figure 3. Cellular TRAIL is required to augment the apoptotic response to rhTRAIL in USP18 silenced cells. (A) IMR90-E1A cells were transfected with combinations of the three different siRNAs as indicated. Twenty-four hours after transfection, cells were treated or not, for a further 20 h, with rhTRAIL (100 ng/ml). Cell death was scored by trypan blue staining and DEVDase assay. (B) T98G cells were transfected with combinations of the three different siRNAs as indicated. Twenty-four hours after transfection, cells were treated or not, for a further 20 h, with rhTRAIL (100 ng/ml). Cell death was scored by trypan blue staining and DEVDase assay. (C) USP18-dependent regulation of TRAIL mRNAs expression in glioblastoma cells. qRT-PCR analysis was performed to quantify TRAIL mRNA in T98G clones expressing the shRNA against USP18 or the control shRNA. Cells were lysed and mRNAs extracted. Samples were normalized as described in MM. (D) T98G cells were transfected with a siRNA against TRAIL or a control. Twenty-four hours later cells were treated or not, for 24 h with increasing doses of rhTRAIL. Cell death was evaluated by trypan blue staining.

Downregulation of USP18 augmented cell death and caspase activity in response to rhTRAIL in E1A cells. The simultaneous downregulation of TRAIL abrogated the sensitizing effect of USP18 silencing (Fig. 3A).

Similarly, a TRAIL-dependent effect of USP18 downregulation on rhTRAIL-dependent apoptosis was also observed when T98G glioblastoma cells were transiently co-transfected with RNAi specific for USP18 and TRAIL (Fig. 3B).

Having confirmed that TRAIL levels can influence rhTRAIL-induced apoptosis we analyzed whether, in T98G cells with downregulated USP18 expression, cellular TRAIL levels were augmented. qRT-PCR analysis was performed and, as illustrated in Figure 3C, expression of TRAIL was increased in cells (sh#3 and sh#12) with reduced USP18 expression.

If the cellular levels of TRAIL, as modulated by USP18 play an important role in sensitizing cells to TRAIL-induced apoptosis, similarly TRAIL-induced apoptosis should be influenced by siRNA against TRAIL, also independently form USP18. Hence, we again took advantage from T98G cells, which express a detectable level of TRAIL and, after downregulating its expression using the specific siRNA (Fig. 4), we evaluated the activation of apoptosis in response to rhTRAIL. Figure 3D illustrates that downregulation of cellular TRAIL levels impairs apoptosis when is induced by incubation with rhTRAIL.

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Figure 4. Comparative analysis of DISC components expression in cells silenced for TRAIL. Analysis of the expression levels of the indicated DISC elements in T98G cells transfected with RNAi against TRAIL or control. Lysates were prepared and subjected to immunoblot analysis using the indicated antibodies. Actin was used as loading control.

As a first step to understand how cellular TRAIL can influence TRAIL responsiveness, we analyzed the expression levels of different DISC elements in T98G cells with downregulated TRAIL expression, as obtained by the specific siRNA. Figure 4 shows that the level of cellular TRAIL does not influence the expression of the tested DISC components.

TRAIL levels correlates with the responsiveness to TRAIL-induced apoptosis

Cancer cells frequently accumulate mutations in pro-apoptotic genes to acquire resistance in the tumor microenvironment. Since our studies have discovered that cellular levels of TRAIL affect the apoptotic response to TRAIL, a correlation between TRAIL expression and resistance to TRAIL-induced cell death should be noted in cancer cell lines.

To explore this possibility, we took advantage from microarray experiments performed by Wagner and co-authors.31 In their studies cancer cell lines of various origin were clustered on the basis of TRAIL sensitivity, in resistant and sensitive. Next gene expression profiles were acquired in order to unveil the genetic origins of the resistance.31

To test for a correlation between TRAIL sensitivity and TRAIL expression levels, we used the robust non-parametric Wilcoxon rank sum test. As is shown in Figure 5A the test failed in identifying a correlation between the expression levels of TRAIL and the responsiveness to TRAIL induced apoptosis (Fig. 5A). Since the cancer cell lines used in the study are of heterogeneous origin, we also analyzed the data according to the different tissue origin. As shown in Figure 5B, when cancer cells were subdivided considering the tissue of origin, there is a slight increase, although not statistically significant, of TRAIL levels in the sensitive compared with the resistant cells. The only exception was noted in the lung cancer cells.

