Fig 2: Subcutaneous ASCs dynamically disassemble and reassemble their FAs. (A) Schedule of the nocodazole washout assay. (B) ASCs were incubated for 5 h with 10 µM nocodazole followed by washout, where the microtubules (MTs) were allowed to regrow for 0, 30 and 75 min. Cells were stained for paxillin (green), p-FAK (red), and DNA (DAPI, blue). Representatives of FA disassembly/reassembly are shown. Scale: 15 µm. (C–E) Kinetics of FA disassembly during MT regrowth and FA reassembly after MT regrowth. Quantification of the mean fluorescence intensity of paxillin (C), p-FAK (D), and FA size (E) (270 FA per condition) is depicted. The results are based on three independent experiments and presented as scatter plots showing mean ± SEM. Unpaired Mann–Whitney U-test, ** p < 0.01, *** p < 0.001. a.u., arbitrary units.

Fig 3: The activation of FAK/AKT signaling may be triggered by facilitating the interaction of SMOC2 and integrin ß1 in MEFs. (a) Western blot assay results showed the effect of SMOC2 and/or siFAK on the phosphorylation levels of FAK and AKT promoted by BMP9 in MEFs at 30 h. (b–c) Quantification of the western blot results showed the effect of SMOC2 and/or siFAK on the phosphorylation levels of FAK and AKT promoted by BMP9 in MEFs. (d) ALP assay results showed the effect of LY290042 on the activity of ALP induced by BMP9 in MEFs. (e) ALP assay results showed the effect of SMOC2 and/or LY290042 on BMP9-induced ALP activity in MEFs. (f) Quantification results of the ALP assay showed the effect of LY290042 on the activity of ALP induced by BMP9 in MEFs. (g) Quantification results of the ALP assay showed the effect of SMOC2 and/or LY290042 on BMP9-induced ALP activity in MEFs. (h) Alizarin Red S staining showed the effect of SMOC2 and/or LY290042 on BMP9-induced mineralization in MEFs. (i) Quantification results of Alizarin Red S staining showed the effect of SMOC2 and/or Ly294002 on the effect of BMP9-induced mineralization in MEFs. (j) Confocal laser scanning immunofluorescence staining results showed the effect of SMOC2 on the phosphorylation of AKT induced by BMP9 in MEFs at 30 h. (k) ChIP assay shows the enrichment of GTF2I at the promoter region of smoc2 in MEFs. (l) Immunoprecipitation assay (IP) results showed that SMOC2 may interact with integrin ß1 in MEFs. Compared with the control group, *p < 0.05; **p < 0.01; compared with the AdBMP9 group, #p < 0.05, ##p < 0.01; compared with the AdBMP9 and LY290042 group, ^p < 0.05, ^^p < 0.01.

Fig 4: Mechanisms of action of SMOC2 in BMP-induced osteogenic differentiation. The activation of FAK/AKT signaling may be triggered by facilitating the interaction of SMOC2 and integrin ß1.

Fig 5: Determination of the signal pathways in brain tissues.(A) Primary neurons from embryonic day 17. WT rats and Tspan7-/- rats were cultured for 6 d and then were stained by double immunofluorescence with antibodies of anti-MAP2 (green) and anti-integrin ß1 (red). Scale bar = 10 µm. (B) Plasma membrane fractions, cytosol fractions, and total lysates of brain tissues from female WT and Tspan7-/- rats at 6 mo old were prepared and separated on SDS–PAGE, respectively. Integrin ß1 in the membrane, cytosol fractions, and total lysates and phosphorylated and total FAK, SRC, PSD95, SYN, GluR1, and GluR2 in total lysates were detected with Western blot. (C) Integrin ß1–immunolabelled area in primary neurons was quantified. (D, E, F, G, H, I, J, K, L) Expression levels of integrin ß1 in the membrane, cytosol fractions, and total lysates and phosphorylated FAK, SRC, PSD95, SYN, GluR1, and GluR2 in total lysates were quantified with a software of ImageJ. n = 6. Data information: In (C, D, E, F, G, H, I, J, K), data were analyzed by two-tailed unpaired t tests. Error bars represent SEM, *P < 0.05, **P < 0.01, ***P < 0.001, n.s, not significant. (C) Each point represents data from one cell in (C), and at least nine cells were analyzed.Source data are available for this figure.