Fig. 5

ASXL3 is a direct target of BET inhibitors and predicts drug sensitivity. a The log2 fold change of nearby gene expression in ASXL3-depleted cells, and cells treated with either DMSO, dBET6 (300 nM), JQ1 (1 μM), or IBET-151 (1 μM). b The Venn-diagram shows the overlap of downregulated genes in cells treated with dBET6, JQ1, or IBET-151 from (a). c The bar plot shows ASXL3 expression (RPM) in NCI-H1963 cells treated with different BET inhibitors, n = 2, two-tailed unpaired Student’s t test. **P < 0.01; *P < 0.05. d Real-time PCR was performed to determine the relative gene expression of EIF4E and ASXL3 in NCI-H1963 cells treated with dBET6, JQ1, or IBET-151 for 4 h or 8 h. DMSO was used as negative control, n = 3, two-tailed unpaired Student’s t test. **P < 0.01; *P < 0.05. e BRD4 ChIP-seq was performed with NCI-H1963 SCLC cells treated either DMSO or dBET6 for 2 h. The representative track shows the occupancy of BRD4 at ASXL3 locus. f Whole-cell lysates were used for western blot with BRD2, BRD3, BRD4, Pol II, CDK9, and CCNT1 antibodies in human SCLC cell line NCI-H1963 treated with dBET6 for 0, 2, 8, and 24 h, n = 3. g Whole-cell lysates were used for western blot with ASXL3, JMJD6, NSD3, and CHD4 antibodies in human SCLC cell line NCI-H1963 treated with dBET6 for 0, 2, 8, and 24 h. HSP90 was used as an internal control, n = 3. h Whole-cell lysates were used for western blot with BRD2, BRD3, BRD4, ASXL3, JMJD6, NSD3, and CHD4 antibodies in human SCLC cell line NCI-H1963 treated with different concentrations of dBET6 for 8 h; HSP90 was used as an internal control, n = 3. i 8 different human SCLC cell lines NCI-H748, NCI-H1882, NCI-H1963, NCI-H209, NCI-H1436, NCI-H1105, NCI-H889, and NCI-H2171 were treated with different concentrations of dBET6 for 72 h. The cell viability was determined by CellTiter-Glo Luminescent Cell Viability Assay, n = 6. j The protein levels of ASXL3 and BRD4 from the previously cell lines mentioned (i) were determined by western blot, n = 3. k The representative track shows H3K27Ac and H3K4me1 levels at ASXL3 locus in ASXL3-high (NCI-H1963) and ASXL3-low (NCI-H2171) SCLC cells. l The Venn-diagram shows SE-associated genes in ASXL3-high and ASXL3-low cells from (k)