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Figure 5. Correlation studies between TRAIL expression and resistance to TRAIL-induced apoptosis. (A) Boxplots/stripchart of TRAIL expression levels in cells resistant or susceptible to rhTRAIL induced apoptosis. Data are from the Wagner data set (n = 111) excluding the intermediate susceptible cell lines (n = 8). (B) Same analysis as in (A) but data are stratified for cancer tissue origin. Single dots in the stripchart represent individual observations (single cell line array experiments). Dark horizontal lines represent the median, with the box representing the first and third quartiles, the whiskers the maximum and minimum. Values below 1.5 IQR (interquartile range) of the first quartile or above 1.5 IQR of the third quartile are represented as single dots above or below the whiskers. (C) Boxplots/stripchart of TRAIL-R1, TRAIL-R2, Caspase-8, and BID expression levels in cells resistant or susceptible to rhTRAIL induced apoptosis. Data are from the Wagner data set (n = 111). (D) The same as in (A) but the analysis was performed with a subset of the Wagner data set in which the expression levels of TRAIL-R1, TRAIL-R2, Caspase-8, and BID, were above or equal to the median of the susceptible sub-population. For every binary comparison resistant vs. susceptible, a Wilcoxon rank sum test was performed and the corresponding W statistic and P value were reported.

It is well known that alterations in specific elements of the death signaling pathway can confer resistance to TRAIL.1,7,32 Hence, we reasoned that, being TRAIL the upstream element of the pathway, a correlation between cellular TRAIL levels and the apoptotic susceptibility to rhTRAIL could emerge only when alterations in downstream elements of the extrinsic pathway are not present.

To address this matter, we first analyzed the correlation between the expression levels of several components of the extrinsic apoptotic pathway: TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, OPG, CASP-8, FADD, FLIP, BID, XIAP, and resistance/sensitivity to TRAIL treatment. We found that only TRAIL-R1, TRAIL-R2, CASP-8, and BID expression correlate significantly with TRAIL sensitivity (Fig. 5C).

To explore our assumption, next we focused the attention to cancer cell lines expressing high levels of genes correlated with TRAIL responsiveness (TRAIL-R1, TRAIL-R2, CASP-8, and BID). As a cut-off we selected cancer cells in which the expression levels of these genes were above the median value reached in the susceptible cell lines. The selected cell lines were again subdivided between resistant and susceptible to TRAIL-induced apoptosis.

After these selections, we found that specifically in cells expressing elevated TRAIL-R2 levels, TRAIL expression correlates with sensitivity to rhTRAIL treatment (Fig. 5D). This correlation was not observed in cells where the expression of the other critical elements of the extrinsic pathway, namely TRAIL-R1, CASP-8, and BID was above the median of the susceptible sub-population.

Cells expressing high levels of USP18 disconnected from the IFN response show resistance to rhTRAIL induced apoptosis with higher frequency

Finally we decided to evaluate the correlation between USP18 expression levels and the susceptibility to rhTRAIL induced apoptosis in the 111 cell lines of the Wagner data set. Since the IFN response can strengthen TRAIL induced apoptosis, before evaluating the correlation between USP18 and TRAIL sensitivity, it was necessary to explore whether a positive correlation with the IFN response could be observed. To monitor the activation of the IFN response, in the different cell lines, we used a signature of 10 IFN-inducible genes, excluding USP18.

As expected, a significant correlation was scored between the expression levels of the IFN signature and apoptosis in response to rhTRAIL (Fig. 6A). By contrast USP18 levels in the tested cell lines do not correlate with rhTRAIL susceptibility (Fig. 6B). This result suggests that in some cells USP18 expression could be disconnected from the IFN response.

