Fig 2: 4-PBA and TUDCA suppress post-IRI ER stress in kidney tubules. C57Bl/6 mice (male, 8–10 week old) were subjected to 30 min of unilateral ischemia of left kidney followed by reperfusion for 7 or 14 days. 4-PBA at 20 mg/kg/day, TUDCA at 250 mg/kg/day, or normal saline (NS) was given from day 3 after uIR. Then kidney tissues were collected for biochemical and histological analyses. (A) Representative images of immunohistochemical staining of BiP. Bar = 200 μm. (B) Quantitative analysis of BiP staining. (C) Representative immunoblots of p-PERK, PERK, BiP, CHOP, and GAPDH. GAPDH was used as a loading control. (D) Quantitative analysis of immunoblot signals of p-PERK, BiP and CHOP. For densitometric analysis, the protein level of sham group was arbitrarily set as 1, and the signals of other conditions were normalized with the sham control group to indicate their protein fold changes. (E) Representative images of immunohistochemical staining of CHOP. Arrows point to positive CHOP staining. Bar = 200 μm. (F) Quantitative analysis of CHOP staining. Data are expressed as mean ± SEM. n = 4. *p < 0.05;**p < 0.01;***p < 0.001; ns, not significant.

Fig 3: Interactions of EK and prothrombin with chaperones.Co-immunoprecipitation (IP) and Western blotting (IB) of EK-WT and EK-4Q (A) or PT-WT and PT-N416Q (B) binding to calnexin, BiP and calreticulin (top three panels). EK (A) and PT (B) proteins in V5 pull-down samples were verified. EK, PT and GAPDH in cell lysates were also verified by western blotting (bottom two panels). Relative levels of calnexin, BiP and calreticulin associated with EK-WT and EK-4Q (A) or PT-WT and PT-N416Q (B) were estimated by densitometric analysis of Western blots. Data are means ± S.E. from three and four independent experiments, respectively. p-Values are shown in bar graphs. IP and IB analysis of EK-WT and EK-4Q (C) or PT-WT and PT-N416Q (D) binding to calnexin and BiP in the cells without (-) or with (+) DNJ treatment (top two panels). EK (A) and PT (B) proteins in V5 pull-down samples were verified. EK, PT and GAPDH proteins in cell lysates were also verified. Relative calnexin and BiP levels associated with EK-WT and EK-4Q (C) or PT-WT and PT-N416Q (D) were estimated by densitometric analysis of Western blots. Data are means ± S.E. from three independent experiments. p-Values are shown in bar graphs.

Fig 4: Spermidine decreases the expression of ERS-related proteins.The protein expression of CHOP, GRP78, ATF6, and IRE-1 was measured by immunoblotting. The intensity of each band was quantified by densitometry, and the data were normalized to ß-actin. The data are expressed as the mean ± SEM. *p < 0.05 vs. the BLM(+)/spermidine(–)group. (n = 3).

Fig 5: Suppression of PPAR? inhibits PPZ023-induced cell death in NSCLC cells.a–c A549 and H460 cells were transfected with PPAR? siRNAs and treated with PPZ023 (25 µ?, 24 h). Next, cell viability and LDH assays were performed along with western blot analyses of PPAR?, p-PERK, p-eIF2?, ATF4, CHOP, and cleaved caspase-3; *p < 0.05. ß-Actin was used as the protein loading control. d–f Stable PPAR?-knockdown H460 cell lines were established after H460 cells were transfected with a PPAR? shRNA. These cells were treated with PPZ023 (25 µ?, 24 h), GW9662 (20 µM, 24 h), or PPZ023/GW9662, and cell viability and LDH assays were performed along with western blot analyses of PPAR?, GRP78, p-PERK, p-eIF2?, ATF4, CHOP, and cleaved caspase-3; *p < 0.05. ß-Actin was used as the protein loading control, and Ponceau S stain was used as the exosome loading control.