Figure 5

Effect of TSA in combination with 5-aza-dc on CASP8. (A) DNA was isolated from the TSA and 5-aza-dc treated and untreated cells and MSP was performed with methylated and unmethylated CASP8 PCR primers as described in Methods. The bands detected by methylated primer represented methylated CASP8 (M), and the bands detected by unmethylated primer represented unmethylated CASP8 (UM). (B) RNA was isolated from the TSA and 5-aza-dc treated and untreated cells. RT and Q-PCR was performed with CASP8 primers. The relative level of CASP8 mRNA was normalized to 18 s as described in Methods. Each bar represented the mean of three independent amplifications with standard deviation (SD), *p < 0.05 between indicated groups. (C) Immunofluorescence analysis was performed with cleaved caspase-8 antibody followed by incubation with anti-goat IgG-FITC and mounted with DAPI mounting medium (left panel). The arrows indicate the FITC labeled cleaved caspase-8 (green in the top panel of left), and the cell nucleus was labeled by DAPI (blue in bottom panel of left). The cells with positive staining for cleaved caspase-8 were counted in five different areas and adjusted with total number of cells (right panel). Each bar represents the mean of number of cells positive for cleaved caspase-8 in the five areas with standard deviation (SD), * p < 0.05 between the indicated groups. (D) Double immunofluorescence analysis was performed with Di-Methyl Histone H3 (Lys27) or Acetyl Histone H3 (Lys9) antibodies followed by actin antibody as described in Methods. The nucleus positive stained with Di-Methyl Histone H3 (Lys27) or Acetyl Histone H3 (Lys9) were labeled with FITC as green (indicated by white arrows), and the cytoplasm stained with actin was labeled with Tex-red as red (indicated with light blue arrows).