Cell culture and treatment

The ATCC (Manassas, VA, USA) provided the DU145, PC-3, LNCaP, 22RV-1, and RWPE-1 cell lines, which were then cultivated in accordance with earlier instructions (Yu et al. 2022; Xie et al. 2022b; Xiao et al. 2021). Prior to being used, cell lines were verified to be mycoplasma-free by short tandem repeat (STR) profiling. We did not employ cells that were beyond passage 20. Cells in experimental cultures were exposed to the TM doses listed (MCE, HY-14153A). Dimethyl sulfoxide (Sigma, S-002-M) was used as the vehicle for TM.

Cell proliferation assay

3000 cells per well were seeded into 96-well plates with 100 μL of medium containing various experimental drug concentrations at 37 °C for the appropriate number of hours. The medium was then replaced with 100 μL of a solution containing 10 μL from the Cell Counting Kit (CCK-8; MCE, HY-K0301), and the plates were incubated for an additional two hours at 37 °C and 5% CO2. Ultimately, a Thermo microplate reader was used to measure the OD value at 450 nm.

In order to conduct the colony formation experiments, 1500 cells per well were seeded onto 6-well plates, and the cells were grown at 37 °C for 12 days using varying concentrations of TM as the vehicle. Finally, the cells were fixed with 4% paraformaldehyde (PFA) dyed with crystal violet, and photographed.

Flow cytometry analysis

Using the Cell Cycle Staining Kit (KeyGEN, KGA512) and the associated procedure, the cell cycle analysis was carried out. In short, after 48 h of growth in 6‐well plates (106 cells/well) with either vehicle or TM, the cells were collected and preserved for the night at 4 °C in 70% ethanol. Subsequently, the cells were collected for propidium iodide (PI) staining after the ethanol was removed. The cells were ultimately examined using a BD Biosciences C6 flow cytometer.

The apoptosis analysis was carried out in accordance with the relevant protocol using the Annexin V-PE/7-AAD Staining Kit (KeyGEN, KGA1018). In 6-well plates (106 cells/well), cells were grown with vehicle or TM for 48 h at 37 °C. Following that, the cells were collected for 7-Aminoactinomycin D (7-AAD) staining and ultimately examined using a BD Biosciences C6 flow cytometer.

Scratch assays and transwell experiments

Ibidi Culture-Insert (Ibidi GmbH, Münich, Germany) was used to set up the scratch assay. Cells were seeded into the culture-insert in 12-well plates. To reduce cell growth after the attachment of the cells, the culture-insert was taken out and the culture medium was substituted with media containing vehicle or TM containing 1% fetal bovine serum (FBS). Lastly, a light microscope was used to inspect and take pictures of the scratch after 0, 12, and 24 h. ImageJ was used to quantify cell movement.

Regarding the transwell migration experiment, 100 μL of media devoid of FBS was used to seed 4 × 104 cells into the top chamber of 24‐well transwell plates (6.5 mm insert, 8.0 μm pores). To the bottom compartment, 600 μL of 10% FBS-supplemented media was introduced. A cotton swab was used to gently remove the cells from the chamber's top surface after 24 h. After that, the migrating cells on the chamber bottom were fixed with 4% PFA, stained with crystal violet and photographed.

As to the transwell invasion assays, 24 well transwell plates were filled with an upper chamber containing 100 μl of diluted Matrigel (BD), which was then incubated for two hours at 37 °C with 5% CO2. 100 μL of serum-free media was used to seed about 4 × 104 cells into the top chamber. To the bottom compartment, 600 μL of 10% FBS-supplemented media was introduced. Using a cotton-tipped swab, the Matrigel and non-invading cells were carefully removed from the chamber's top surface after 24 h. After that, the cells on the chamber bottom were fixed and dyed. For quantification, three randomly chosen fields from each chamber were photographed under a light microscope.

Plasmid construction and transfection

Plasmid construction and transfection were performed as stated previously (Yan et al. 2023). The PCDH vector's NheI and EcoRI restriction sites were subcloned with the full-length coding sequences (CDS) of GLI2. DNA sequencing served as validation for each plasmid. As directed by the manufacturer, Lipofectamine 3000 (Invitrogen Life Technologies®, Carlsbad, CA, USA) was used to transfect plasmids. GLI2 expression was assessed by Western blotting using anti-GLI2 (Abclonal, A16863) antibodies and RT-qPCR.

