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. 2021 Jan 7;6(7):2039–2057. doi: 10.1016/j.bioactmat.2020.12.025

Fig. 4.

Fig. 4

152RM induces osteogenic differentiation of MSCs by inhibiting endoplasmic reticulum stress, with downregulation of ATF3 expression. (A) MSCs were induced to differentiate into mature OBs in osteogenic differentiation medium without or with 152RM after 14 days. Representative alkaline phosphatase staining images (left) and quantification of the activity of alkaline phosphatase were performed (right) (n = 5 per group). (B) MSCs were induced to differentiate into mature OBs in osteogenic differentiation medium without or with 152RM after 21 days. Representative Alizarin red staining images (left) and quantification of matrix mineralization (right) (n = 5 per group). (C) RNA-seq analysis showed the alteration of endoplasmic reticulum stress-related gene expression in MSCs cultured with 152RM (n = 3 each). (D) Relative mRNA expression levels of ATF3 in MSCs cultured with 152RM (n = 5 each). (E) Western blot analysis of the expression of ATF3 and CHOP in MSCs (pretreated with 5 ng/mL tunicamycin) after the addition of 152RM (n = 5 per group). (F) Representative immunostaining images of β-catenin (red) MSCs or MSCs pretreated with 5 ng/mL tunicamycin in osteogenic differentiation medium with 152RM after 14 days (n = 5 per group). Scale bar, 100 μm. (G) Representative immunostaining images of osterix (red) MSCs or MSCs pretreated with 5 ng/mL tunicamycin in osteogenic differentiation medium with 152RM after 14 days (n = 5 per group). Scale bar, 100 μm. (H) Representative immunostaining images of osteocalcin (red) MSCs or MSCs pretreated with 5 ng/mL tunicamycin in osteogenic differentiation medium with 152RM after 14 days (n = 5 per group). Scale bar, 100 μm. (I) Representative immunostaining images of Runx2 (red) MSCs or MSCs pretreated with 5 ng/mL tunicamycin in osteogenic differentiation medium with 152RM after 14 days (n = 5 per group). Scale bar, 100 μm. (J) Schematic illustration of the role of 152RM in promoting MSC migration and osteogenic differentiation. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA and Student's t-test were employed. For all panels in this figure, data are representative of three independent experiments.