Reagents
Fetal bovine serum (FBS), charcoal-stripped fetal bovine serum (CSS, depleted androgen and any other steroid), Bovine pituitary extract (BPE), and cell culture mediums were purchased from Gibco (NY, USA). ENZ was purchased from MedChemExpress (NJ, USA). FL3 was synthesized in Dr. Laurent Désaubry’s lab according to a described procedure[30]. DHT was purchased from Meilunbio (Dalian, China). Epidermal growth factor (EGF) was obtained from Peprotech (NJ, USA). Glutamine PenStrep was obtained from Invitrogen. (CA, UAS). Serial dilutions of all drugs were made using DMSO.
Bioinformatics Analysis
The mRNA expression data of PHB1 were extracted from GEO (http://www.ncbi.nlm.nih.gov/geo) and cBioPortal (http://www.cbioportal.org/) database. The data used for Kaplan-Meier survival analysis were from TCGA (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/using-tcga) and cBioPortal (http://www.cbioportal.org/) database.
Immunohistochemistry(Ihc)
The cancer-adjacent normal tissues and PCa tissues were collected from undergoing radical prostatectomy for PCa at Qilu Hospital, Shandong University (Jinan, China) and made into tissue microarray (TMA). TMA was stained with H&E to be reviewed by two pathologists according to the WHO histologic classification of PCa, and the IHC staining was performed with Universal two-step detection kit (PV9000, Zhongshan Golden Bridge Biotechnology Co, Beijing, China). For PHB1 expression in tissues, the staining intensity was scored from 1 to 3: 1, weak; 2, moderate; 3, strong. The information of antibody used is summarized in Supplementary Table S2
Cell Lines And Cell Culture
Human prostate epithelial cell line RWPE-1 was purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). PCa cell lines LNCaP, C4-2B, 22Rv1 and PC-3 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). RWPE-1 cells were cultured in Keratinocyte serum-free medium supplemented with EGF, BPE, and 1% glutamine PenStrep. LNCaP, C4-2B, and 22Rv1 cells were cultured in RPMI-1640 with 10% FBS or charcoal-stripped fetal bovine serum (CSS). PC-3 cell was cultured in F12-K with 10% FBS or CSS. To establish derived LNCaP-AI (androgen-independent) cell line, LNCaP cells were maintained in RPMI-1640 supplemented with 10% CSS for at least 3 months[31]. All cells were cultured at 37°C in an atmosphere of 5% CO2 and used up to 15 passages maximum. After the last experiment, all cells were confirmed mycoplasma free using GMyc-PCR Mycoplasma Test Kit (40601ES10, YeSen Biotech, Shanghai, China).
Qrt-pcr
Total RNA was isolated using TRIzol reagent (Invitrogen, CA, UAS), and 1 µg of total RNA was used as template for the first strand cDNA synthesis using the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan). qPCR reactions were carried out with a FastStart Universal SYBR Green Master Mix (Roche, Mannheim, Germany). Data were normalized by the expression level of β-actin in each sample. The primer sequences used are listed in Supplementary Table S1.
Western Blotting
Whole-cell lysates were prepared by lysing the cells in ice-cold RIPA buffer (P0013C, Beyotime, Shanghai, China) with 1% protease inhibitor cocktail (NCM Biotech, Suzhou, China). 20 µg total protein was separated by SDS-PAGE and transferred to the PVDF membrane, which were then incubated with corresponding antibodies. The information of antibodies is summarized in Supplementary Table S2.
Plasmids, Sirna, And Cell Transfection
Expression vector pENTER-PHB1 and its control vector was designed and constructed by Vigene Bioscience (Shandong, China). siNC and siPHB1 were synthesized by Jikai company (Shanghai, China). The target sequences are listed in Supplementary Table S3. Lipofectamine 3000 (Invitrogen, CA, USA) was used for transient transfection following the manufacturer’s instruction. The transfection efficiency was confirmed using qRT-PCR and Western blotting.
MTS assay
Cell viability and cell proliferation were determined by MTS assay with MTS Cell Proliferation Assay Kit (BestBio, Shanghai, China). LNCaP cells (3.0×103 cells/well), C4-2B cells (2.0×103 cells/well), 22Rv1 (1.5×103 cells/well), or PC3 cells (1.5×103 cells/well) were plated in 96-well plate. After cells adhering to the wall, 10 µl MTS was added to each well at 0, 24, 48, and 72 h, respectively, and incubated at 37°C for 2h. Then the absorbance was measured at 490 nm. Cell viability was expressed as the percentage of absorbance of cells treated with FL3 or Enzalutamide compared with cells treated with DMSO. Experiments were performed in triplicate and repeated three times.
