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. 2022 May 27;6(9):2354–2367. doi: 10.1002/hep4.1997

FIGURE 2.

FIGURE 2

Endoplasmic reticulum stress and unfolded protein response in Huh7.5Z cells. (A) Representative western blot and (B) quantification graph of PERK (n = 4), p‐PERK (n = 4), eIF2α (n = 3), p‐eIF2α (n = 3), and CHOP (n = 4) in Huh7.5Z and Huh7.5 cells. Results demonstrated increased activation of the PERK signaling branch in Huh7.5Z cells. The changes in the fold increase of expression levels in Huh7.5Z cells compared with Huh7.5 cells were: PERK 3.25‐fold, p = 0.049; p‐PERK 1.47‐fold, p = 0.018; p‐eIF2α 1.86‐fold, p = 0.049; CHOP 2.27‐fold, p = 0.033. Western blot quantification data are presented as mean ± SD. Significance was determined by the Student t test. *p < 0.05. (C) Representative western blot and (D) quantification graph of IRE1α (n = 4), p‐IRE1α (n = 4), ATF6α (n = 4), and ATF6αf (n = 4). Results showed significantly increased levels of p‐IRE1α and ATF6αf in Huh7.5Z cells compared with Huh7.5 cells, indicating the activated IRE1α and ATF6α signaling branches. The fold increases were: p‐IRE1α 1.69‐fold, p = 0.018; ATF6α 1.41‐fold, p = 0.01; ATF6αf 2.64‐fold, p = 3 × 10−4. Western blot quantification data are presented as mean ± SD. Significance was determined by the Student t test; *p < 0.05, ***p < 0.001. (E) qPCR analysis showed elevated Eif2ak3 mRNA (encodes PERK) levels in Huh7.5Z cells compared with Huh7.5 cells; qPCR results are presented as mean ± SD. The change in fold increase was 2.62‐fold. Significance was determined by the Student t test; **p < 0.01. ATF6α, activating transcription factor 6α; ATF6αf, the cytosolic fragment of ATF6α; CHOP, CCAAT‐enhancer‐binding protein homologous protein; eIF2α, eukaryotic initiation factor 2α; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; IRE1α, inositol‐requiring transmembrane kinase/endoribonuclease 1α; mRNA, messenger RNA; p‐eIF2α, phosphorylated eIF2α; PERK, protein kinase R‐like endoplasmic reticulum kinase; p‐IRE1α, phosphorylated IRE1; p‐PERK, phosphorylated PERK; qPCR, quantitative polymerase chain reaction; RQ, relative quantification; UPR, unfolded protein response