Figure 3.
Figure 3. Relationship between CD95 expression and NF-κB, STAT1, or p53 activation in LCL cells. (A) CD95 expression in LCL cells transfected with pRT-1-IκBαS32,36A–inducible vector pretreated with (+) or without (-) doxycycline for 36 hours and additionally treated with TNFα for 24 hours or left untreated. Upper panel: CD95 mRNA expression by real-time RT-PCR (fold changes are indicated at the top of each bar compared with the untreated condition; error bars correspond to the standard deviation of 3 experiments). Middle panel: CD95 expression by Western blot. As in Figure 1A, the CD95 signal was normalized to Ponceau red (PR) signal and fold changes in CD95 protein are presented in the histogram. Lower panel: Expression of surface membrane CD95 by flow cytometry in LCL cells transfected with pRT1-IκBαS32,36A. Percentage of CD95+ cells is given in the upper-left corner of the histogram for NGFR-positive (IκBαS32,36A expressing) cells and in the upper-right corner of the histogram for NGFR-negative cells. (B) CD95 expression in LCL cells transfected with pRT-1-STAT1β–inducible vector pretreated with (+) or without (-) doxycycline for 36 hours and additionally treated with IFNγ for 24 hours or left untreated. Upper panel: CD95 mRNA expression by real-time RT-PCR (fold changes are indicated at the top of each bar compared with the untreated condition; error bars correspond to the standard deviation of 3 experiments). Lower panel: Expression of CD95 and STAT1 by Western blot. As in Figure 1A, the CD95 signal was normalized to Ponceau red (PR) signal and fold changes in CD95 protein are presented in the histogram. (C) CD95 expression in LCL cells transfected with pRT-1-p53wt vector pretreated with (+) or without (-) doxycycline for 36 hours and additionally treated with fludarabine for 24 hours or left untreated. Upper panel: CD95 mRNA expression by real-time RT-PCR (fold changes are indicated at the top of each bar compared with the untreated condition; error bars correspond to the standard deviation of 3 experiments). Lower panel: Expression of CD95 and p53 by Western blot. As in Figure 1A, the CD95 signal was normalized to Ponceau red (PR) signal and fold changes in CD95 protein are presented in the histogram. Note that protein loading was decreased in the doxycycline-treated condition, probably due to increase of spontaneous apoptosis of the cells (not shown).

Relationship between CD95 expression and NF-κB, STAT1, or p53 activation in LCL cells. (A) CD95 expression in LCL cells transfected with pRT-1-IκBαS32,36A–inducible vector pretreated with (+) or without (-) doxycycline for 36 hours and additionally treated with TNFα for 24 hours or left untreated. Upper panel: CD95 mRNA expression by real-time RT-PCR (fold changes are indicated at the top of each bar compared with the untreated condition; error bars correspond to the standard deviation of 3 experiments). Middle panel: CD95 expression by Western blot. As in Figure 1A, the CD95 signal was normalized to Ponceau red (PR) signal and fold changes in CD95 protein are presented in the histogram. Lower panel: Expression of surface membrane CD95 by flow cytometry in LCL cells transfected with pRT1-IκBαS32,36A. Percentage of CD95+ cells is given in the upper-left corner of the histogram for NGFR-positive (IκBαS32,36A expressing) cells and in the upper-right corner of the histogram for NGFR-negative cells. (B) CD95 expression in LCL cells transfected with pRT-1-STAT1β–inducible vector pretreated with (+) or without (-) doxycycline for 36 hours and additionally treated with IFNγ for 24 hours or left untreated. Upper panel: CD95 mRNA expression by real-time RT-PCR (fold changes are indicated at the top of each bar compared with the untreated condition; error bars correspond to the standard deviation of 3 experiments). Lower panel: Expression of CD95 and STAT1 by Western blot. As in Figure 1A, the CD95 signal was normalized to Ponceau red (PR) signal and fold changes in CD95 protein are presented in the histogram. (C) CD95 expression in LCL cells transfected with pRT-1-p53wt vector pretreated with (+) or without (-) doxycycline for 36 hours and additionally treated with fludarabine for 24 hours or left untreated. Upper panel: CD95 mRNA expression by real-time RT-PCR (fold changes are indicated at the top of each bar compared with the untreated condition; error bars correspond to the standard deviation of 3 experiments). Lower panel: Expression of CD95 and p53 by Western blot. As in Figure 1A, the CD95 signal was normalized to Ponceau red (PR) signal and fold changes in CD95 protein are presented in the histogram. Note that protein loading was decreased in the doxycycline-treated condition, probably due to increase of spontaneous apoptosis of the cells (not shown).

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