Figure 2

Peroxide sensitivity in CCDC6 silenced GC1 cells. (A) Left side: percentage of cell viability evaluated by MTT analysis on GC1 shCCDC6 or sh ctrl cells not treated or treated for 1 hour with H2O2. Right side: CCDC6 and Cytochrome C immunoblots of GC1 shCCDC or sh ctrl cells, not treated or treated with H2O2. (B) Protein extracts from cytosol (C) and mitochondria (M) of shctrl and shCCDC6 cells untreated or treated with H2O2 as indicated were assayed for Cytochrome c by western blot analysis. Tubulin was used as cytosolic marker and COX IV as a mitochondrial marker. Densitometric acquisition are shown from three separate experiments. *p > 0,05 vs untreated shCTRL and shCCDC6 cells (C) WB analysis from GC1 shCCDC6 or sh ctrl lysates from cells not treated or treated with H2O2. The blots are representative of three independent experiments. (D) Caspase 3 activity was evaluated in GC1 cells, shCCDC6 or sh ctrl, not treated or treated with H2O2. The plotted values represent the mean +/- s.e.m. of three independent experiments. (E) Whole cell lysates from GC1 shCCDC6 or sh ctrl cells, and from GC1 cells overexpressing CCDC6T434A or the empty vector, treated with H2O2 (10 μM) or untreated were immunoblotted with anti-CCDC6 or anti-myc. Anti- γH2AX and total H2AX are shown. (F) GC1 cells, depleted or not depleted for CCDC6, were exposed to 50 μM H2O2 for 30 minutes and ROS intracellular levels were evaluated by the DCFH-DA fluorometric method. (G) GC1 cells overexpressing CCDC6T434A or the empty vector were exposed to 50 μM H2O2 for 30 minutes and ROS intracellular levels were evaluated by the DCFH-DA fluorometric method. In F and G data are representative of three separate experiments. # p > 0.05 vs control. Immunoblot of anti-tubulin is shown in A, C and E.