Fig 1: Immunofluorescence of type-H vessels (Endomucin+ & CD31+), osteoprogenitor cells (Osterix+) and HIF-1α. A, Representative immunofluorescent images of type-H vessels in the central region of scaffolds. Endomucin+, red; CD31+, green; DAPI, blue. B, Representative immunofluorescent images of Osterix+ osteoprogenitor cells in the central region of scaffolds. Osterix+, red; DAPI, blue. C, Representative immunofluorescent images of HIF-1α+ cells in the central region of scaffolds. HIF-1α+, red; DAPI, blue. Scale bar = 50 μm. D, Quantative analysis of fluorescent intensity of CD31, Endomucin, Osterix and HIF-1A expression, respectively. * Significant difference, P < 0.05; ** Very significant difference, P < 0.01; *** Highly significant difference, P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 2: HOXA13 promotes osteogenic differentiation ability of MC3T3-E1 cells. (A) Transfection efficacy of oe-HOXA13 determined by RT-qPCR and western blot analysis (unpaired t-test, *p < 0.05). (B) mRNA and protein expression of RUNX2, OCN and OSX in cells on day 7 determined by RT-qPCR and western blot analysis, respectively (two-way ANOVA, *p < 0.05). (C) ALP concentration in cells measured by ALP staining (unpaired t-test, *p < 0.05). (D) Calcareous accumulation in cells on day 21 evaluated by ARS staining (unpaired t-test, *p < 0.05). (E,F) Protein level of ß-catenin in cells after HOXA13 (E) or miR-128 (F) overexpression determined by western blot analysis (unpaired t-test, *p < 0.05).
Fig 3: Isolation and osteogenic differentiation of BMSCs (A) Normal and osteoporotic BMSCs were isolated from normal and osteoporotic bone marrow tissues, respectively (the scale bar is 50 µm). (B) Flow cytometry was performed to identify isolated BMSCs detecting CD34, CD45, CD73, CD90, and CD105. (C) Normal and osteoporotic BMSCs were induced towards osteogenic differentiation for 21 days, and the protein levels of ALP, OCN, Runx2, and Osterix were examined using Immunoblotting on days 0, 7, and 14 of osteogenic induction (C); ALP staining was performed on day 0, 7, and 14 of osteogenic induction (D); the formation of mineralized nodules were examined using Alizarin red staining on days 0 and 21 of osteogenic induction (E); the expression of miR-4739 was examined using qRT-PCR on day 0, 3, and 7 of osteogenic induction in normal BMSCs and osteoporotic BMSCs (F). n=3, **p < 0.01 compared to OP-BMSC. aa p<0.01 compared between 0 days OP-BMSC and 3 days OP-BMSC; bb p < 0.01 compared between 0 days normal-BMSC and 3 days normal-BMSC; cc p<0.01 compared between 0 days OP-BMSC and 14 days OP-BMSC; dd p < 0.01 compared between 0 days normal-BMSC and 14 days normal-BMSC.
Fig 4: MALAT1 promoted osteogenic differentiation of human renal interstitial fibroblasts (hRIFs). (A) Agarose gel electrophoresis showed the PCR products from the 5' and 3' RACE of MALAT1 in hRIFs. (B) RNA fluorescence in situ hybridization (FISH) for MALAT1 in isolated hRIFs. (C) Alizarin Red staining in transfected hRIFs 14 days after osteogenic induction (100 ×). (D) The relative MALAT1 expression level was determined by qRT-PCR in transfected hRIFs 7 days after osteogenic induction. (E) Relative alkaline phosphatase (ALP) activity in transfected hRIFs 7 days after osteogenic induction. (F–I) Relative mRNA expression levels of osteogenic markers (Runx2, Osterix, OPN, OCN) were determined by qRT-PCR in transfected hRIFs 7 days after osteogenic induction. (J) Protein expression levels of osteogenic markers (Runx2, Osterix, OPN, OCN) were determined by WB in transfected hRIFs 7 days after osteogenic induction. GAPDH was used as the internal control. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 5: Comparative analysis of pharmacological effects of SR and kaempferol on the differentiation of osteoclasts and osteoblasts. (A) TRAP-positive cells were stained using a TRAP kit. (B) TRAP activity was measured using an ELISA reader. (C) A number of TRAP-positive cells with >3 nuclei were counted using an inverted microscope (magnification, ×100). (D) RAW 264.7 cell viability was measured using a CCK-8 assay kit. (E) Protein expression levels of NFATc1 and c-Fos were measured by western blotting. (F) The bands were normalized to that of ß-actin, which was used as a loading control. (G) MC3T3-E1 cell viability was analyzed by CCK-8 assays at 3 and 7 days. (H) Protein expression levels of BMP-2, p-Smad 1/5, RUNX-2 and Osterix were measured by western blotting. (I) BMP-2, RUNX-2 and Osterix levels were normalized to that of ß-actin. p-Smad 1/5 levels was normalized to that of Smad 1/5/9. Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ## p < 0.01 vs. the normal group (untreated cells, RAW 264.7 cells); * p < 0.05, ** p < 0.01 vs. the RANKL only treatment group. † p < 0.05, †† p < 0.01 vs. the normal group (untreated cells, MC3T3-E1 cells); $ p < 0.05 vs. the osteogenic medium-treated cells.
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