Author response image 1. Antioxidant treatments do not affect the induction of CES protein expression in MIN6 treated with Tg for 18h.
MIN6 cells were either kept in DMEM growth medium (10% heat inactive FBS, 2 mM glutamine, and 25 mM glucose) as control, or were cultured in the medium supplement with Tg alone, Tg and N-acetylcysteine (NAC, 1 mM), or Tg and catalase (CAT, 500 unit/mL) for 18h. After treatments, cells were washed with cold PBS and lysed with the RIPA buffer (150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholic acid and 100 mM Tris-HCl (pH 7.5) containing protease and phosphatase inhibitor from Roche. Cells were then sonicated on ice, lysates were clarified by centrifugation at 4°C and the protein concentrations were determined by the BCA assay (BioRad). (Top panel) The antioxidant effects were evaluated by the caspase 3 activity assay. We found that the caspase 3 activation was significantly reduced by both antioxidant treatments compared to untreated cells. However, the induction of CSE protein expression was unaffected by the treatments (bottom panel), indicating that Increased ROS production does not contribute in induction of CSE protein levels via the ATF4 transcription program during ER stress in MIN6.