Figure 2. The retinoblastoma Y79 cell line was treated with 5 µM Azacytidine (AZC) and the epithelial cell adhesion molecule (Ep-CAM) expression was studied by flow cytometry analysis (FC), Immunohistochemistry (IHC) and real time quantitative reverse transcriptase polymerase chain reaction (Q-RT–PCR). A-E: M1 represents the EpCAM percentage positivity in the FC graphs (solid line). FC analysis demonstrated 47.73%, 44.2%, 42.96%, and 18.27% of Y79 cells positive for Ep-CAM on days 5, 8, 10, and 15 of AZC treatment, respectively. F-I: Immunohistchemistry pictures stained with Di-amino benzidine (DAB) and was counter stained with hematoxylin. All the pictures were captured at 20 times magnification. EpCAM was not stained in Y79 cells not treated with AZC (F); Strong membrane positive expression of Ep-CAM was seen in Y79 cells treated AZC on 5th day (G); Strong membrane positivity of Ep-CAM in the retinoblastoma primary tumor represented as positive control (H); Y79 cells were stained negative when isotypic antibodies were used as the negative control (I). Q-RT–PCR shows the mRNA expression of Ep-CAM in Y79 cells after 5 days of AZC treatment (J). After day 5, Y79 cells were withdrawn from AZC exposure and subsequently passaged in the complete medium up to 30 days, and Ep-CAM mRNA expression was retained up to 21 days as follows: 210-fold, 132-fold, 56-fold, and 20-fold on days 8, 10, 15, and 20 days, respectively. The error bars represents the standard deviation of triplicate values (J).