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. 2023 Jul 7;14:4022. doi: 10.1038/s41467-023-39515-0

Fig. 3. Microgel-mediated volumetric confinement induces mechanoresponsive Plakoglobin expression in mESCs.

Fig. 3

a Schematic cell culture formats: conventional 2D-tissue culture plastic, agarose microgel encapsulation, 2D-tissue culture plastic overlaid with a bulk agarose gel and suspension/hanging drop culture. b Confocal immunofluorescence images of naive pluripotent (2i/LIF) mESCs cultured as hanging drops or microgel-encapsulated. Cells were stained for Plakoglobin (C-term) and KLF4 (naive pluripotency marker) after 48 h in culture. Representative of 3 independent experiments. c, d Confocal immunofluorescence images of naive pluripotent (2i/LIF) mESCs on tissue culture plastic (2D), in microgels (3D confined) or as suspension culture (3D unconfined) after 48 h. Cells were stained for (c) Plakoglobin and (d) β-catenin. e Quantification of Plakoglobin and β-catenin intensities per colony normalized by Hoechst. Error bars indicate the mean and standard deviations. p-values were determined by a two-tailed unpaired t-test with Welch’s correction; 3 independent experiments. f mESCs were cultured for 48 h on tissue culture plastic or on plastic overlaid with 1.5% agarose. Immunofluorescence analysis of Plakoglobin (N-term) showed an increase upon gel-mediated confinement in 2D. Error bars indicate the mean and standard deviations. n = 27 (Plastic), n = 50 (Plastic + 1.5% agarose). The p-values were calculated using a two-tailed unpaired t-test with Welch’s correction; 3 independent experiments.