Transwell Assay
Transwell invasion/migration assays were performed using Transwell Chambers with or without coated Matrigel (Corning, NY, USA). RPMI-1640 or F12-K medium with 10% FBS was added to the lower chamber as a chemoattractant for cells. LNCaP (10.0×104 cells/well), C4-2B (10.0×104 cells/well), 22Rv1 (7.0×104 cells/well), or PC3 cells (7.0×104 cells/well) were plated in the upper chamber with RPMI-1640 or F12-K medium. After 24h of incubation, the migrated and invasive cells were fixed with 4% paraformaldehyde (Biosharp, Beijing, China) and stained with crystal violet solution (Solarbio, Beijing China). 5 fields of view were randomly selected for counting transmembrane cells under the light microscope using a 40 × magnification.
Plate Colony Formation Assay
The clonogenic ability of PCa cells was measured by plate colony formation assay. LNCaP (300 cells/well), C4-2B (300 cells/well), 22Rv1 (200 cells/well) or PC3 cells (200 cells/well) were plated in 6-well plate. 14d after initial seeding, cells were fixed with 4% paraformaldehyde and stained with crystal violet solution. Colonies containing more than 50 cells were counted and plotted. Experiments were repeated independent three times.
Subcellular Fractionation
Nucleus and cytosol proteins were separated using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Nantong, China) following the manufacturer’s instruction. plasma membrane extracts were prepared using Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies, Eden Prairie, USA) according to manufacturer’s manual.
Immunofluorescence (If)
IF was performed as previously described[32]. Pre-treated LNCaP (4 × 104 cells/well) and C4-2B (2 × 104 cells/well) cells were seeded on glass coverslips in 24-well plates, respectively. Mitochondrial labelling was performed using MitoTracker Deep Red FM (40743ES, Yeasen Biotech, Shanghai, China) following the manufacturer’s instruction. Nucleus was stained with DAPI (H-1200, VECTASHIELD, CA, USA). Antibodies used is summarized in Supplementary Table S2. Cells were observed under Laser Confocal Microscope LSM 980 (Zeiss, Oberkochen, Germany). Images taken were appropriately processed with the ZEN software program. Experiments were repeated three independent times.
Co-immunoprecipitation (Co-ip)
Co-IP assays were performed according to the instruction of BeyoMag™ Protein A/G Magnetic Beads for IP (Beyotime, Shanghai, China). 10 µg antibody in a 1:50 dilution was incubated with 20 µl Protein A/G Magnetic Beads for 1h at temperature. The cell lysate or nuclear extract, leaving 50 µl as input, was incubated with antibody-coated immunomagnetic beads at 4℃ overnight. The immuno-complex was collected and was analyzed by Western blotting. The information of antibodies is summarized in Supplementary Table S2
Tumor Xenografts
5-week-old male BALB/c nude mice were purchased from Weitonglihua Biotechnology (Beijing, China). A total of 6.0 × 106 C4-2B cells were mixed with Matrigel (1:1) and injected subcutaneously into the mice (n = 6/group). After the mice were surgically castrated, they were randomized into 4 groups (n = 6/group) and treated with indicated drugs and concentrations. ENZ and FL3 were administered by oral gavage and subcutaneously every two days, respectively. Body weight and tumor volume was measured and recorded twice a week. Tumor tissues were harvested and weighed after 15 times of treatment. Tumor volume was calculated with the following formula: tumor volume = length × width2 × 0.5. All animal experiments followed a protocol approved by the Shandong University Animal Care Committee (Document No. ECSBMSSDU2019-2-019).
Flow Cytometry
Dead Cell Apoptosis Kit (Invitrogen, CA, USA) was used according to the manufacturer's instruction. C42B cells treated with FL3 or DMSO were harvested and dealed with Annexin V-FITC and propidium iodide, followed by apoptosis analysis with CytoFLEX S (1720610S-2, BECKMAN COULTER, CA, USA). Experiments were repeated three independent times.
Pattern Drawing
The pattern diagram was drawn using the Figdraw software (ResearchHome, Zhejiang, China).
Statistical analysis
For in vitro experiment, experiments were carried out at least in triplicate to confirm reproducibility and presented as mean ± SD. In the xenograft studies, tumor sizes were served as the primary response measure when the mice were sacrificed. Statistical analysis was carried out with GraphPad Prism 8.0 using a t-test and two-way ANOVA. Significance was determined at *P < 0.05, **P < 0.01 and ***P < 0.001.