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. 2014 Jul 14;9(7):e102022. doi: 10.1371/journal.pone.0102022

Figure 3. Knockdown of c-FLIPL affects apoptosis and proliferation of Th cells.

Figure 3

Freshly isolated Thp cells were transfected with c-FLIPS, c-FLIPL or non-targeting (NT) siRNA, left to rest for 20–24 h and then activated and stimulated with IL-12 (Th1) or IL-4 (Th2). A. The knockdown efficiency of c-FLIPS and c-FLIPL siRNAs. Samples for western blotting analysis were harvested 24 h after priming. GAPDH was used as a loading control. Bars show relative levels of c-FLIPL (upper panel) and c-FLIPS (lower panel) obtained by quantifying and normalizing against the levels of GAPDH. The value of NT Th1 was set as 1. B. the knockdown of c-FLIPL affects the proliferation of Th1 and Th2 cells. Transfected cells were left to rest for 20–24 h and then stained with CFSE and activated and stimulated with IL-12 (Th1) or IL-4 (Th2). The proliferation of CFSE stained cells was analyzed by flow cytometry at days 2, 3 and 4 after initiation of the culture. Histogram shows representative data of three independent biological replicate cultures at day 4. C. Bars show proliferative indexes calculated from three independent biological replicate cultures. Statistical significance was calculated using paired student's t-test, * p<0.05. D. Analysis of CD69 expression by flow cytometry at 24 h time-point. Results are calculated from three independent biological replicate cultures. E. c-FLIPL knockdown Th cells have elevated levels of apoptotic cells. Transfected cells were left to rest for 24 h, cultured and activated in Th1 or Th2 conditions (as described above) followed by staining with Annexin-FITC and prodium iodide (PI). Representative data of three independent biological replicate cultures is shown. F. Bars represent the average of percentage of early apoptotic (Annexin-FITC+PI-) cells (±SEM). Results were calculated from three independent biological replicate cultures. Statistical significance was calculated using the paired student's t-test, * p<0.05. B–F. NT, cFLIPS and cFLIPL refer to the used siRNAs. SEM =  standard error of mean.