RNA isolation, RNA-sequencing (RNA-seq), RT-qPCR and Western blotting

The methods previously described (Hong et al. 2023) were followed for RNA isolation, RNA-seq, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting. TRIzol Reagent (Invitrogen) was used to extract total RNA from PCa cells treated with vehicle or TM for 48 h, and RNA seq analysis was performed by Hangzhou Kaitai Biotechnology Co., Ltd. (Hangzhou, China).

Using the Evo M-MLV kit (AG11606), 1 μg of RNA was transcriptionally converted to complementary DNA (cDNA) for RT-qPCR. SYBR Green reaction mix (AG11732) and a Light Cycler 96 System (Roche, Basel, Switzerland) were used for the qPCR. Ct, the cycle threshold, was used to compute the relative expression using the 2−ΔΔCt method. Supplementary Table 1 contains a list of sequences of all the particular primers.

About Western blotting, the protease inhibitor cocktail-containing RIPA Lysis and Extraction Buffer (Fudebio, FD008) was used to lyse the cells. After that, proteins were separated by SDS–polyacrylamide gel electrophoresis and put onto membranes made of nitrocellulose. The membranes were blocked for 1 h at 25℃ with 5% fat-free milk, and then they were treated with primary antibodies for an overnight period at 4 °C: GLI2 antibody (Abclonal, A16863), PTCH1 antibody (Abclonal, A0826), SUFU antibody (Abclonal, A13429), SMO antibody (Abclonal, A3274), SHH antibody (Abclonal, A18863), Cyclin D1 antibody (Abclonal, A11022), Cyclin D2 antibody (Abclonal, A1773), Cyclin E1 antibody (Abclonal, A22461), CDK4 antibody (Abclonal, A2352), GLI1 antibody (Abclonal, A14675), c-Myc antibody (Abclonal, A1309), Bcl-2 antibody (Abclonal, A0208), GAPDH antibody (CST, #2118), β-actin antibody (CST, #4970) and β-tubulin antibody (CST, #2146). The membranes were then incubated with HRP-conjugated, anti-rabbit secondary antibody (Sigma, GENA934) for 1 h at room temperature.

Mouse model and in vivo treatments

The animal care committee of the Central People's Hospital in Zhanjiang authorized the animal research (approval number: ZJDY2023-61). Subcutaneous injections of 1 × 106 DU145 or PC-3 cells were given to male athymic nude mice (Jiangsu JICUI Laboratory Animal) that were five weeks old. Mice were injected intraperitoneally with vehicle or TM (2.5 mg/kg) every two days for seven days after implantation. PEG 300 and Tween-80 were used as the TM solvents for the in vivo investigation. Following medication delivery, the tumor volume and body weight of nude mice were monitored every two days for the following three weeks. The formula V = L × W2/2 (V = volume, mm3; L = major axis length, mm; W = minor axis length, mm) was used to estimate the tumor volume. Using a digital caliper, the length of the main and minor axes was measured. Following a three-week period of medication treatment, the tumors were removed, quantified, photographed, embedded in 4% PFA, and sectioned into paraffin.

Immunohistochemistry (IHC) assays

We used the paraffin slices of the xenograft tumor for IHC. The following tools were used for IHC: HRP-conjugated goat anti-rabbit IgG (Fudebio, FDR007), GLI2 antibody (Abclonal, A16863), cleaved caspase-3 antibody (CST, 9661), Ki67 antibody (CST, #9027), SHH antibody (Abclonal, A18863) and Instant Immunohistochemistry Kit I (KeyGEN, KGOS300). Under a light microscope, stained slices were viewed and photographed.

Statistical analysis

Software for graphics was GraphPad Prism 9, and statistical analysis was done using SPSS. The mean ± SD was used to represent the experimental outcomes. For statistical analysis, the student's t test was used. P-values less than 0.05 (*), less than 0.01 (**), or less than 0.001 (***) were regarded as statistically significant.