Figure 4

Induction of ER-stress following depletion of SREBP is blocked by serum lipids or oleate. (A) Cells depleted of SREBP1 and SREBP2 (siBP1 + 2) were placed in medium with 10% FCS or 1% LPDS, treated with 100 nM 4-OHT or solvent (ethanol) for 24 hours. Lysates were analyzed for phosphorylation of PERK and eIF2α. (B) Cells were depleted of SREBP1 and SREBP2 and treated with 100 nM 4-OHT or solvent in medium containing 1% LPDS supplemented with BSA or BSA-coupled oleate (300 μM oleate) for 24 hours. Phosphorylation of PERK and eIF2α was determined. (C) cDNA from cells treated as in B was used to determine CHOP expression by qRT-PCR. Graph shows mean ± SEM of three independent replicates. (D) Effect of oleate treatment on XBP-1 splicing. Cells treated with 50 nM thapsigargin (TG) were used as control. Line indicates removal of unrelated lanes from scanned gel image. (E) Induction of apoptosis (cleaved poly (ADP-ribose) polymerase (PARP)) in cells treated with BSA, BSA-oleate or BSA-stearate (both 300 μM fatty acid). Actin is shown as a loading control. (F) Expression of stearoyl-CoA desaturase (SCD) protein following Akt activation and SREBP silencing. (G) Parental RPE cells were treated with 1 μM of A939572 in medium with 10% FCS or 1% LPDS. Induction of CHOP was determined by qRT-PCR. (H) Phosphorylation of PERK (upper band) and eIF2α in cells treated with A939572 as in G. (I) Effect of SREBP depletion on CHOP induction was determined in empty vector (pBabe-EV) or SCD expressing cells (pBabe-SCD). (J) Expression of SCD mRNA in empty vector (pBabe-EV) or SCD expressing cells (pBabe-SCD). **P < 0.01.