Figure 6

Depletion of TIA proteins activates cell proliferation and anchorage-independent growth. (a) Post-transfected HeLa cells with siRNAs against control (C; white diamond), TIA-1 (1; grey square), TIAR (R; grey circle) or TIA-1 plus TIAR (1+R; black circle) were seeded in six-well plates and the number was counted on the days indicated for 6 days. Each time point represents the means ± standard error of the mean (SEM; n = 3 to 6; *P < 0.01). (b) Cells grown for 6 days as in (a) were monitored by methyl thiazolyl tetrazolium (MTT) assays. The represented values were normalized and are expressed relative to control (C), whose value is fixed arbitrarily to 1, and are means ± SEM (n = 3 to 5; *P < 0.001). (c) Analysis of cell-cycle phases by flow cytometry after propidium iodide staining. The data are means ± SEM (n = 3; *P < 0.01; **P < 0.05). (d) Representative photographs of colonies generated from transfected HeLa cells. One week after seeding 2,000 cells in a 6-well plate, the cells were fixed in paraformaldehyde (5%), stained with crystal violet (0.01%) and quantified by light microscopy. The represented values are the means ± SEM from three independent experiments performed in duplicate for at least eleven independent colonies (*P < 0.001). A colony was defined as a cell cluster when containing at least 86 cells. Scale bars represent 200 μm. (e) Soft-agar colony formation. In each 6-well plate, 5,000 post-transfected HeLa cells as above were seeded into soft-agar matrix and incubated for 14 days. The cell number was counted under a light microscope after staining with crystal violet as before. The represented values are means ± SEM for three independent experiments performed in duplicate for at least 36 counted fields (*P < 0.001; **P < 0.01). In this case, a colony was defined as a cell cluster containing at least 4 cells. Scale bars represent 100 μm.