Figure 4.
Figure 4. EVL-mediated actin polymerization is necessary for mechanosensing, and EVL is sufficient to promote mechanosensory signaling under myosin suppression. (a) Dot plot shows quantification of total cell area of control (LKO vector) + GFP, EVL KD + GFP, EVL KD + GFP-ΔGF-PFN EVL, and EVL KD + GFP-shResist EVL MCF7 cells plated on 8-, 35-, or 64-kPa hydrogels. Data are collected from three independent experiments; all data points are shown (n ≥ 75 per condition; P values were determined using regression analysis; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM). (b) Representative inverted TIRF images of p-FAK staining in control (LKO vector) + GFP, EVL KD + GFP, EVL KD + GFP-ΔGF-PFN EVL, and EVL KD + GFP-shResist EVL MCF7 cells. Boxed insets are inverted images of expression constructs. Scale bars are 10 µm. (c) Dot plot shows quantification of p-FAK area. Data are collected from three independent experiments; all data points are shown (n ≥ 43 per condition; P values were determined using regression analysis; *, P ≤ 0.05; ****, P ≤ 0.0001; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM). (d) Representative inverted TIRF images of paxillin staining in untreated and Y-27632–treated control (GFP) and GFP-EVL–overexpressing MCF7 cells. Insets are inverted images of expression constructs. Scale bars are 10 µm. (e) Dot plots show quantification of paxillin area. Data are collected from four independent experiments; all data points are shown (n ≥ 51 per condition; P values were determined using regression analysis; **, P ≤ 0.01; ****, P ≤ 0.0001; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM). (f) Representative inverted TIRF images of p-pax staining in untreated and Y-27632–treated control (GFP) and GFP-EVL–overexpressing MCF7 cells. Insets are inverted images of expression constructs. Scale bars are 10 µm. (g) Dot plots show quantification of p-pax area. Data are collected from four independent experiments; all data points are shown (n ≥ 51 per condition; P values were determined using regression analysis; *, P ≤ 0.05; **, P ≤ 0.01; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM). (h) Representative inverted TIRF images of p-FAK staining in untreated and Y-27632–treated control (GFP) and GFP-EVL–overexpressing MCF7 cells. Insets are inverted images of expression constructs. Scale bars are 10 µm. (i) Dot plots show quantification of p-FAK area. Data are collected from three independent experiments; all data points are shown (n ≥ 43 per condition; P values were determined using regression analysis; *, P ≤ 0.05; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM).

EVL-mediated actin polymerization is necessary for mechanosensing, and EVL is sufficient to promote mechanosensory signaling under myosin suppression. (a) Dot plot shows quantification of total cell area of control (LKO vector) + GFP, EVL KD + GFP, EVL KD + GFP-ΔGF-PFN EVL, and EVL KD + GFP-shResist EVL MCF7 cells plated on 8-, 35-, or 64-kPa hydrogels. Data are collected from three independent experiments; all data points are shown (n ≥ 75 per condition; P values were determined using regression analysis; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM). (b) Representative inverted TIRF images of p-FAK staining in control (LKO vector) + GFP, EVL KD + GFP, EVL KD + GFP-ΔGF-PFN EVL, and EVL KD + GFP-shResist EVL MCF7 cells. Boxed insets are inverted images of expression constructs. Scale bars are 10 µm. (c) Dot plot shows quantification of p-FAK area. Data are collected from three independent experiments; all data points are shown (n ≥ 43 per condition; P values were determined using regression analysis; *, P ≤ 0.05; ****, P ≤ 0.0001; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM). (d) Representative inverted TIRF images of paxillin staining in untreated and Y-27632–treated control (GFP) and GFP-EVL–overexpressing MCF7 cells. Insets are inverted images of expression constructs. Scale bars are 10 µm. (e) Dot plots show quantification of paxillin area. Data are collected from four independent experiments; all data points are shown (n ≥ 51 per condition; P values were determined using regression analysis; **, P ≤ 0.01; ****, P ≤ 0.0001; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM). (f) Representative inverted TIRF images of p-pax staining in untreated and Y-27632–treated control (GFP) and GFP-EVL–overexpressing MCF7 cells. Insets are inverted images of expression constructs. Scale bars are 10 µm. (g) Dot plots show quantification of p-pax area. Data are collected from four independent experiments; all data points are shown (n ≥ 51 per condition; P values were determined using regression analysis; *, P ≤ 0.05; **, P ≤ 0.01; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM). (h) Representative inverted TIRF images of p-FAK staining in untreated and Y-27632–treated control (GFP) and GFP-EVL–overexpressing MCF7 cells. Insets are inverted images of expression constructs. Scale bars are 10 µm. (i) Dot plots show quantification of p-FAK area. Data are collected from three independent experiments; all data points are shown (n ≥ 43 per condition; P values were determined using regression analysis; *, P ≤ 0.05; n.s., not significant; exact P values for all two-way comparisons are found in Table S3; mean ± SEM).

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