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Journal: Cell Reports
Article Title: Caveolin-1 Modulates Mechanotransduction Responses to Substrate Stiffness through Actin-Dependent Control of YAP
Figure Lengend Snippet: CAV1 Modulates YAP Activity (A) Variations in gene expression in the RNA-seq analysis between cells grown on stiff and soft substrates, showing all identified genes (left bar) and YAP-target genes alone (right bar). Genes significantly upregulated by ECM stiffness are boxed, and the enrichment for YAP target genes was analyzed using the Fisher exact test. Genes highlighted green are those that were upregulated on the stiff substrate, whereas those highlighted red were downregulated. (B) qRT-PCR analysis of Ctgf and Ankrd1 expression in WT and Cav1KO MEFs grown on stiff and soft substrates for 24 hr. Data are normalized to WT cells grown on a stiff substrate. n = 3. (C) TEAD transcriptional activity in WT and Cav1KO MEFs expressing the 8xGTIIC-luciferase reporter and grown on stiff or soft substrates for 24 hr. Luciferase activity was measured and normalized as described in . Data are normalized to WT MEFs grown on stiff substrate. n = 4. (D) Confocal immunofluorescence images of YAP expression in WT and Cav1KO MEFs grown on stiff or soft substrate. F-actin was stained with fluorophore-conjugated phalloidin (red; left column), and nuclei were stained with Hoechst (blue in merged images; third column). The right column shows zoomed views of the YAP ROI (boxed in white in the YAP images). (E) Percentage of cells from analysis as in (D) with predominantly nuclear YAP (N), predominantly cytosolic YAP (C), or an even nuclear-to-cytosolic distribution (N/C). Randomly selected images from 3 independent experiments were analyzed (60–200 interphase cells per condition). (F) SEEK computational gene co-expression analysis, showing expression correlation ( Z score) between YAP target genes and the rest of the genome. (G) Confocal immunofluorescence images of YAP in WT and Cav1KO MEFs plated on different micropatterns with a fibronectin-coated grid that allows cells to spread to a predefined size of 2,025 μm 2 or 300 μm 2 . Nuclear contours are outlined with dotted gray lines. (H) ImageJ quantification of YAP subcellular distribution in cells plated on micropatterns of 3 grid sizes (300 μm 2 , 1,024 μm 2 , and 2,025 μm 2 ). Data are presented as the nuclear to total cell staining intensities; 10–20 cells were analyzed from 2 biological replicates per condition. The boxplots show the median, 1 st and 3 rd quartiles, and 90 th and 10 th percentiles (whiskers). (I) qRT-PCR analysis of the YAP targets Ctgf and Ankrd1 in cells subjected to cyclic mechanical stretching (CS; see ) and unstretched cells. n = 4. Data in (B), (C), (E), and (I) are presented as means ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, and ∗∗∗∗ p < 0.0005. See also Figure S1 and .
Article Snippet: Monoclonal antibodies were sourced as follows: anti-YAP (sc-101199) and anti-TEF-1 (sc-376113) from Santa Cruz Biotechnology; anti-CAV1 XP (#3267) and anti-YWHAH (14-3-3 η (D23B7); #5521) from Cell Signaling (Danvers, Massachusetts, United States); anti-Flag M2 (F-3167) from Sigma-Aldrich; and anti-glyceraldehyde-3-phosphate dehydrogenase (MAB374) and anti-cortactin (p80/85, clone 4F11) from Millipore (Burlington, Massachusetts, United States).
Techniques: Activity Assay, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Luciferase, Immunofluorescence, Staining