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. 2020 Oct 1;34(19-20):1345–1358. doi: 10.1101/gad.340661.120

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© 2020 King et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 2. — Increased Yap/Taz transcriptional activity and EMT phenotype in cells selected for extracellular protein-dependent growth. (A) Gene set enrichment analysis (GSEA) plots showing enrichment in expression of Yap target genes, as measured by RNA sequencing of KRPC^S cells, relative to expression in KRPC cells cultured for 3 wk in complete medium + 3% BSA. (B) Relative mRNA expression of Yap, Taz, and target genes as measured by qRT-PCR in KRPC cells cultured in ±Leu ±3% BSA for 16 h or 3 wk. Bars represent mean ± SD from three biological replicates. (C) Immunofluorescence micrographs showing localization of Yap (antibody also cross-reacts with Taz) in parental KRPC and KRPC^S cells. (D) Immunoblot of cytosolic and nuclear extracts from parental KRPC and KRPC^S cells. (E) Phase contrast micrographs of parental KRPC and KRPC^S cells. Scale bars = 200 µm. (F) GSEA plot showing enrichment in expression of EMT genes in KRPC^S cells, relative to expression in KRPC cells cultured for 3 wk in complete medium + 3% BSA. (G) Light micrographs showing migration of parental or KRPC^S cells across control (top panels) or matrigel-coated (bottom panels) membranes. Bar plot represents mean ± SEM of invasion index (ratio of cells that migrated across matrigel membrane to control membrane) across three biological replicates (***) P = 0.0005, unpaired two-tailed t-test.