Journal: Advanced Science

Article Title: IL‐4/STAT6‐signaling Influences Local Inflammation and Regeneration Processes During Acute Pancreatitis and Promotes Fibrosis by a Direct Activation of Pancreatic Fibroblasts During Chronic Pancreatitis

doi: 10.1002/advs.202515585

Figure Lengend Snippet: CD206 M2‐like macrophages differentiate in the absence STAT6 but retain proinflammatory properties (A) Immunofluorescent labeling of CD206, CCR2 and CD3 in CP tissue. (B) Quantification of CD206 + , CCR2 + and CD3 + cells in the pancreas of CP mice ( n = 10). (C) Heat map illustrating different levels of inflammation related proteins between wild type and Stat6‐/‐ mice. The numbers in the boxes represents the change compared with untreated control mice, the amount of all red labeled proteins were significantly changed in Stat6‐/‐ compared to wild type mice after onset of CP ( n = 5). (D) Quantitative gene expression analysis by RT‐qPCR of Arg1 , Fizz , Ym1 and Il10 in CP tissue of wild type and Stat6‐/‐ mice ( n = 5). (E) Immunofluorescent labeling of MMP9 (red) and α‐amylase (green) in CP tissue and quantification of MMP9 + cells in the pancreas of CP mice. (F) Quantitative RT‐PCR of Nos2 , Il1b , Mmp9 and Il6 in CP tissue of wild type and Stat6‐/‐ mice ( n = 10). (G) Measurement of TGF‐β in serum of untreated control mice and CP mice ( n = 9). (H) Quantitative RT‐PCR of Tgfbi and Csf1 in CP tissue of wild type and Stat6‐/‐ mice ( n = 5). Data in B and D‐H are shown as mean +/‐ SEM. Differences were tested for statistical significance by unpaired students t‐test or Mann–Whitney for non‐normally distributed samples for independent samples, significance levels of p < 0.05 are marked by an asterisk.

Article Snippet: Recombinant Mouse IL‐4 Protein (#404‐ML, R&D Systems), Recombinant Mouse IL‐13 Protein (#413‐ML, R&D Systems), Lipopolysaccharide Escherichia coli O26:B6 (L8274, Sigma Aldrich), Recombinant Mouse IL‐6 Protein (406‐ML, R&D systems), Recombinant Mouse TGF‐β Protein (7666‐MB, R&D systems), Recombinant mouse IL‐10 (575804, BioLegend).

Techniques: Labeling, Control, Gene Expression, Quantitative RT-PCR, MANN-WHITNEY

Journal: Journal of molecular cell biology

Article Title: METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.

doi: 10.1093/jmcb/mjad005

Figure Lengend Snippet: Figure 3 METTL3 is involved in primary mouse RPE cells undergoing EMT. ( A and B ) The mRNA ( A ) and protein ( B ) levels of EMT-related markers ( E-cadherin, N-cadherin, and α-SMA ) and METTL3 in primary mouse RPE cells with and without 10 ng/ml TGF- β2 for 48 h. ( C ) Phase- contrast microscopy images of RPE cells showed the spindle fibroblast-like morphology induced by TGF- β2 application. Scale bar, 50 μm. ( D ) Immunofluorescence analysis demonstrated increased METTL3 in RPE cells treated with TGF- β2. Scale bar, 25 μm. ( E ) Primary RPE

Article Snippet: For the EMT group, cells were passaged at a 1:3 ratio and treated with recombinant mouse TGF- β2 ( R&D Systems ) at a concentration of 10 ng/ml to model the EMT of RPE in vivo .

Techniques: Microscopy

Journal: Journal of molecular cell biology

Article Title: METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.

doi: 10.1093/jmcb/mjad005

Figure Lengend Snippet: Figure 4 METTL3-mediated m 6 A modification enhances the stability of HMGA2 mRNA. ( A ) MeRIP–qRT-PCR with anti-m 6 A antibody was performed to determine the m 6 A enrichment of HMGA2 mRNA with or without METTL3 knockdown in primary RPE cells. ( B ) Western blotting analysis showed that HMGA2 protein level was induced by TGF- β2 and inhibited upon METTL3 knockdown. ( C ) Immunofluorescence analysis confirmed the reduced expression of HMGA2 in METTL3-knockdown RPE cells ( n = 15 for each group ) . Actin cytoskeleton was visualized by phalloidin staining, which targets F-actin. Scale bar, 25 μm. ( D ) Schematic representation of pmirGLO dual-luciferase vectors fused with WT or MUT HMGA2 3 ′ UTR. ( E ) 3 ′ UTR WT or 3 ′ UTR MUT reporters were transfected into RPE cells along with empty vectors or METTL3 expression plasmid. Relative luciferase activity was measured after 48 h. ( F ) After transfection with either si-METTL3 or si-NC for 48 h, primary RPE cells were treated with Act-D to block transcription. HMGA2 mRNA was analyzed at the indicated times. Data present mean ± SD of three independent experiments. Student’s t-test for two independent groups, one-way ANOVA tests for luciferase reporter assay and repeated measures, two-way ANOVA tests for HMGA2 mRNA decay experiment, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For the EMT group, cells were passaged at a 1:3 ratio and treated with recombinant mouse TGF- β2 ( R&D Systems ) at a concentration of 10 ng/ml to model the EMT of RPE in vivo .

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Staining, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Blocking Assay, Reporter Assay