Anti mouse and anti rabbit secondary antibodies

Purified EFD was thoroughly dissolved in dimethyl sulfoxide (DMSO) and stock solution was stored at −20 °C. Stock solutions of the positive controls (Doxorubicin and Cabazitaxel obtained from Sigma, Ronkonkoma, NY, USA) were also prepared in DMSO. Cell Signaling Technology (Beverly, MA, USA) supplied primary antibodies i.e., Bcl2, P53, Nf-kB, PARP and cleaved Caspase 3 while anti-mouse and anti-rabbit secondary antibodies were procured from GE healthcare (PitTSBurgh, PA, USA). PCa cell lines i.e., PC3 [CRL-1435] and DU-145[HTB-81] were ordered from ATTC; Manassas, VA, USA. DMSO, BPA, TCA, and TSB used in the experiments were purchased from Sigma while tween 20 from Merck (Rahway, NJ, USA). Medium 99, FBS, RPMI-1640, DMEM, PBS, and MTT powder were procured from Merck Millipore (Burlington, MA, USA). All sized silica gel plates and chromatography columns were ordered from Merck, Darmstadt, Germany.

Lipofectamine RNAiMAX reagent and OPTI‐MEM were purchased from Life Technologies; 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) was purchased from USB Corporation; CellTiter Blue was purchased from Promega Corporation; Primary antibodies were purchased from Genetex; antimouse and anti‐rabbit secondary antibodies were purchased from GE Healthcare; fluorescein isothiocyanate–conjugated anti‐rabbit immunoglobulin G antibody was purchased from Jackson Immuno Research Laboratories; foetal bovine serum (FBS), trypsin‐EDTA, Dulbecco modified Eagle medium (DMEM), and penicillin and streptomycin (P/S) were purchased from Gibco‐Invitrogen Corporation; 10‐, 15‐ and 4‐cm Petri dishes and 96‐ and 24‐well plates were purchased from Orange Scientific; MTT, Tris base, glycerol, NP‐40, sodium dodecyl sulphate (SDS) and tetramethylethylenediamine were purchased from USB Corporation; bovine serum albumin was purchased from Sigma‐Aldrich; Horseradish peroxidase‐linked anti‐rabbit IgG was purchased from GE Healthcare; Tris HCl (pH 6.8) was purchased from Severn Biotech Ltd; HCl was purchased from Scharlau Chemie; repel solution and cover oil were purchased from GE Healthcare; and an enhanced chemiluminescence substrate kit was purchased from Visual Protein Corporation. All chemicals and biochemicals used in this study were of an analytical grade.

Briefly, 20 μg of cellular proteins were subjected to SDS-PAGE (NuPAGE precast gel, Thermo Fisher Scientific, USA) for electrophoresis and followed by dry transfer onto nitrocellulose membranes (iBlot Transfer Stack, Thermo Fisher Scientific, USA). The membranes were blocked with 5% BSA in TBS containing 0.1% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 60 minutes and incubated with the following primary antibodies: anti-human SIRT1 (1:2000 dilution), acetylated-p53 at K382 (Ac-p53; 1:500 dilution), AMPKα (1:4000 dilutuion), and p21 (1:2000 dilution) antibodies purchased from Cell Signaling Technology (Beverly, MA, USA), and p300 (1:2000 dilution) and total p53 (1:2000 dilution) from Santa Cruz (Dallas, USA). Anti-human GAPDH (1:5000 dilution), and β-actin (1:10000 dilution) antibodies were used as loading controls and purchased from Millipore (Billerica, USA) and Sigma Aldrich (St Louis, USA), respectively. Anti-rabbit and anti-mouse secondary antibodies (1:10000 dilutions) were bought from GE Healthcare (Buckinghamshire, United Kingdom). The immunoreactive bands were detected by an enhanced chemiluminescence system (Proteinsimple, Santa Clara, USA). Related signals were quantified using Proteinsimple image software (Santa Clara, USA).

Cells were washed twice with cold phosphate-buffered saline (PBS) (4 °C), and whole cell lysates were prepared in sodium dodecyl sulphate (SDS) lysis buffer (1% SDS, 10% glycerol, 100 mM Tris pH 7.4, Protease Inhibitor Cocktail (Calbiochem, San Diego, CA, USA), 1 mM Na₃VO₄, 1 mM NaF), sonicated and heated for 10 min at 90 °C. Protein concentration was determined with Bio-Rad DC Protein Assay (Bio-Rad Laboratories Inc., Hercules, CA, USA). The isolation and preparation of EpCAM⁺ cells from tumors and normal colon mucosa have been described previously [48 (link)]. The protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore). Specific proteins were detected with primary antibodies (see Supplementary Materials Table S2 for the full list of antibodies used in the present study). The mouse monoclonal antibody against β-actin (A5441, Sigma-Aldrich) was used as loading control. Anti-rabbit and anti-mouse secondary antibodies, conjugated with horse-radish peroxidase, and ECL-Plus reagent were purchased from GE Healthcare (Little Chalfont, UK), and they were used according to the manufacturer’s instructions.

Transfected cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate) with EDTA-free protease inhibitor cocktail (Roche). Clarified protein lysates were mixed with 5× protein sample buffer (10% SDS, 25% 2-mercaptoethanol, 50% glycerol, 0.01% bromophenol blue, 300 mM Tris-Cl [pH 6.8]) and denatured at 95°C. Proteins were then separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto polyvinylidene fluoride membranes, and detected by WesternBright ECL reagent (Advansta, USA). Primary anti-Flag and anti-β-actin antibodies from Sigma-Aldrich (USA), and secondary anti-mouse and anti-rabbit antibodies from GE Healthcare (USA) conjugated with horseradish peroxidase (HRP) have been used.