Regulation of mitochondrial quality and quantity through p62 in adipocyte-specific Pld2 -ablated or PLD2-pharmacoinhibited mice. (A and B) Representative Western blot images (top) and quantification (bottom) of p62, UCP1, and β-Actin or GAPDH in ingWAT (A) and BAT (B) from HFD-induced control and Pld2 Ad-KO mice for 12 wk (n = 4/group). (C) Representative Western blot images (top) and quantification (bottom, left) of p62, β-Actin, and quantitative analysis (bottom, right) of p62 mRNA expression in primary brown adipocytes treated with isoprenaline (1 µM), CL (10 µM), and CAY10594 (10 µM) at the indicated time points (n = 3/group). (D and E) Representative Western blot images of p62, UCP1, and β-Actin (D) or mRNA expression of thermogenic genes (E) in primary brown adipocytes transfected with a negative control siRNA (N.C.) or siRNA against p62 and treated with CAY10594 (10 µM) at 24 h. (F) Differentiated primary brown adipocytes were treated with CAY10594 (10 µM) for 0, 3, or 24 h. Immunostained (left) with anti-p62 (red), anti-TOM20 (green), and DAPI (blue) and quantitative analysis (right) of p62 and TOM20 colocalization (n = 3/group). Scale bars, 10 µm. (G and H) Representative images of transmission electron microscopy (G, left), mitochondria number (G, right), and relative ratio of mitochondrial DNA (mt-Co1) to nuclear DNA (Ndufv1; H) of BAT from HFD-induced control and Pld2 Ad-KO mice for 12 wk (n = 6/group). LD, lipid droplet; M, mitochondria. Scale bars, 2 µm (top), 1 µm (bottom). (I and J) Representative images of transmission electron microscopy (I, left), mitochondria number (I, right), and relative ratio of mitochondrial DNA (mt-Co1) to nuclear DNA (Ndufv1; J) of BAT from vehicle- or CAY10594-injected obese mice (n = 6/group). Scale bars, 2 µm (top), 500 nm (bottom). (K) Representative TMRM analysis (left) and TMRM low or high population (right) of stimulated primary brown adipocytes with vehicle- or CAY10594 (10 µM) at the indicated times with palmitate (Pal, 500 µM) stimulation (n = 3/group). (L) Representative seahorse extracellular flux assays measuring OCR in primary brown adipocytes treated with vehicle or CAY10594 (10 µM) for 5 d (n = 3/group). (M) Representative measurements of the percentage change in OCRs in primary brown adipocytes treated with vehicle or CAY10594 (10 µM) for 5 d after injection of oligomycin, as an index of uncoupled respiration. The OCR before oligomycin injection was set as 100% (n = 3/group). (N) Representative seahorse extracellular flux assays measuring OCR in primary brown adipocytes from control and Pld2 Ad-KO mice (n = 3/group). (O) Representative measurements of the percentage change in OCRs in primary brown adipocytes from control and Pld2 Ad-KO mice after injection of oligomycin, as an index of uncoupled respiration. The OCR before oligomycin injection was set as 100% (n = 3/group). The data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student’s t test (A–C [bottom], E, F and G [right], H, I [right], J, and K [right]). Data are representative of at least two (A–D, F, and K) or three independent experiments (E, H, and J). Veh, vehicle; R/A, Rotenone/Antimycin A; FCCP, Trifluoromethoxy carbonylcyanide phenylhydrazone.