Figure 5

Growth kinetics and morphological characteristics of human tumor HepG2 cells trnsnfected with the pcDNA3.1, pcDNA3.1- rps4 (negative control), or pcDNA3.1- mrp4 vector construct. (A) Efficiency of vector transfection into HepG2 cells. At 24 and 48 hrs after transfection of the pcDNA3.1 (Mock) or pcDNA3.1-gfp (inserted instead of rps4), phase-contract (PC) and fluorescence (GFP) micrographs were taken to evaluate the efficiency of vector transfection. It is manifest that a considerable number of cells are strongly stained with GFP. Bar, 20 μm. (B) After the vector transfection, HepG2 cells transfected by the pcDNA3.1 (Mock) or pcDNA3.1-rps4 (negative control) continue to increase their cell number in growth medium. In the case of HepG2 cells transfected by the pcDNA3.1-mrp4, increase in the cell number is significantly suppressed during the first 48 hrs of incubation. However, once the decreased number of cells begins to increase during 48-72 hrs after transfection. The results represent the mean±SD in three independent experiments. (C) TUNEL assay. HepG2 cells transfected with the pcDNA3.1 (Mock), pcDNA3.1- rps4, or pcDNA3.1-mrp4 were incubated for 48 hrs and subjected to a TUNEL assay, as described in Methods. From morphological assessment of apoptosis detected by FITC-staining (FITC) and phase-contrast (PC) images of the same field, it is evident that HepG2 cells transfected with the pcDNA3.1 (Mock) or pcDNA3.1- rps4 are not stained with the FIFC-conjugated antibody, but that HepG2 cells ectopically expressing the pcDNA3.1-mrp4 are strongly stained with the antibody. As shown in the most-right column, when HepG2 cells transfected with the pcDNA3.1 (Mock) was pretreated with DNase-I, they were stained with the FITC-conjugated antibody because of DNA fragmentation as observed in the process of apoptosis. Bar, 50 μm.