Fig. 7: γ-MSH induces hepatic autophagy and nuclear TFEB activity.
From: Melanocortin 3 receptor regulates hepatic autophagy and systemic adiposity
a Representative fluorescent images of primary hepatocytes from Mc3r+/+ transgenic mice carrying GFP-LC3 under 2 nM D-Trp8-γ-MSH (γ-MSH) or 10 nM rapamycin (Rap) after staining with Lysotracker Red and DAPI. b Time-lapse images of primary hepatocytes from Mc3r+/+ transgenic mice carrying GFP-LC3 under 2 nM γ-MSH after staining with Lysotracker Red. c Representative western blot images of primary hepatocytes from Mc3r+/+ and Mc3rTB/TB after 2 nM γ-MSH treatment. d Representative super resolution microscope images of primary hepatocytes from Mc3r+/+ transgenic mice carrying GFP-LC3 under 100 nM MC3R-specific (versus MC4R) agonist (NDP42) for 60 min after staining with Lysotracker Red compared to control. e Measurements of total area of GFP-LC3 after 60 min of stimulation (d). f Representative western blot images of primary hepatocytes treated with serum starvation (HBSS) for 60 min, 1 µM Torin for 60 min, or 100 nM NDP42 (MC3R-specific agonist versus MC4R) for 30 min. g Quantification of LC3II normalized by β-actin in (f). Groups were analyzed between Control (n = 5), Starvation (n = 3), Torin (n = 5), and NDP42 (n = 5) independent experiments. h Representative western blot images of liver lysates from Mc3r+/+ with intraperitoneal γ-MSH injection. i Quantification of LC3II normalized by β-actin in (h) (n = 10) independent observations. j Representative western blot images of nuclear fractions (top panels) and cytosolic fractions (bottom panels) from primary Mc3r+/+ hepatocytes after 1 h starvation (HBSS) or 2 nM γ-MSH treatment. k qPCR measurement from Mc3r+/+ and Mc3rTB/TB hepatocytes after 2 nM γ-MSH (n ≥ 5 independent observations). l Heatmap of Log10 normalized counts for TFEB target genes and lysosomal genes in mouse liver RNAseq analysis across Mc3r+/+, Mc3rTB/TB, and Mc3rHep/Hep genotypes on a normal chow diet (n = 3) independent observations. Data are represented as mean ± SEM. Groups were compared by paired two-tailed Student’s t-test (e), unpaired two-tailed Student’s t-test (i, k), one-way ANOVA followed by Fisher’s LSD test (g). p < 0.05; ** p < 0.01; *** p < 0.001, Scale bar, 10 µm (a, b, d). Source data are provided as a Source Data file.
