Fig. 1
SMARCA4-deficient SCCOHT cells are vulnerable to inhibition of CDK4/6 kinase activities. a Schematic outline of the shRNA screens for kinases whose inhibition is selectively lethal to SMARCA4-deficient SCCOHT cells (BIN-67) but not to SMARCA4-proficient control cells (IOSE80, OVCAR4). Cells were infected with the lentiviral shRNA library (T0) and cultured for selection for 14 days (T1). The relative abundance of shRNAs in the cell populations was determined by next-generation sequencing. b Analysis of the shRNA screens using the MAGeCK statistical software package31. CDK6 (magenta) and CDK4 (blue) are the first two ranked genes that were negatively selected in BIN-67 cells. All genes were ranked based on their RRA (robust rank aggregation, top) or raw p values (bottom) generated from the MAGeCK analysis. c, d Validation of CDK6 and CDK4 in SCCOHT cells (BIN-67, SCCOHT-1, COV434) and SMARCA4-proficient controls (IOSE80, OVCAR4). c Colony-formation assay of the indicated cell lines expressing pLKO control or shRNAs targeting CDK6 or CDK4 after 10–15 days of culturing. For each cell line, all dishes were fixed at the same time, stained, and photographed. d Western blot analysis of CDK6 and CDK4 and phosphorylated RB at serine 795 (pRB-S795) in the cells described in c. HSP90 was used as a loading control. e–j SCCOHT cells are more vulnerable to inhibition of CDK4/6 kinase activities, compared to SMARCA4-proficient control cells. e BIN-67 cells stably expressing pLX304-GFP, pLX304-CDK6, or pLX304-CDK6D163N were infected with viruses containing pLKO control or a shRNA targeting the 3’UTR of CDK6, selected for integration, and cultured for 14 days. All dishes were fixed at the same time. f Western blot analysis for CDK6, pRB-S795, and HSP90 in the cells described above. g, i BIN-67 (g) and OVCAR-4 (i) cells expressing pLX317-GFP, pLX317-CDK4, or pLX317-CDK4D158N were infected with viruses containing pLKO control or a shRNA vector targeting the 3’UTR of CDK4, selected for integration, and cultured for 14 days. For each cell line, all dishes were fixed at the same time. h, j Western blot analysis for CDK4, pRB-S795, and HSP90 in the cells described above
