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. 2020 Jan 29;9(2):319. doi: 10.3390/cells9020319

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Angiogenesis-related mRNA modulation and associated signaling activation in TEX-treated endothelial cells. (A) EC were cultured for 48 h in matrigel in the presence of AS- or AS-Tspan8-TEX (30 µg/mL). Growth and spriting was evaluated by light microscopy (scale bar: 20 µm); (B) clusters of mRNA (STRING functional protein analysis) of AS-Tspan8-TEX-treated EC and indication of overrepresented edges according to UniProt Keywords; (C) protein array of chemokines, cytokines, growth factors, receptors, adhesion molecules, and proteases of AS- and AS-Tspan8-TEX-treated EC. Signal strength of duplicates was evaluated by imageJ and is presented adjusting signal strength of EC = 1. The signal strength particularly of cytokines being very low, only >3-fold changes between untreated vs. AS- or AS-Tspan8-TEX-treated EC were considered as significant: *. (D) Western blot (WB) of chemokine receptors, PI3K-p85 and paxillin in untreated and AS- or AS-Tspan8-TEX-treated EC and immunoprecipitation of CXCR2 with CXCL1 in untreated and AS-Tspan8-TEX-treated EC; lysate control and immunoprecipitation with control IgG are included; (E) WB of proteins playing a central role in cytokine/growth factor activation and transcription of related genes. (F,G) Overrepresented edges according to biological processes in AS- and AS-Tspan8-TEX-treated EC, STRING functional protein analysis being performed under less stringent conditions (signal strength ≥500, fold increase: ≥1.5-fold); (F) mRNA upregulated and (G) mRNA downregulated after AS- or AS-Tspan8-TEX treatment. (H) Signaling engaged molecules were selected by Panther Gene list. Altered signaling molecule and signaling regulating mRNA in AS- and AS-Tspan8-TEX treated EC including the incidence of overrepresented edges was evaluated by String functional protein analysis. Upregulated mRNA is indicated by a red circle. (I) Recovery of vasculo-/angiogenesis-related mRNA in AS- and AS-Tspan8-TEX-treated EC, genes affected by AS- and AS-Tspan8-TEX: underlined; inhibitory genes: white bars, mRNA interfering with smooth muscle development: SM. (J) Altered vasculo-/angiogenesis related mRNA were analyzed by STRING for engagement into selective vasculo-/angiogenesis-related functions; upregulated mRNA is marked by a red circle. (K) Altered signaling-related RNA recovery selectively for vasculo-/angiogenesis related mRNA was evaluated according to (J). (L) Transcription factor and transcription initiating molecule mRNA recovery in AS- and AS-Tspan8-TEX-treated EC; >2-fold differences: filled bars, >3-fold differences: red *. (M) Transcription-related molecule overrepresentation as evaluated by STRING; upregulated mRNA is marked by a red circle. Full name of gene symbols: Table S10.

Angiogenesis-related mRNA modulation and associated signaling activation in TEX-treated endothelial cells. (A) EC were cultured for 48 h in matrigel in the presence of AS- or AS-Tspan8-TEX (30 µg/mL). Growth and spriting was evaluated by light microscopy (scale bar: 20 µm); (B) clusters of mRNA (STRING functional protein analysis) of AS-Tspan8-TEX-treated EC and indication of overrepresented edges according to UniProt Keywords; (C) protein array of chemokines, cytokines, growth factors, receptors, adhesion molecules, and proteases of AS- and AS-Tspan8-TEX-treated EC. Signal strength of duplicates was evaluated by imageJ and is presented adjusting signal strength of EC = 1. The signal strength particularly of cytokines being very low, only >3-fold changes between untreated vs. AS- or AS-Tspan8-TEX-treated EC were considered as significant: *. (D) Western blot (WB) of chemokine receptors, PI3K-p85 and paxillin in untreated and AS- or AS-Tspan8-TEX-treated EC and immunoprecipitation of CXCR2 with CXCL1 in untreated and AS-Tspan8-TEX-treated EC; lysate control and immunoprecipitation with control IgG are included; (E) WB of proteins playing a central role in cytokine/growth factor activation and transcription of related genes. (F,G) Overrepresented edges according to biological processes in AS- and AS-Tspan8-TEX-treated EC, STRING functional protein analysis being performed under less stringent conditions (signal strength ≥500, fold increase: ≥1.5-fold); (F) mRNA upregulated and (G) mRNA downregulated after AS- or AS-Tspan8-TEX treatment. (H) Signaling engaged molecules were selected by Panther Gene list. Altered signaling molecule and signaling regulating mRNA in AS- and AS-Tspan8-TEX treated EC including the incidence of overrepresented edges was evaluated by String functional protein analysis. Upregulated mRNA is indicated by a red circle. (I) Recovery of vasculo-/angiogenesis-related mRNA in AS- and AS-Tspan8-TEX-treated EC, genes affected by AS- and AS-Tspan8-TEX: underlined; inhibitory genes: white bars, mRNA interfering with smooth muscle development: SM. (J) Altered vasculo-/angiogenesis related mRNA were analyzed by STRING for engagement into selective vasculo-/angiogenesis-related functions; upregulated mRNA is marked by a red circle. (K) Altered signaling-related RNA recovery selectively for vasculo-/angiogenesis related mRNA was evaluated according to (J). (L) Transcription factor and transcription initiating molecule mRNA recovery in AS- and AS-Tspan8-TEX-treated EC; >2-fold differences: filled bars, >3-fold differences: red *. (M) Transcription-related molecule overrepresentation as evaluated by STRING; upregulated mRNA is marked by a red circle. Full name of gene symbols: Table S10.