Bax inhibition through alternative means is protective against rotenone toxicity.

<p><b>a)</b> M17 cells were pretreated with BIP (0, 200, or 500 µM) for four hours prior to treatment with rotenone at 1 µM. After 48 hours, cell death was assessed by LDH release into the culture media. LDH release into media was normalized to maximal LDH release for each well. Cells treated with BIP were more resistant to rotenone compared to untreated cells. Error bars reflect SEM. Results reflect three independent experiments with at least two replicates per experiment. ***p<0.001 (Bonferroni's multiple comparison test). <b>b)</b> shRNA targeting Bax showed considerable knockdown of Bax protein expression. Naïve M17 cells were infected with a pLKO.1 lentiviral construct containing Bax-specific shRNA sequence or with an empty pLKO.1 lentiviral construct (with no shRNA sequence; C). Infected cells were selected for in the presence of puromycin. Protein lysates from these infected cells were immunoblotted with a polyclonal antibody against Bax (top blot). Immunoblotting against tubulin (bottom blot) shows comparable protein loading. <b>c)</b> pLKO.1 control or Bax-shRNA M17 cells were treated with rotenone at varying concentrations for 48 hours. Cell death was assessed by LDH release. Bax-knockdown cells showed considerable protection against rotenone compared to control cells at all concentrations tested. Error bars reflect SEM. Results reflect three independent experiments with at least two replicates per experiment. ***p<0.001 (Bonferroni's multiple comparison test). <b>d)</b> shRNA targeting Bax also showed knockdown of Bax protein in both empty vector control and 14-3-3θ stable cell lines. Control and 14-3-3θ stable lines were infected with an empty pLKO.1 virus (C) or with the Bax-specific shRNA lentivirus. Protein lysates from these cells were immunoblotted with a polyclonal antibody against Bax and tubulin. <b>e)</b> Empty vector stable and 14-3-3θ stable cells infected with either empty pLKO.1 or Bax shRNA viruses were treated with rotenone at varying concentrations for 48 hours, and cell death was assessed by LDH release. Knockdown of Bax in 14-3-3θ stable cells provided additional reduction of rotenone toxicity. Error bars reflect SEM. Results reflect four independent experiments with at least two replicates per experiment. *p<0.05, **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test).