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. Author manuscript; available in PMC: 2018 Oct 1.
Published in final edited form as: Kidney Int. 2017 May 3;92(4):836–849. doi: 10.1016/j.kint.2017.03.005

Figure 1. MiR-378a-3p regulates NPNT expression in cultured human podocytes.

Figure 1

Figure 1

(A) Real-time PCR reveals induction of miR-378a-3p expression in cultured human podocytes after TGF-β stimulation (5 ng/ml) at 48 h compared to baseline. ** p<0.01. (B) Real-time PCR reveals repression of NPNT mRNA in cultured human podocytes after TGF-β stimulation (5 ng/ml) at 48 h compared to baseline. (C) Western blot for NPNT in cultured human podocytes after TGF-β stimulation (5 ng/ml) compared to baseline for the time points indicated. Quantification of NPNT protein expression is given in the histogram. (D) Real-time PCR for relative NPNT mRNA expression in cultured human podocytes (POD), human mesangial cells (MC) and human glomerular endothelial cells (GEC). ** p<0.01. (E) Western blot for NPNT in cultured human podocytes at baseline and 72 h after a 4 h transfection with a miR-378a-3p mimic (5 nM) indicates complete suppression of NPNT protein compared to control after 72 h. Quantification of NPNT protein expression is given in the histogram. (F) Real-time PCR reveals upregulation of NPNT in cultured human podocytes after transfection with a miR-378a-3p inhibitor. Stimulation with TGF-β in the presence of a miR-378a-3p inhibitor decreased NPNT expression comparable to stimulation with TGF-β alone. ** p<0.01. (G) Luciferase reporter assay to validate miR-378a-3p binding to NPNT was performed in human embryonic kidney cells 293 (HEK cells). Cells were lysed 24 h after transfection and subsequently used for luciferase activity. Luciferase reads were normalized with beta-Gal values. ** p<0.01.