JNCI Journal of the National Cancer Institute

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• Supplementary Table 1 - Sequences of genomic and cDNA primers
• Supplementary Fig. 1 - Specificity of anti-integrin á7 (ITGA7) serum and membrane localization of inducible integrin á7. A) Immunoblot analysis of anti-ITGA7 serum for specificity against ITGA7. Proteins in lysates of 1573 and PC-3 cells were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with preimmune serum (lanes 1 and 2), anti-ITGA7 serum (lanes 3 and 4), anti-ITGA7 monoclonal antibody (lanes 5 and 6), or anti-ITGA7 serum depleted of anti-ITGA7 by incubation with ITGA7 peptides (lanes 7 and 8). B) Immunofluorescence localization of inducible ITGA7. Immunofluorescence micrograph of membrane staining for ITGA7 in PITT1 cells induced to express integrin á7 with tetracycline at 1 mg/mL. C) Coimmunoprecipitation of integrin â1 (ITGB1) and ITGA7. Lysates from tetracycline-induced PITT cells (lane 1) were immunoprecipitated with preimmune (lane 2) or anti-ITGA7 serum (lane 3), and proteins were separated by SDS-PAGE and immunoblotted with anti-ITGB1. IP = immunoprecipitate; WB = immunoblot.
• Supplementary Fig. 2 - Mutations of integrin á7 in human cancer tissues. A) Diagram and description of integrin á7 structural alterations from truncation mutations. Top) Organization of the integrin á7 exons. Red and black boxes = mutually exclusive exons; green boxes = regular exons; open boxes = unrelated translation from a frameshift produced by nucleotide insertion. Bottom) Description of each mutation. Each structural alteration is shown with an exon number, a description of the mutation (amino acid and nucleotide), number of sample(s) examined, specimen source, type of malignancy, whether matched normal sample was sequenced (for prostate cancer, hepatocellular carcinoma cancer, glioblastoma multiforme, and leiomyosarcoma), zygosity, template used for sequencing, other mutations present in the same samples, pathologic grade, tumor stage, and length of relapse-free survival. NA = not applicable; NK = not known; ND = not determined because of lack of matched normal samples; (s) = separate allele; m = month after primary tumor resection; * = three prostate cancer samples with homozygous mutations and six with heterozygous mutation; ** = A93T, D697G, H722Y, L948P(s), and M812V(s). B) Histogram of integrin á7 mutations. Histograms are shown for representative sequences that have point mutations leading to a stop codon (sense primer, Left), insertion leading to a frameshift (antisense primer, Middle), missense mutation from a case containing only missense mutation (sense primer, Right). Mutation is indicated with an arrow. The histograms of sequences from matched normal samples are shown at the top.
• Supplementary Fig. 3 - Expression of integrin á7 in tumor cells and representative images of colony formation and anchorage-independent growth assays from Fig. 1. A) Immunoblots of cell clones using rabbit antibodies against integrin á7 (top panel) and mouse monoclonal antibody against â-actin (bottom panel) were performed on cells transfected with pCMVscript or pCMV-integrin á7, including PC-3 cells (P4 and P5 = vector control and IT4 and IT8 = ITGA7), Du145 cells (DP1 and DP2 = vector control, ITDu3 and ITDu4 = ITGA7), and SK-UT-1 cells (PSK1 and PSK3 = vector control and ISK3 and ISK7 = ITGA7). H1299 and H358 were transfected with vectors expressing either scramble small interfering RNA (siRNA) or integrin á7 specific siRNA. B) Representative images of hematoxylin staining of colony formation assays on cells from panel A. C) Representative images of anchorage-independent growth in soft agar of cells from panel A.