Fig 2: MUC1-C induces PBRM1, ARID2, and BRD7 expression.A DU-145/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for PBRM1, ARID2, and BRD7 mRNA levels by qRT-PCR using primers listed in Supplementary Table S1. The results (mean ± SD of 4 determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). B Lysates from DU-145/tet-CshRNA and DU-145/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. C Lysates from DU-145/CsgRNA, DU-145/MUC1sgRNA#1, and DU-145/MUC1sgRNA#2 cells were immunoblotted with antibodies against the indicated proteins. D LNCaP-AI/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for PBRM1, ARID2, and BRD7 mRNA levels by qRT-PCR. The results (mean ± SD of 4 determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). E LNCaP-AI/tet-CshRNA and LNCaP-AI/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. F Lysates from LNCaP/tet-MUC1-C cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. G Lysates from NCI-H660/CshRNA and NCI-H660/MUC1shRNA cells were immunoblotted with antibodies against the indicated proteins.

Fig 3: MUC1-C→E2F1 pathway induces ARID2 and BRD7 expression.A Schemas of the ARID2 and BRD7 promoter regions with positioning of putative E2F binding motifs. B, C Soluble chromatin from DU-145 cells was precipitated with anti-E2F1, anti-MUC1-C or a control IgG (left). Soluble chromatin was precipitated with anti-MUC1-C (ChIP) and then reprecipitated with anti-E2F1 or a control IgG (re-ChIP) (right). The DNA samples were amplified by qPCR with primers for the ARID2 (B) and BRD7 (C) promoter regions. The results (mean ± SD of 3 determinations) are expressed as the relative fold enrichment compared to that obtained with the IgG control (assigned a value of 1). D, E DU-145/tet-MUC1shRNA cells were treated with vehicle or DOX for 7 days. Soluble chromatin was precipitated with anti-E2F1 or a control IgG. The DNA samples were amplified by qPCR with primers for the PBRM1 (D) and BRD7 (E) promoter regions. The results (mean ± SD of 3 determinations) are expressed as the relative fold enrichment compared to that obtained with the IgG control (assigned a value of 1). F DU-145/CshRNA and DU-145/E2F1shRNA cells were analyzed for ARID2 and BRD7 mRNA levels by qRT-PCR. The results (mean ± SD of 4 determinations) are expressed as relative mRNA levels compared to that obtained for CshRNA cells (assigned a value of 1). G Lysates from DU-145/CshRNA and DU-145/E2F1shRNA (left) or LNCaP-AI/CshRNA and LNCaP-AI/E2F1shRNA (right) cells were immunoblotted with antibodies against the indicated proteins.

Fig 4: BRD7 deficiency sensitizes tumor cells to CHK1 inhibitors via accumulation of CHK1.A Silencing of BRD7 sensitizes tumor cells to CHK1 inhibitors. H1650 cells transfected with siRNA targeting BRD7 or scrambled control siRNA were plated in triplicate in 96-well plates and treated with various concentrations of AZD7762, PF477736, and LY2603618 for 72 h for the CCK8 assay. Data from three independent experiments are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. B Silencing of BRD7 promotes CHK1 inhibitor-induced apoptosis. H1650 cells transfected with siRNA targeting BRD7 or scrambled control siRNA were treated with AZD7762, PF477736, or LY2603618 for 24 h and then subjected to immunoblotting (IB) with the indicated antibodies. C Simultaneous CHK1 knockdown reverses the BRD7 deficiency-induced chemosensitization to CHK1 inhibitors. Cells transfected with the indicated siRNAs were plated in triplicate in 96-well plates, treated with various concentrations of the three CHK1 inhibitors for 72 h, and then subjected to the CCK8 assay. The cells remaining in the plates were subjected to IB with the indicated antibodies (bottom). Data from three independent experiments are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; ns: not significant. Given that BRD7 knockdown significantly increased cell proliferation in H1650 cells, to maintain the equivalent number of cells in each group in response to drug treatment, 1 × 103 or 1.2 × 103 cells per well were used for siRBD7 group and 3 × 103 cells per well for other groups. LEX: longer exposure.

Fig 5: BRD7 deficiency promotes DNA replication, cell proliferation, and survival in a CHK1-dependent manner.A Silencing of BRD7 increases CHK1 levels and simultaneous silencing of BRD7 and CHK1 downregulates CHK1 levels. H1650 cells transfected with indicated siRNA were harvested at 48 h, followed by immunoblotting (IB) with the indicated antibodies. B Silencing of BRD7 increases DNA replication, while simultaneous silencing of BRD7 and CHK1 suppresses DNA replication. H1650 cells transfected with indicated siRNA were performed EdU incorporation assay using BeyoClick™ EdU Cell Proliferation Kit. The percentage of EdU-positive cells in the total number of cells was determined from at least five random fields. Silencing BRD7 increases the percentage of S phase of cell cycle (C), cell proliferation (D) and cell survival (E) in a CHK1-dependent manner. H1650 cells were transfected with the indicated siRNA oligos for 48 h and then split for flow cytometry (C), CCK8 assay (D), and clonogenic survival assay (E). Data from three independent experiments are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001; ns: not significant.