Steap3

Liver cancer cells with different matrix stiffness were fixed in 4% (v/v) paraformaldehyde (Solarbio, Beijing) solution at room temperature for 15 min, permeabilized with 0.2% (v/v) Triton X-100 (Solarbio, Tongzhou, BJ) for 15 min, washed with PBS three times, and blocked with 2% (w/v) BSA (Solarbio, Beijing) at 37 °C for 30 min. Then, cells were incubated with primary antibodies against SLC7A11 (Proteintech, 1:100), STEAP3 (Proteintech, 1:100), PD-L1 (Proteintech, 1:100), PD-L2 (Thermo, 1:100) and at 4 °C overnight, followed by an incubation with goat anti-rabbit IgG (H&L) Alexa Fluor 488 secondary antibodies (Beyotime, Jiangsu) and donkey anti-rabbit Alexa Fluor 555 secondary antibodies (Beyotime, Jiangsu) at 37 °C for 30 min. Nuclei were stained with DAPI (Cell Signaling, Danvers, MA). Images were obtained by using a laser confocal fluorescent microscope (Leica, Germany).

The slides were subjected to 30 min of H₂O₂ solution (0.3%) treatment to inhibit the activity of endogenous peroxidase. Permeabilization was conducted by treating tissues with 0.5% Triton X-100 for 30 min, followed by incubation of tissues with citrate buffer at working strength to identify the relevant antigen. Following three times of rinsing with PBS, 5% normal goat serum was used to block the slides. Samples were subsequently subjected to cross-reaction with STEAP1 antibody (Proteintech: 20199-1-AP, China), STEAP2 (Proteintech: 20201-1-AP, China), STEAP3 (Proteintech: 17186-1-AP, China), STEAP4 (Proteintech: 11944-1-AP, China), SMO (Proteintech: 20787-1-AP, China), GLI1 (Abcam: ab134906, USA), PTCH1 (Abcam: ab53715, USA), Ki-67 (Proteintech: 27309-1-AP, China), PCNA (Proteintech: 10205-2-AP, China), E-Cadherin (Proteintech: 20874-1-AP, China), N-Cadherin (Proteintech: 22018-1-AP, China). Dilution of antibodies was performed at a ratio of 1:100, followed by two hours of incubation with the sample at ambient temperature. The slides were then subjected to 30 min of incubation with a secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (H + L). Besides, counterstaining of nuclei was carried out with DAPI.