Fig. 4: LSH depletion leads to decreased chromatin association of macroH2A and to an increase of nuclear size.
From: LSH mediates gene repression through macroH2A deposition
a Western blot analysis for detection of indicated proteins in whole-cell extract or in the chromatin fraction of Lsh WT and KO MEFs. b, c Microscopic images to visualize the nuclear size of Lsh WT and KO MEFs, or WT and KO MEFs treated with control siRNA (si-Ctrl) or two combined macroH2A siRNA (si-mH2A1 + 2) by staining with Lamin B1 and DAPI (b). Scale bar, 20 μm. Quantification of nuclear area of samples as shown in c. ***adjusted p < 0.0001, n.s. means not significant. d Western blot analysis to detect restoration of LSH protein level in Lsh−/− (KO) murine ES cells after transfection with wild-type LSH (WT) or ATP mutant LSH (K 237A) vector compared to Lsh WT and KO mouse ES cells. e, f Microscopic images to visualize the nuclear size in Lsh WT and KO mouse ES cells, and KO ES cells restored with wild-type LSH (WT) or ATP mutant LSH (K 237A) by staining with Lamin B1 and DAPI (e). Scale bar, 20 μm. Quantification of nuclear area of samples is shown in f. ***adjusted p < 0.0001, n.s. means not significant. g Flag-IP of indicated proteins confirmed by western blot analysis in U2OS cells with (Flag-LSH) or without (Ctrl) stable expression of Flag-LSH fusion protein. h GFP-IP of indicated proteins confirmed by western blot analysis in U2OS cells with a stably integrated vector for GFP or macroH2A2-GFP expression. An arrowhead indicates macroH2A2-GFP protein and an asterisk indicates a nonspecific cross-reaction band. Data are represented as dot plots with mean ± SD (c, f). One-way ANOVA with Tukey’s multiple comparison test (c, f). Source data are provided as a Source Data file.
