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. Author manuscript; available in PMC: 2019 Aug 1.
Published in final edited form as: Mol Cancer Ther. 2018 May 2;17(8):1781–1792. doi: 10.1158/1535-7163.MCT-17-1076

Figure 2. Statin treatment is cytostatic and cytotoxic in VHL-deficient CC-RCC.

Figure 2

LIVE/DEAD assay measuring live cell numbers via Calcein staining (a) and dead cell numbers via PI staining (b) reveals that Simvastatin treatment inhibits RCC4 cell proliferation at nanomolar and micromolar doses, and triggers cell death at higher micromolar doses. RCC4±VHL cells were treated with Simvastatin or vehicle control (80%DMSO/20%Ethanol) for 6 days. Calcein-positive cells in (a) were normalized to the vehicle control. Statistical analysis in (a-b) was performed using a paired t-test between the matched cell lines at each dose (* p < 0.05, ** p < 0.01), SEMs are shown. (c) Representative images of Live/Dead assay. (d) VHL-deficient CC-RCC are more sensitive to Simvastatin treatment than renal cancer cell lines endogenously expressing VHL. Cell lines were treated with 5 μM Simvastatin for 6 days and the live cell number was assessed by Calcein staining. The results were normalized to vehicle-treated cells. Statistical significance was determined using a one-way ANOVA followed by Tukey’s post-hoc analysis (*** p < 0.001), SEMs are shown. The experiment was conducted in duplicate and repeated two times. (e) Western blot confirming VHL expression in cell lines used in (d), HIF1α and HIF2α expression is also shown. α-tubulin serves as a loading control.