Figure 5.

Knockdown of TIGAR by siRNA reduces the antiproliferative properties of urolithin A. (A) HCT116 WT cells were left untreated (ctrl) or transfected with siRNA (200 nM final concentration, scrambled (scr) or TIGAR-specific sequences #1–3, as indicated). After recovery and 24-h treatment with solvent (0.1% DMSO) or 30 µM urolithin A cells were lysed and subjected to western blot analysis for TIGAR, p21 and actin as loading control. Representative blots and densitometric analyses of three biological replicates are depicted. (B) HCT116 WT cells were transfected with the siRNAs (scr/mix#1–3) as indicated. Eight hours after transfection, cells were seeded into 96-well plates and treated with different concentrations of urolithin A. After 72-h incubation, a biomass staining via crystal violet was performed. Residual biomass was plotted against the concentration of urolithin A, and IC₅₀ values were derived from the fitted curve (n = 3, mean ± SD, *P < 0.05; scr versus si). (C) HCT WT cells were transfected with scrambled siRNA or siRNAs (mix #1–3) targeting TIGAR, seeded in 96-well plates and treated with urolithin A and oxaliplatin at the indicated concentrations for 72 h. After biomass staining isobolograms for 50% reduction in biomass were drawn and combinatorial indices determined. Presented data are representative for three independent experiments with consistent results.