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Figure 6. Correlation studies between the IFN response, USP18 expression and resistance to TRAIL-induced apoptosis. (A) Boxplots/stripchart of the IFN signature expression in cells resistant or susceptible to rhTRAIL induced apoptosis. The Interferon signature represents the average of the expression levels of 10 interferon-responsive genes (IRF9, ISG15, UBE2L6, UBA7, STAT1, MX1, OAS1, TRAIL, XAF1, and IRF7). Data are from the Wagner data set (n = 111). (B) Boxplots/stripchart of the USP18 expression levels in cells resistant or susceptible to rhTRAIL induced apoptosis. (C) Scatterplot of USP18 expression levels vs. the IFN signature in cell-resistant or -susceptible. The Pearson correlation index for USP18/IFN in the two subpopulations (resistant and susceptible) is reported. The area of the graph highlighted with dotted squares clusters cell lines with high USP18 expression levels and low IFN activation.

Hence, we performed a correlation study between USP18 and the IFN signature in the different cell lines and in relation to rhTRAIL susceptibility. The dot-plot in Figure 6C illustrates such analysis and demonstrates that in the susceptible cell lines USP18 expression shows a good correlation with the IFN response (Pearson = 0.60, P = 0.0003), whereas in the resistant cells the correlation is much weaker (Pearson = 0.23, P = 0.014).

Next we focused our attention on those cell lines, which express high levels of USP18 (expression levels between 2 and 4), in the absence of a robust IFN response (expression levels of IFN signature from 0 to 2). Twelve cell lines (Fig. 6C) shared these features and only one entered apoptosis in response to rhTRAIL (8% of the cell lines). By contrast 29% of the 111 cell lines analyzed are responsive to TRAIL-induced apoptosis. Hence we can conclude that USP18 expression, when uncoupled with the IFN response correlates with resistance to rhTRAIL treatment.

Discussion

In this work, by investigating the ability of the deISGylase USP18 to influence rhTRAIL-induced apoptosis, we have discovered a role of the cellular TRAIL in influencing the apoptotic response to rhTRAIL.

It is well known that type I IFNs can sustain the pro-apoptotic activity of TRAIL.23 We have confirmed this observation and also demonstrated that, through the downregulation of USP18, a negative regulator of the IFN signaling, it is possible to sustain the spontaneous interferon response29 and to strengthen apoptosis induced by rhTRAIL. Analysis of gene expression profiles in several cancer cell lines corroborated that cells expressing high levels of USP18 exhibit resistance to rhTRAIL-induced apoptosis.

Several studies have proved that TRAIL is an important player of the apoptotic response to IFNs.24,25 TRAIL itself is an interferon inducible gene.2 Interestingly, analysis of gene expression signatures from several tumors has revealed that the interferon response is frequently upregulated in cancer. Under the same circumstances expression of TRAIL is instead downregulated, thus possibly limiting the anti-proliferative potency of IFNs.27

Surprisingly we have found that TRAIL expression, which is augmented in cells with downregulated USP18 is an important determinant also when apoptosis is triggered by ectopically added rhTRAIL. This conclusion is sustained by the observation that: (1) the simultaneous downregulation of USP18 and TRAIL abrogates the increase in apoptosis in response rhTRAIL, and (2) downregulation of TRAIL alone reduces apoptosis in response to rhTRAIL

By analyzing the gene expression profiles of cancer cells resistant or responsive to rhTRAIL, a statistic significant correlation between TRAIL levels and apoptosis can be evidenced only in cells with elevated levels of TRAIL-R2 expression. This result is not surprisingly since, being TRAIL the uppermost element of the signaling pathway, alterations in the downstream effectors could impair apoptosis also in the presence of elevated levels of cellular TRAIL. Surprisingly the correlation was not observed with TRAIL-R1. Although we cannot exclude that with an enlarged number of samples a correlation could be found also with this receptor. It is important to note that TRAIL-R1 and TRAIL-R2 show some peculiarities; for example, for the mechanisms controlling the trafficking to the PM,33 the recruitment to the membrane rafts,34 and their internalization.

Since TRAIL-R2, compared with TRAIL-R1 is internalized with lower frequency32,35 it could be argued that a complex among cellular TRAIL and TRAIL-R2 could be more stable and could facilitate the activity of rhTRAIL.

Several additional hypotheses could be formulated about the mechanisms through which cellular TRAIL influences TRAIL-induced apoptosis. Cellular TRAIL could interact with and engage DRs that are not exposed at the cell surface36 or it can promote with stronger potency the re-localization into the membrane rafts.37 Certainly, we can exclude that cellular TRAIL can influence the expression levels of the major DISC components. Finally cellular TRAIL could engage additional signaling pathways thus influencing responsiveness to rhTRAIL.

