Supplementary Fig. 2. Regulation of mitochondrial biogenesis and representative

photomicrographs of confocal microscopy detection for double immunofluorescence of UCP1

and VEGF-A in ingWAT of DJ-1 KO mice. (A) Transcript levels of indicated genes required for

mitochondrial biogenesis (Nrf1, Tfam, CoxIV, and Cox8b) from tissue lysates of ingWAT. (B)

Western blot (left) and densitometry analysis (right) of indicated proteins involved in

mitochondrial electron transport chain (COX-IV, ATP5B and ETFA) in ingWAT extracts. (C)

Representative red MitoTracker staining on ingWAT sections. Nuclei were counterstained with

DAPI (blue). Scale bar, 50 µm. (D) Immunoblotting (top) and quantitation (bottom) of indicated

proteins implicated in mitochondrial fusion (Mfn1) and fission (Drp1) in ingWAT tissue lysates.

(E) Double immunofluorescence detection with antibodies against brown adipocyte marker

UCP1 (green) and VEGF-A (red) on representative sections of ingWAT. Nuclei were stained

with DAPI (blue). Co-localization is shown by merged images. Scale bar, 50 µm. Results are

representative of at least three independent experiments. Graphical data show densitometry

analyses normalized to actin (loading control). Gene expression levels were normalized to β-

actin. Data are presented as the means ± S.D. Data were analyzed by using ANOVA Tukey’s

multiple comparisons or Student’s t-test (if two groups). *p < 0.05, **p < 0.01 compared to WT

counterpart (A, B, D); †p < 0.05, ††p < 0.01 compared to WT-ND group (A, B, D).