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. 2009 Oct 1;4(10):e7239. doi: 10.1371/journal.pone.0007239

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Rodrigues-Ferreira et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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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.