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Figure S1. Evaluation of loss of heterozygosity (LOH) at the Numb locus in Numb-negative breast cancer patients and phenotypic characterization of primary matched normal and mammary tumor cultures. (A) Seven polymorphic STS markers (D14S 71, D14S77, D14S268, D14S277, D14S43, D14S70, and D14S785) localized on chromosome 14q23 were selected from the genomic database (NCBI accession no. Z16844, Z24162, Z23878, Z16997, X56973, Z16819, and X569055, respectively) according to both their high frequency of heterozigosity in a control population and to their proximity to the Numb locus. Specific primer pairs flanking the (CA)n repeat were chosen for each STS marker. PCR reactions were assembled in a final volume of 50 µl according to the Taq-gold polymerase manufacturer's instructions (Perkin-Elmer) with the following modifications: 10 pmol of each primer pair was used; dNTP final concentration in the PCR reaction was 200 µM for dATP/dGTP/dTTP and 10 µM for dCTP; 0.025 µl/reaction of {alpha}-32PdCTP (3000 Ci/mol); 20-50 ng of genomic DNA. Genomic DNA was extracted from matched paraffin-embedded normal and tumor tissues and tested separately. PCR conditions were as follows: 94°C 30 s, 58°C to >53°C 30 s (decrease of 0.5/cycle), 72°C 30 s for 10 cycles; 94°C 30 s, 53°C 30 s, and 72°C 30 s for 20 cycles. PCR reaction was denatured with 5_µl of loading buffer (10X: 98% formamide; 1mM EDTA; 0.1 % bromophenol blue; and 0.1 % Xylene cyanol), for 5 min and run on a 7% acrylamide gel, TBE 1X and 32 % formamide. The following primers were used: D14S277 (5'-ctccccattgctttcact-3'; 5'-ttgaagattcagataaggt-3'); D14S43 (5'-ctggaacactcaggcgag-3'; 5'-gccactttctactttggg-3'); D14S71 (5'-tgtgcaccaatgcctcct-3'; 5'-gcccggccagaaatgctt-3'); D14S77 (5'-gcctgagtcactgtgcc-3'; 5'-cagacagaaattaaccagag-3'); D14S268 (5'-agcttcctactgtgtaaaacga-3'; 5'-ggctggggctgcaccttgta-3'); D14S70 (5'-agctaatgacttagacacgttgta-3'; 5'-atcaatttgctagtttggca-3'); D14S785 (5'-gctctgtctcac-3'; 5'-gatcattgacataggaaacac-3'). The position of each primer on the chromosome region containing the Numb locus is shown. 20 class-1 and 10 class-3 breast tumours were analyzed for LOH. Out of 40 tumors analyzed, LOH was detected in only one class1(type-0) mammary tumor. However, the sequence of transcripts originating from the non-deleted Numb allele in that tumor did not show any alteration, with respect to the wild-type sequence (not depicted). In addition, we selected two class1(type-0) tumors, which showed presence of Numb transcripts by ISH, and isolated areas of high tumor cellularity (>90%). Numb transcripts, cloned from these tumors by RT-PCR, did not show any mutation with respect to the wild-type sequence (not depicted). We concluded that genetic alterations at the Numb locus are unlikely to account for lack of Numb expression in tumors. (B) Normal and tumor mammary epithelial cells were grown in appropriate selective medium, as described inMaterials and methods. Normal cell cultures typically show two major morphological types of cells (top left): (1) small, smooth-edged, refractile, polygonal cells, which maintain active proliferation and seemingly represent some form of "stem cell population;" and (2) larger, flatter, irregular-shaped cells, which do not seem to undergo many cell divisions. The latter might represent cells already programmed to stop multiplying after a few more population doublings. Tumor cells usually display a much higher morphological heterogeneity (top right), ranging from a typical epithelial appearance to an irregular spindle-like phenotype, resembling a more "dedifferentiated" status. By the second passage, normal and tumor primary cultures were proven to be of pure epithelial origin by immunofluorescence staining for keratin expression (bottom panels). Myoepithelial cell component was <_1-2 as="as" assessed="assessed" by="by" img="img" src="/math/agr.gif" alt="{alpha}">-smooth muscle actin staining (unpublished data). A mixture of mAbs recognizing the major cytokeratins (CK 1, 4, 5, 6, 8, 10, 13, 18, and 19; Sigma-Aldrich; C2562) and an mAb against {alpha}-smooth muscle actin (Sigma-Aldrich; clone 1A4) were used on ethanol-fixed cells grown on glass coverslips. Results are representative of all matched pairs used in this work.