Fig 1: LncRNA-Smad7 KD leads to decreased H3K27me3 modification at the Bmp2 promoter region and lncRNA-Smad7 interacts with hnRNPK. (A) IGV tracks showed H3K27me3 ChIP-seq of WT and lncRNA-Smad7 KIpA progenitor cells on CM D3.3 (mm10). (B) ChIP-qPCR analysis of H3K27me3 signals at the promoter regions of Bmp2 on CM D3.3. The IgG was set for the control group, and –5.8 kb region of Lefty1 was used as a negative control for H3K27me3 enrichment. (C) Combination of mass spectrometry (MS) and ChIRP indicated the interacted proteins of lncRNA-Smad7 in mESCs with two independent experiments (related to Supplementary Table S6). The KO cells are set as the negative control. (D) EMSA showed the association of (UG)25 containing and (CA)12 containing segments of lncRNA-Smad7 with hnRNPK in vitro. 1.6 pmol RNA were used as a fixed amount in this experiment. (E) IGV tracks showed ChIRP-DNA seq of lncRNA-Smad7, CUT & Tag of hnRNPK, and ChIP-seq of H3K27me3 and PRC2 complex in mESCs at Bmp2 loci (GSE183465, GSE103258, GSE127117) (mm10). (B) All data are representative of three independent experiments. Data are presented as mean ± S.D., n = 3, *P< 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t test). See also Supplementary Figure S6.
Fig 2: Saporin-CD11b reduces macrophages increase during DO. (A, B) RT-qPCR for CD68 mRNA (A) and ELISA for CD68 protein (B) in regenerating bone tissue from saporin-CD11b-treated mice or control IgG-treated mice. (C, D) FACS for CD68+ cells in regenerating bone tissue saporin-CD11b-treated mice or control IgG-treated mice by representative flow charts (C), and by quantification (D). (E–I) ELISA for IL-1ß (E), IL-6 (F), TNFa (G), BMP2 (H) and TGFß (I) from FAC-sorted CD68+ macrophages. DP-day 0: day 0 of distraction phase, CP-day 0: day 0 of consolidation phase, CP-day 28: day 28 or consolidation phase. *p<0.05. NS: non-significant. N=8.
Fig 3: Effect of the bioprinted scaffolds on osteogenic differentiation of BMSCs. a) ALP staining after 7 and 14 days of culture. The OM group contained only osteogenic supplements. b) Alizarin Red staining after 21 days of co-culture. c) Quantitative analysis of Alizarin Red staining. d) BMP2 secretion from scaffolds according to ELISA. **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 4: Cell proliferation of SAMR1 and SAMP6 BMSCs (A, B), bmp2 mRNA expression in SAMR1 and SAMP6 BMSCs (C, D) and the concentrations of BMP2 examined using ELISA (E, F).(A, B) The cells were treated with 0 µM, 0.1 µM, 0.5 µM and 1.0 µM of fluvastatin for 1, 3, 7 and 14 days, and cell proliferation was assessed using WST-1-based colorimetory. No significant differences in cell proliferation were associated with differences in concentrations of fluvastatin in SAMR1 and SAMP6 on any day. (C, D) The expression level of bmp2 mRNA was shown as FC (fold chnage). (??Ct method, baseline = 1day on control in SAMR1) *P < 0.05 bmp2 mRNA expression was significantly increased at fluvastatin concentrations more than 0.1 µM at 3 and 7 days of culture in SAMR1, and at concentrations more than 0.5 µM at 3and 7 days in SAMP6. (E, F) In ELISA assay, BMP2 concentrations were significantly increased at concentrations more than 0.1 µM at 3 days of culture in SAMR1, and at concentrations more than 0.5 µM at 3 days of culture in SAMP6.
Fig 5: The 3D-bioprinted scaffold with cells exhibiting DOX-controlled BMP2 expression. a) Construction of the transfected cells that can express BMP2 based on the Tet-on expression control system, and fabrication of 3D bioprinting scaffold that contains composite of PCL/MBG/DOX and engineering cells within the bioink. b) Mechanisms of antibacterial properties and BMP2 controlled release ability of the bioprinting dual-functional scaffold.
Supplier Page from Abcam for Mouse BMP2 ELISA Kit