Figure 5.
Lack of α-catenin–vinculin interaction endows cells with rigidity-independence. (A) Left: α-Catenin staining in WT MDCK cells before and after EMT. Right: Quantification of the percentage of cells with α-catenin cell edge localization 24, 48, and 72 h after the addition of TGF-β. (B) Schematic of the experiment designed to test α-catenin’s role in rigidity dependence after EMT in MDCK and NMuMG cells. (C) Representative images of MDCK, NMuMG (after EMT), and MEF cells stained with Annexin-FITC on 0.2-kPa gels coated with FN, 6 h after plating (PI staining not depicted). (D) Quantification of the percentage of Annexin V– and PI-positive WT and α-catenin KD MDCK cells on 0.2-kPa gels coated with FN. (E) Quantification of the percentage of Annexin V– and PI-positive WT and α-catenin KD NMuMG cells on 0.2-kPa gels coated with FN. (F) Quantification of the percentage of Annexin V– and PI-positive WT MEFs, α-catenin KD MEFs, and α-catenin KD MEFs expressing WT GFP-α-catenin or GFP-α-catenin L344P, plated for 24 h on 0.2-kPa FN-coated matrices. (G) MTT measurement of WT MEFs, α-catenin KD MEFs, and α-catenin KD MEFs expressing WT GFP-α-catenin or GFP-α-catenin L344P, plated for 24 h on 0.2-kPa FN-coated matrices. In the Annexin/PI graphs, positive and negative controls, the cells were heated at 65°C for 15 min for the former and left untreated for the latter and plated on FN-coated plastic Ibidi wells. For the MTT assay, the positive growth control refers to WT and α-catenin KD MEF cells plated on a FN-coated 96-well plate (plastic). Statistical analysis of the α-catenin cell-edge localization (A) was tested by nested ANOVA followed by Tukey’s post hoc test, and percentage of apoptotic cells and MTT assay (D–G) were tested by ANOVA followed by Tukey’s post hoc test (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

Lack of α-catenin–vinculin interaction endows cells with rigidity-independence. (A) Left: α-Catenin staining in WT MDCK cells before and after EMT. Right: Quantification of the percentage of cells with α-catenin cell edge localization 24, 48, and 72 h after the addition of TGF-β. (B) Schematic of the experiment designed to test α-catenin’s role in rigidity dependence after EMT in MDCK and NMuMG cells. (C) Representative images of MDCK, NMuMG (after EMT), and MEF cells stained with Annexin-FITC on 0.2-kPa gels coated with FN, 6 h after plating (PI staining not depicted). (D) Quantification of the percentage of Annexin V– and PI-positive WT and α-catenin KD MDCK cells on 0.2-kPa gels coated with FN. (E) Quantification of the percentage of Annexin V– and PI-positive WT and α-catenin KD NMuMG cells on 0.2-kPa gels coated with FN. (F) Quantification of the percentage of Annexin V– and PI-positive WT MEFs, α-catenin KD MEFs, and α-catenin KD MEFs expressing WT GFP-α-catenin or GFP-α-catenin L344P, plated for 24 h on 0.2-kPa FN-coated matrices. (G) MTT measurement of WT MEFs, α-catenin KD MEFs, and α-catenin KD MEFs expressing WT GFP-α-catenin or GFP-α-catenin L344P, plated for 24 h on 0.2-kPa FN-coated matrices. In the Annexin/PI graphs, positive and negative controls, the cells were heated at 65°C for 15 min for the former and left untreated for the latter and plated on FN-coated plastic Ibidi wells. For the MTT assay, the positive growth control refers to WT and α-catenin KD MEF cells plated on a FN-coated 96-well plate (plastic). Statistical analysis of the α-catenin cell-edge localization (A) was tested by nested ANOVA followed by Tukey’s post hoc test, and percentage of apoptotic cells and MTT assay (D–G) were tested by ANOVA followed by Tukey’s post hoc test (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

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