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