Anti tfam

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Big Cabin, OK, USA). Collagenase and trypsin were purchased from Sigma (St. Louis, MO, USA). Iso and metformin were purchased from Sigma. The following antibodies were used in this study: anti-Mfn2, anti-PINK1 (Abcam, Cambridge, UK), anti-Beclin1, anti-P62, anti-LC3B, anti-PGC 1α, anti-TFAM, anti-NRF1, anti-ANP, anti-β-MHC, anti-Col-1, anti-TGF-β-1, anti-GAPDH and anti-β-actin (Proteintech, Chicago, IL, USA).

Paraffin sections were dewaxed and rehydrated. For detection, a tyramine signal amplification-based multiplexed immunofluorescence assay was performed using the triple standard multiplex immunofluorescence Kit (Cat# AFIHC025; AiFang Biological, China). The following protocol was followed:
The cells were fixed in 4% paraformaldehyde for 0.5 h. Then permeabilized with a solution containing 0.2 µL Triton, 0.25 g bovine serum albumin (BSA), and 5 mL phosphate belanced solution (PBS) for 1 h at 25 ℃. The following primary antibodies were used: anti-SFPTC (Cat# PA5-71680; Thermo; USA; 1:50), anti-FBXL5 (Cat# DF14329; Affinity; China; 1:100), anti-IREB2 (Cat# R1706-12; HUABIO, China; 1:100), anti-MFRN2 (Cat# orb457153; Biorbyt; UK; 1:100), anti-DNA (Cat# 690014S; PROGEN; Germany; 1:200), and anti-TFAM (Cat# 22586-1-AP; Proteintech; China; 1:200), anti-EP1 (Cat# A2913; Abclonal; China; 1:200), anti-EP2 (Cat# A9053; Abclonal; China; 1:200) and anti-EP4 (Cat# 24895-1-AP; Proteintech; China; 1:200) were used. Fluorescent secondary antibody for 1 h at 25 ℃ and 4',6-diamidino-2-phenylindole (DAPI) for 15 min at 25 ℃ before measured using fluorescence microscope.

For immunoblotting, antibodies used in this study were purchased from EMD Millipore (Oakville, Canada), Sigma Aldrich (Oakville, Canada), Proteintech (Illinois, USA), and Cell Signaling Technology (Whitby, Ontario). The following primary antibody was used from EMD Millipore (Oakville, Canada): anti-androgen receptor (Millipore Cat# 06-680, 1:1,000). The following primary antibodies were used from Sigma Aldrich (Oakville, Canada): anti-fast skeletal myosin (M4267, 1:15,000), anti-slow skeletal myosin (Sigma-Aldrich Cat# M8421, 1:10,000), and anti-beta actin (Sigma-Aldrich Cat# A2066, 1:10,000). The following primary antibodies were used from Proteintech (Illinois, USA): anti-PGC1α (Proteintech Cat# 66369-1-Ig, 1:5,000), anti-NRF-2 (Proteintech Cat# 16396-1-AP, 1:500), and anti-TFAM (Proteintech Cat# 22586-1-AP, 1:1,000). The following HRP-conjugated secondary antibodies were used from Cell Signaling Technology (Whitby, Ontario): goat anti-rabbit IgG, HRP-linked (Cell Signaling Technology Cat# 7074, 1:5,000) and horse anti-mouse IgG, HRP-linked (Cell Signaling Technology Cat# 7076, 1:5,000).