Figure 3.

Establishment and phenotype of MCF-10A cells or HCT116 cells with stable silencing of CEACAM1. (a) HCT116 cells stably transfected with CEACAM1 shRNA vector no. 26 (CEACAM1-KD 026), with CEACAM1 shRNA vector no. 28 (CEACAM1-KD 028) or with a control shRNA vector (CTRL) were lysed and analyzed for CEACAM1 or NBS1 expression levels by western blotting. One of two experiments with similar results is shown. (b) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (CEACAM1-KD 025), with CEACAM1 shRNA vector no. 26 (CEACAM1-KD 026) or with a control shRNA vector (CTRL) were analyzed for CEACAM1 mRNA levels by quantitative real-time PCR. Error bars indicate s.d.; n=2. (c) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (MCF10A^{CEACAM1−KD 025}) or with a control shRNA vector (MCF10A^CTRL) were incubated in the presence or the absence of NCS 1.47 nM for the indicated time points, labeled with BrdU (10 μM, 20 min), fixed with ice-cold ethanol and treated for analysis of the cell cycle phase distribution by flow cytometry using a FACSCalibur apparatus (Becton-Dickinson Biosciences, Le Pont-De-Claix Cedex, France). At least 10⁴ events were recorded and data analysis was done with CellQuest Pro software (Becton-Dickinson Biosciences). (d) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (CEACAM1-KD 025), with CEACAM1 shRNA vector no. 26 (CEACAM1-KD 026) or with a control shRNA vector (CTRL) were seeded in 60 mm Petri dishes at the density of 1500 cells/dish. On the following day the cells were treated with the indicated dose of etoposide for 1 h, washed with phosphate-buffered saline, and allowed to grow in new medium at 37 °C. After 7 days, the colonies were fixed with methanol, stained with crystal violet, and counted.