(A) Western blot and densitometry analysis for PINK1 expression normalized to β-actin in THP-1–derived human macrophages cultured in the absence (–) or presence (+) of TGF-β1 (5 ng/mL) for 48 hours (n = 5 per group). (B) Western blot and densitometry analysis for MFN2 and Parkin normalized to GAPDH in renal macrophages isolated from WT mice 7 days after sham or UUO surgery (n = 3 per group). (C) Western blot and densitometry analysis for MFN2 and Parkin normalized to TIM23 in mitochondrial lysates from BMDMs (n = 4 per group) cultured in the absence (–) or presence (+) of TGF-β1 (5 ng/mL) for 48 hours. β-Actin (a cytosolic marker) was used to confirm the purity of mitochondrial fractions. (D and E) Representative confocal microscopy images (D) and quantification of mitochondria colocalized with LC3 (E). Pink1^+/+ and Pink1^–/– BMDMs cultured in absence or presence of TGF-β1 were analyzed using MitoTracker (red) dye, anti-LC3 (green), and Hoechst (blue) dye. White arrows indicate colocalization of LC3 with mitochondria. Over 100 cells from 6 random fields per experimental group, each in triplicate, were analyzed using ImageJ. Scale bar: 10 mm. (F) Mitophagy assessment in Pink1^+/+ and Pink1^–/– F4/80⁺ renal macrophages from mice fed with control (Ctl, n = 5 per group) or adenine (AD, n = 7 per group) diet for 28 days and stained with MitoTracker and LysoTracker dyes using flow cytometry. (G) Mitophagy assessment in Pink1^+/+ and Pink1^–/– BMDMs (n = 3 per group) cultured in the absence or presence of TGF-β1 (5 ng/mL) for 48 hours using flow cytometry. (H) Representative histograms for the detection of mitophagy showing the lyso dye–labeled positive lysosomes gated for Mtphagy dye–stained mitochondria. Data are mean ± SEM representative of 3 independent experiments and analyzed by Student’s unpaired 2-tailed t test (A, B, and C) or 1-way ANOVA (E, F, and G). *P < 0.05. **P < 0.01, ***P < 0.001.