Fig. 7. ATO stabilizes L3MBTL2 protein and augments the L3MBTL2-induced condensates.

(A) HEK293T cells were cotransfected the indicated plasmids for 48 hours and then were analyzed by immunoprecipitation using Ni-NTA and anti-FLAG beads followed by Western blotting. WCL, whole cell lysate. (B) U2OS and 143B cells were treated with increasing concentration of ATO for 12 hours and then were analyzed by Western blotting. (C) Representative images of immunofluorescence staining for endogenous L3MBTL2 in U2OS cells treated with ATO (1 μM) or vehicle for 12 hours. Scale bars, 5 μm. (D) Quantification of the numbers of L3MBTL2 condensate in cells from (C). Data are means ± SD of n = 20 cells. (E) Percentage of n = 100 cells with L3MBTL2 condensates in cells from (C). Data are means ± SD of n = 3 biologically independent experiments. (F) Representative images of immunofluorescence staining for endogenous L3MBTL2 in the indicated 143B stable cells. Scale bars, 5 μm. (G) Quantification of the numbers of L3MBTL2 condensate per nucleus in the indicated 143B stable cells (F). Data are means ± SD of n = 20 cells. (H) Percentage of n = 100 cells with L3MBTL2 condensates in the indicated 143B stable cells from (F). Data are means ± SD of n = 3 biologically independent experiments. (I to K) Representative fluorescence microscopy images of condensates formed by L3MBTL2-EGFP with Ring1-mCherry (I), PCGF6-mCherry (J), or RYBP-mCherry (K) in U2OS cells treated with ATO (1 μM) for 12 hours. Scale bars, 5 μm. Data in (A to C, F, and I to K) are representative of n = 3 biologically independent experiments. (L and M) Quantification of the size (L) and number (M) of the condensates from (I to K). Data are means ± SD of n = 10 cells. (N) HEK293T cells were transfected with the indicated constructs, along with the 9xGal-TK-luc reporter and the Renilla control reporter for 24 hours. Then, cells transfected with Gal4-L3MBTL2 were treated with or without ATO for 12 hours and analyzed for luciferase activity.