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. 2024 Jan 11;11(11):2306722. doi: 10.1002/advs.202306722

Figure 4.

Figure 4

Osmolarity‐induced molecular crowding increases anabolic response. a) Schematic diagram showing cellular response to altered molecular crowding and the methods used to investigate the changes to cellular response. b) BMP signaling in C2C12 cells as measured by luciferase reporters after stimulating (BMP7, 10 ng mL−1) and culturing them in 330, 370, 410 and 450 mOsm at the indicated time points, n = 3 biological replicates. c) TGFβ signaling in OA hPCs after culturing them in OA or healthy‐like mOsm for 6 h, with or without TGFβ1 (10 ng mL−1) as measured by luciferase reporters, n = 3 donors and 3 biological replicates, per donor. d) TGFβ signaling in OA and healthy hPCs without changing osmolarity after 6 h of supplementing with or without TGFβ1 (10 ng mL−1) as measured by luciferase reporters, n = 2 OA and 3 healthy donors, 3 biological replicates per donor. e) FRAP measurements of SOX9‐mGFP(A76E) mobility in OA hPCs in cytoplasm and nucleus after exposing them to OA or healthy‐like osmolarities. 3 donors per condition, n > 120 cells, per condition (all donors combined). f) ACAN, g) COL1 and h) COL2A mRNA expression as measured by qPCR in OA and healthy hPCs exposed to 330, 370, 410, and 450 mOsm for 24 h, n = 3 donors. i) COL2/COL1 ratio as quantified by fluorescence intensity in OA hPCs after treating with either 330 or 400 mOsm for 5 d, n > 49 cells. j) COL2/COL1 ratio as quantified by fluorescence intensity in OA and healthy hPCs after culturing them in iso‐osmotic medium (330 mOsm) for 5 d, 3 donors per condition, n > 150 cells. k) Stiffness of OA hPCs exposed to OA or healthy‐like osmolarities for 24 h, 2 donors, n = 64 cells. l) TRPV4 gene expression in OA hPCs after exposing them to OA and healthy‐like osmolarity for 24 h. n = 3 donors. Statistics: c‐e,l) Unpaired t‐test, i–k) Mann‐Whitney U‐test, *p < 0.05, **p < 0.01, ***p < 0.001, shades and error bars b–d, f–h,l) indicate SD, whiskers and boxes in i–k) indicate SD and 25%, 75% percentile, respectively. All statistical significance is compared to normalized control, unless otherwise indicated.