Fig. 2

Cell viability, cytotoxicity and cell cycle progression in TfRCC cell lines treated with mTOR inhibitors. a, b Cell viability, as measured by MTT assay for TfRCC cell lines and the benign renal epithelial cell line HRCE after 72 h of treatment with up to 1000 nM concentrations of the dual mTORC1/2 inhibitor, AZD8055 (a), or selective mTORC1 inhibitor, sirolimus (b). Viability in TfRCC cells was suppressed by approximately 80–90% with AZD8055 and 30–50% with sirolimus relative to the untreated (0 nM drug) condition. Both drugs inhibited growth to a greater degree in TfRCC cells than in benign renal cells. c, d Cell cytotoxicity, as measured by LDH release by UOK120 and UOK146 TfRCC cell lines after 48 h of treatment with 1 μM of AZD8055 (c) or sirolimus (d). Only slight cytotoxicity in UOK120 cells and no cytotoxicity in UOK146 cells was observed after AZD8055 treatment, while sirolimus treatment had no cytotoxic effect. Multi protein inhibitor LY294002 [100 μM] was used as a positive control. e, f Relative fraction of cells in S-phase of the cell cycle, as measured by BrdU incorporation in UOK120 (e) and UOK146 (f) cell lines treated for 24 h with low (50 nM) and high (500 nM) concentrations of AZD8055 or sirolimus. Dose-dependent reductions in S-phase in both cell lines with either drug mirror the magnitude of reductions observed in cell viability (a, b), supporting a predominantly cytostatic mechanism of growth inhibition for both drugs. *p < 0.05; **p < 0.01; ***p < 0.001; NS = non-significant