Fig. 6: Efficacy of CDK4/6 inhibitor is independent of MCM3 expression.

(a) MCM3 protein expression was evaluated following treatment with CDK4/6 inhibitor (CDK4/6i) in endocrine-resistant cells, including tamoxifen- (TamR-1), aromatase inhibitor- (LetR-1) and fulvestrant-resistant (FulvR-1), and in cell lines resistant to combined CDK4/6i and endocrine therapy (MPF-R and TPF-R), by Western blotting using whole cell lysate. β-actin was used as loading control. A representative of two biological replicates is shown. (b) Representative micrographs (40x magnification) showing MCM3 immunhistochemistry staining of FulvR-1 and MPF-R tumor xenografts treated with CDK4/6i (50 mg/Kg) combined with fulvestrant (100 mg/Kg; n = 7 and 9) or vehicle (castor oil and 25% w/v HPB cyclodextrin; n = 8 and 10). (c) Evaluation of growth of endocrine-resistant cells following treatment with CDK4/6i as measured by colorimetric crystal violet assay at 96 h. Data are representative of three independent experiments and are shown as ± s.e.m in triplicates. (d) Transfection of MPF-R cells with 2 separate MCM3 targeting siRNAs (siMCM3.2 and siMCM3.6) resulted in a marked reduction of MCM3 protein levels as evaluated by Western blotting 96 h post siRNA transfection. β-actin was used as loading control. A representative of two biological replicates is shown. Knockdown of MCM3 expression caused no significant changes in growth, proliferation, or apoptosis as measured by colorimetric crystal violet (e), BrdU incorporation (f), and cell death (g) assays, respectively, at 96 h after transfection. Data were confirmed in 3 independent experiments and are shown as ± s.e.m in triplicates. *p < 0.05.