Figure 5From: Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growthDepletion 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.Back to article page