Fig. 1

Reduced cyclin D1 in SMARCA4-deficient non-small cell lung cancer (NSCLC) cells causessensitivities to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. a, b SMARCA4-deficient NSCLC cell lines express reduced cyclin D1 levels. Western blot analysis for the indicated proteins (a) and CCND1 messenger RNA (mRNA) expression (b) of a panel of NSCLC cell lines. HSP90 was used as a loading control. Relative CCND1 mRNA expression (relative to GAPDH) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). A4: SMARCA4, A4/2: SMARCA4/2, Pro: proficient, Def: deficient, K: KRAS mutation. Empty triangles indicate RB-deficient cell lines. Turquoise color indicates cell lines with KRAS mutation. Error bars: mean ± standard deviation (s.d.) of biological replicates (n = 3); two-tailed t test, *p < 0.05. c, d SMARCA4-deficient NSCLC cells are highly sensitive to palbociclib treatment, similar to KRAS mutation cells. c Half-maximal inhibitory concentration (IC₅₀) of palbociclib in the above cell line panel was determined by measuring cell viability using CellTiter-Blue assay. Error bars: mean ± s.d. of biological replicates (n = 4); two-tailed t test, *p < 0.05, **p < 0.01. d Colony formation assays of the representative cell lines. Cells were cultured in the absence or presence of palbociclib at the indicated concentrations for 10–14 days. For each cell line, all dishes were fixed at the same time. e, f Palbociclib treatment in SMARCA4-deficient NSCLC cells induces strong G1 cell cycle arrest. H1299 (e) and H1703 (f) cells treated with palbociclib for 24 h were fixed, stained with propidium iodide and analyzed by flow cytometry using the Guava easyCyte HT System. g, h Ectopic expression of cyclin D1 confers drug resistance to palbociclib in H1299 (g) and H1703 (h) cells. Upper, colony formation assays; lower, immunoblot of cells with stable ectopic expression of GFP or CCND1 and treated with palbociclib (H1299, 300 nM; H1703, 33 nM). i, j Cyclin D1 knockdown sensitizes HCC827 (i) and PC9 (j) cells to palbociclib. Upper, colony formation assays in the absence or presence of 300 nM palbociclib; lower, immunoblot of cells expressing pLKO control or short hairpin RNAs (shRNAs) targeting CCND1