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. 2020 Aug 13;219(10):e201908036. doi: 10.1083/jcb.201908036

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© 2020 Déjardin et al.

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Figure 4. — Nesprin tension regulates catenin nuclear translocation. (A) Left: MDCK cells stably expressing β-catenin-GFP with and without mCherry-DNKASH, with and without HGF addition. Right: β-Catenin nucleus/cytoplasmic balance (GFP intensity ratio) as a function of HGF and DNKASH (n = 43–, 89+-, 65++); one replicate. (B) Left: MDCK cells stably expressing α-catenin-GFP with and without mCherry-DNKASH, with and without HGF addition. Right: α-Catenin nucleus/cytoplasmic balance (GFP intensity ratio) as a function of HGF and DNKASH (n = 35–, 36+-, 31++); two replicates. (C) Relative cytoplasmic, nuclear envelope, and nuclear levels of α-catenin in cells plated at high density (HD; 10×), low density (LD; 1×), at the wound front (WH, wound healing), upon HGF exposure, and upon HGF exposure and mCherry-DNKASH expression. n = 10 cells for each condition and compartment; two replicates. (D) Top: Front of collectively migrating α-catenin-GFP MDCK cells (arrows indicate direction of migration) before (t = 0) and after cytochalasin D (t = 250 min) treatment. Bottom: α-catenin-GFP intensity along dotted line above through time; one replicate. (E) Left: MDCK cells stably expressing β-catenin-GFP with transient expression of NLS-iRFP-α-catenin (arrowheads) and without (asterisks). Right: β-catenin nucleus/cytoplasmic balance (GFP intensity ratio) as a function of NLS-iRFP-α-catenin expression (n = 112-, 66+); two replicates. (F) Left: MDCK cells stably expressing β-catenin-GFP with transient expression of shRNA against α-catenin and lyn-mCherry (arrow) and without (asterisk). Right: β-catenin nucleus/cytoplasmic balance (GFP intensity ratio) as a function of shRNA/lyn-mCherry cotransfection (n = 102-, 44+); two replicates. (G) Left: MDCK cells stably expressing TOP-dGFP and transiently expressing NLS-iRFP-α-catenin after 10 h with and without LiCl. Right: dGFP intensity as a function of NLS-α-catenin-iRFP expression and LiCl (n = 17—, 39-+, 17+-, 39++); two replicates. (H) Working model. See text for details. Scale bars = 10 µm. Mean ± SEM. Two-tailed Kruskal-Wallis (A–C and H) and Mann-Whitney (E and F) tests. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.