Ponasterone A (PA)-inducible expression of Bax; the effect of cathepsin inhibitors on Bax expression and viability studies. Three separate groups of WT, Asp33Ala, p18, Glu6Ala, or empty vector-transfected 293 cells were used for inducible Bax expression and viability studies as described. (A) Representatives of time course-inducible Bax expressions. Following addition of 6 μM PA, proteins were extracted at the indicated times and subjected to SDS-PAGE and Western blotting with mouse-specific anti-Bax antibody and anti-actin antibody for loading control. (B) Cell viabilities were measured by trypan blue exclusion method following PA-inducible expression of Bax. DNA fragmentations were determined to confirm that cells underwent apoptosis. (C) Twenty-four-hour PA induction of Bax was performed in the presence of 20 μM calpeptin (inhibits calpain) or 40 μM ALLM (inhibits both calpain and cathepsin) or 20 μM cathepsin inhibitor I (CI) or 2 μg/mL adriamycin as described in “Materials and methods.” Lane alignment: 0, vector control; 1, WT; 2, Asp33Ala; 3, p18; 4, Glu6Ala. The representatives of 3 independent experiments were shown. (D) Cell viabilities following 24-hour PA induction of Bax in the presence of CI. (E) Cell viabilities following PA induction of Bax in the presence of ALLM. (F) Cell viabilities following 24-hour combination of PA induction and adriamycin treatment. Error bars in B, D-F indicate means ± SD (n = 3).