Fig 2: M2 macrophage-derived exosomes inhibit HSCs activation(A) LX2 cells were incubated with PKH26-labeled M2 macrophage-derived exosomes, and the exosomes internalization was observed by an inverted fluorescence microscope. The white arrows indicate PKH26-labeled exosomes (scale bar = 50µm).(B) The mRNA levels of aSMA, COL1A1, and COL3A1 were analyzed by RT-qPCR in LX2 cells treated with PBS, WASH, and M2 macrophage-derived exosomes (10 µg/mL or 50 µg/mL) (n = 3).(C) The mRNA levels of aSMA, COL1A1, and COL3A1 were analyzed by RT-qPCR in TGFß1-treated LX2 cells treated with M2 macrophage-derived exosomes or PBS (n = 3).(D) Levels of aSMA, COL1A1, and COL3A1 protein were analyzed by western blotting studies in LX2 cells treated with PBS, WASH, and 50 µg/mL M2 macrophage-derived exosomes (n = 3).(E) Levels of aSMA, COL1A1, and COL3A1 protein were analyzed by western blotting studies in TGFß1-treated LX2 cells treated with M2 macrophage-derived exosomes or PBS (n = 3).(F) Expression of aSMA was analyzed by immunofluorescence staining in LX2 cells treated with PBS, WASH, and 50 µg/mL M2 macrophage-derived exosomes (scale bar = 100µm).(G) Expression of aSMA was analyzed by immunofluorescence staining in TGFß1-treated LX2 cells treated with M2 macrophage-derived exosomes or PBS (scale bar = 100µm). WASH is PBS obtained after washing exosomes twice; M2exo: M2 macrophage-derived exosomes. Data are expressed as mean ± SEM The statistical significance of the difference between the compared groups was analyzed by one-way ANOVA. *, **and ***denotes p values less than 0.05, 0.01, and 0.001, respectively, in comparison to PBS groups; whereas #, ##, and ### represent p values less than 0.05, 0.01, and 0.001, respectively, in comparison to WASH groups or TGFß1+PBS groups.

Fig 3: Knockdown of CAMSAP1 inhibits hepatic stellate cell activation(A) Levels of CAMSAP1 mRNA was analyzed by RT-qPCR in TGFß1-treated LX2 cells subjected to CAMSAP1 knockdown with 100 nM CAMSAP1 siRNA or Control siRNA (n = 4).(B) Levels of aSMA, COL1A1, and COL3A1 mRNA were analyzed by RT-qPCR in TGFß1-treated LX2 cells subjected to CAMSAP1 knockdown with 100nM CAMSAP1 siRNA or Control siRNA (n = 4).(C) Levels of aSMA, COL1A1, and COL3A1 protein were analyzed by western blotting analysis in TGFß1-treated LX2 cells subjected to CAMSAP1 knockdown with 100nM CAMSAP1 siRNA or Control siRNA (n = 3).(D) Expression of aSMA was analyzed by immunofluorescence staining in TGFß1-treated LX2 cells subjected to CAMSAP1 knockdown with 100nM CAMSAP1 siRNA or Control siRNA (scale bar = 100µm).(E) A schematic diagram showing that M2 macrophage-derived exosomes inhibit the activation of HSCs and this inhibition is mediated by miR-411-5p. MiR-411-5p can directly down-regulate the expression of CAMSAP1 to inactivate stellate cells, and knockdown of CAMSAP1 also inhibits the activation of HSCs. Data are expressed as mean ± SEM The statistical significance of the difference between the compared groups was analyzed by one-way ANOVA. *, **, and *** denote p values less than 0.05, 0.01, and 0.001, respectively, in comparison to Control groups; #, ##, and ### denote p values less than 0.05, 0.01, and 0.001, respectively, in comparison to TGFß1+Control siRNA groups. siR-CAMSAP1: CAMSAP1 siRNA.

