Figure 2

Inhibition of SREBP function induces ER-stress. (A) Schematic overview of the ER-stress pathway. (B) RPE-myrAkt-ER cells were transfected with siRNA targeting SREBP1 (siBP1), SREBP2 (siBP2) or both (siBP1 + 2). Scrambled siRNAs were used as controls (siCtr). At 72 hours post-transfection, cells were placed in medium containing 1% LPDS and treated with 100 nM 4-OHT or solvent (ethanol) for 24 hours. Phosphorylation of PERK (threonine 980) and eIF2α (serine 51) was determined. Actin was used as a loading control. (C) cDNA from cells treated as in B was analyzed for expression of SREBP1, SREBP2 and C/EBP-homologous protein (CHOP) by quantitative reverse transcriptase PCR (qRT-PCR). Graphs show mean ± standard error of the mean (SEM) of three independent replicates. (D) Splicing of XBP-1 was determined by RT-PCR. Bands representing the unspliced (XBP-1us) and spliced transcript (XBP-1 s) are marked. (E) Cleaved ATF6 (50 kDa) was detected by immunoblotting. Treatment with 50 nM thapsigargin (TG) or 6 μM tunicamycin (TM) was used as control. (F) Cells depleted of SREBP1 and SREBP2 were treated with 100 nM 4-OHT or 10 mM of 4-phenyl butyric acid (PBA) for 24 hours as indicated. Phosphorylation of PERK and eIF2α was determined. (G) CHOP expression in cells treated in parallel to F. Graphs show mean ± (SEM) of three independent replicates. (H) Effect of PBA treatment on XBP-1 splicing. 50 nM thapsigargin (TG) was used as control. (I) Effect of SREBP depletion on protein synthesis. Graph shows mean and range of two independent experiments. *P < 0.05; **P < 0.01.