E cadherin

The cell protein was separated by 10% SDS-polyacrylamide gels electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk powder for 2 hours, the membrane was incubated with primary antibodies against E-cadherin (1:5000, Proteintech Group, Chicago, IL, USA), vimentin ((1:2000, Proteintech Group), N-cadherin (1:2000, Proteintech Group) and β-actin (1:10000, Proteintech Group) at 4°C overnight. Then the membrane was incubated with rabbit or mouse secondary antibodies at room temperature for 1 hour, and the protein bands were detected using ECL detection system (Tanon-5200Multi, Shanghai, China).

Total protein in PC cells was extracted using radioimmunoassay precipitation lysis buffer (Beyotime Biotechnology, Suzhou, China) containing phenylmethylsulfonyl fluoride (Servicebio, Wuhan, China). Protein concentrations in the samples were determined using the bicinchoninic acid method (Servicebio, Wuhan, China). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gels (Meilune, Dalian, China)and thereafter, transferred to polyvinylidene fluoride membranes (Thermo Scientific, USA). The membranes were blocked with skim milk powder (Beyotime Biotechnology, Suzhou, China) and incubated with primary antibodies (GADD45A [1:500], Cat No. A1797, Abconal, China; CDK1 [1:500], Cat No. 19532-1-AP, Proteintech, China; CCNB1 [1:500], Cat No. 28603-1-AP, Proteintech, China; N-cadherin [1:500], Cat No. 22018-1-AP, Proteintech, China; E-cadherin [1:500], Cat No. 20874-1-AP, Proteintech, China; and β-actin [ACTB], 1:500; Cat No. 20536-1-AP, Proteintech) for 16 hours at 4℃. After washing twice with Tris-buffered saline containing 0.1% Tween‐20, the membranes were incubated with secondary antibody and visualized using an enhanced chemiluminescence reagent. ACTB was used as the loading control to calculate the relative protein expression.

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).

MM102 (HY-12220A) and PFA (HY-15484) were purchased from MedChemExpress (NJ, USA). Cisplatin was purchased from the Pharmacy of Rhode Island Hospital (NDC 0703-5747-11, RI, USA). Antibodies to C-cas3 (9664), MLL1 (14197), p53 (2524), p-p53 (ser15) (9284), ATM (2873), p-ATM (4526), ATR (2790), p-ATR (2853), Chk1 (2360), p-Chk1 (2348), Chk2 (2662), p-Chk2 (2661), and RIPA lysis buffer (9806) were purchased from Cell Signaling Technology (MA, USA). Antibodies to WDR5 (ab56919), histone H3 (ab1791), histone H2AX (ab124781), and γ-H2AX (ab81299) were from Abcam (MA, USA). E-cadherin was bought from Proteintech (20874-1-AP, IL, USA). Antibody to NGAL was purchased from R&D systems (AF1857, MN, USA). MLL1 siRNA (Assay ID 501621), Lipofectamine 2000 (11668019), and SuperSignal chemiluminescent substrate (34580) were from Thermo Fisher Scientific (MA, USA). H3K4me3 antibody (07-473), TUNEL Kit (11684795910), CCK8 (96992), DAPI (D9542), Creatinine Assay Kit (6M01K06250), and Urea Assay Kit (7C08K03750) were purchased from Sigma-Aldrich (MO, USA). Klotho antibody (sc-515942), WDR5 siRNA (sc-61799), E-cadherin siRNA (sc-35243), p53 siRNA (sc-29436), and negative siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (CA, USA).

Western blotting was performed as previously described [41 (link)]. The antibodies used for western blotting include P27 (CST, 3686T), CDK4 (CST, 12790S), CyclinB1 (CST, 12231S), P21 (CST, 2947T), CyclinD1 (CST, 2978S), E-cadherin (Proteintech, 60335-01), ITGB8 (Abcam, ab243023), H3K27ac (Abcam, ab4729) and β-actin (CST, 8480S).

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).