Fig 2: The Wnt signaling inhibitor XAV939 reversed the effects of ED-71. (A). ALP staining of MC3T3-E1 cells in OM, DEX, DEX+XAV939, DEX+ ED-71, DEX+ED-71+XAV939 groups at 7 days. Bar, 500µm. (B). AR staining images of these groups after 21 days of culture. Bar, 500µm. (C). The mRNA levels of COL1, ALP, RUNX2, OCN and ß-catenin detected by RT-qPCR after 7 days of culture. GAPDH was used as an internal control. (D). Western blotting detection of ß-catenin, RUNX2, OCN, Wnt5a, Active ß-catenin, FZD4 and GAPDH after 14 days of culture. (E). The statistical analysis of Western blotting. Data were given as the mean ± S.D. from three independent experiments. *P < 0.05, **P < 0.01. ***P < 0.001.

Fig 3: Treatment with Zeb1-packaged cationic liposomes has therapeutic effects on OVX-induced bone loss.a Representative micro-CT images of trabecular bone (top panels) and cortical bone (bottom panels) of the tibia in Sham and OVX mice that were i.v. injected with 4 µg Lipo.-Vector-GFP or Lipo.-ZEB1-GFP for 6 consecutive weeks (designed Sham + Lipo.-Vector-GFP, OVX + Lipo.-Vector-GFP, and OVX + ZEB1-GFP mice, respectively; n = 5, each). Scale bar, 0.2 mm. b Quantification of bone architectures in the tibia as shown in a (n = 5 independent experiments). c Runx2 and Osterix immunostaining of the tibia as described in a (n = 5, each). Scale bar, 100 µm. d Quantification of Runx2+ and Osterix+ cells in the tibia as shown in c (n = 5 independent experiments). All data are represented as mean ± SD. **P < 0.01, *P < 0.05. Differences are tested using one-way ANOVA with Tukey’s post hoc test (b, d). The source data are provided as a Source Data file.

Fig 4: (A) Comparative cell proliferation assay of BMSCs seeded on the TCPs and DBMPs. (B) Live/dead cell analysis for the TCPs and DBMPs on which BMSCs had been seeded for 3 days. Representative images show the live (green) and dead (red) BMSCs in the TCPs and DBMPs and the viability analysis for the cells on the TCPs and DBMPs. Bar = 100 µm. (C) Pro-inflammatory cytokine (TNF-a, IL-6, and IL-1ß) release by stimulation with TCPs, DBMPs, or LPS detected by ELISA assay. (D) Immunofluorescence staining of the Runx2 expression in BMSCs after culturing with TCPs or DBMPs. Comparative analysis of Runx2-positive cells after BMSCs were cultured on TCPs or DBMPs. Bar = 15 µm. (E) Alizarin red staining of BMSCs after culturing with TCPs or DBMPs. Comparative analysis of the alizarin red staining area after BMSCs cultured on TCPs or DBMPs. Bar = 20 µm. (F) qRT-PCR analysis shows that the expression of osteogenic (Runx2 and Alp) genes after BMSCs cocultured on TCPs or DBMPs. n = 4 for each group. Data are showed as means ± standard deviation (*p < 0.05, **p < 0.01, and ***p < 0.001).

Fig 5: miR-23a and miR-30a mediate inhibition of osteoblast differentiation induced by SM-EVs.a, b Effect of miRNA mimics on osteoblast differentiation. hFOB1.19 cells were plated and next day transfected with 100 nM of control (Con miR), miR-21, mR-23a, miR-25, miR-30a, or miR-93 mimics. After 2 days, the transfection media were replaced. Five days later, alkaline phosphatase (ALP) activity was determined in the culture media (a) and RUNX2 protein expression in cell lysates determined by Western blot (b). c, d Functional knockdown of miR-23a and miR-30a reverse the attenuating effects of extracellular vesicles isolated from the serum of patients with systemic mastocytois (SM-EVs) on osteoblast differentiation. hFOB1.19 cells were seeded and then treated or not (white bars are untreated cells) with pooled extracellular vesicles isolated from the serum of healthy volunteers (HV-EVs) (black bars), pooled SM-EVs from patients with serum tryptase values lower than 110 ng/mL (blue bars; SM-EV: Tryp < 110 ng/mL), or SM-EVs from patients with serum tryptase values >110 ng/mL (red bars; SM-EV: Tryp > 110 ng/mL), as described in “Methods.” Cells were then transfected with the miRNA inhibitors (inhib.) for 2 days. Transfection media were replaced and 5 days later, ALP activity was measured in the media (c) and RUNX2 expression in cells detected by Western blotting (d). In b and d, the bar graphs represent the relative fold changes in band intensity normalized to ß-actin and compared to the respective controls (first bar). All data (a–d) represent the mean ± SEM (n = 5 biological replicates) of representative experiments and repeated at least twice with similar results. In a–d, **p < 0.01; ***p < 0.001; ****p < 0.0001; and n.s., not significant using an unpaired Student t-test (two-tailed) for the indicated comparisons or compared to the first bar (b, d). The blots are representative images from one experiment.