4.7. Molecular Analysis by Western Blotting and Antibodies

Briefly, 1 × 10⁶ of MDA-MB-468 and BT-20 cells, and 1.5 × 10⁶ of HCC70 cells were seeded in (Ø90mm) Petri dishes and allowed to attach overnight, then treated with berberine at 0.5 and 1 µM, and then incubated for 120 and 144 h in 37 °C with 5% CO₂. DMSO (0.005%) was used as control. For whole cell protein lysates, the samples were prepared according to the established protocol [32]. Briefly, the lysates were collected using the Laemmli 1X buffer. Proteins dosage was done by BCA Protein assay kit reducing Agent Compatible (Perbio, ref 23250). Proteins concentrations were read as absorbance values at 562 nm using Infinite 200 (Tecan, Lyon, France) with Magellan data analysis software. Equivalent amounts of protein lysates (20 µg), were separated by electrophoresis in acrylamide gel (Bio-Rad TGX 4–15%) starting with 15 mA then increasing to 25 mA, then blotted onto nitrocellulose membrane (Bio-Rad, Marnes la Coquette, France). The membranes were then saturated with 5% BSA+TBS/0.1% Tween for 1 h at RT.

The blotted membranes were incubated with different primary antibodies for 2 h at RT, and then washed four times with TBS/0.1% Tween 5 min each, at RT. The membranes were then followed by incubations with secondary antibodies labeled with horseradish peroxidase (Jackson Immuno Research Laboratories, Interchim, Clichy, France) for 1 h at RT.

The proteins were visualized using ECL kit (Amersham Pharmacia Biotech, Orsay, France). The quantification was performed using a LAS-3000 Luminescent Image analyser and Multi Gauge software (Fuji, FSVT, Courbevoie, France). Actin was detected for normalisation between samples using anti-beta-actin primary antibody at the dilution of 1:2000 (Sigma-Aldrich, Saint Quentin Fallavier, France). MGMT, PCNA, cleaved-PARP (cl-PARP), cleaved-caspase-7 (cl-caspase-7) and cleaved-caspase-8 (cl-caspase-8) antibodies were used at 1:1000 dilutions. Cyclin D1 antibody was used at 1:10000 dilution. Histone H2AX and phosphorylated-H2AX (P-H2AX) antibodies were diluted at 1:2000. Cyclin B1 antibody was used at 1:1000 dilution. All the antibodies were purchased from Cell Signaling Technology, Ozyme, Saint Quentin en Yveline, France.