Depletion of SREBP1 and SREBP2 causes reactive oxygen species (ROS) accumulation. (A) Levels of reactive oxygen species (ROS) in cells depleted of SREBP1 (siBP1) and SREBP2 (siBP2) or both (siBP1 + 2) and treated with 100 nM 4-OHT or solvent for 24 hours in medium with 1% LPDS. Graph shows mean ± SEM of three independent experiments. (B) Cells were treated as in A but in the presence or absence of 10 mM of the antioxidant N-acetyl cysteine (NAC). Lysates were analyzed for phosphorylation of PERK (* = unspecific band). (C) Expression of CHOP in cells treated as in B. Graph shows mean ± SEM of three independent replicates. (D) Effect of NAC on XBP-1 splicing. Treatment with 50 nM thapsigargin (TG) was used as control. (E) ROS levels in SREBP-depleted cells treated with 4-OHT or solvent in medium with 10% FCS or 1% LPDS for 24 hours. Graph shows mean and range of two independent experiments. (F) Total ROS levels in cells depleted of SREBP and treated with 4-OHT or solvent in medium containing 1% LPDS supplemented with BSA or BSA-coupled oleate (300 μM oleate) for 24 hours. Graph shows mean and range of two independent experiments. (G) Mitochondrial ROS levels in cells treated as in F. Graph shows mean ± SEM of three independent experiments. (H) Mitochondrial respiration of control and SREBP depleted cells was determined using a Seahorse Bioanalyzer. Cells were treated with 4-OHT (solid lines) or solvent (dashed lines) for 24 hours in medium with 1% LPDS. Mitochondrial respiratory capacity was determined in the presence of FCCP. (I) Mitochondrial respiration after addition of BSA (0.3%, dashed lines) or BSA oleate (300 μM oleate, solid lines). *P < 0.05; **P < 0.01; ns = non-significant.