Fig. 1: GH induces TGF-β/SMAD pathway in podocytes.

A, B qRT-PCR analysis showing the expression of TGF-β1 in human podocytes treated with or without GH in concentration (100–500 ng/ml) and time- (up to 48 h) dependent manner. mRNA levels were normalized to β-actin and presented as fold change on the y-axis. Mean ± SD. (n = 5). ****p < 0.0001 by one-way ANOVA post hoc Dunnett test. C, D Immunoblotting analysis for indicated genes from podocytes treated with GH in concentration (100–500 ng/ml) and time- (up to 48 h) dependent manner. (n = 3). E Immunofluorescence analysis for TGF-β1 (purple color) and counterstained with DAPI [4′,6-diamidino-2-phenylindole (blue color)] in podocytes treated with or without GH (500 ng/ml, 48 h). Scale bar = 50 μm, magnification ×500. F, G Estimation of TGF-β1 in conditioned media (CM) from podocytes treated with GH in concentration (100–500 ng/ml) and time- (up to 48 h) dependent manner. Mean ± SD. (n = 5). H, I SMAD4 luciferase activity in podocytes treated with GH in concentration (100–500 ng/ml) and time- (up to 48 h) dependent manner. Mean ± SD. (n = 6). F–I ****p < 0.0001 by one-way ANOVA post hoc Dunnet test. J, K qRT-PCR and immunoblotting analysis show TGFBR1 and TGF-β1 in mice podocytes treated with or without GH (1.5 mg/kg b.w). Mean ± SD. (n = 3). ****p < 0.0001 by Student’s t test. Each data point represents the average value of mice from each group. n = 6. L Representative images for TGFBR1 and TGF-β1 expression assessed by 3,3′-diaminobenzidine (DAB) staining in mice glomerular sections treated with GH. Scale bar = 100 μm, magnification ×200. (n = 3). Quantification of TGF-βR1 and TGF-β1 staining area in the glomerulus (right panel) represented as a dot plot. Mean ± SD. (n = 6). ****p < 0.0001 by Student’s t test. M Quantification of TGF-β1 in urine from mice administered with GH. Mean ± SD. (n = 6). ****p < 0.0001 by Student’s t test. Each data point represents the average value of a single mouse from each group.