Figure 2.

Loss of p62 in NESCs Results in Reduced Respiratory Chain Function, but p62 Is Dispensable for Mitophagy

(A) Mitochondrial respiratory chain enzyme activities in Co1^NES (black), Co2^NES (white), Pat1^NES (red), and Pat2^NES (orange). Complex I (CI; NADH coenzyme Q reductase), complex I + III (CI+III; NADH cytochrome c reductase), complex II (CII; succinate dehydrogenase), complex II + III (CII+III; succinate cytochrome c reductase), and complex IV (CIV; cytochrome c oxidase) are shown. Data are presented as mean ± SD and differences were tested by a two-tailed t -test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, n = 3.

(B) Oxygen consumption rates from Co1^NES (black), Co2^NES (white), Pat1^NES (red), and Pat2^NES (orange), treated with CCCP or dinitrophenol (DNP). Data are expressed as mean ± SD and differences were tested by a two-tailed t test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, n = 3.

(C) Mitochondrial ATP production rates in Co1^NES (gray) and Pat1^NES (red) supplemented with glutamate/succinate (Glu+Succ), pyruvate/L-malate (Pyr+Mal), or palmitoylcarnitine/L-malate (PalCar+Mal). Data are expressed as units per unit of citrate synthase (CS).

(D) Representative confocal images of control fibroblasts transfected with YFP-Parkin (green) and treated with CCCP (20 μM) (top) or O/A (10 μM/1 μM) (bottom) for 2 h. Fibroblasts were immunostained against Tom20 (red) and p62 (blue). n = 3.

(E) Representative confocal images of Co1^NES cells treated for 2 h with or without 10 μM CCCP or O/A (5 μM/1 μM) and with or without 50 nM bafilomycin, using antibodies against p62 (green) and TOM20 (red) and DAPI stain (blue). Scale bar: 20 μm. n = 3.

(F) Representative confocal images of 14-days-differentiated neurons treated for 2 h with or without 10 μM CCCP or O/A (5 μM/1 μM) and with or without 50 nM bafilomycin, using antibodies against p62 (green) and TOM20 (red) and DAPI staining (blue). Scale bar: 20 μm. n = 3.

(G and H) Representative western blot analysis of (G) Co1^NES and Pat1^NES cells treated for 12 h or (H) Co1^NEU and Pat1^NEU after 20 days of differentiation treated for 2 h with or without 10 μM CCCP or O/A (5 μM/1 μM) and with or without 50 nM bafilomycin for 2 h. n = 3.

See also Figures S2–S4 and Table S2 for p62 primers.