Figure S4 from Tumor Suppressors Condition Differential Responses to the Selective CDK2 Inhibitor BLU-222

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Figure S4. Loss of RB1 shifts phase of arrest and sensitivity to BLU-222. A, Heatmap of RNA sequencing data comparing differential gene expression between BLU-222-treated Kuramochi (125 nM BLU-222) and OVCAR-8 (250 nM BLU-222) cells normalized to respective DMSO-treated controls. B, Cell cycle profiling from univariate flow cytometric analysis from OVCAR-8 cells at baseline, after 1 to 5 days of treatment with 500 nM BLU-222, and after 1 to 5 days of drug washout. C, Western blot for abrogation of G2/M checkpoint signaling in OVCAR-8 cells following doxycycline (Dox) induction of the RB1 expression vector and treatment with BLU-222 compared with RB1 deficient controls. D, Live cell proliferative monitoring of OVCAR-8 cells following Dox induction of the RB1 expression vector (RB1 Dox +) with non-induced controls (RB1 Dox -) and treatment with serial concentrations of palbociclib (n = 3–4 per group; error bars represent SEM). E, Table summarizing effect of RB1 induction on IC50 to BLU-222 in OVCAR-8 cells. F, Live cell proliferative monitoring of OVCAR-8 cells following doxycycline induction of the RB1 expression vector (RB1 Dox +) with non-induced controls (RB1 Dox -) and transfection with siNT, siCCNE1, or siCCNA2 [n = 3–4 per group; two-way ANOVA, significance (shown as #) for combined RB1 and siRNA effect; error bars represent SEM]. G, Live-cell imaging endpoint stills for the OVCAR-8 cells in D (scale bar = 100 µm). H, Western blot of RB1-E2F pathway activity in Kuramochi cells with RB1 knockdown or siNT control and treatment with BLU-222. I, Cell cycle profiling from univariate flow cytometric analysis from Kuramochi cells with RB1 knockdown or siNT control and treatment with BLU-222. J, Western blot of RB1-E2F pathway activity in MDA-MB-157 cells with and without deletion (DEL) of RB1 and treatment with BLU-222. K, Cell cycle profiling from univariate flow cytometric analysis from MDA-MB-157 cells with and without deletion (DEL) of RB1 and treatment with BLU-222. L, Table summarizing effect of RB1 knockdown/deletion on IC50 to BLU-222 in Kuramochi and MDA-MB-157 cells, respectively. ####, P < 0.0001. PI, propidium iodide.

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