Ascl4

The cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (Selleckchem). The protein concentration was tested with a BCA kit, and appropriate amounts of protein were prepared for SDS-PAGE and then transferred to PVDF membrane (Millipore, MA, USA). The membranes were blocked for 1 h with 5% non-fat dry milk and then incubated with rabbit anti-SIRT1 (1:1000; #9475; Cell Signaling Technology Europe, Netherlands); TFAM (1:1000; #8076; Cell Signaling Technology Europe, Netherlands); CATALASE (1:1000; #12,980; Cell Signaling Technology Europe, Netherlands); SOD2 (1:1000; #MA1-106; Invitrogen); UQCRC2 (1:1000; #ab14745; Abcam); MTCO2 (1:1000; #ab110258; Abcam); ASCL4 (1:500; #sc-365230; Santacruz); Actin-b (1:5000, A5441; Sigma) mABs were used as loading controls. The results were imaged using a gel image analysis system (Bio-Rad, California, USA) according to the manufacturer’s instructions.

Immunohistochemistry was performed using affinity-purified antibodies against ASCL4 (Santa Cruz, Dallas, TX). Paraffin-embedded tissue sections were dewaxed in xylene, rehydrated, and washed in Phosphate -buffered saline, pH 7.4. For antigen retrieval, paraffin sections were heated in a microwave oven (900 watts) in 10 mM citrate buffer followed by treatment with 3% H₂O₂ and blocking with 20% normal goat serum. Sections were then incubated with antibody against ASCL4 (1:100 dilution), followed by incubation with a biotinylated rabbit secondary antibody (1:1000, Vector Labs, Burlingame, CA) An avidin-biotin complex was formed and developed using diaminobenzidine chromagen, followed by a counter-stain with hematoxylin.
Immunohistochemical staining was examined and scored independently and in a blinded manner by two pathologists using the Allred immunohistochemistry score system [42 (link)]. Intensity levels, from 0 (as negative) to 3 (as strong), of cytoplasmic expression and percentage score, 0 (as 0%) to 5 (as 100%) were recorded semiquantitatively, resulting in a combined score for statistical analysis.