Fig. 3: BAP1 inactivation suppresses ASCL1 expression.

A NCI-H1963 cells were treated with iBAP-II for 24 h. The protein levels of ASCL1 were determined by western blot. HSP90 was used as the internal control, n = 2. B The mRNA levels of ASCL1, GRP, DMPK, RNF183, SCN3A, MYCL, and CACNA1A were determined by real-time PCR in NCI-H1963 cells treated with either 10 or 20 μM of iBAP-II. DMSO was used as the negative control, n = 3. C The occupancy heatmaps show ASCL1 binding levels to chromatin after iBAP-II treatment. The Venn-diagram (D) and average plot (E) show the ASCL1 levels at chromatin in NCI-H1963 cells treated with either DMSO or iBAP-II. F Pathway analysis shows the most significant signaling pathways enriched in genes nearest to the loss of ASCL1 peaks (n = 11,744) induced by iBAP-II treatment. G Representative track example that shows ASCL1 occupancy between NCI-H1963 cells treated with either DMSO or iBAP-II. H NCI-H1963 cells were transduced with two distinct RING1B shRNAs. The protein levels of RING1B, H2AK119Ub, and BAP1 were determined by western blot. β-tubulin was used as the internal control, n = 2. I The mRNA levels of ASCL1 were determined by real-time PCR in NCI-H1963 cells transduced with either non-targeting gRNA or two distinct RING1B shRNAs, n = 3. J The representative tracks show the chromatin occupancy of ASXL3, BAP1, RING1B, H3K27me3, and H2AK119Ub levels at the ASCL1 gene locus in NCI-H1963 cells. K Representative tracks show the occupancy of BAP1, RING1B, H3K27me3, and H2AUb levels at chromatin. L RNA-seq was performed with NCI-H1963 cells treated with iBAP-II or transduced with non-targeting shRNA and two different RING1B gRNAs, n = 2. M The representative tracks show the expression levels of RNF2 and ASCL1 in cells transduced with either non-targeting gRNA or two distinct RING1B shRNAs. N The average plots show the levels of H3K4me1 (GSE145028), H3K27me3 (GSE164247), H2AK119Ub (upper), H3K27Ac (GSE145028), H3K4me3 (GSE164247), and Pol II (lower) at the gene body of all 739 genes that were downregulated in iBAP-II treated cells and upregulated in RING1B-depleted cells. O RNA-seq was performed with cells treated with either DMSO or EZH2 inhibitor GSK126 (2 μM) for 96 h, n = 2. The Log2FC heatmap shows the gene expression changes in the same order as L.