Fig. 4
Cyclin D1 deficiency in SCCOHT cells is caused by SMARCA4 loss. a–c RNA-Seq analysis in BIN-67 and SCCOHT-1 cells stably expressing pReceiver control or pReceiver-SMARCA4 identified CCND1 as the top ranked cell cycle regulator upregulated upon SMARCA4 restoration (n = 3). a, b Venn diagrams showing the genes upregulated (a) or downregulated (b) upon SMARCA4 restoration (fold change >3, adjusted p < 0.05). c Heatmap of the top 50 genes upregulated upon SMARCA4 restoration in both BIN-67 and SCCOHT-1 cells. Red arrow points to CCND1. d, e SMARCA4 restoration in SCCOHT cells upregulated CCND1 mRNA (d) and cyclin D1 protein (e) levels. d Relative expression levels of CCND1 mRNA (normalized to GAPDH) in SCCOHT cells were measured by qRT-PCR. Error bars: mean ± s.d. of biological replicates (n = 3; two-tailed t test, **p < 0.01). e Western blot analysis for the indicated proteins in the cells described above. f, g SMARCA4 knockdown in SMARCA4-proficient cells suppressed CCND1 mRNA (f) and cyclin D1 protein (g) levels. f Relative expression levels of CCND1 mRNA (normalized to GAPDH) in IOSE80 and OVCAR4 cells were measured by qRT-PCR. Error bars: mean ± s.d. of biological replicates (n = 3; two-tailed t test, ****p < 0.0001, **p < 0.01). g Western blot analysis for the indicated proteins in the cells described above. h, i SMARCA4 occupancy in the CCND1 promoter region. Chromatin immunoprecipitation experiments were performed in SCCOHT cells (SCCOHT-1, BIN-67) expressing pReceiver or pReceiver-SMARCA4 (h) and in SMARCA4-proficient cells (IOSE80, OVCAR4) expressing pLKO or shRNA targeting SMARCA4 (i), using an antibody against SMARCA4 or IgG. qPCR was used to analyze SMARCA4 occupancy using the primer sets for CCND1, CCND3, and CCNE1 locus as indicated. p promoter, TSS transcription start site, error bars: mean ± s.d. of measurement replicates of a representative experiment (n = 3; two-tailed t test, *p < 0.05, **p < 0.01)
