Fig 2: RUNX3 promoted osteogenic differentiation of ADSCs via upregulating hsa_circ_0005752. A. The mRNA levels of RUNX3 and hsa_circ_0005752 were determined by qRT-PCR in OM-induced ADSCs after transfection with pcDNA3.1 empty vector, pcDNA3.1-RUNX3 single or together with sh-NC or sh- hsa_circ_0005752. B-C. The osteogenic differentiation was assessed by the ALP staining (B), and ARS staining (C) in OM-induced ADSCs after transfection with pcDNA3.1 empty vector or pcDNA3.1-RUNX3 single or together with sh-NC or sh- hsa_circ_0005752. D. The protein levels (MDM2, p53, Runx2, ALP, Osx, and OCN) were measured by western blot in OM-induced ADSCs after transfection with pcDNA3.1 empty vector, pcDNA3.1-RUNX3 single or together with sh-NC or sh- hsa_circ_0005752. Data indicated the mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.01.

Fig 3: miR-496 inhibited ADSCs osteogenic differentiation. A: The miR-496 expressions were detected by qRT-PCR after transfection of miR-496 mimics/inhibitors or individual control.B-C: Alkaline phosphatase (ALP) and the mineralization were determined by the ALP staining (B) or ARS staining (C). Scale bar = 50 µm. D–E: Runx2, ALP, Osx, OCN, and p53 mRNA levels were determined by qRT-PCR (D) or Western blot (E) in OM-treated ADSCs after miR-496 overexpression or knockdown. Data indicated the mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.01.

Fig 4: Hsa_circ_0005752 promoted osteogenic differentiation of ADSCs. A: Hsa_circ_0005752 levels were determined by qRT-PCR in ADSCs during osteogenic differentiation at 0, 3, 7, and 14 days. B: Hsa_circ_0005752 was measured by qRT-PCR after transfections of hsa_circ_0005752, sh-hsa_circ_0005752, or their corresponding control. C–D: Alkaline phosphatase (ALP) or the mineralization was determined by the ALP staining (C) or Alizarin Red S (ARS) staining (D). Scale bar = 50 µm. E–F: Runx2, ALP, Osx, OCN, and p53 mRNA levels were determined by qRT-PCR (E) or Western blot (F) in OM-treated ADSCs after hsa_circ_0005752 overexpression or knockdown. Data indicated the mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.01.

Fig 5: Tumor resident but not bone marrow resident TdTOSX+ cells increase tumor growth.(A) Model showing isolation of TdTOSX+ cells from B16-F10 primary tumors injected into doxy-fed Osx-cre;TdT mice and re-inoculation of TdTOSX+ cells together with B16-F10 tumor cells into WT recipient mice at the ratio 5:1. Mice injected with B16-F10 alone were used as controls. (B) Tumor growth of experimental model described in (A) determined by caliper measurement. n = 3/group, experiment repeated twice. Significance was determined by one-way ANOVA with Tukey post-hoc test. (C) Fluorescence images of bone sections showing presence of TdTOSX+ within the bone and bone marrow of naïve Osx-cre;TdT, Osx-cre;TdT mice inoculated subcutaneously with B16-F10 tumors or WT;TdT mice used as negative control. Sections were counterstained with DAPI (blue), magnification 200X. (D–E) Quantification of TdTOSX+ cells in the bone marrow of tumor-free and B16-F10 tumor bearing (D) Osx-cre;TdT mice (doxy-fed) and (E) Osx-creERT2;TdT (TAM-treated) determined by FACS. Experiment repeated twice. Significance was determined by student t-test statistical analysis. (F) Tumor growth of WT mice inoculated with bone marrow-derived TdTOSX+ cells from B16-F10 bearing doxy-fed Osx-cre;TdT mice together with B16-F10 tumor cells (ratio 5:1). Mice injected with B16-F10 alone were used as controls. n = 3/group. Significance was determined by two-way ANOVA followed by Tukey post-hoc test. ***p<0.001, ****p<0.0001.