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. 2009 Jan 26;184(2):241–252. doi: 10.1083/jcb.200807019

Figure 3.

Figure 3.

Increased S-glutathionylation of Fas, caspase-8 activity, and cell death in cells lacking Grx1. (A) Assessment of S-glutathionylation of Fas after knockdown of Grx1. C10 cells were transfected with Grx1 siRNA or control (Ctr) siRNA and treated with FasL + M2 for the indicated times. S-glutathionylated proteins were immunoprecipitated using antiglutathione antibody (IP: PSSG). Samples treated with 50 mM DTT to reduce S-glutathionylated proteins (+DTT) were used as reagent controls. The content of Fas, Grx1, and actin in whole cell lysates (WCL) used as the input for IP are shown in the bottom panels. (B) Assessment of S-glutathionylation of Fas after loading of cells with biotinylated glutathione. siRNA-transfected cells were labeled with 5 mM biotinylated glutathione ethyl ester for 1 h before treatment with FasL. After 2 h of FasL + M2 treatment, glutathionylated proteins in lysates were immunoprecipitated using antibiotin antibody followed by immunoblot detection of Fas. The bottom panel shows total Fas expression in whole cell lysates as a loading control. (C) Evaluation of caspase-8 and -3 enzymatic activities in cells after knockdown of Grx1. C10 cells were transfected with control or Grx1 siRNA before stimulation with FasL + M2 for 1 h, and lysates were prepared for evaluation of caspase activity. Results are expressed as mean + SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P < 0.05 compared with respective control siRNA groups (ANOVA). (D) Impact of Grx1 knockdown on cell survival. Cells were transfected with control or Grx1 siRNA and exposed as described in A. Cell survival was assessed using the MTT assay. Results are expressed at a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Grx1 siRNA–treated cells was 11% lower than the control siRNA–treated cells in the absence of stimulation with FasL. *, P < 0.05 compared with respective control siRNA groups (ANOVA). (E) Assessment of S-glutathionylation of Fas in lung fibroblasts lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as described in A. All samples were run on the same gel, and the lanes were cut and reassembled for consistency. Note that S-glutathionylation of Fas in these primary cells is relatively protracted compared with results obtained in the C10 cell line. Black line indicates that intervening lanes have been spliced out. (F) Assessment of S-glutathionylation of Fas in CD4+ T lymphocytes lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as in A. The bottom panels show content of Fas and Grx1 in whole cell lysates. (G) Caspase-8 and -3 activities in primary lung fibroblasts isolated from WT or Glrx1−/− mice in response to exposure to FasL + M2 for the indicated times. Results are expressed as mean ± SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P < 0.05 compared with respective WT groups (ANOVA). Note that activation of caspases in these primary cells is relatively protracted compared with results obtained in the C10 cell line. (H) Comparative assessment of FasL-induced cell death in WT or Glrx1−/− primary lung fibroblasts. Cells were exposed as indicated, and survival was assessed using the MTT assay. Results are expressed as a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Glrx1−/− cells was 13% lower than their WT counterparts in the absence of stimulation with FasL. *, P < 0.05 compared with the WT FasL-treated group (ANOVA). Note that FasL-induced cell death in the primary cells is relatively protracted compared with results obtained in the C10 cell line.