Journal: The Journal of Biological Chemistry

Article Title: The ER stress sensor inositol-requiring enzyme 1α in Kupffer cells promotes hepatic ischemia-reperfusion injury

doi: 10.1016/j.jbc.2021.101532

Figure Lengend Snippet: IRE1α deficiency diminishes iNos expression in Kupffer cells. A–C, control or MφKO mice were subjected to sham or hepatic warm ischemia/reperfusion (n=3–4 per group). A, immunoblot analysis of iNos protein in liver lysates. β-Actin was used as the loading control. B, qRT-PCR analysis of the mRNA abundance of Nos2 in isolated Kupffer cells. C, immunoblot analysis of iNos protein in freshly isolated Kupffer cells. HSP90 was used as the loading control. The relative levels of iNos protein were quantified. D–G, Kupffer cells were isolated from control and MφKO mice. Cells were then treated with 100 ng/ml LPS for the indicated time intervals ( D and E ), or with 0.1 or 1 μg/ml LPS for 12 h ( F and G ). D and F, immunoblot analysis of IRE1α and iNos protein. E and G, qRT-PCR analysis of the mRNA abundance of Xbp1s and Nos2 . H, primary Kupffer cells were treated with PBS (veh) or 100 ng/ml LPS with or without 10 μM 4μ8C. Immunoblot analysis of the indicated proteins. The relative levels of iNos protein were quantified. All data represent the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by one way or two-way ANOVA.

Article Snippet: For iNOS inhibition, mice were injected with 1400W (35 mg per kg body weight; Targetmol, #T3491) or 10% DMSO, 40% PEG300, 5% Tween 80 in saline as vehicle control at 1 h prior to hepatic I/R surgery.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Isolation

Journal: The Journal of Biological Chemistry

Article Title: The ER stress sensor inositol-requiring enzyme 1α in Kupffer cells promotes hepatic ischemia-reperfusion injury

doi: 10.1016/j.jbc.2021.101532

Figure Lengend Snippet: Blocking IRE1α RNase attenuates Kupffer-cell-mediated inflammation during hepatic I/R injury. A, immunohistochemical staining of Ly6G + neutrophils in liver sections of the indicated group. Quantification of Ly6G + cells is shown per field. Scale bar, 100 μm. B, ELISA analysis of serum levels of the indicated inflammatory cytokines. C and D, qRT-PCR analysis of the mRNA abundance of the indicated UPR ( C ) or inflammatory genes ( D ) in freshly isolated Kupffer cells. E, immunoblot analysis of the indicated proteins in lysates of freshly isolated Kupffer cells. HSP90 was used as the loading control. The relative levels of NLRP3, c-Casp1 and iNos proteins were quantified. All data represent the mean ± SD. ∗ p < 0.05 and ∗∗ p < 0.01 by one-way ANOVA. NLRP3, nucleotide-binding domain, leucine-rich repeat containing protein 3; UPR, unfolded protein response.

Article Snippet: For iNOS inhibition, mice were injected with 1400W (35 mg per kg body weight; Targetmol, #T3491) or 10% DMSO, 40% PEG300, 5% Tween 80 in saline as vehicle control at 1 h prior to hepatic I/R surgery.

Techniques: Blocking Assay, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation, Western Blot, Control, Binding Assay

Journal:

Article Title: Regulation of induction of nitric oxide synthase and the inhibitory actions of dexamethasone in the human intestinal epithelial cell line, Caco-2: influence of cell differentiation

doi: 10.1038/sj.bjp.0702827

Figure Lengend Snippet: Inducible NOS protein expression in Caco-2 cells assessed by Western blot. The inducible NOS protein expression was determined by Western blot using a polyclonal iNOS antibody. Each panel is a representative of three different experiments. (a) Caco-2 cells (passages 32 to 39) were used 10 days after confluence. Cells were treated for 18 h with vehicle alone (Con), cytomix (CM), cytomix and cycloheximide (CHX) (5 μg ml−1, added concurrently), cytomix and actinomycin D (ActD) (5 μg ml−1, added concurrently), or cytomix and pyrrolidine dithiocarbamate (PDTC) (200 μM, 2 h pre-treatment). (b) Caco-2 cells were used 10 days after confluence. Cells were treated for 18 h with vehicle alone (Con), cytomix (CM) or cytomix and dichloroisocoumarin (DCI) (200 μM, 2 h pre-treatment). (c) Caco-2 cells were used 10 or 15 days after confluence as indicated. Cells were treated for 18 h with vehicle alone (Con), cytomix (CM), or cytomix and dexamethasone (Dex) (5 μM). (d) Caco-2 cells were used 10 days after confluence. Cells were treated for 18 h with vehicle alone (Con), cytomix (CM), or cytomix and tyrphostin A25 (200 μM), cytomix and tyrphostin B42 (200 μM), cytomix and PDTC (100 μM, 2 h pre-treatment), or cytomix, PDTC (100 μM) and A25 (20 μM).

Article Snippet: After blocking with PBS (pH 7.4), 0.25% tween 20 (v v −1 ) and 5% non-fat dried milk, membrane was probed with anti-iNOS polyclonal antibody (1/500) (Autogen Bioclear, Calne, U.K.) for 1 h at room temperature, washed with PBS-tween 20 and then incubated with horseradish peroxidase-conjugated second antibody (1/4000) for 1 h at room temperature.

Techniques: Expressing, Western Blot