Wpmy 1

Human prostate cancer cell lines LNCaP, 22RV1, DU145, PC3 and normal prostate epithelial cell WPMY-1 were used in this study. LNCaP cells were purchased from the American Type Culture Collection (Manassas, USA) which was confirmed by short tandem repeat (STR) analysis. 22RV1, DU145, PC-3 and WPMY-1 were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China) where they were authenticated by mycoplasma detection, DNA-Fingerprinting, isozyme detection and cell vitality detection. The four prostate cancer cells were cultured in RPMI-1640 medium (Corning, USA) with 10% FBS (Hyclone, USA) and WPMY-1 in DMEM medium (Corning, USA) with 10% FBS in a humidified atmosphere containing 5% CO2 at 37°C.

Prostate cancer cell lines VCaP, 22Rv1, CRL-1740, CRL-2422, PC3M and normal prostate stromal cell WPMY-1 were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Following resuscitation, the cells were cultured with Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco Inc., Carlsbad, CA, United States) containing 10% fetal bovine serum (FBS; Gibco) in an incubator (Thermo Fisher Scientific, MA, United States) in a saturated humidity atmosphere with 5% CO₂ at 37°C. After cell confluence had reached 90%, the cells were detached with 0.25% trypsin and passaged at a ratio of 1:3.

Human normal prostate stromal immortalized cell line WPMY-1 and prostate cancer cell lines (DU 145, LNCaP, and PC-3, purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China)) were used in our research. The cells were cultured in 1640 culture medium containing 10% FBS and maintained in an incubator at 37 °C with 5% CO₂. shCtrl, shCDKL3, shSTAT1 lentiviruses (BR-V108 construct with GFP tag) and CDKL3- or CBL- overexpression vectors (LV-013 construct with GFP tag) were purchased by Shanghai YBR Bioscires Co., Ltd and the sequences were listed in Table S2. For cell transfection, lentiviruses (1 × 10⁸ TU/mL) were transfected into HEK293T cells upon the application of Lipofectamine 2000. After 48 h, the supernatant containing the recombinant lentiviruses was collected, filtered, and used to treat target cells. Following infection, cells were grown in media as usual. Puromycin (2 μg/ml) was added 48 h after infection. Infection efficiency was evaluated by observing green fluorescent protein (labeled on lentivirus vector) expression using a fluorescence microscope.

Human colorectal cancer cells HCT116, Caco‐2, breast cancer cells MDA‐MB‐231, non‐small cell lung cancer cells A549, prostatic stromal myofibroblast cells WPMY1, and HEK293FT were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCT116 p53−/− (379.2) and p53+/+ (4016) were homologous recombinant cell lines as a gift from the laboratory of Dr. Bert Vogelstein.^[52] Cells were cultured in Dulbecco's MEM medium containing 10% (v/v) FBS, 1% penicillin‐streptomycin (all from Life Technologies) at 37 °C in a 5% CO₂ atmosphere. For subculture of suspended HCT116, cells were centrifuged at 1000 rpm for 3 min and then counted to reseed at a proper density (ultra‐low attachment 6‐well plates, 10⁵–10⁶ cells per well). All cell cultures were regularly tested for Mycoplasma contamination. Irradiation of X‐ray (dose rate 300 cGy min⁻¹) was delivered to cells using a Varian Clinaci X linear accelerator (Varian Medical Systems Inc.).

The normal human prostate stroma cell line WPMY-1 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM medium (HyClone, USA) with 10% fetal bovine serum (Gibco, USA).

Cigarettes (Hongjinlong brand, the content of nicotine, tar and carbon monoxide was 0.6 mg, 8 mg and 11 mg/cigarette, respectively) were obtained from China Tobacco Hubei Industrial LLC. (Hubei, China). Swertiamarin (purity>98%) was purchased from Aladdin Co, Ltd. (Shanghai, China). RWPE-1 and WPMY-1 were obtained from China Center for Type Culture Collection (Shanghai, China). The antibodies of SHH (ab19897), IHH (ab52919), GLI-1 (ab49314), SMO (ab113438), α-SMA (ab5694), E-cad (ab1416), Col-I (ab34710), Col-III (ab7778), Snail (ab180714) and ZEB1 (ab81972) were obtained from Abcam, Co. (UK). The antibody of TGF-β1 (21898-1-AP) was purchased form PTG, Co. (Wuhan, China). ELISA kits for the analysis of COX-2 (JYM0885Ra), IL-1β (JYM0419Ra), IL-6 (JYM0646Ra) and TNF-α (JYM0635Ra) were purchased from ELISA Lab, Co. Ltd. (Hubei, China). The commercial kits for the analysis of iNOS (A014-1-2), NO (A012-1-2), TAOC (A015-2-1), T-SH (A063-1-1), MDA (A003-1-2), SOD (A001-1-2), CAT (A007-1-1), GPx (A005-1-2), GSH/GSSG (A061-1-2) and Hyp (A030-2-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).