Overexpression of Oct-2, Spl, and C/EBPα in K-562 cells. (A) Expressions of I antigen in the K-562 cells with overexpression of Oct-2, Sp1, and C/EBPα. The pcDNA3.1 expression vector harboring the respective full coding cDNAs of Oct-2, Sp1, or C/EBPα was transfected into the K-562 cells. K-562 cells were split into 6-well culture plates at a density of 2 × 105 cells/mL, and transfected with 1.0 μg of the constructed and mock pcDNA3.1 expression vector. The antibiotic-selected cells were then analyzed for the expression of I antigen using flow cytometry. Open and shaded areas are representative of the cells detected with anti-I mAb (1:5 dilution) and FITC-conjugated secondary antibody, and with FITC-conjugated secondary antibody only, respectively. (B) Expression profiles for IGnT transcripts in the K-562 cells with C/EBPα overexpression. The expressions of the IGnTA, IGnTB, and IGnTC transcripts in the K-562 cells transfected with the C/EBPα expression vector or mock pcDNA3.1 plasmid were analyzed using real-time PCR. Quantities of the IGnT transcripts were normalized to that of β-actin transcript in each sample. Data were obtained from 3 detections; standard deviations are shown. (C) Transcriptional activity of C/EBPα on IGnTC gene 5′ promoter. K-562 cells were cotransfected with the −318/+62-bp reporter construct and the C/EBPα expression vector or with the same reporter vector and mock pcDNA3.1 plasmid. After transfection for 48 hours, the cells were harvested to analyze the activity of the expressed luciferase. Results are presented as averages of luciferase activities from 3 repetitions; standard deviations are shown.