Fig 4: M2 macrophage-derived exosomes inhibit HSCs activation via miR-411-5p(A) The heatmap shows the miRNA expression profiles in the exosomes isolated from serum samples of NASH patients (n = 3) or healthy controls (n = 3).(B) The overlapping area of the Venn diagram indicating four downregulated miRNAs in the three GEO datasets.(C) The expression of miR-411-5p in rat livers was analyzed by RT-qPCR (n = 5).(D) Expression of miR-411-5p in the exosomes secreted by M0 and M2 macrophages was analyzed by RT-qPCR (n = 3).(E) Expression of miR-411-5p in the exosomes secreted by M1 and M2 macrophages was analyzed by RT-qPCR (n = 3).(F) Levels of aSMA, COL1A1, and COL3A1 mRNA were analyzed by RT-qPCR in LX2 cells treated with 20 nM miR-411-5p mimics or NC mimics (n = 3).(G) Levels of aSMA, COL3A1, and COL1A1 mRNA were analyzed by RT-qPCR in TGFß1-treated LX2 cells with miR-411-5p mimics or NC mimics (n = 3).(H) Levels of aSMA and COL3A1 protein were analyzed by western blotting analysis in LX2 cells treated with 20 nM miR-411-5p mimics or NC mimics (n = 3).(I) Levels of aSMA and COL3A1 protein were analyzed by western blotting analysis in TGFß1-treated LX2 cells with miR-411-5p mimics or NC mimics (n = 3).(J) Expression of aSMA was analyzed by immunofluorescence staining in LX2 cells treated with 20 nM miR-411-5p mimics or NC mimics (scale bar = 100µm).(K) Expression of aSMA was analyzed by immunofluorescence staining in TGFß1-treated LX2 cells with miR-411-5p mimics or NC mimics (scale bar = 100µm). Data are expressed as mean ± SEM The statistical significance of the difference between the compared groups was analyzed by Student’s t-test. *, **, and *** denote p values less than 0.05, 0.01, and 0.001, respectively, in comparison to Control, M0exo, M1exo or NC-mi groups, whereas #, ##, and ### represent p values less than 0.05, 0.01, and 0.001, respectively, in comparison to TGFß1+NC-mi groups. NC-mi: NC mimics; miR-mi: miR-411-5p mimics. See also Figures S1 and S2.

Fig 5: Loss of Adgrg6 in mature osteoblast lineages is dispensable of adolescent idiopathic scoliosis (AIS) development.(A–C) Representative X-ray images of Cre (-) control (A), Cre (+) control (B), and Bglap-Cre; Adgrg6f/f mutant (C) mice at P120. (D, E) Longitudinal analyses of Cobb angle values of Cre (-) control mice (D) and Bglap-Cre; Adgrg6f/f mice (E) at P40 and P120. Cre (-) control mice, n = 8 mice at P40 and P120; Bglap-Cre; Adgrg6f/f mutant mice, n = 8 and 7 at P40 and P120, respectively. Thresholds of scoliosis (Cobb angle >10°) are indicated with two red dot lines. No Bglap-Cre; Adgrg6f/f mice showed scoliosis at P40 (0/8) or P120 (0/7) (E). (F–J) MicroCT scanning of the thoracic region of the spine shows normal morphology of the vertebral bodies in both Cre (-) controls (F) and the Bglap-Cre; Adgrg6f/f mice (G). Transverse sections of the microCT three-dimensional reconstruction of the thoracic vertebral body show a comparable bone mass in the control (H) and mutant mice (I). The bone volume per total volume (BV/TV) of the control and mutant mice is shown in (J). n = 4 mice for each group. Bars are plotted with mean and 95% CI. The statistical difference is evaluated by a two-tailed Student's t-test. The p-value is shown. ns: not significant. (K, L) Real-time RT-PCR analysis of RNA isolated from long bone shows that the expression of Adgrg6 is very low in bony tissues compared with the expression of Col1a1 (K). However, the expression of Adgrg6 was efficiently knockdown in Bglap-Cre; Adgrg6f/f mice (L). RNA was isolated and pooled from three mice of each experimental group. Bars are plotted with mean and SD. The statistical difference is evaluated by a two-tailed Student's t-test. The p-value for each comparison is shown. Scale bars: 10 mm in (A); 1 mm in (G) and (I). Figure 5—source data 1.Characterization of mice with Adgrg6 ablation in mature osteoblast lineages.