Journal: Burns & Trauma

Article Title: Type 3 deiodinase activation mediated by the Shh/Gli1 axis promotes sepsis-induced metabolic dysregulation in skeletal muscles

doi: 10.1093/burnst/tkae066

Figure Lengend Snippet: Dio3 expression increased mainly in skeletal muscles and lung tissues upon early sepsis. ( a ) Representative immunoblots of Dio3 expression in primary peripheral tissues, including the liver, intestine, kidney, heart, lung, and skeletal muscles, of control rats or rats subjected to CLP modelling (n = 6). ( b, c ) Quantitative analysis of (a). ( d ) Representative images of Dio3 IHC staining in skeletal muscles of rats with or without CLP modelling and their quantification (n = 6; scale bar: 50 μm&100 μm). ( e ) Representative images of Dio3 IHC staining in lung tissues of rats with or without CLP modelling and their quantification (n = 6; scale bar: 50 μm&100 μm). ( f ) Immunoblotting demonstrating the expression of Dio3 in PC9 and C2C12 cells exposed to gradient LPS concentrations for 24 h (0, 1, 10, 20, 50, and 100 μg/ml; n = 3). ( g ) Quantitative RT-PCR analysis of Dio3 mRNA expression in PC9 and C2C12 cells treated with 100 μg/ml LPS (n = 3). ( h ) Immunoblotting demonstrating the expression of Dio3 in human skeletal muscle biopsy samples (acute stage, biopsy taken within 1 week of admission; convalescence, biopsy taken shortly before discharge during the convalescent stage). * P < 0.05; NS, not significant. Two-tailed Student’s unpaired t-test and Mann–Whitney test for (b, c) or two-tailed Student’s unpaired t-test for (d, e and g). Quantitative data are represented as mean ± SD. NS not significant, Dio3 type 3 deiodinase, CLP cecal ligation and puncture, LPS lipopolysaccharide, IHC immunohistochemistry

Article Snippet: C2C12 myotube nuclear lysates, post 1% formaldehyde cross-linking, were sonicated to fragment chromatin, followed by immunoprecipitation with Gli1 (sc-515751, Santa Cruz, USA) antibody or control IgG.

Techniques: Expressing, Muscles, Western Blot, Control, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Ligation

Journal: Burns & Trauma

Article Title: Type 3 deiodinase activation mediated by the Shh/Gli1 axis promotes sepsis-induced metabolic dysregulation in skeletal muscles

doi: 10.1093/burnst/tkae066

Figure Lengend Snippet: Shh signaling is involved in the reactivation of Dio3. ( a ) Comparative analysis of altered genes between the con group and C2C12 cells exposed to LPS for 4 h (one technical replicate of three biological replicates per group). ( b ) Analysis of the KEGG pathway from upregulated genes in the same setting as in (a). ( c ) Analysis of volcano plot from altered genes in the same setting as in (a). ( d ) Representative immunoblots of Shh expression in the skeletal muscle tissues of control rats or rats subjected to CLP modelling and their quantification (n = 6). ( e ) Representative images of the IF staining of Shh in skeletal muscles (scale bar: 50 μm) and quantification of Shh-positive area per selective field in rats with or without CLP modelling (n = 6). ( f ) Immunohistochemical staining of Dio3 in skeletal muscle biopsy samples from patients (scale bar: 50 μm&100 μm) and its quantification (n = 10; acute stage, biopsy taken within 1 week of admission; convalescence, biopsy taken shortly before discharge during the convalescent stage). ( g ) Immunoblotting demonstrating the expression of Shh and Dio3 in biopsy samples from the skeletal muscle of critically ill patients and its quantification (n = 10). ( h ) Spearman’s rank correlation analysis for the expression of Shh and Dio3 in the skeletal muscles of critically ill patients (n = 10). ( i ) Immunoblotting demonstrating the expression of Shh and Dio3 in C2C12 cells treated with gradient concentrations of rShh (0, 10, 200, and 500 ng/ml; n = 3) and its quantification. * P < 0.05. Two-tailed Student’s unpaired t-test for (d), Mann–Whitney test for (e, f, and g), spearman correlation analysis for (h), or one-way ANOVA followed by Tukey’s multiple comparisons test for (i). Quantitative data are represented as mean ± SD. NS not significant, LPS lipopolysaccharide, Shh sonic hedgehog, CLP cecal ligation and puncture, Dio3 type 3 deiodinase

Article Snippet: C2C12 myotube nuclear lysates, post 1% formaldehyde cross-linking, were sonicated to fragment chromatin, followed by immunoprecipitation with Gli1 (sc-515751, Santa Cruz, USA) antibody or control IgG.

