Computational protocol: EGCG antagonizes Bortezomib cytotoxicity in prostate cancer cells by an autophagic mechanism

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Protocol publication

[…] PC3 cells were seeded at 9 × 103 cells per well of a 96-well plate and allowed to attach overnight. Thereafter, cells were treated with 200 μL of DMEM/Ham’s F12 medium supplemented with increasing concentration of EGCG (0–350 μM), BZM (0–20 nM), MG132 (0–1.4 μM) or the aforementioned concentrations of BZM or MG132 in combination with 5 or 50 μM EGCG. After 48 hours of treatment, PC3 cell viability was determined with the WST-1 assay (Roche, Lewes, UK). For this, the spent medium was removed and replaced with fresh medium (100 μL/well) supplemented with 10% WST-1 reagent. This assay is based on the reduction of tetrazolium salt WST-1 to formazan by mitochondrial dehydrogenases in metabolically active cells. After a 1 hour incubation at 37 °C, formazan dye production was determined at an absorbance of 450 nm with the EnSpire® multimode plate reader (PerkinElmer, Waltham, MA, USA). Dose-response curves were generated and IC50 (50% inhibiting concentration) values were determined by non-linear regression analysis (four parameter logistic curve) using SigmaPlot software (version 12.0). IC50 values were used in all subsequent experiments. [...] PC3 cells (5 × 105 cells/dish) were seeded in 60 mm dishes. After 48 hours, treated and untreated cells were washed twice with ice-cold PBS and scraped into ice-cold RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 1% Triton X-100, 150 mM NaCl) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein concentration was estimated by the DC Protein assay kit (Bio-Rad, Hercules, CA, USA), using bovine serum albumin (Sigma-Aldrich) as a standard. Total cell lysates (75 μg of protein in Laemmli buffer) were resolved by 12% acrylamide SDS-PAGE under reducing conditions and electrophoretically transferred to PVDF membranes (EMD Millipore, Merck KGaA, Darmstadt, DE). Transfer efficiency was routinely monitored by 0.1% Ponceau S (Sigma-Aldrich). Non-specific binding to the PVDF membrane was minimized by blocking for 1–5 hours at ambient temperature with TBS-T buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% (w/v) non-fat dry milk or 1% Blocking reagent solution (Roche, Basel, CH). PVDF membranes were incubated overnight with primary antibodies in 5% non-fat dry milk or 1% Blocking reagent solution at 4 °C with gentle shaking. Membranes were probed with the following primary antibodies: rabbit polyclonal anti-GRP78/BiP (GL-19) (dilution 1:100) and mouse monoclonal anti-p21waf1/Cip1 clone CP74 (dilution 1:200) from Sigma-Aldrich; rabbit polyclonal anti-ubiquitin (dilution 1:200), mouse monoclonal anti-NF-kB p65 (F-6) (dilution 1:200), mouse monoclonal anti-PARP-1 (F-2) (dilution 1:1,000) and mouse monoclonal anti-β-actin (dilution 1:500) from Santa-Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal anti-caspase 8 (1C12) (dilution 1:500), rabbit polyclonal anti-caspase 9 (dilution 1:1,000), mouse monoclonal anti-CHOP (L63F7) (dilution 1:1,000) and rabbit monoclonal anti-LC3B (D11) (dilution 1:1,000) from Cell Signaling Technology (Danvers, MA; USA); rabbit monoclonal anti-p-eIF2α (dilution 1:500) from Abcam (Cambridge, UK). Following the incubation with primary antibody, membranes were probed with horseradish peroxidase-conjugated anti-mouse (dilution 1:5,000) or anti-rabbit secondary antibodies (dilution 1:200,000) (Sigma-Aldrich) for 1 hour at ambient temperature. Immunoreactive bands were detected using the BM Chemiluminescence Western Blotting Substrate (POD) (Roche). Densitometric analysis of protein bands was performed using Quantity One® 1-D Analysis software (version 4.6.3) (Bio-Rad). […]

Pipeline specifications

Software tools SigmaPlot, Quantity One 1-D
Applications Miscellaneous, DNA fingerprinting
Diseases Neoplasms, Prostatic Neoplasms, Drug-Related Side Effects and Adverse Reactions