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. 2012 May 7;209(5):1029–1047. doi: 10.1084/jem.20110996

Figure 5.

Figure 5.

Autophagy limits CHIKV-induced apoptosis. (A–D) WT, Bax−/− Bak−/−, or Atg5−/− MEFs were infected with CHIKV (MOI = 1) for 24 h and were stained for LC3 and activated caspase-3. (A) Representative ImageStreamX dot plots from WT, Bax−/− Bak−/−, or Atg5−/− infected MEFs are depicted and the gating strategy is indicated. (B–D) The relative percentage of autophagic cells (LC3 BDIhi, cleaved caspase-3lo; defined by R1), apoptotic cells (LC3 BDIlo, cleaved caspase-3hi; defined by R2), or cells with evidence for both processes (LC3 BDIhi, cleaved caspase-3hi; defined by R3) is shown. Error bars indicate mean ± SD of three independent experiments. (E and F) WT, Bax−/− Bak−/−, or Atg5−/− MEFs were infected with CHIKV (MOI = 1) at the indicated time points, and activated caspase-3 (a-CASP3), activated caspase-9 (a-CASP9), or activated caspase-8 (a-CASP8) were stained from parallel cultures. Representative microscopic images are shown (E), and the percentage of positive cells were determined for >100 cells per condition (F). Error bars show mean ± SD of three independent experiments. Bars, 25 µm. (G and H) WT, Bax−/− Bak−/−, or Atg5−/− MEFs were infected with GFP-expressing recombinant CHIKV (MOI = 1) and stained with anti–active capase-3, -8, and -9 antibody. Cells were gated as uninfected (R1) and infected (R2) cells according to GFP intensity (G) while simultaneously assessing the active capase-3, -8, and -9 staining (H). Similar results were observed in three independent experiments. (I) HFFs were infected with CHIKV (MOI = 1) for 24 h, and the percentage of activated caspase-3 cells was determined for >100 cells per condition. Error bars show mean ± SD of three independent experiments.