In conclusion in this manuscript, by investigating the role of USP18 in the apoptotic response to TRAIL, we have discovered an important contribution of cellular TRAIL in the apoptotic response to rhTRAIL. Further studies are necessary to define the specific molecular events that are influenced by cellular TRAIL, which are responsible for increase apoptotic responsiveness.

Materials and Methods

Cell culture and apoptosis

T98G and IMR90-E1A were propagated in the Dulbecco’s modified Eagle medium supplemented with l-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum at 37 °C in 5% CO2, as previously described.29,38 Stealth RNA interference RNAi for USP18, TRAIL, and non-targeting shRNA were purchased from Invitrogen. Cells were transfected 24 h after plating by adding the medium OptiMem, containing Lipofectamine 2000 (Invitrogen) plus the stealth RNAi oligos. IFN-α2a (Jena Bioscience) was used at 1000 units/ml, final concentration. In all trypan blue exclusion assays, 400 cells from three independent samples were counted for each data point. Data were represented as arithmetic mean ± SD for at least three independent experiments. The DEVDase activity was evaluated using the Apo-ONE assay (Promega). For experimental data the Student t-test was employed. P < 0.05 was chosen as statistical limit of significance. We marked with *P < 0.05, **P < 0.01, and ***P < 0.001. Unless otherwise indicated, all the data in the figures were represented as arithmetic mean ± SD of at least three independent experiments.

Generation of T98G cells stably expressing USP18-wt, USP18C64S or USP18shRNA

The USP18 point mutant C64S29 was cloned into a pLPC retroviral vector. USP18shRNA and non-targeting control shRNA were obtained by cloning double stranded oligos into the pSUPER.retro puro, retroviral vector, purchased from OligoEngine. Retroviral supernatants were produced after transfection of 293T packaging cell line, using the calcium phosphate method. At 72 h after transfection, viral supernatants were collected, filtered by 0.45 μm filter and used as medium to infect target cells, after addition of 8 μg/ml polybrene. The infected cells were incubated at 32 °C for 24 h and selected with puromycin (1 μg/ml), until the formation of single clones.

Immunoblotting

Proteins obtained after an SDS denaturating lysis and sonication were transferred to a 0.2 µm pore sized nitro-cellulose membrane (Schleicher and Schuell) using a semidry blotting apparatus (Amersham Pharmacia Biotech) (transfer buffer: 20% methanol, 48 mM Tris, 39 mM glycine, and 0.0375% SDS). The nitrocellulose membranes were saturated for 1 h in Blotto-Tween 20 (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5% nonfat dry milk, and 0.1% Tween 20) and incubated overnight at room temperature using the primary antibodies. Membranes were then rinsed three times with Blotto-Tween 20 and incubated with peroxidase-conjugated goat anti-rabbit (Sigma) or goat anti-mouse (Sigma) for 1 h at room temperature. After 4 washes by Blotto-Tween 20, the membranes were rinsed in phosphate buffered saline and developed with Super Signal West Pico, as recommended by the vendor (Pierce).

Quantitative reverse transcription-PCR

cDNAs were synthesized from 1 μg of total RNA, obtained by Trizol (Invitrogen) extraction, using the First-Strand cDNA Synthesis kit (Invitrogen). Real-time PCR was performed using the KAPA SYBR® FAST Master Mix (Kapabiosytems) on a CFX96 Real-Time System (Bio-Rad). The obtained data were analyzed using the ΔΔCt method. The geometric average of HPRT1 (hypoxanthine phosphoribosyltransferase 1) GAPDH and ACTB (β-actin) was used for normalization. Data were expressed as fold change from the T98G cells expressing the control shRNA.

Bioinformatic analysis

For gene arrays analysis the free software R with the GEOquery package for downloading gene expression data from Geo portal (http://www.ncbi.nlm.nih.gov/geo) was used. In the case of multiple probe set for a given gene the best probe set was selected using the PLANdbAffy database.39 The calculation of the Wilcoxon test P values, the graphics, and all the following analysis were also performed with R.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by AIRC (IG-10437) and FIRB (Progetto RBAP11S8C3_002). I Manini was supported by CIB and Talents (Area science park Trieste) fellowships.

10.4161/cbt.26525

Footnotes

References

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