Fig 2: NSD3 KO by CRISPR/Cas9 exerts tumor-suppressive functions in pancreatic cancer cells.Single stable PanCa-1 primary pancreatic cancer cells, bearing the lenti-CRISPR/Cas9 construct encoding NSD3 sgRNA (“koNSD3”) or the lenti-CRISPR/Cas9 empty vector (“koC”), were established; expression of NSD3 mRNA and protein was examined by qRT-PCR (A) and western blotting (B) assays, respectively. Cells were further cultured for applied time periods, cell viability (CCK-8 assays, C), proliferation (nuclear EdU staining assays, D), as well as cell migration (“Transwell” assays, E) and invasion (“Matrigel Transwell” assays, F) were tested, with results quantified); caspase-3 activity (G), mitochondrial depolarization (JC-1 staining assays, H) and cell apoptosis (TUNEL staining assays, I) were tested as well. Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05 vs. “koC” cells. Experiments in this figure were repeated five times with similar results obtained. Scale bar = 100 μm (D, H).

Fig 3: NSD3-T1232A accelerates pancreatic cancer cell proliferation, migration and invasion.Single stable PanCa-1 primary pancreatic cancer cells bearing the lenti-CRISPR/Cas9 construct encoding NSD3 sgRNA (“koNSD3”) were further transduced with NSD3-T1232A-Flag construct, the wild-type NSD3-Flag construct or empty vector (“Vec”); Expression of listed proteins was examined by western blotting assays (A). Cells were further cultured for applied time periods, cell viability (CCK-8 assays, B), proliferation (EdU staining assays, C), cell migration (“Transwell” assays, D), and invasion (“Matrigel Transwell” assays, E) were tested. Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05. Experiments in this figure were repeated five times with similar results obtained. Scale bar = 100 μm (C–E).

Fig 4: 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)

Fig 5: NSD3 is overexpressed in pancreatic cancer tissues and cells.TCGA-PAAD cohorts show relative NSD3 mRNA expression in 171 cases of pancreatic ductal adenocarcinoma (PDAC) tissues (“Cancer”) and 179 cases of normal pancreatic tissues (“Normal”) (A). Kaplan-Meier survival analyses of NSD3-low (N = 89) and NSD3-high (N = 89) PDAC patients (B). Badea Pancreas Statistics shows relative NSD3 mRNA expression in PDAC cancer tissues (“Cancer”) and matched surrounding normal pancreatic tissues (“Normal”) (C). NSD3 mRNA and protein expressions in pancreatic cancer tissues (“Can”) and matched surrounding normal pancreatic tissues (“Nor”) from 12 (N = 12) local PDAC patients were tested by qRT-PCR (D) and western blotting (E, F) assays. NSD3 mRNA (G) and protein (H) expressions in described pancreatic cancer cells and epithelial cells (“priEpi”) were tested. Data were presented as mean ± standard deviation (SD). *P < 0.05 vs. “Normal” tissues (A–C). *P < 0.05 vs. “Nor” tissues (D, F). *P < 0.05 vs. “priEpi” cells (G and H).