Skip to main content
. 2012 Jan 12;7(1):e28568. doi: 10.1371/journal.pone.0028568

Figure 5. Signalling leading to CCT020312-dependent EIF2A phophorylation.

Figure 5

A) Schematic showing EIF2A kinases and their regulation. Pathway agonists thapsigargin, poly (I∶C) and NaAS2O3 and their locus of action are indicated. B) Effect of EIF2AK3/PERK ablation on EIF2A phosphorylation by CCT020312. U-2OS human osteosarcoma cells were transfected with either of two different EIF2AK3/PERK siRNA oligonucleotides (PERK-1, PERK-2) or an irrelevant control (NT) for 72 hours. Cells were treated with 10 µM CCT020312 (CCT) or 2 µM thapsigargin (Tg) for the indicated times. Lysates were analysed by immunoblot as indicated. C) Quantitation of PERK mRNA expression in siPERK transfected cells. PERK mRNA was quantified in siRNA tranfected cells using SYBR Green based quantitative PCR. The comparative cycle threshold method was used to determine the fold change in PERK mRNA relative to cells tranfected with irrelevant oligonucleotide. GAPDH was quantified in parallel and used to normalise between samples. Bars represent the mean fold change in triplicate technical replicates. D) Ablation of EIF2AK3/PERK prevents CCT020312-mediated cyclin D loss and accumulation of underphosphorylated pRB. HCT116 human colon cancer cells were transfected with EIF2AK3/PERK siRNA oligo ‘2’ or non-targeting siRNA (NT). After 72 hours cells were treated with 10 µM CCT020312, 2 µM thapsigargin (Tg) or DMSO for times indicated. Cell lysates were analysed by immunoblotting as indicated.