Fig 1: Depletion of IRE1α reduces ER stress and the production of inflammatory cytokines in HK‐2 cells following hypoxia‐reoxygenation (H/R). HK‐2 cells depleted of IRE1α and vehicle‐infected HK‐2 cells were cultured under normal conditions or subjected to H/R (4 h of hypoxia followed by 12 h reoxygenation). Representative western blots of ER stress markers (A) upstream (GRP78, PERK, ATF6) and (B) downstream (pJNK, CHOP) of IRE1α; β‐actin was used as the loading control. Quantitative analysis of western blots of ER stress markers (C) upstream (GRP78, PERK, ATF6) and (D) downstream (p‐JNK, CHOP) of IRE1α. E, qRT‐PCR analysis of IRE1α, CHOP and ACTB (internal control). F, IL‐6 secretion to culture supernatant, and qRT‐PCR analysis of IL‐6 and ACTB (internal control). G, MCP‐1 secretion to the culture supernatant, and qRT‐PCR analysis of MCP‐1 and ACTB (internal control). Con: HK‐2 cells without H/R; H/R 12 h:HK‐2 cells with 4 h of hypoxia followed by 12 h reoxygenation; IRE1αShRNA: IRE1α shRNA‐infected HK‐2 cells; Vehicle: vehicle‐infected HK‐2 cells. (NS: not significant, **P < .01, ***P < .001, ****P < .0001)
Fig 2: Depletion of PERK inhibits lipogenic gene expression in HCMV-infected HF cells.mRNA levels of lipogenic enzymes were determined by quantitative RT-PCR using total RNA extracted from mock- and HCMV-infected cells that had been treated with shGFP or shPERK at 72 hpi. ACC1, acetyl CoA carboxylase 1; ACL, ATP-citrate lyase; FAS, fatty acid synthetase; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase.
Fig 3: Depletion of PERK inhibits the induction of lipid synthesis in HCMV infection.(A) shLuc and shPERK treated HF cells, grown on fibronectin coated coverslips, were infected with HCMV and stained with lipophilic dye BODIPY 493/503 at 48 hpi. (B) and (C) Total lipid synthesis was assayed in HF cells. HF cells were treated with shGFP or shPERK, and then mock- or HCMV-infected for 48 hours, then the cells were labeled with 14C-acetate for 3 hours; total lipids were extracted and counted by scintillation counter.
Fig 4: PERK expression is highly increased in HCMV-infected cells and critical for HCMV growth.(A) PERK protein levels in HCMV infection. Whole cell extracts from mock- or HCMV-infected cells at indicated times post infection were analyzed to determine the levels of PERK by Western. (B) Efficiency of shRNA treatment in depleting the levels of PERK protein in HF cells. Lentiviral vectors were used to introduce a control shRNA, shGFP, and two independent shRNAs targeted to PERK mRNA (shPERK, #1 and #2). Whole cell extracts were collected for Western analysis three days after shRNA treatment. (C) HCMV viral growth curves in the presence (shGFP) and absence of PERK (shPERK, #1 and #2) in HF cells. (D) HCMV viral protein expression in the presence (shGFP) and absence of PERK (shPERK #1) in HF cells.
Fig 5: ER stress is activated in the OSCC tissues. A Representative immunohistochemical images of the ER stress marker (PERK, ATF6, and GRP78) staining in the paraneoplastic tissues (NT) and OSCC tissues (TT) of patients with OSCC (scale bar = 50 μm) (n = 3). B–D Quantification of PERK (B), ATF6 (C), and GRP78-positive (D) staining in (A) (n = 3). E Western blot analyses of PERK, ATF6, and GRP78 protein expression in the paraneoplastic tissues (NT) and OSCC tissues (TT) of patients with OSCC. F–H Quantification of the protein levels of PERK (F), ATF6 (G), and GRP78 (H) in (E). I–K The level of PERK (I), ATF6 (J), and GRP78 mRNA (K) in the paraneoplastic tissues (NT) and OSCC tissues (TT) of patients with OSCC (n = 3) (**P < 0.01 and ***P < 0.001)
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