Fig. 7
Contact normalization affects Src-transformed cell transcriptome. RNA-seq analysis was performed on Src transformed (MEC-Src) cells cultured with themselves, nontransformed (MEC) cells, and cadherin deficient (MEC-CdhKo) cells in the layered culture system. a A total of 49,315 unique gene transcripts were detected, with 21,738 expressed at a level of at least 1 transcript per million (TPM) in any of the cell types. b Analysis of transcripts from nontransformed (MEC) and Src transformed (MEC-Src) cells cultured with themselves indicate that oncogenic Src kinase altered the expression of 3391 transcripts by at least threefold (p < 0.05 by t-test, n = 3). Src transformation increased the expression of 2240 genes and decreased the expression of 1151 genes as indicated. c Transcripts from Src transformed (MEC-Src) cells cultured with themselves were compared with transcripts from MEC-Src cells cultured with nontransformed (MEC cells) to identify genes affected by contact normalization. Six hundred and fifty four out of the 3391 genes affected by Src transformation were inversely affected by at least 40% (p < 0.08 by t-test, n = 3). Contact normalization increased the expression of 326 genes that were originally inhibited by Src, and decreased the expression of 328 genes that were originally increased by Src. d Transcripts from Src transformed (MEC-Src) cells cultured with nontransformed (MEC) cells were compared with transcripts from MEC-Src cells cultured with cadherin deficient (MEC-CdhKo) to identify genes affected by contact normalization in a cadherin dependent manner. Eighty four of the 326 genes affected by contact normalization were differentially affected by cadherin expression by at least twofold (p < 0.05 by t-test, n = 3). Contact normalization decreased the expression of 16 of these genes, and increased the expression of 68 genes. e Onion diagram of genes affected by Src transformation and contact normalization. Src increased the expression of 2240 transcripts (light red), and decreased the expression of 1151 transcripts (light green) that were not affected by contact normalization (p < 0.05 by t-test, n = 3). The expression of 326 Src induced transcripts were suppressed (medium red) and 328 Src suppressed transcripts were induced by contact (medium blue) with by cadherin deficient nontransformed (MEC-CdhKo) cells (p < 0.08 by t-test, n = 3). The expression of 16 Src induced transcripts were suppressed (dark red) and 68 Src suppressed transcripts (dark blue) were induced by contact with cadherin expressing (MEC) nontransformed cells, but not cadherin deficient (MEC-CdhKo) cells (p < 0.05 by t-test, n = 3). f Genes affected by contact normalization in a cadherin dependent manner were categorized by cellular location. Transcript expression levels are shown as the percent of Src transformed (MEC-Src) cultured with nontransformed cadherin competent (MEC) cells compared with Src transformed cells cultured with nontransformed cadherin deficient (MEC-CdhKo) cells (mean + SEM, n = 3). The expression of putative tumor promoters and suppressors were decreased and increased by contact normalization, respectively. g PDPN interacts with 40 gene products affected by Src. STRING protein–protein interaction analysis reveals network nodes representing proteins (circles) and edges (lines) indicating physical and functional protein associations. PDPN interacts with 18 proteins encoded by transcripts increased (light red) and 6 transcripts suppressed (light blue) by Src, but not affected by contact normalization. Cadherin deficient and competent nontransformed cells decreased the expression of 3 Src induced transcripts (medium red), and increased the expression of 8 transcripts (medium blue) suppressed by Src that encode proteins interacting with PDPN in adjacent transformed cells. Cadherin competent cells decreased the expression of PDPN induced by Src (dark red), and increased the expression of 3 transcripts suppressed by Src (dark blue) that were not affected by cadherin deficient cells. Edge line thickness represent interaction strength score