Fig. 4: Autophagy is impaired in hepatocytes lacking Usp39. | Nature Communications

Fig. 4: Autophagy is impaired in hepatocytes lacking Usp39.

From: Spliceosome component Usp39 contributes to hepatic lipid homeostasis through the regulation of autophagy

Fig. 4

a Pathway enrichment analysis of differentially expressed genes using RNA-seq data for control and Usp39-HKO male mice. b qPCR analysis of autophagy-related genes in the livers (n = 3 per group). c qPCR was performed to analyze expression of autophagy-related genes in Usp39 knockdown and control AML12 cells (n = 3). d Immunoblotting was performed to analyze protein levels of autophagy-related genes in 5-week-old control and Usp39-HKO male mice (n = 3 per group) fasted for 16 h. e, f Immunoblotting was performed to analyze protein levels of autophagy-related genes in Usp39 knockdown and control AML12 cells (n = 3) (e) and primary cells (n = 4) (f) supplemented with 0.4 mM oleic acid for 6 h. Immunoblotting band intensities were quantified by Image J, which were shown in Fig. S5g–i. g Electron micrographs in the livers of control (n = 11) and Usp39-HKO (n = 10) male mice showing autophagic vesicles with quantification. Scale bars, 2 µm. h Confocal image of the liver sections in control and Usp39-HKO mice showing co-localization of Plin2 (green) and Lamp2 (red). Scale bars, 10 µm. i Confocal images of primary hepatocytes transfected with Ad-EGFP-LC3 and control vectors followed by serum deprivation to 2 h, showing EGFP-LC3 puncta/cell (n  =  56 cells for control, n  =  64 cells for HKO). Scale bars, 10 µm. j Co-localization of EGFP-LC3 puncta and BODIPY C12 (white arrows) in primary hepatocytes transfected with Ad-EGFP-LC3 and control vectors for 72 h, cells were then cultured in medium supplemented with 0.4 mM oleic acid (n  =  37 cells for control, n  =  39 cells for HKO). Scale bars, 10 µm. Immunofluorescence staining and co-localization was quantified by Image J. Images are representative of at least three independent experiments. The p-value was obtained by IPA (a) and others performed unpaired two-sided Student’s t-test, and the results are presented as the mean ± S.D. NS, stands for non-significant. OA, Oleic acid. MW, molecular weight. Source data are provided as a Source data file.

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