Total protein was extracted from cultured cells by adding 2X sample buffer, 20 mM Tris HCl pH 7. 4, 5 mM mag nesium chloride, 10 ug/ml complete protease Rapamycin clinical trial inhibitor cocktail, 1 mM phenylmethylsulfonylfluoride. DNA was shared by pipetting up and down for 3 minutes at room temperature. Samples were boiled at 95 C for 15 minutes, centrifuged at 13,000 rpm for 10 seconds and then sub jected to 14% SDS PAGE. After blocking overnight at 4 C in a buffer containing PBS, 0. 1% Tween 20 and 5% low fat milk powder, nitro cellulose membranes were incubated for 90 minutes with primary antibodies. Antibodies against DNMT1, DNMT3a, DNMT3b, APC, RASSF1A and B actin were used. Membranes were washed three times for 10 minutes in a buffer containing PBS and 0.
1% Tween 20 and were incubated with a peroxidase coupled secondary antibody to visualize responsive bands after incubation with West Pico lumi nescence substrate. Densitometry analysis was performed by peak intensity analysis on a GeneGnome image capture and analysis system. Bands were normalized to B actin expression which was used as an internal loading control. Immunohistochemistry Formalin fixed and paraffin embedded xenograft tumour samples were cut into 5 um sections deparaffinised using graded alcohols. Antigen retrieval was performed by heat induced epitope retrieval in pH 9 antigen retrieval buffer at 95 C for 60 minutes. Endogenous peroxidase blocking was carried out for 10 minutes with peroxidase blocking reagent. Subsequently, the primary antibody against DNMT1 and DNMT3a was applied for 30 minutes at RT.
For detection of the primary anti bodies the ready to use REAL EnVision Detection System was used in accordance with the manu facturers instructions. The EnVision staining system is based on an HRP labeled dextran polymer, which is con jugated to secondary antibodies eliminating the nonspe cific staining background resulting from endogenous avidin biotin activity. Visualization was performed using diaminobenzidine as the chromogen substrate being a part of the REAL EnVision Detection System. Slides were counterstained with hematoxylin. The stained slides were digitalized using the ImageAccess 9 Enterprise software. Significance was calculated using the t test for paired samples. P 0. 05 was regarded as significant.
Results Panobinostat inhibits DNMT activity and expression in vitro After only 6 h of treatment, incubation of HepG2 and Hep3B cells led to a rapid and significant decrease in total DNMT activity by 46. 7% and 47. 4%, respectively. At later points in time, DNMT activity was stably reduced by approximately 20% in both cell lines, except for the 24 and 72 h time point in GSK-3 HepG2, where an in hibition of more than 40% was observed. Expression of DNMT1, DNMT3a and DNMT3b were then investigated by quantitative real time RT PCR.