Tbs tween 20

Tumour tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Paraffin sections were cut into sections (4 μm thick) and then deparaffinized and rehydrated prior to antigen retrieval. Sections were blocked with 10% bovine serum albumin in TBS‐Tween 20 (Sigma‐Aldrich) for 1 h at room temperature. Sections were then incubated overnight at 4℃ with primary antibodies against P53 (21891–1‐AP) and PINK1 (23274–1‐AP). The following morning, sections were incubated with secondary antibodies for 30 min at 37℃. Then, slides were incubated with diaminobenzidine for 5 min, and then counterstained with Gill's haematoxylin for 30 s. Images were captured on a microscope (Discover ECHO Company).

MCF7 cells were seeded in 10 cm dishes for 72 hrs. Then, cells were lysed in RIPA buffer (Sigma), containing proteinase inhibitors (Roche) and kept at 4°C for 30 minutes. Lysates were collected by centrifugation for 10 minutes at 10,000 × g, and protein concentration were determined using the BCA protein assay kit (Pierce). Samples were diluted into SDS-PAGE sample buffer and were boiled for 5 minutes before being separated by SDS-PAGE, using a 4–15% gradient Mini-PROTEAN TGX Gel (Biorad). Samples were then transferred onto a nitrocellulose membrane (Biorad), blocked in 5% milk in TBS-Tween 20 (Sigma) and probed with antibodies directed against DNA-PK (ThermoScientific, #MS-423-P0) and β-actin (Santa Cruz Biotechnology, #sc-1616), using a secondary antibody at a dilution of 1 to 5000. Bound antibodies were detected using the Supersignal West Pico Chemiluminiscent substrate (ThermoScientific).

Whole-cell lysates were obtained as previously described (Di Tinco et al., 2021 (link)). A measure of 30 µg of protein extract for each sample was quantified by Bradford protein assay (Bio-Rad), and separation was performed by SDS-polyacrylamide gel electrophoresis on Mini-PROTEAN^® TGX™ Stain-Free Precast gels. Gels were then UV-activated using a ChemiDoc MP Imaging System (Bio-Rad), and proteins were subsequently transferred to 0.2-μm nitrocellulose membranes (Bio-Rad). The membranes were then imaged using the ChemiDoc Imaging System (Bio-Rad) for total protein normalization. Membranes were incubated overnight with the following primary antibodies: mouse anti-α-SMA (Invitrogen), mouse anti-fibronectin (Invitrogen), rabbit anti-PPARγ (Cell Signaling Technology), rabbit anti-PDGFRβ (Cell Signaling Technology), and rabbit anti-GLI1 (Invitrogen), all diluted 1:1,000 in 0.1% TBS-Tween 20 (Sigma-Aldrich) and then were incubated with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (1:3,000; Thermo Fisher Scientific) for 30 min at room temperature. The membranes were visualized using a ChemiDoc Imaging System (Bio-Rad). Finally, the relative expression levels of each evaluated marker were then obtained by normalizing the density of the protein bands to the corresponding stain-free blot image using Image Lab software (Version 6.1, Bio-Rad).