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

Figure 3.

Figure 3.

Nesprin tension is differentially sensitive to induction of partial and complete EMT. (A) Top: FRET index map of a wounded MDCK monolayer expressing the CB construct. Bottom: FRET index as a function of the distance from the front. (B) FRET index of the CB construct and the CH mutant at the front and back (500 µm) of the monolayer in MDCK (left; n = 70 CB front, 130 CB back, 73 CH mutant front, 91 CH mutant back) and NIH 3T3 (right; n = 363 CB front, 311 CB back, 125 CH mutant front, 143 CH mutant back) cells; three replicates. Solid line to guide the eye. (C) FRET index of the CB construct in MDCK cells as a function of cell migration velocity from experiments at low and high cell densities, upon epithelial wounding, and collectively migrating from outside (out) to inside (in) 40-µm-wide channels. R2 was derived from a least-squares linear regression; P value was derived from an extra sum-of-squares F test with slope = 0 as the null hypothesis (n = 240 1×, 255 10×, 111 front, 110 back, 64 in, 78 out, 1 replicate). (D) FRET index of the CB construct in MDCK cells as a function of internuclear distance from experiments at low and high cell densities, upon epithelial wounding, and upon entering 40-µm-wide channels. Solid line is the linear fit. R2 was derived from a least-squares linear regression; P value was derived from an extra sum-of-squares F test with slope = 0 as null hypothesis (n = 22 1×, 22 10×, 22 front, 22 back, 33 in, 18 out, 1 replicate). (E) Direct fluorescence and FRET index maps of the CB construct in MDCK cells after 5 h with or without HGF. (F) Internuclear distance of CB construct–expressing MDCK cells through time with or without HGF addition at time 0 h (n +HGF = 26 0 h, 27 5 h; n –HGF = 30 0 h, 32 5 h); two replicates. (G) Single-cell area of CB construct–expressing MDCK cells through time with or without HGF addition (n +HGF= 25 0 h, 24 5 h; n –HGF = 29 0 h, 35 5 h); two replicates. (H) FRET index of the CB construct and CH mutant in MDCK cells through time with or without HGF addition (n +HGF = 20 CB 0 h, 22 CB 5 h, 9 CH mutant 0 h, 7 CH mutant 5 h; n -HGF = 26 CB 0 h, 26 CB 5 h, 13 CH mutant 0 h, 10 CH mutant 5 h); two replicates. Scale bars = 20 µm. Mean ± SEM. Two-tailed Mann-Whitney tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.