Figure 4

Down-regulation of serpinE2 expression in human colorectal cancer cells inhibits colony formation. A- HCT116 and LoVo cells were stably infected with lentiviruses encoding for a control shRNA (against TGFP) or encoding serpinE2-specific shRNAs (shSerpinE2#15 or shSerpinE2#16). Stable cell populations were thereafter lysed and RNA isolated to determine serpinE2 or β2MIC gene expression by Q-PCR. The relative level of each mRNA was calculated using the standard curve method and normalized to the corresponding β2MIC mRNA level. B- LoVo or HCT116 cells expressing either shTGFP or shSerpinE2 (#16) were seeded in a 6-well plate at 200 000 cells per well. Cells were harvested, clumps disrupted using a syringe and counted. Values are means of 4 experiments ± SE. C- Cell populations were cultured in soft agarose for 2-3 weeks before MTT staining. The number of colonies was determined using Image J software. The number of shTGFP-expressing cells (control) was set at 100%. Results are the mean ± SE of at least 3 independent experiments. D- Phase-contrast microscopy of foci from two-week post-confluent shTGFP and shSerpinE2(#16)-expressing LoVo cells stained with crystal violet. *, significantly different from shTGFP cell populations (set at 1) at p < 0.05 (Student's t test); **, significantly different from control at p < 0.002 (Student t test).