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. 2021 Oct 28;12:6229. doi: 10.1038/s41467-021-26454-x

Fig. 2. N-cadherin clustering and β-catenin recruitment in mimetic PEG hydrogels and stiffness dependent YAP nuclear localization via N-cadherin signaling.

Fig. 2

a HAVDI ligation caused sparse cells to adopt the N-cadherin and β-catenin patterning of confluent cells, as evident from confocal images of DAPI (blue), N-cadherin (green) and β-catenin (purple) staining in hMSCs cultured on either sparse (non-confluent) or confluent conditions on Scram/RGD or HAVDI/RGD hydrogels of Young’s modulus 20 kPa. The white arrow highlights real cell-cell adhesions between neighboring hMSCs; the yellow arrow highlights N-cadherin/HAVDI binding for an isolated hMSC on a HAVDI/RGD hydrogel. Scale bar: 20 μm. b–d Representative subcellular distributions of N-cadherin and β-catenin in three different cases corresponding to the numbered areas shown in a, showing that HAVDI ligation caused sparse cells to adopt the N-cadherin and β-catenin patterning of confluent cells. Scale bars: 5 μm. b Isolated cells on Scram/RGD hydrogels, which do not mimic cell-cell adhesions, did not show substantial N-cadherin clustering or β-catenin recruitment. c Isolated cells cultured on HAVDI/RGD hydrogel, which mimicked cell-cell adhesions, showed N-cadherin clustering or β-catenin recruitment that was indistinguishable from that in d confluent cells on Scram/RGD hydrogels. Yellow lines in b–d indicate the pixel regions of interest used to generate intensity profiles of N-cadherin (green) and β-catenin (purple). e Confocal images of YAP immunostaining on hMSCs cultured on 2, 11, 20 and 41 kPa Scram/RGD or HAVDI/RGD hydrogels for 3 d. Scale bars, 20 μm. f Quantification of YAP n/c ratios on Scram/RGD or HAVDI/RGD hydrogels of increasing stiffness with or without blebbistatin (myosin inhibitor) as shown in e (from left to right n= 82, 87, 79, 74, 126, 71, 60, 65, 71, 68, 53, 35, 105, 82, 39, 63 cells examined over 14, 13, 16, 18, 17, 16, 27, 19, 15, 13, 19, 24, 9, 9, 23, 27 images respectively, p values were obtained using one-way ANOVA followed by Tukey’s post hoc test, mean ± s.e.m.). Source data are provided as a Source Data file.