Figure 2
From: Periplocin and cardiac glycosides suppress the unfolded protein response
Periplocin suppresses activation of the global UPR pathway. (a) Chemical structure of periplocin. (b) HEK293 cells were pretreated with the indicated doses of periplocin for 1 h, and were then incubated with TM (0.5 µg/ml) for 6 h. The expression of each gene was assessed by semi-qPCR. Unspliced or spliced XBP1 mRNA are indicated. GAPDH was used as the loading control. (c) HEK293 cells were pretreated with the indicated doses of periplocin for 1 h, and were then incubated with TM (0.5 µg/ml) for 6 h. Each expression of spliced XBP1 mRNA and total XBP1 mRNA was quantified by real time qPCR. The ratio of spliced XBP1 mRNA/total XBP1 mRNA (XBP1 s/t) are shown as mean fold changes ± S.D. (n = 3 technical replicates). IC50s for each of these compounds are shown. Significant differences are indicated as *p < 0.05, ***p < 0.001, assessed by one-way ANOVA with Dunnett’s post-test; n. s., not significant. (d) HEK293 cells were pretreated with the indicated doses of periplocin, GSK2656157 (GSK, 1 µM) or ISRIB (0.5 µM) for 1 h, and were then incubated with TM (0.5 µg/ml) for 6 h. Cell lysates were resolved by a 5–20% gradient gel and immunoblotted with the indicated antibodies. β-actin was used as the loading control. One representative from two independent experiments is shown. (e) HEK293 cells were pretreated with periplocin (2 µM) or the ATF6α inhibitor Ceapin-A7 (6 µM) for 1 h, and were then incubated with TM (2 µg/ml) for 4 h. The proteasome inhibitor MG132 (10 µM) was added for 1 h prior to cell lysis to avoid degrading ATF6α fragments. Full length of ATF6α (FL), cleaved ATF6α (CL), and non-glycosylated ATF6α (*) are indicated. β-actin was used as the loading control. One representative from two independent experiments is shown. (f) A schematic representation of the p(ERSE)2-Luc plasmid is shown (top). HEK293 cells were transiently transfected with p(ERSE)2-Luc and pCMV/β-gal. After 24 h, cells were pretreated with periplocin (0.2 µM) or Ceapin-A7 (6 µM) for 1 h, and were then incubated with TM (0.5 µg/ml) for 16 h. Luciferase activity in cell lysates was measured and normalized by the β-galactosidase activity. Values are shown as mean fold activity ± S.D. (n = 3 biological replicates). Significant differences are indicated as ***p < 0.001, assessed by one-way ANOVA with Dunnett’s post-test. (g) A schema for the putative action of periplocin. Uncropped images of gels/blots are shown in Supplementary Information, Fig. S7.
