Fig. 6.

Effects of caspase-12 inhibition on H₂O₂-induced cell death in H9c2 cells.

A, Immunoblots of FKBP8 in knockdown H9c2 cells. H9c2 cells were transfected with FKBP8-specific siRNA (siFKBP8) or non-targeting control siRNA (siCTRL) for 96 h. The graph shows densitometric analysis. The value of FKBP8-to-GAPDH ratio in siCTRL was set equal to 1. Data are presented as the mean ± SEM (n = 3). *P < 0.05 versus siCTRL. B, Transfected cells were incubated with Z-ATAD-FMK (3 μmol/L) or DMSO for 2 h before H₂O₂ administration (50 μmol/L). Six h after H₂O₂ stimulation, cellular viability was determined by Cell-Titer Blue assay. The value in the cells transfected with siCTRL and incubated without H₂O₂ nor Z-ATAD-FMK was set to 1. Values represent the mean ± SEM from 3 independent experiments with triplicates. *P < 0.05 versus all other groups. C, Z-ATAD-FMK inhibited apoptotic cell death caused by H₂O₂ in FKBP8 knockdown H9c2 cells. Representative images of double staining of TUNEL (green) and DAPI (blue). White arrows indicate TUNEL positive nuclei. The graph shows quantitative analysis of TUNEL positive nuclei. Values are presented as the mean ± SEM (n = 5). *P < 0.05 versus all other groups. ^†P < 0.05 versus all other groups except the group transfected with siCTRL and incubated with H₂O₂. Scale bar, 50 μm. D, Western blot analysis of caspase-12, caspase-3 and KDEL. The graphs show densitometric analysis. Data are normalized to GAPDH and presented as the mean ± SEM from 4 to 6 independent experiments. *P < 0.05 versus all other groups. ^†P < 0.05 versus all other groups except the siCTRL-transfected group followed by H₂O₂ without Z-ATAD-FMK. ^‡P < 0.05 versus the groups without H₂O₂. ^§P < 0.05 versus all other groups except the siFKBP8-transfected group followed by H₂O₂ with Z-ATAD-FMK. ^|| P < 0.05 versus all other groups except the siFKBP8-tranfected group followed by H₂O₂ without Z-ATAD-FMK. Open and closed bars represent siCTRL- and siFKBP8-transfected groups, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)