Figure 9

Effects of anti-HER2 antibody BH1 and tumor necrosis factor-α on DNA synthesis and cell cycle in SK-BR-3 cells. (A) Effects on DNA synthesis were tested by 24-h BrdU incorporation assay. Cells were plated on coverslips in 12-well plates, treated as described in Figure 5, and were exposed to BrdU during 24 h prior to indicated days. Nuclear BrdU was detected by indirect immunofluorescence assay. Nuclei were counterstained with DAPI, examined using Zeiss Axio Imager.A1 microscope. Representative photographs (3–5 frames per coverslip) were acquired. Percent BrdU incorporation was calculated after manual counting of BrdU⁺ and BrdU^- nuclei. * Denotes p-values ≤ 0.05 (BH1 vs. IgG1, BH1 + TNF-α vs.IgG1 + TNF-α). T-bars: SD. (B-D). Effects on cell cycle were analyzed by flow cytometry. SK-BR-3 cells were treated with 20 μg/ml BH1 or IgG isotype control antibody in the presence or absence of 1000 U/ml TNF-α. Cells were fixed on day 1 (B), day 3 (C) and day 6 (D), stained with propidium iodide and the cell cycle distribution was analysed with flow cytometry. Fraction of cells present in G2/M-phase (G2/M), S-phase (S) and G1-phase (G1) are shown in purple, green and orange respectively. Fraction of apoptotic cells observed as a SubG1 peak is shown in black.