High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis
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1 High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis Supplementary Material Supplementary Figure S1. Representative CRBP-1 immunostaining of non-neoplastic lung tissue and of major histological patterns of invasive lung adenocarcinomas and percentages of expression. A, normal bronchial epithelium. B, normal lung. C, acinar. D, papillary, with evident tumor cells growing on the surface of fibrovascular cores. E, micropapillary, with small papillary clusters
2 growing within air spaces and F, solid patter. G, bar graph showing the percentage of CRBP-1 High and CRBP-1 Low expression adenocarcinomas according to the histological subtype (1). Original magnification: A, x400; B-F, x125. H, bar graph showing the percentages of tumor keratin (K) 1, 5/6, 14 and nox4 expression in CRBP-1 High and CRBP-1 Low adenocarcinomas.
3 Supplementary Figure S2. Representative EGFR mutational status, CRBP-1 gene copy number gain and promoter methylation in lung adenocarcinomas. A, representative pyrograms of mutated 19 and 21 EGFR exons in lung adenocarcinoma. B, pie chart of CRBP-1 gene copy number gain in lung adenocarcinoma tissues. C, bar graph of percentages of methylated and unmethylated CRBP-1 lung adenocarcinomas. D, representative lanes of the methylation status. M and U lanes correspond to specific amplified products of methylated and unmethylated DNA, respectively. Methylated samples are marked with asterisks. Normal lung (NL) as negative and DNA (Qiagen, P ) as positive control were used to ensure primer specificity; N no template control.
4 Supplementary Figure S3. CRBP-1 transcript and protein in wild, empty vector and CRBP-1- transfected A549 adenocarcinoma cells. A, RT-PCR shows evident CRBP-1 transcript in transfected A549 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as housekeeping gene. B, representative blots of protein expression in CRBP-1-transfected A549 lung adenocarcinoma cells. Membranes were reblotted with anti-tubulin to ensure equal loading, and Hek-293 cells as positive control. C, transcript levels of epithelial-to-mesenchimal transition genes in CRBP-1 + compared to CRBP-1 - A549 cells (CTRL).
5 Suppplementary Table S1. Primers Sequences Gene Primer Sequence Accession Number GAPDH Sense: 5 -ACGGATTTGGTCGTATTGG-3 NM_ Antisense: 5 -GATTTTGGAGGGATCTCGC-3 ß2-microglobulin Sense: 5 -GATTCAGGTTTACTCACGTC-3 NM_ Antisense: 5 -GTTCACACGGCAGGCATACT-3 CRBP-1 Sense: 5 -CGCTTGTGG CCAAACTGGCTC-3 NM_ Antisense: 5 -ACACATCCTGCTGATTGGTTGG- 3 CRABP-2 Sense: 5 -GCCCAACTTCTCTGGCAACTG-3 NM_ Antisense: 5 -GTCTGCTCCTCAAACTCCTCCC-3 RARα Sense 5 -GGTCGGCGATGGTGAGGGT-3 NM_ Antisense 5 -TGGGCAAATACACTACGACAACAG-3 RARß Sense: 5 -GCAGAGCGTGTAATTACCTTGAA-3 NM_ Antisense: 5 -GTGAGATGCTAGGACTGTGCTCT-3 RARγ Sense: 5 -CTGCCAGTACTGCCGGCTAC-3 NM_ Antisense: 5 -TCTGCACTGGAGTTCGTGGTATACT-3 RXRα Sense: 5 -TCCTCCTCCCACCGCTCCATC-3 NM_ Antisense: 5 -CAGCTCCGTCTTGTCCATCTG-3 FABP-5 Sense: 5 -CCTGTCCAAAGTGATGATGG-3 NM_ Antisense: 5 -CAGCATCAGGAGTGGGATG-3 PPARß/δ Sense: 5 -ATGGAGCAGCCACAGGAGGAAGCC-3 NM_ Antisense: 5 -GCATGAGGCCCCGTCACAGC-3 K1 Sense: 5 -ATTTCTGAGCTGAATCGTGTGATC-3 NM_ Antisense: 5 -CTTGGCATCCTTGAGGGCATT-3 K10 Sense: 5 -TGATGTGAATGTGGAAATGAATGC-3 NM_ Antisense: 5 -GTAGTCAGTTCCTTGCTCTTTTCA-3 K5 Sense: 5 -CAGCGTCAAATTTGTCTCCAC-3 NM_ Antisense: 5 -TTGGTCTAGACTACTCTCCAG-3 K7 Sense: 5 -TGAATGATGAGATCAACTTCCTCAG-3 NM_ Antisense: 5 -TGTCGGAGATCTGGGACTGC-3 K14 Sense: 5 -CCAGTTCTCCTCTGGATCGCAG-3 NM_ Antisense: 5 -GATCTTCCAGTGGGATCTGTGTCCA-3 Involucrin Sense: 5 -GGGTGGTTATTTATGTTTGGGTGG-3 NM_ Antisense: 5 - GCCAGGTCCAAGACATTCAAC-3 Nestin Sense: 5'-GCGGTGGCTCCAAGACTTC-3' NM_ Antisense: 5'-ACTGGGAGCAAAGATCCAAGAC-3' SMAD-4 Sense: 5'-TTCTGGAGGAGATCGCTTTTG-3' Antisense: 5'-TTGCCTATGTGCAACCTTGCT-3' NM_ NANOG Sense: 5'-GCAATGGTGTGACGCAGAAG-3' NM_ Antisense: 5'-AGGTTCCCAGTCGGGTTCA-3' SOX-2 Sense: 5'-CCGTTCATCGACGAGGCTAA-3' NM_ Antisense: 5'-TCATGAGCGTCTTGGTTTTCC-3' Vimentin Sense: 5'-TCCAAGTTTGCTGACCTCTCTG-3' Antisense: 5'-CAGTGGACTCCTGCTTTGCC-3' NM_ SNAIL Sense: 5'-TGCATATTCGGACCCACACA-3' NM_ Antisense: 5'-TGTTGCAGGAGGGCAAGAA-3' TGF-β Sense: 5'-GGACACCAACTATTGCTTCAG-3' NM_ Antisense: 5'-TCCAGGCTCCAAATGTAGG-3' MMP-2 Sense: 5'-TGGCAAGTACGGCTTCTGTC-3' NM_ Antisense: 5'-TGGCAAGTACGGCTTCTGTC-3' MMP-9 Sense: 5'-TGCGCTACCACCTCGAACTT-3' NM_ Antisense: 5'-GATGCCATTGACGTCGTCCT-3' References 1. Kadota K, Suzuki K, Kachala SS, Zabor EC, Sima CS, Moreira AL, et al. A grading system combining architectural features and mitotic count predicts recurrence in stage I lung adenocarcinoma. Mod Pathol 2012;25:
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Supplementary Information Titles Journal: Nature Medicine
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