Skip to main content
. 2015 Mar 10;6(2):e00133-15. doi: 10.1128/mBio.00133-15

FIG 2 .

FIG 2 

SpyA promotes cell death and caspase-3 activation in a manner independent of MAPK and NF-κB signaling. (A) LIVE/DEAD staining illustrating BMDM viability after 4 h of GAS (AP) infection. Cells were infected at an MOI of ~25 or remained uninfected (UI). Scale bar, 100 µM. (B) Numbers of dead cells were counted from multiple field of views (n = 12). (C) Relative levels of LDH release (percentages compared to uninfected [UI] cells). ΔspyA-infected BMDMs exhibit reduced 2-h cell cytotoxicity at an MOI of 10 or 25. Infection with the SpyA-complemented strain restored LDH release to the WT level (n = 4). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s two-tailed unpaired t test). (D to H) Western blot analysis of whole BMDM lysates after 2 h of GAS infection. β-Actin was used as a loading control. (D) Phospho-ERK1/2 (42 or 44 kDa). (E) Phospho-p-p65 (65 kDa). (F) Apoptosis-inducing factor (AIF; 58 or 68 kDa). (G) Cytochrome c (15 kDa). (H) Caspase-3 (FL, full length [35 kDa]; CLVD, cleaved [17 or 19 kDa]). (I) Capsase-3 and caspace-7 (Caspace-3/7) activity assay. Error bars, SEM. *, P < 0.05; n = 4 (Student’s two-tailed unpaired t test).