Fig 2: Celastrol induced ER stress in HCC cells(A) ER stress related gene expression in HepG2 cells treated with celastrol were analyzed by real-time qPCR, *P < 0.05, student's t-test. (B) Immunoblot analysis of whole-cell lysates from HepG2 cells treated with celastrol and probed with monoclonal antibodies for p-IRE1 and spliced XBP1. For immunoblot analysis, each membrane was stripped and re-probed with monoclonal β-actin. “0 μM” indicates equal molar DMSO control.

Fig 3: TG reverses the anti-ERS effect of rutin on H9C2 cells treated with HG. Western blotting analysis was used to determine the expression levels of ERS related proteins. TG, thapsigargin; ERS, endoplasmic reticulum stress; HG, high glucose; NG, normal glucose; MA, mannitol; HSPA5, heat shock protein A5; IRE1α, insulin response element 1α; XBP1, X-box binding protein 1; CHOP, C/EBP-homologous protein; ATF6, activating transcription factor 6 **P<0.01, ***P<0.001 vs. MA. #P<0.05, ##P<0.01, ###P<0.001 vs. HG. ΔP<0.05, ΔΔP<0.01, ΔΔΔP<0.001 vs. HG + Rutin.

Fig 4: IRE1α signaling is adaptive in cultured GECs subjected to proteotoxic stress.A, B Incubation with TM for 24 h stimulated the upregulation of total IRE1α, robust XBP1 splicing, and mild apoptosis (caspase-3 cleavage) in control (Ctrl) GECs. Quantification of IRE1α phosphorylation was normalized to the actin signal. IRE1α phosphorylation was absent in IRE1α KO GECs but was not affected by 4μ8C, compared to control GECs. IRE1α KO and 4μ8C-treated GECs show undetectable XBP1 splicing and greater apoptosis (immunoblots). C–F TM increased the expression of ER chaperones in control GECs. C, D Upregulation of GRP94, BiP and MANF was impaired in IRE1α KO GECs, and these results were generally recapitulated by 4μ8C. E, F The chaperone ERdj3 is a glycosylated protein. TM inhibits glycosylation and induces an underglycosylated band of ~34 kDa (both bands were used for quantification). Upregulation of ERp57 and ERdj3 was not affected by IRE1α inhibition. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Three experiments performed in duplicate.

Fig 5: XBP1 promotes lactate production and the expression of glycolytic and inflammatory genes during B. abortus infection or LPS treatment. WT and XBP1 KO RAW 264.7 cells were infected with B. abortus (Ba) for 48 h (A and B) or treated with 100 ng/mL Salmonella LPS for 24 h (C and D). Supernatant lactate was quantified (A and C) and relative expression of the indicated genes was assessed by RT-qPCR (B and D). Expression levels of Glut1, Pfkfb3, and Irg1 were normalized to uninfected controls. Because it is not detected in unstimulated cells, IL-6 expression was normalized to the infected or LPS-stimulated WT cells. The data are presented as means of triplicate wells ± the SD. *, P ≤ 0.05; **, P ≤ 0.01; ns, no statistical difference (Student two-tailed t test).