Ab134057

To extract whole‐cell proteins, cells were lysed in 1 × RIPA buffer (Solarbio, Beijing, China) containing a cocktail solution of protease inhibitors (Sangon Biotech) on ice for 30 minutes and then centrifuged (12 000 × g for 10 minutes at 4°C). Subsequently, the Bradford method was used to normalize protein concentrations, and a representative Western blotting protocol was adopted. In brief, 20 μg of protein per channel was separated through 10% SDS‐PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with a blocking solution (5% skim milk in Tris‐buffered saline with 0.1% Tween‐20, TBS‐T) for 1 hour, the membranes were incubated with the primary antibodies (including rabbit anti‐human NR5A1: ab168380, GATA4: ab134057, DMRT1: ab126741, GAPDH: ab37168, Abcam, Cambridge, UK, at 1:2000; and rabbit anti‐human CYP17A1: orb213833, HSD3B1: orb5478, STAR:orb129747, CYP11A1: orb213832, Biorbyt, San Francisco, CA, USA, at 1:1000) overnight at 4°C. Next, the membranes were incubated with the goat anti‐rabbit secondary antibody (ab150079, Abcam, 1:10000) for 1 hour at RT in the dark, and specific bands were detected using a two‐colour Infrared Laser Imaging System (LI‐COR Odyssey, USA).

Anti-hypoxic activity and CSC markers were detected by FCM Analysis. CSCs were incubated with the supernatant fluids of MΦ, LPS + MΦ, pMΦ, and MSCs. Cells were then harvested after 0, 6, 12, and 24 h in hypoxia (0.5% O₂), respectively. Apoptosis of CSCs was determined using the Annexin V-PE/7-ADD as previously described. Three steps were followed to determine CSC markers. In step one, the cells were fixed with 4% paraformaldehyde (Fushen, Shanghai, China, Cat#FS030101) for 10 min and then permeabilized with 0.1% PBS-Tween20 (Beyotime, Shanghai, China, Cat#ST825) for 20 min. In step two, the cells were washed thrice with PBS and resuspended with 100 µL PBS, and then primary antibody was added for 30 min at room temperature. The secondary antibody was added for 30 min at room temperature, and the cells were washed thrice and resuspended with 100 µL PBS. Then, the cells were washed and resuspended in PBS and identified by FCM; the results were analyzed using FlowJo software. The primary antibody against GATA-4 (Cat#ab134057) and TnI (Cat#ab47003) and the secondary antibodyAlexa Fluor^® 488 goat anti-rabbit IgG (H + L) (Cat#ab150077) were purchased from Abcam (Cambridge, UK), and used at the recommended concentrations in experiments carried out in triplicate.