Journal: Nature Communications

Article Title: DNA-PK-mediated CRTC2 phosphorylation promotes NHEJ and suppresses antitumor immunity via relocation to repair complexes

doi: 10.1038/s41467-026-73228-4

Figure Lengend Snippet: a Schematic diagram of I-SceI-induced NHEJ repair in EJ5-GFP U2OS cells. b NHEJ repair efficiency (left) in response to CRTC2 overexpression, and western blot analysis of I-SceI and CRTC2 expression (right) in EJ5-GFP U2OS cells ( n = 3 biologically independent experiments). c NHEJ repair efficiency (left) in response to CRTC2 knockdown, and western blot analysis of I-SceI and CRTC2 expression (right) in EJ5-GFP U2OS cells ( n = 3 biologically independent experiments). Immunofluorescence staining ( d ) and quantitative analysis ( e ) of 53BP1 foci formation at 0, 0.5, 2, 4, 8, and 12 h after IR (6 Gy) in control and CRTC2-knockout HCCLM3 cells ( n = 40 cells per group). Scale bars, 10 µm. f Schematic diagram of VDJ rearrangement and primer locations in pro-B cells. g Pro-B cell sorting process with CD19, B220, IgM, and CD43 from spleen of Crtc2 +/+ and Crtc2 −/− mice (left) and mRNA level of distal and proximal VDJ rearrangement detection by qPCR (right) ( n = 3 mice per group). h Schematic diagram of mouse immunoglobulin class switch recombination (CSR) in mature B cells. i Serum IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA levels in Crtc2 +/+ and Crtc2 −/− mice ( n = 10 mice per group). j Splenic B cells cultured under LPS or LPS/IL4 stimulation following CSR to IgG3 (left) and IgE (right) ( n = 3 mice per group). All data are shown as the mean ± SEM. Comparisons between two groups were analyzed by Student’s t test. Comparisons between three or more groups with two or more conditions were assessed by two-way ANOVA. All statistical tests were conducted as two-sided. are provided with this paper.

Article Snippet: To induce class switching in mouse B cells, they were cultured for 96 h in medium supplemented with LPS (37.5 μg/ml, Solarbio, Cat# 8880) or a mixture of IL4 (10 ng/ml, MCE, Cat# HY-P7080) and LPS 4, 8.

Techniques: Over Expression, Western Blot, Expressing, Knockdown, Immunofluorescence, Staining, Control, Knock-Out, FACS, Cell Culture

Journal: Hepatology Communications

Article Title: Arrb2 in hepatocytes promotes M2 macrophage polarization, ameliorates hepatic ischemia–reperfusion injury through upregulating metabolite 6-ketoLCA

doi: 10.1097/HC9.0000000000000916

Figure Lengend Snippet: 6-ketoLCA upregulated by Arrb2 in hepatocytes promotes M2 macrophage polarization, alleviating hepatic IRI. (A–E) Liver and serum samples from liver-specific Arrb2-CKO and WT mice with or without 6-ketoLCA treatment after I/R (n=6). (A) HE and TUNEL staining were analyzed microscopically. Scale bar: 200 μm; Suzuki score and necrosis area were examined. (B) Quantitative analysis of the bile acid metabolite 6-ketoLCA in liver tissue. (C) Liver function levels (ALT, AST, TBA, GGT) were analyzed between groups. (D) qRT-PCR analysis of mouse liver tissue mRNA expression for inflammatory factors (IL-6, TNF-α, IL-10, TGF-β). (E) iNOS, CD86, CD206, and CD163 expression in PMMs was assessed by WB analysis. (F) The CCK8 assay measured the viability of RAW264.7 cells exposed to varying concentrations and durations of 6-ketoLCA (n=3). (G, I) Co-culture of RAW264.7 and AML12 cells. (G) ELISA for culture medium supernatant inflammatory factors (IL-6, TNF-α, IL-10, TGF-β). (I) BCL2, BAX, and GAPDH expression in AML12 cells was assessed by WB analysis. (H, J) Flow cytometry detected the proportions of M1 (CD86, PE) and M2 (CD206, APC) macrophages treated with IL-4 and 6-ketoLCA. The ratio of M1/M2 macrophages was measured. All results are presented as mean ± SD, and statistical significance was assessed. * p <0.05, ** p <0.01, and *** p <0.001. Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; CKO, conditional knockout; GGT, gamma-glutamyl transferase; HE, hematoxylin and eosin; I/R, ischemia/reperfusion; IL-6, interleukin 6; IL-10, interleukin 10; IRI, ischemia–reperfusion injury; qRT-PCR, quantitative reverse transcription polymerase chain reaction; PMMs, primary mouse macrophages; TBA, total bile acids; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling; WB, western blotting; WT, wild type.

Article Snippet: RAW264.7 were cultured in 10% FBS RPMI-1640 medium with 6-ketoLCA (100 ng/mL) and IL-4 (20 ng/mL) to induce polarization to M2-like macrophage. shRNA targeting TGR5 (GeneCopoeia, Rockville, MD, USA) was transfected into RAW264.7 cells utilizing Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol to achieve TGR5 knockdown.

Techniques: TUNEL Assay, Staining, Quantitative RT-PCR, Expressing, CCK-8 Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Knock-Out, Reverse Transcription, Polymerase Chain Reaction, End Labeling, Western Blot