Ptch1

Cells receiving various treatments and collected at various time points were subjected to western blot analysis, as previously described [35 (link),37 (link)]. Primary antibodies against IL-24 (1:2000; Introgen Therapeutics, Houston, TX, USA), GLI1, GLI2, PTCH2, SMO, γ-H2AX, PARP, Capase3, pATM^S1981, ATM, pCHK2^T68, CHK2, MRE11 (1:1000; Cell Signaling Technology Inc.), PTCH1 (Abcam, Cambridge, MA, USA), RAD50 (Santa Cruz Biotechnology, Dallas, TX, USA), and beta-actin (1:2000; Sigma Chemicals, St. Louis, MO, USA) were purchased and used as recommended by the manufacturers. Proteins were detected using the appropriate secondary antibodies (Santa Cruz Biotechnology, Inc., and Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) and an enhanced chemiluminescence kit (Thermo Scientific). Protein levels were detected using a chemiluminescence imaging system (Syngene, Frederick, MD, USA) and quantified using GelQuant software (V1.7.8, University of California- San Francisco, CA, USA).

The tumors were fixed with 4% paraformaldehyde and embedded in paraffin. The sections (5μm) were incubated with antibodies: HBx, SHH, PTCH-1, SMO, and GLI-1 (Abcam, USA). The pictures were captured by Fluorescence Inversion microscope system (Olympus, USA)

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, Rockford, IL, USA), and then the protein concentration was measured by bicinchoninic acid (BCA) protein assay with BSA as standard. Protein was submitted to electrophoresis in a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrically transferred onto a polyvinylidene difluoride transfer membrane. Membranes were blocked in 5% fat-free milk and primary antibodies were incubated overnight at 4 °C followed by peroxidase-conjugated secondary antibody incubation for 1 h at room temperature (RT). Proteins were visualized using the enhancing chemiluminescence ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The following primary antibodies were used: anti-β-actin(1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Ptch1 (1:100, Abcam, Cambridge, MA, USA) and Gli2 (1:100, Abcam, Cambridge, MA, USA).

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.