Figure 2
From: A novel specific PERK activator reduces toxicity and extends survival in Huntington's disease models
MK-28 is a PERK activator in an HD cellular model. (A) Scheme of the PERK pathway. (B) Treatment of STHdhQ7/7 and STHdhQ111/111 cells (expressing WT and pathogenic Htt respectively) with MK-28 (0–5 μM) or Thap (2 μg/ml) for 1.5 hours caused an increase of eIF2α-P levels relative to total eIF2α-P for both cell types. Calnexin was used as loading control. See full length blots in Supplementary Fig. S6. In the graph, results are compared to untreated STHdhQ7/7 cells (=100) and expressed as medians +- interquartile range of 4 independent experiments. P values treated vs. untreated in black (STHdhQ7/7 MK-28 0.1 μM vs. untr = 0.0004, STHdhQ7/7 MK-28 1 μM vs. untr = 0.02, STHdhQ7/7 MK-28 5 μM vs. untr = 0.0005) and between the two untreated cell lines in grey (Untr. STHdhQ111/111 vs. untr. STHdhQ7/7 = 0.0001). (C) Treatment of MEF cells with MK-28 or Thap (2 μg/ml) for 3 hours caused a shift of PERK consistent with its phosphorylation (PERK-P). The dashed line is for better visualization of the shift. The graph shows an estimation of PERK-P/total PERK (population over the dashed line/total population), expressed as medians +- interquartile range of 4 independent experiments. P values MK-28 1 μM vs. Cntl = 0.02, MK-28 5 μM vs. Cntl = 0.003. (D) Similar to (B) but for ATF4, with actin as loading control. Quantified only for STHdhQ7/7. P value MK-28 10 μM vs. untr = 0.009. (E,F) Cell lysates of STHdhQ7/7 and STHdhQ111/111 cells were subjected to qPCR for CHOP (E) and GADD34 mRNA (F), compared to a housekeeping gene, GAPDH. The graphs show values relative to untreated STHdhQ7/7 cells and are medians +- interquartile range of 3 independent experiments. P values: CHOP: STHdhQ7/7 MK-28 5 μM vs. untr = 0.04, STHdhQ7/7 MK-28 50 μM vs. untr = 0.0009, STHdhQ111/111 MK-28 5 μM vs. untr = 0.005, STHdhQ111/111 MK-28 50 μM vs untr = 0.01, GADD34: STHdhQ111/111 MK-28 5 μM vs. untr = 0.04.
