Fig. 2: Inhibition of BAP1 reduces ASXL3 protein stability in small cell lung cancer cells.

A Three different human SCLC cell lines (NCI-H1963, NCI-H748, and NCI-H1882) were treated with iBAP-II (0, 10, and 20 μM) for 24 h. The protein levels of ASXL1, ASXL2, ASXL3, BAP1, HCFC1, FOXK1, and FOXK2 were determined by western blot. β-tubulin was used as the internal control. Human SCLC cell line NCI-H1963 cells were treated with 50 μg/ml cycloheximide (CHX) at different times in the presence of either DMSO or iBAP-II. The ASXL3 protein levels were determined by western blot (B) and further quantified by ImageJ (C), n = 2. D The log2 fold change of the expression levels of genes nearby ASXL3 peaks grouped as TSS and nonTSS regions annotated by Homer in ASXL3-depleted cells, and cells treated with either iBAP or iBAP-II, n = 2. E Gene Set Enrichment Analysis (GSEA) of iBAP-II gene signature enrichment in ASXL3 or BAP1-depleted conditions. A total of 383 downregulated genes in iBAP-II (20 μM) versus DMSO treatment (adj.p < 0.01, logFC < −1) is defined as the iBAP signature gene set. GSEA of iBAP signature genes in ASXL3-depleted cells compared with control cells by shRNA. GSEA of iBAP signature genes in BAP1-depleted cells compared with control cells by sgRNA. F Representative track example that shows BAP1 occupancy in NCI-H1963 cells treated with either DMSO or iBAP-II. G Pathway analysis shows the most significant signaling pathways enriched in genes nearest to the loss of BAP1 peaks (n = 1999) induced by iBAP-II treatment. H ChIP-Seq analysis representing 70% (2470/3529) of all BAP1 peaks from BAP1 chromatin occupancy being collectively reduced upon iBAP treatment. Occupancy and log2FC heatmaps of BAP1 in DMSO and iBAP-II conditions are centered on these BAP1 peaks (n = 2470). When comparing the same ranking of peaks, BAP1 occupancy also decreased from ASXL3 depletion via shRNA. Peaks were sorted based on the mean signals from DMSO and iBAP-II samples in each row. I The average plot shows BAP1 levels at ASXL3-occupied TSS (left) and nonTSS (right) regions. J The box plot shows the H2AK119Ub levels between DMSO and iBAP-II treated NCI-H1963 cells.