Techniques: Western Blot, Expressing, Control, Staining, Muscles, Immunohistochemical staining, Two Tailed Test, MANN-WHITNEY, Ligation

Journal: Burns & Trauma

Article Title: Type 3 deiodinase activation mediated by the Shh/Gli1 axis promotes sepsis-induced metabolic dysregulation in skeletal muscles

doi: 10.1093/burnst/tkae066

Figure Lengend Snippet: Shh regulates Dio3 expression via Gli1 in myoblasts under an inflammatory backdrop. ( a ) Immunoblotting demonstrating the expression of Dio3 in C2C12 cells treated with rShh (500 ng/ml) or Gant61 (10 or 20 μM) and its quantification (n = 3). ( b ) Immunoblotting demonstrating the expression of Dio3 in C2C12 cells treated with rShh (500 ng/ml) or 10 μm Gant61 for 24 or 48 h (n = 3) and its quantification. ( c ) Quantitative RT-PCR analysis of Gli1 and Gli2 mRNA expression in C2C12 cells treated with 100 μg/ml LPS (n = 3). ( d ) Representative immunoblots of Shh, Gli1, and Gli2 expression in C2C12 cells exposed to gradient LPS concentrations (0, 1, 20, and 100 μg/ml; n = 3) for 24 h, with quantification presented. ( e ) Representative images of Gli1 IF staining in C2C12 cells treated with or without LPS and its co-location analysis conducted by ImageJ (scale bar: 20 μm&50 μm). ( f ) Gli1 motif and schematic diagram of the putative binding site of Gli1 in mouse Dio3 promoter regions predicted by the JASPAR database. ( g ) Agarose gel electrophoresis map of DNA samples amplified by PCR, which are immunoprecipitated and purified by Gli1 antibody binding to C2C12 cell lysates with or without treatment of LPS or rShh and its quantification. ( h ) Quantitative RT-PCR analysis of Dio3 expression in the promoter binding site of ChIP samples in the same setting as in (g). ( i ) Immunoblotting demonstrating Dio3 expression in H293T, HepG2, and PC9 cells treated with rShh and its quantification (n = 3). ( j ) Immunoblotting demonstrating the expression of Dio3 in C2C12 cells treated with 100 μg/ml LPS or 10 μM Gant61 (n = 3) and its quantification. * P < 0.05. Two-tailed Student’s unpaired t-test for (c, h, and i) or ANOVA followed by Tukey’s multiple comparisons tests for (a, b, d, g, and j). Quantitative data are represented as mean ± SD. NS not significant, Shh sonic hedgehog, Dio3 type 3 deiodinase, LPS lipopolysaccharide, GLI GLI family zinc finger

Article Snippet: C2C12 myotube nuclear lysates, post 1% formaldehyde cross-linking, were sonicated to fragment chromatin, followed by immunoprecipitation with Gli1 (sc-515751, Santa Cruz, USA) antibody or control IgG.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Staining, Binding Assay, Agarose Gel Electrophoresis, Amplification, Immunoprecipitation, Purification, Two Tailed Test

Journal: Burns & Trauma

Article Title: Type 3 deiodinase activation mediated by the Shh/Gli1 axis promotes sepsis-induced metabolic dysregulation in skeletal muscles

doi: 10.1093/burnst/tkae066

Figure Lengend Snippet: IL-6 and ROS mediate Shh activation via STAT3. ( a ) Correlation analysis between rT3 and IL-6 (n = 62). ( b ) Representative immunoblots of IL-6, STAT3, and p-STAT3 expression in skeletal muscle samples of rats subjected to sham or CLP modelling (n = 6) and its quantification. ( c ) Quantitative RT-PCR analysis of Dio3 and Shh expression in C2C12 cells treated with 500 ng/ml IL-6 or 5 μM Stattic (n = 3). ( d ) Representative western blots of Shh, Dio3, STAT3, and p-STAT3 expression in C2C12 cells treated with 500 ng/ml IL-6 or 5 μM Stattic (n = 3). ( e ) Representative western blots of Shh, Dio3, STAT3, and p-STAT3 expression in C2C12 cells treated with 100 μg/ml LPS or 5 μM Stattic or 500 ng/ml rShh (n = 3). ( f ) Representative immunoblots of IL-6, STAT3, and p-STAT3 expression in C2C12 cells treated with 100 μg/ml LPS for different time courses (n = 3). ( g ) Quantitative analysis of ( f ). ( h ) Representative DCFH-DA staining fluorescent images of C2C12 cells treated with 100 μg/ml LPS for different time courses (n = 10) and its quantification. ( i ) Representative western blots of STAT3, p-STAT3, Shh, and Dio3 expression in C2C12 cells treated with 100 μg/ml LPS or 10 mM NAC (n = 3). * P < 0.05. Spearman correlation analysis for (a), two-tailed Student’s unpaired t-test and Mann–Whitney test for (b, h), or one-way ANOVA followed by Tukey’s multiple comparisons test for (c, g, and i). Quantitative data are represented as mean ± SD. NS not significant, IL-6 interleukin 6, rT3 reverse triiodothyronine, STAT3 signal transducer and activator of transcription 3, Dio3 type 3 deiodinase, Shh sonic hedgehog, LPS lipopolysaccharide, NAC N-acetylcysteine

Article Snippet: C2C12 myotube nuclear lysates, post 1% formaldehyde cross-linking, were sonicated to fragment chromatin, followed by immunoprecipitation with Gli1 (sc-515751, Santa Cruz, USA) antibody or control IgG.

Techniques: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR, Staining, Two Tailed Test, MANN-WHITNEY