Vimentin

The paraffin-embedded tissues were sectioned at 4 mm thickness and arranged on glass slides in sequence. In brief, slides were baked at 60°C for 6 h, followed by deparaffinization with xylene, rehydrating in graded ethanol, and 3% hydrogen peroxide to block the endogenous peroxidase activity. The sections were submerged in citrate or EDTA buffer and microwaved for antigen retrieval. Goat serum (ZSGB-BIO, China) was used to block nonspecific background and then the sections were incubated at 4°C with specific primary antibodies against LSD1 (1:100 Abcam), E-cadherin (1:500 Abcam), N-cadherin (1:200 Proteintech), Vimentin (1:1,000 Proteintech), Twist (1:200 Abcam), followed by the secondary antibody conjugated with streptavidin-biotin-horseradish peroxidase complex (Biotinylated Anti-Rabbit IgG, SP-9001; Biotinylated Anti-Mouse IgG, SP-9002, ZSGB-BIO, China). The slides were scanned by using a computerized image system composed of an Olympus CCD camera (Tokyo, Japan) connected to a Nikon eclipse Ti-s microscope (Tokyo, Japan) and captured by NIS-Elements F3.2. All of the slides were assessed by two urological pathologists.

Initially, a radio-immunoprecipitation assay (RIPA) lysis buffering solution (Beyotime, Shanghai, China) was applied to abstract proteins. Following the separation of proteins by sodium salt-polyacrylamide gel electrophoresis, target proteins were electrophoretically moved to nitrocellulose films, blocked via 5% milk-TBST for 2 h under room temperature. Subsequently, they were washed three times in TBST buffering solution and cultivated with the first anti-substances, viz., COL5A1, COL1A1, E-cadherin, N-cadherin, snail, GADPH rabbit polyclonal antibody (Proteintech, USA), vimentin, and vinculin mouse polyclonal antibody (Proteintech, USA) at 4°C overnight. Subsequently, the films were washed three times in TBST again, each for 10 min. Goat anti-rabbit IgG H&L (HRP) and goat anti-mouse IgG H&L (HRP) (Abcam, USA) were added then for cultivation. Afterwards, the membranes were cleaned in TBST as per the steps mentioned above to analyze relative protein expression with Image-Pro Plus software 6.0.

Total proteins from cell lines and tissues were extracted with RIPA buffer and then quantified by BCA analysis. Subsequently, 20 µg of total protein per sample (10 µL per lane) was separated using sodium lauryl sulphate–polyacrylamide gel electrophoresis (10% polyacrylamide gel) before the proteins were transferred to a PVDF membrane. After incubation with primary antibodies overnight, the membranes were then incubated with secondary antibody. Finally, target protein bands were detected using a chemiluminescence system. The antibodies used targeted the following proteins: AKT (Cell Signaling #9272s), Caspase-9 (Proteintech 10380-1-AP), Caspase-3 (Proteintech 66470-2-Ig), Caspase-8 (Proteintech 66093-1-Ig), p-ATK (Cell Signaling #4060), BAX (Proteintech 60267-1-Ig), Vimentin (Proteintech 10366-1-AP), N-cadherin (Proteintech 22018-1-AP), E-cadherin (Proteintech 20874-1-AP), GAPDH (Signalway Antibody #21612) and METTL3 (ABclonal A8370).

Western blotting was performed according to previously described standard methods.²⁸ Antibodies were used to against AKR1C2 (Abcam), phospho‐AKT Ser473 (Affinity), total AKT (Proteintech), cleaved‐PARP(Affinity), caspase3 (Cell Signaling Technology), E‐cadherin (Proteintech), N‐cadherin (Proteintech), Vimentin (Proteintech), Androgen receptor (AR) (Invitrogen), GAPDH (Proteintech). Dilution ratio used was according to the manufacturer's instructions. GAPDH was used as an internal reference.

Antibodies specific for c-Met (#25869-1-AP); c-Cbl (#25818-1-AP); Fibronectin (#1G10F9); Vimentin (#10366-1-AP); E-cadherin (#20874-1-AP); Tubulin (#10094-1-AP); DYKDDDDK (#66008-3-Ig); β-actin (#66009-1-Ig); mTOR (#20657-1-AP) were obtained from proteintech (Wuhan, China). The Phospho-AKT (Ser473) (#4060) and AKT (#9272) antibodies were both supplied by Cell Signaling Technology (Danvers, MA). An anti-phosphotyrosine antibody was obtained from Abbkine. Antibodies used were goat polyclonal to ORP5 (Abcam, ab59016); mouse monoclonal to N-cadherin (Servicebio, GB12135); p-mTOR (59. Ser 2448) (Santa Cruz Biotechnology, sc-293133). For immunoblotting, horseradish peroxidase-conjugated secondary antibodies were obtained from Beyotime. For immunofluorescence, CoraLite488––conjugated Affinipure Goat Anti-Mouse IgG(H + L) was obtained from proteintech.
Chloroquine Sulfate and MG-132 were from APExBIO (MA, USA). Cycloheximide (CHX) was from MedChemExpress (shanghai, China).

Total protein lysate from cells or tissues was prepared using standard RIPA lysis buffer (Sigma Chemicals, St. Louis, MO, USA). To minimize protein dephosphorylation, phosphatase-inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added into the lysis buffer. Protein concentration was then measured using a bicinchoninic acid assay (Thermo Scientific, Bonn, Germany). Fifty to 80 μg of each protein lysate was separated by 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were then blocked in 5% skim milk solution in TBST for 1 h at room temperature and then incubated with primary antibodies raised against PTPRD (1:500; LifeSpan BioSciences), ALDH1 (1:300; Proteintech Group, Chicago, USA), STAT3 antibody (1:500; Proteintech Group), pSTAT3 (Tyr705) (1:500; Cell Signaling Technology, Inc., Danvers, MA, USA), OCT-4 (1:300; Proteintech Group), E-cadherin (1:500; Proteintech Group), and vimentin (1:500; Proteintech Group) at 4°C overnight, and subsequently with an IRDye 800 CW-labeled secondary antibody (1:5,000). Protein bands were quantified by optical density analysis and normalized to GAPDH.