Fig. 4: Parkin deletion slightly impairs mitophagy but extended Pgc1α protein stability in adipocytes.

a,b Western blot analysis of autophagy markers p62 and LC3 in the eWAT of Control f/f and Parkin^Adi mice fed with HFD (n = 6 mice per genotype) and in differentiated adipocytes of Control (Scr) and Parkin^KD (n = 3 biological replicate). The bar graphs at the bottom are the densitometric quantification of protein level normalized to Actin or GAPDH [Unpaired Student’s t test two-tailed. For p62/Actin Control f/f-HFD vs. Parkin^Adi-HFD, p = 0.0065, 95%CI = −0.9381 to −0.1982, R squared = 0.5394]. c Adenoviral GFP and mCherry Fis1 probe was used for mitophagy quantification. d Fluorescent microscopy analysis of mitophagy signals in Control (Scr) and Parkin^KD adipocytes. The graph on the right is the quantification of mitolysosome number in differentiated 3T3-L1 Control (Scr) and Parkin^KD adipocytes cultured in the normal medium or in HBSS for 4 h (n = 3 biological replicate) [Unpaired Student’s t test two-tailed. For Control (Scr) vs. Control (Scr) HBSS, p = 0.0106, 95%CI = 0.1274–0.5309, R squared = 0.8369; for Control (Scr) HBSS vs. Parkin^KD HBSS, p = 0.0426, 95%CI = −0.4017 to −0.01107, R squared = 0.6827]. e Western blot and densitometry analysis of Pgc1α, OXPHOS proteins, and Vdac1 (normalized to Hsp90) in eWAT of Control f/f and Parkin^Adi mice fed with HFD (n = 6 mice per genotype) [Unpaired Student’s t test two-tailed. For Pgc1α, p = 0.0446, 95%CI = 0.01360–0.9214, R squared = 0.3450; for Sdhb, p = 0.0103, 95%CI = 0.1254–0.7323, R squared = 0.4979; for Vdac1, p = 0.0338, 95%CI = 0.02981–0.6094, R squared = 0.3765]. f Western blot and densitometry analysis of Pgc1α and OXPHOS proteins in the differentiated Control (Scr) and Parkin^KD adipocytes (n = 3 biological replicate) [Unpaired Student’s t test two-tailed. For Pgc1α, p = 0.0018, 95%CI = 0.2430–0.5338, R squared = 0.9322; for Atp5α, p = 0.0201, 95%CI = 0.1810–1.225, R squared = 0.7776; for mtCo1, p = 0.0060, 95%CI = 0.4386–1.399, R squared = 0.8759; for Sdhb, p = 0.0061, 95%CI = 0.2690–0.8624 R squared = 0.8751]. g Western blot and densitometry analysis of Pgc1α (normalized to GAPDH) in the differentiated 3T3-L1 Control (Scr) and Parkin^KD adipocytes treated with 100 μg/ml CHX at the indicated time. (n = 3 biological replicate) [Two-way ANOVA F (1,4) = 21.27, p = 0.0099, 95%CI = −1.900 to −0.4720]. Data are presented as mean ± SEM. AU = arbitrary units, CHX = cycloheximide. * p < 0.05, ** p < 0.01.