Fig. 5
A mRNA expression levels of CDH1 in MCF7 cells (n = 3) grown on control culture plates (Ctrl) or on PAN/G10 membranes (PAN) was determined by qRT-PCR. mRNA levels are relative to that of GAPDH and are expressed as mean ± SD. B Schematic representation of part of the E-cadherin gene CDH1 encompassing a CpG island (from − 406 to + 1329). Inset shows the detailed sequence of the region analyzed by Methylation Sensitive PCR (MSP). The position of the Transcription Starting Site (TSS) is marked as “+1”, the position of cytosines in CpG dinucleotide are colored in red and their position is indicated. The positions of primers used for analysis of methylated or unmethylated DNA by MSP are shown in the inset. C MSP was performed using bisulfite-converted genomic DNA (gDNA) from MCF7 cells grown on control culture plates (MCF7-Ctrl) or on PAN/G10 membranes (MCF7-PAN). As positive control (MSP-control (+) we used bisulfite-converted gDNA from normal human tissue samples and as negative control (MSP-control (−)), gDNA was excluded from the PCR reaction. The PCR products obtained after PCR amplification using the two primer sets designed for recognition of methylated (116 bp) or unmethylated DNA (96 bp) were run in a 2% agarose gel and the image was acquired using a GelDoc system (Biorad). D 100% Stacked bars graph depicting the percentage of methylated (red) and unmethylated (yellow) DNA. Band quantification was performed after background removal using ImageJ software. E–H Immunofluorescence and confocal microscopy images showing H3K9ac (E, F) and H4ac (G, H) expression in cell membrane of MCF7 breast cancer cells grown on control coverslips or on PAN/G10 membranes. I Quantitative measurement of mean fluorescence intensity per nuclear area after background removal. Data were obtained from three different images with more than 100 cells per image using ImageJ (NIH) software. Bars represent mean ± SD. J Western blotting analysis showing H3K9ac, expression in MCF7 cells grown on normal Petri dishes (n = 3, Ctrl) or on PAN/G10 membranes (n = 3, PAN/G10). GAPDH was used as loading control