Journal: bioRxiv

Article Title: The IRE1/XBP1 signaling axis promotes skeletal muscle regeneration through a cell non-autonomous mechanism

doi: 10.1101/2021.08.19.457023

Figure Lengend Snippet: Left side TA muscle of Ern1 fl/fl and Ern1 cKO mice was given intramuscular injection of 1.2% BaCl 2 solution whereas right side TA muscle was injected with saline alone. After 5 days, the TA muscles were harvested and processed by western blotting and qPCR. ( A ) Representative immunoblots presented her demonstrate the levels of p-p65, p65, p100, p52 and unrelated protein GAPDH in uninjured and 5d-injured TA muscle of Ern1 fl/fl and Ern1 cKO mice. Quantification of levels of ( B ) p-p65 and p65 ( C ) p100 and p52 protein (n=3 or 4 mice per group). Data are presented as mean ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. * P≤0.05, values significantly different from uninjured TA muscle of Ern1 fl/fl mice. # P ≤ 0.05, values significantly different from 5d-injured TA muscle of Ern1 fl/fl mice. ( D ) Relative mRNA levels of Tnfa , Tnfr1 , Tnfr2 , Il4 , Il6 , Il10, Ifng, Tnfsf12 , Tnfrsf12a in 5d-injured TA muscle of Ern1 fl/fl and Ern1 cKO mice (n=6 mice per group). Data are presented as mean ± SEM. * P≤0.05, values significantly different from 5d-injured TA muscle of Ern1 fl/fl mice. U, uninjured; I, injured.

Article Snippet: Approximately 100 μg protein was resolved in each lane on 10% SDS-polyacrylamide gels, electrotransferred onto nitrocellulose membranes, and probed with the following antibodies: anti-phospho-IRE1α (1:500; Novus, NB 100-2323), anti-IRE1α (1:500; Cell Signaling Technology, #3294), anti-sXBP-1 (1:1000; Cell Signaling Technology, #12782), anti-Pax7 (1:100; DSHB Cat# pax7, RRID:AB_528428), anti-eMyHC (1:200, DSHB Cat# F1.652 RRID:AB_528358), anti-MyoD (Santa Cruz Biotechnology sc-377460), anti-Myogenin (1:100; DSHB Cat# F5D), anti-phospho-p65 (1:500; Cell Signaling Technology, #3033), anti-p65 (1:500; Cell Signaling Technology, # 8242), anti-p100/p52 (1:500; Cell Signaling Technology, #4882), anti-Notch1 (Santa Cruz Biotechnology, #SC-6015), anti-Hes6 (Santa Cruz Biotechnology, #SC-25396), and anti-GAPDH (1:2000; Cell Signaling Technology, #2118).

Techniques: Injection, Western Blot

Journal: Nature immunology

Article Title: Type I IFNs and TNF cooperatively reprogram the macrophage epigenome to promote inflammatory activation

doi: 10.1038/ni.3818

Figure Lengend Snippet: Type I IFNs block TNF-mediated tolerization of inflammatory genes without affecting LPS signaling. ( a ) Experimental design: IFN, treatment with IFN-α (25 ng/ml); IFN-L, treatment with IFN-α, followed by LPS challenge (10 ng/ml); IFN/T, treatment with IFN-α and TNF (10 ng/ml); IFN/T-L, treatment with IFN-α and TNF, followed by LPS. ( b ) Bar graphs represent cumulative values for a given gene class in RNA-seq analysis (left) from three replicates (% of maximum value). Error bars indicate SEM. The dot plots (right) show percent of genes in each class that were up-regulated, down-regulated or not changed by IFN-α (>1.5-fold, T-L vs. IFN/T-L). Each dot represents 1% of genes; red = upregulated, blue = downregulated, grey = not changed. ****p < 0.0001, *p<0.05, analysis of variance and Dunnett’s multiple comparison post hoc test. ( c ) Heatmap showing inflammatory Class 1 genes whose tolerization is reversed by IFN-α treatment. ( d ) Immunoblot analysis of IκBα, p105/p50, cRel, p100/p52, RelB and phosphorylated IKKβ, ERK and STAT1 in primary macrophages cultured for 24 h with TNF (10 ng/ml) with or without IFN-α (25 ng/ml), and challenged for the indicated times with LPS (10 ng/ml). Data are representative of four experiments. ( e ) RT-qPCR analysis of TNF and IL6 primary transcripts normalized relative to HPRT. Data are representative of five independent donors and error bars indicate SEM. ( f ) ChIP assays for recruitment of Pol II to IL6 promoter in the indicated conditions. Data are representative of 4 different donors.

Article Snippet: Antibody to phosphorylated IKKβ (2697), Erk (4377) and STAT1 (9171), and antibody to IκBα (4812), p105/p50 (3035), cRel (4727), p100/p52 (3017), and RelB (4922) were from Cell Signaling.

Techniques: Blocking Assay, RNA Sequencing Assay, Western Blot, Cell Culture, Quantitative RT-PCR