Fig. 2: GH induces TGF-β/SMAD pathway-mediated Notch signaling in podocytes.

A qRT-PCR analysis showing the expression of Notch1 and Jag1 in human podocytes treated with or without a conditioned medium (CM; 50%) from podocytes treated with or without GH for 48 h. Mean ± SD. (n = 6). ****p < 0.0001 by Student’s t test. mRNA levels were normalized to β-actin and presented as fold change on the y-axis. B Immunoblotting analysis of podocytes treated with CM (50%) from podocytes treated with or without GH for 48 h. (n = 3). C Immunoblotting analysis for indicated genes in podocytes treated with or without GH (250 and 500 ng/ml), TGFβ-1 (5 ng/ml), GH (500 ng/ml) + SB431542 (100 nM/ml), and GH (500 ng/ml) + AG490 (10 μM/ml) for 48 h. (n = 3). n-NICD1 (nuclear-NICD1), n-HES1 (nuclear HES1), and n-HEY1(nuclear HEY1). D Podocytes transfected with siRNA targeting TGFBR1 or scramble RNA (Scr-RNA) were subjected to immunoblotting for indicated genes. (n = 3). E Immunofluorescence for the nuclear colocalization of NICD1 (red color), HES1 (purple color), and counterstained with DAPI (blue color) in podocytes treated with or without GH for 48 h. Magnification ×630. Scale bar = 20 μm. (n = 3). F HES1 reporter activity was measured in podocytes exposed to GH for 48 h. Mean ± SD. G qRT-PCR analysis for Notch1 and JAG1 in podocytes isolated from a mouse treated with or without GH (1.5 mg/kg b.w), GH + SB431542 (1 mg/kg b.w), and GH + AG490 (1 mg/kg b.w). Mean ± SD. (n = 6). F, G ****p < 0.0001 by one-way ANOVA post hoc Dunnett test (n = 6). H Representative immunoblots for indicated genes in podocytes isolated from mice treated with or without GH. (n = 3). β-Actin and lamin-B1 served as an internal control.