Figure 1.
ATIP3 down-regulation in invasive breast carcinomas.
A–B. Comparison of MTUS1 (U133A Affymetrix 212096_s_at) probeset data intensities in (A) normal breast tissue and 151 invasive breast tumors classified according to histological grade (I, II, III), (B) breast tumors classified according to the occurrence of distant metastasis or axillary lymph node (ALN) metastasis and (C) molecular subgroups of breast tumors defined by immunodetection of surrogate markers ER/PR+ (luminal), HER2+ (HER2−like) and ER−, PR−, HER2− (triple negative, TN). Probeset intensities were calculated using Affymetrix Raw MAS5.0 default settings. The number of samples is indicated below in brackets. *p<0.001; **p<0.0001 compared to normal tissue; ap<0.0001 compared to grade I; bp<0.005 compared to grade II; cp<0.0001 compared to ER+/PR+; dp<0.05 compared to HER2+. D. Immunohistochemistry on a breast tumor section of histological grade I and adjacent normal breast tissue (upper panel), and two representative grade III breast cancer sections (lower panel) using anti-MTUS1 monoclonal antibodies. A bar represents 100 µm. E–G. Correlation between MTUS1 (212096_s_at) probeset intensities and real-time RT-PCR expressed relative to internal control EEF1G in 29 representative breast tumor samples. Oligonucleotides were designed to amplify total ATIP transcripts (“MTUS1”) (E), ATIP3 transcripts (F), or ATIP1 transcripts (G).
Table 1.
MTUS1 expression levels in invasive breast carcinomas.
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.
Figure 3.
ATIP3 suppresses tumor growth in vivo.
A. Number of tumor xenografts (in percent) developing after s.c. injection of GFP-ATIP3 expressing MCF7 clones (HC1, HC6, HC7) or GFP-transfected MCF7 cells (stable clone or pool) into immunodeficient mice. B. Time-course of tumor progression in mice injected with GFP or GFP-ATIP3 cell clones as in A. Tumor size was measured twice weekly as indicated in the methods. C. Mean weight (in grams) of tumors excised on day 38 after injection of MCF7 cells expressing GFP (clone and pool, n = 18) and GFP-ATIP3 (clones HC1, HC6, HC7, n = 11). D. Pictures of representative tumors excised on day 38 after injection of MCF7 cell clones as in C. E. Representative histologic analysis of tumors grown from GFP and GFP-ATIP3 expressing MCF7 cells, after hematoxylin-eosin staining. A bar represents 100 µm. F. Representative photomicrographs of cancer cells as in E, stained by hematoxylin-eosin (magnification×63). Arrows show cancer cells undergoing active mitosis. A bar represents 20 µm. Histogram shown on the right is a quantification of the number of mitotic cells per field counted under an inverted microscope. The number of fields analyzed is indicated below in brackets. **p<0.0001.
Figure 4.
ATIP3 associates with microtubules.
A–B. Representative photomicrographs of confocal microscopy analysis showing the cellular distribution of (A) endogenous ATIP proteins in SK-MES cancer cells and (B) transiently transfected (24 h) GFP-ATIP3 fusion protein, stained with anti-MTUS1 (green) and anti-alpha-tubulin (red) antibodies. A bar represents 10 µm. C. Microtubule co-sedimentation assay performed on MDA-MB-231 stable transfectants expressing GFP-ATIP3 or GFP (upper panel) and SK-MES, MDA-MB-468 and CAMA-1 tumor cells expressing endogenous ATIP3 (lower panel). Immunoblots were revealed using anti-GFP or anti-MTUS1 antibodies, and reprobed with anti-alpha-tubulin antibodies. Molecular weights are indicated on the left. L: total cell lysate; S: supernatant; P: pellet.
Figure 5.
A. Representative photomicrographs from live cell imaging of HeLa cells stably expressing mCherry-histone 2B (in red) and transiently transfected with GFP-ATIP3 (in green). Time (in hrs) is indicated above or below each panel. B. Time (in min) necessary to achieve mitosis in control, GFP-transfected and GFP-ATIP3 transfected cells. The number of cells analyzed is indicated below in brackets. **p<0.0001. C. Time (in min) between chromosome condensation and the onset of anaphase in GFP-transfected and GFP-ATIP3 transfected cells. The number of cells analyzed is indicated below in brackets. **p<0.0001.