8p22 MTUS1 Gene Product ATIP3 Is a Novel Anti-Mitotic Protein Underexpressed in Invasive Breast Carcinoma of Poor Prognosis
Figure 2
ATIP3 inhibits cancer cell proliferation.
A. ATIP3 silencing in MDA-MB-468 cells transfected with control siRNA or specific ATIP3 siRNA#1 or siRNA#2 for 72 hours. Left panel: real-time RT-PCR using ATIP3-specific primers relative to EEF1G, normalized to non transfected cells (NT). Middle panel: immunoblotting with anti-MTUS1 antibodies showing ATIP3 at 170 kDa, and reprobing with anti-alpha-tubulin antibodies for internal control. Right panel: MTT assay. Results are expressed as percent of MTT incorporation in control siRNA-transfected cells at time 96 hours. Shown are the results of two representative experiments out of four performed in quadruplicate. **p<0.0001. B. Cell proliferation in stable MDA-MB-231 transfectants. Left panel : immunoblotting of total cell lysates from MDA-MB-231 cells non transfected (NT) or stably expressing GFP or GFP-ATIP3 (clone 3A1) at levels comparable to those in MDA-MB-468. Blots were probed with anti-MTUS1 (upper panel), anti-GFP (middle panel), anti-alpha-tubulin antibodies (lower panel). Arrows indicate the migration of GFP-ATIP3 (200 kDa), ATIP3 (170 kDa), GFP (25 kDa), alpha-tubulin (50 kDa). Middle and right panels: measurement of BrdU incorporation and MTT assay. Shown is one representative experiment out of three performed in quadruplicate. **p<0.0001; *p<0.001. C. Colony formation of GFP- or GFP-ATIP3-transfected MCF7 cells plated at different densities. Shown is one representative experiment out of five performed in duplicate. Right panel : quantification of the number of colonies per well. **p<0.0001. D. Immunoblotting of total lysates from MCF7 cells left untransfected (NT) or stably transfected with GFP or GFP-ATIP3 (clones HC1 and HC6), revealed as in (B). Middle panel : MCF7 stable transfectants grown in soft agar for four weeks. Shown are photomicrographs of one representative experiment out of three performed in triplicate. A bar represents 50 µm (upper panel) and 10 µm (lower panel). Right panel : quantification of the number of colonies per field counted under an inverted microscope. **p<0.0001.