Vegfa

Recombinant Tat101 was purchased from ImmunoDiagnostics. Recombinant PDGF-BB was purchased from R&D Systems. Jagged-1 and DAPT were purchased from Sigma. Antibodies were obtained from the following sources: Ki67 (abcam), NICD (cell signaling), Actin (sigma), α-SM, Notch3 (cell signaling), VEGF-A (abcam); goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology. Flag-tagged CSL-VP16 plasmids were obtained from Dr. Aly Karsan (University of British Columbia, Vancouver, Canada) and control Notch3 siRNA (sc-29798), RBPJ siRNA (sc-41446), and scrambled siRNA (sc-37007) were from Santa Cruz Biotechnology.

The proteins were extracted by ice cracking. SDS-PAGE was used to separate the proteins. Subsequently, the target proteins were transferred onto a polyvinylidene difluoride membrane at low temperatures, followed by treatment with 5% milk for 50 min. Immediately afterward, the membranes were incubated overnight at 4°C with a primary antibody (ATAD2, 1 : 1000, #78568, Cell Signaling; VEGFA, 1 : 10000, ab52917, Abcam; and GAPDH, 1 : 5000, abs132004, Absin). In the following day, the membranes were removed and subjected to the process of PBST washing, treatment with a secondary antibody (anti-rabbit IgG, HRP-linked antibody, 1 : 5000, #7074 Cell Signaling), PBST rinsing, and, finally, exposure to the chromogenic apparatus by dropwise addition of the chromogenic solution for color development.

Western blotting was performed as previously described [16 (link)]. Briefly, cell lysates were prepared from parental wild type or miR-214KO PC3 and MDA-PCa-2b cells using lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Cell lysates (40 μg) were separated and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4 °C with anti-PCNA, VEGFR2, E-Cadherin, N-Cadherin, Vimentin, PTK6, and GAPDH (Cell Signaling Technology), CD31 (Life Technologies), VEGFA (Abcam), CXCR4, SESN3, PD-L1, ALK, and SAA1 (Abclonal Technology, and the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. All primary antibodies were used at a concentration of 1:1000. Following the incubation, membranes were washed, and protein bands were detected with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

The tissue microarrays (TMAs) containing representative cores of tumor tissue were constructed as described in our previous study [4 (link)]. Subsequently, TMAs were stained with the following antibodies: HIF1A (1:500, Abcam, Cambridge, United Kingdom, code ab463); SLC2A1 (1:200, DAKO, Gdynia, Poland, code A3536); CA9 (1:1000, Abcam, Cambridge, United Kingdom, code ab15086); VEGFA (1:250, Abcam, Cambridge, United Kingdom, code m68334), and assessed semi-quantitatively. Evaluation of hypoxia-markers expression employed estimation of the percentage of positively staining cells (0–5%, 6–25%, 26–50%, more than 50%) and the intensity of staining (no staining, low intensity, intermediate intensity, high intensity). Combined intensity and percentage of immunopositive cells enabled the calculation of the expression coefficient and classification of each patient into a low-expression (score 0–7) or high-expression (score 8–12) group, as described previously and presented in Table 1 [22 (link)]. The stainings were examined under light microscopy by two independent pathologists blinded to the clinical data. The percentage of positive cells was estimated manually. In discrepant cases, the final scores were reconciled following discussion.

Cells were washed three times using cold PBS and lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate) containing protease inhibitors. Approximate 30 μg of protein was separated with 10% SDS–PAGE gel and blotted onto nitrocellulose membranes. Then membranes were blocked with 5% skim milk at room temperature for 1 hour and then incubated with primary antibodies against GAPDH (Shanghai Kangchen), RICTOR (Bethyl Laboratories), VEGFA (Abcam), Akt (Cell signaling Technology), mTOR (Cell signaling Technology), HIF1α (Abcam) and HIF2α (Abcam) at 4°C overnight, followed by TBST wash and 1 hour incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature. Protein bands were visualized by a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA).

Prior to treatment, the wells of six-well plates were seeded with 1 × 106 cells. A protease inhibitor-containing radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China) was used to lyse the cells. Proteins were separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Beyotime Biotechnology, Shanghai, China) and subsequently transferred to polyvinylidene fluoride membranes. After washing membranes for 15 min with a quick-blocking reagent (Beyotime Biotechnology, Shanghai, China), they were incubated overnight with a primary antibody VEGFa (1:500, Abcam, UK), Ang II (1:1000, Abcam, UK), LC3 I/II (1:1000, Abcam, UK), Beclin-1 (1:1000, CST, USA), Atg 5 (1:1000, CST, USA), or GAPDH (1:1000, Engibody, China). Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000, Beyotime Biotechnology, Shanghai, China) were used to detect the bands using ECL Plus reagent (Bio-Rad Laboratories). The experiment was performed in triplicate.