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

Figure 5.

Figure 5

Osmolarity‐induced higher molecular crowding decrease catabolic signaling. a) Schematic diagram showing cellular response to altered molecular crowding and the methods used to investigate the changes to cellular response. b) IL1β signaling in HEK‐293T cells as measured by luciferase reporters after stimulating (IL1β, 10 pg mL−1) and culturing them in 330, 370, 410 and 450 mOsm at the indicated time points, n = 3 biological replicates). c) IL1β signaling as measured by luciferase reporters in OA hPCs exposed to OA and healthy‐like osmolarity, with or without IL1β (1 pg mL−1) for 6 h, n = 3 donors, 3 biological replicates. d) IL1β signaling as measured by luciferase reporters in OA and healthy hPCs cultured in iso‐osmotic medium (330 mOsm), with or without IL1β (10 pg mL−1) for 6 h, n = 3 donors, 3 biological replicates. e) Nitric oxide produced by OA hPCs after exposing them to OA and healthy‐like osmolarities for 24 h, n = 4 donors. f) MMP3, g) MMP13 and h) ALPL 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) MMP activity in OA hPCs after treating them with OA and healthy‐like osmolarity for either 24 h or 48 h, n = 3 donors, 3 biological replicates per donor. j) Heat‐map showing MMPs and TIMPs expression as measured by dot blots in OA hPCs treated with OA or healthy‐like osmolarity for 48 h. Ratio of healthy/OA‐like (400/330) osmolarity is plotted in the heat‐maps, blue and red indicate lower and higher expression, respectively. n = 3 donors. Heat‐maps show multiplex analysis of expression of cytokines associated with k) pro‐inflammation and l) pain and growth pathways in OA and healthy hPCs after treating them with OA or healthy‐like osmolarities for 24 h. Ratio of healthy/OA‐like (400/330) osmolarity is plotted in the heat‐maps, n = 4 donors. Statistics: unpaired t‐test, *p < 0.05, **p < 0.01, ***p < 0.001, shades in b, f–h) and error bars in c–e,i) indicate SD. All statistical significance is compared to normalized control, unless otherwise indicated.