Fig. 3 | Nature Communications

Fig. 3

From: CDK4/6 inhibitors target SMARCA4-determined cyclin D1 deficiency in hypercalcemic small cell carcinoma of the ovary

Fig. 3

Cyclin D1 deficiency in SCCOHT cells results in vulnerability to CDK4/6 inhibition. a SMARCA4 loss in SCCOHT is associated with cyclin D1 deficiency and reduced CDK4 expression. Western blot analysis of key cell cycle regulators in a cell line panel: non-transformed ovarian epithelial (IOSE80), ovarian carcinoma (OVCAR4, OVCAR8), SCCOHT (BIN-67, SCCOHT-1, COV434), and ER+ breast cancer (CAMA-1, MCF7). l.e. long exposure. b SCCOHT cells express low levels of CCND1 mRNA. Relative CCND1 expression (normalized to GAPDH) in the cell panel described above were measured by qRT-PCR. Error bars: mean ± s.d. of biological replicates (n = 3). c SCCOHT cells have lower total CDK4 kinase activities compared to SMARCA4-proficient control cells. Normalized amount of immunoprecipitated CDK4 kinase complexes from IOSE80 and SCCOHT cell lines were subjected to in vitro kinase assays using recombinant RB. Upper, western blot analysis of immunoprecipitated CDK4 input; lower, kinase assay radiography. di Ectopic cyclin D1 expression increases RB phosphorylation and confers resistance to palbociclib in SCCOHT cells. d, e Western blot analysis of immunoprecipitations using an antibody against CDK4 or IgG in SCCOHT-1 (d) and BIN-67 (e) cells stably expressing GFP or CCND1. f, g Western blot analysis of SCCOHT-1 (f) and BIN-67 (g) cells with stable ectopic expression of GFP, CDK4, or CCND1. h, i Colony-formation assay of SCCOHT-1 (h) and BIN-67 (i) cells (described in f, g) treated with palbociclib. j, k Spontaneously palbociclib-resistant clones of SCCOHT-1 expressed elevated cyclin D1 and RB phosphorylation. j Cell viability assay of SCCOHT-1 parental cells and resistant clones (R1 and R2) treated with palbociclib for 9 days. Error bars: mean ± standard deviation (s.d.) of biological replicates (n = 4). k Western blot analysis for the indicated proteins in the cells described above. l, m Cyclin D1 knockdown in palbociclib-resistant SCCOHT-1 cells resensitizes them to palbociclib. l Cell viability assay of SCCOHT-1 R1 cells expressing pLKO control or two independent shRNAs targeting cyclin D1 treated with palbociclib for 9 days. Error bars: mean ± standard deviation (s.d.) of biological replicates (n = 4). m Western blot analysis for the indicated proteins in the cells described above

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