Effect of caspase 9 inhibition on apoptosis of HMC induced by palmitate. (A) Cells were treated with control (Con) or palmitate (Palm) in the presence and absence of the cell permeable caspase 9 inhibitor (C9I, Z-LEHD-FMK) or the caspase 8 inhibitor (C8I, Z-IETD-FMK), both at 40 μM. Addition of the caspase inhibitors was concurrent with addition of the FFA. After 24 and 48 h, caspase 9 enzyme activity was measured and expressed as fold-change over control. (B) Palmitate and Z-LEHD-FMK were added to HMC as above, and cleavage of caspase-3 was measured by ELISA as described in Experimental Procedures. (C) The number of pyknotic nuclei was assessed in cells treated with palmitate with and without Z-LEHD-FMK. (D) DNA fragmentation, assessed by ELISA as the number of nucleosomal fragments in the cytosol, was measured in cells treated with palmitate and by Z-LEHD-FMK. For A-D, data are mean ± SEM for 3 independent experiments. **, P < 0.01, *, P < 0.05 versus control by ANOVA in (A, B and D) or Chi-square test in (C).