To harvest cells in hydrogels, gels containing cells were incubated in cold PBS containing 50 mM EDTA (Sigma-Aldrich) to chelate calcium for 5 min while pipetting to break up the gels. Cells were centrifuged at 500g for 10 min, and the supernatant was removed. For SDS–polyacrylamide gel electrophoresis of whole-cell lysates, cells were lysed in Pierce radioimmunoprecipitation assay buffer (catalog no. 89900, Thermo Fisher Scientific) supplemented with protease inhibitor cocktail tablets (catalog no. 11836170001, Roche) and PhosSTOP phosphatase inhibitor cocktail tablets (catalog no. 04906845001, Roche) following the manufacturer’s instructions. The concentration of harvested protein from the cell lysates was quantified using the Pierce Bicinchoninic Acid Protein Assay Kit (catalog no. 23227, Thermo Fisher Scientific). Laemmli sample buffer (catalog no. 1610747, Bio-Rad) was added to lysates, and samples were boiled for 10 min before loading 25 μg of protein in each lane of a polyacrylamide gradient gel (catalog no. 4561086, Bio-Rad). Proteins in the polyacrylamide gels were transferred to nitrocellulose at 100 V for 1 hour, blocked with 5% milk in TBS-T [137 mM NaCl, 2.7 mM KCl, 19 mM tris base, 0.1% Tween (pH 7.4)], incubated in primary antibodies overnight, and then incubated in IRDye 680– or IRDye 800–conjugated secondary antibodies for 1 hour. Blots were visualized using a Li-COR Odyssey imaging system (Li-COR Biotechnology). The following antibodies were used for immunoblotting: AkT antibody (catalog no. 2920S, Cell Signaling Technology), phosphorylated Akt antibody (catalog no. 4060S, Cell Signaling Technology), TRPV4 (catalog no. ab191580, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog no. ab181602, Abcam).

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