Idh2 Deficiency Exacerbates Acrolein-Induced Lung Injury through Mitochondrial Redox Environment Deterioration

Figure 1

Idh2 knockdown in LLC cells and their vulnerability to acrolein. (a) idh2 protein expression levels were measured by immunoblotting using anti-idh2 antibody. RT-PCR analysis of gene expression in WT and idh2 knockdown (idh2^KD) LLC cells. β-Actin was used as an internal control for the experiment. (b) Viability of control and idh2^KD LLC cells. Cells were cultured for 2 days at 37°C, exposed to 25 μM acrolein for 1 h, and cell viability was then evaluated using MTT assay. Data are presented as the mean ± SD of four independent experiments. versus WT cells exposed to acrolein. (c) The ratio of cells undergoing apoptosis was measured by FACS. (d) Apoptosis was measured with FITC-labeled annexin in conjunction with PI. Cells were analyzed by flow cytometry. The lower right quadrants represent apoptotic cells. (e) Evaluation of apoptosis with annexin V by FACS. (f) Immunoblot analysis of apoptosis-related proteins. Control and idh2^KD cells were exposed to 25 μM acrolein for 1 h. Cell extracts were electrophoresed on 10–15% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with antibodies against cleaved caspase-3 (c-Casp3), cleaved caspase-9 (c-Casp9), cleaved PARP (c-PARP1), cytochrome c, p53, p-p53, p38, p-p38, JNK, p-JNK, and BAX. β-Actin was used as an internal control.

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