Journal: International Journal of Stem Cells

Article Title: PML Regulated HIF1AN Ubiquitination and Activated PI3K/AKT Pathway to Promote Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation

doi: 10.15283/ijsc24110

Figure Lengend Snippet: Promyelocytic leukemia protein (PML) was up-regulated in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). (A) Flow cytometry was used to determine BMSCs surface markers (CD44 and CD90) and hematopoietic cells surface markers (CD34 and CD45). BMSCs were cultivated in an osteogenic medium (OM) for 7 days or 14 days, and cells were divided into Control and OM groups. (B, C) The osteogenic differentiation potential of BMSCs was measured by Alizarin red S (ARS) and alkaline phosphatase (ALP) stainings (scale bar=50 μm), ARS staining was used to check the calcium deposition in BMSCs, and the ALP staining was employed to detect the number of ALP-positive BMSCs. (D) The protein expressions of PML, HIF1α, HIF1AN, SOD3, RUNX2, OCN, and ALP were determined by using Western blot. Data were exhibited as mean±SD from three independent experiments. *p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: When cells grow to 80% confluence, they were cultivated in the osteogenic medium (OM) (MDADGM; iXCells) which encompassed 1% FBS, 50 mg/mL ascorbic acid, 10 mM b-glycerophosphorus sodium, and 0.1 mg/mL dexamethasone for further incubation for 2 weeks.

Techniques: Flow Cytometry, Control, Staining, Western Blot

Journal: International Journal of Stem Cells

Article Title: PML Regulated HIF1AN Ubiquitination and Activated PI3K/AKT Pathway to Promote Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation

doi: 10.15283/ijsc24110

Figure Lengend Snippet: Promyelocytic leukemia protein (PML) knockdown inhibited the osteoblast differentiation of bone marrow mesenchymal stem cells (BMSCs). Short hairpin RNA (sh)-PML was transfected into BMSCs for PML knockdown, and sh-NC served as the negative control. (A) The transfection efficiency was assessed by Western blot. Cells incubated with osteogenic medium (OM) and divided into four groups: Control, OM, OM+sh-NC, and OM+sh-PML. (B) The PML expression was determined using Western blot. (C, D) The osteogenic differentiation potential of BMSCs was measured by Alizarin red S (ARS) and alkaline phosphatase (ALP) stainings (scale bar=50 μm), ARS staining was used to check the calcium deposition in BMSCs, and the ALP staining was employed to detect the number of ALP-positive BMSCs. (E) Western blot was used to detect the HIF1AN, HIF1α, SOD3, RUNX2, OCN, and ALP expressions. Data were exhibited as mean±SD from three independent experiments. **p<0.01 and ***p<0.001.

Article Snippet: When cells grow to 80% confluence, they were cultivated in the osteogenic medium (OM) (MDADGM; iXCells) which encompassed 1% FBS, 50 mg/mL ascorbic acid, 10 mM b-glycerophosphorus sodium, and 0.1 mg/mL dexamethasone for further incubation for 2 weeks.

Techniques: Knockdown, shRNA, Transfection, Negative Control, Western Blot, Incubation, Control, Expressing, Staining

Journal: International Journal of Stem Cells

Article Title: PML Regulated HIF1AN Ubiquitination and Activated PI3K/AKT Pathway to Promote Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation

doi: 10.15283/ijsc24110

Figure Lengend Snippet: HIF1AN overexpression suppressed the osteoblast differentiation of bone marrow mesenchymal stem cells (BMSCs). pcDNA (oe)-HIF1AN was transfected into BMSCs for HIF1AN overexpression, and oe-NC served as the negative control. Cells were divided into the Control, osteogenic medium (OM), OM+oe-NC, and OM-oe-HIF1AN groups. (A) Western blot was conducted to analyze the expression of HIF1AN. The overexpressed BMSCs were incubated with OM, and the sh-NC as the negative control. (B) HIF1AN expression was evaluated by using western blot. (C, D) The osteogenic differentiation potential of BMSCs was measured by Alizarin red S (ARS) and alkaline phosphatase (ALP) stainings (scale bar=50 μm), ARS staining was used to check the calcium deposition in BMSCs, and the ALP staining was employed to detect the number of ALP-positive BMSCs. (E) Protein expressions of HIF1α, SOD3, and osteogenesis-related markers (RUNX2, OCN, and ALP) were assessed by western blot. Data were exhibited as mean±SD from three independent experiments. *p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: When cells grow to 80% confluence, they were cultivated in the osteogenic medium (OM) (MDADGM; iXCells) which encompassed 1% FBS, 50 mg/mL ascorbic acid, 10 mM b-glycerophosphorus sodium, and 0.1 mg/mL dexamethasone for further incubation for 2 weeks.

Techniques: Over Expression, Transfection, Negative Control, Control, Western Blot, Expressing, Incubation, Staining

Journal: International Journal of Stem Cells

Article Title: PML Regulated HIF1AN Ubiquitination and Activated PI3K/AKT Pathway to Promote Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation

doi: 10.15283/ijsc24110

Figure Lengend Snippet: Promyelocytic leukemia protein (PML) promoted HIF1AN ubiquitination degradation and facilitated bone marrow mesenchymal stem cells (BMSCs) osteoblast differentiation. (A) The interaction between PML and HIF1AN was verified by co-immunoprecipitation (Co-IP) assay. (B) PML overexpression BMSCs were treated with a proteasome inhibitor (MG-132), and Co-IP was employed to detect HIF1AN ubiquitination. PML overexpression BMSCs treated with cycloheximide (CHX) or MG-132. (C) Analysis of HIF1AN the half-life and degradation rate on PML up-regulation in CHX treated BMSCs. (D) Western blot is used to assess the level of HIF1AN protein. (E) The location of PML and HIF1AN was evaluated by immunofluorescence staining (scale bar=25 μm). (F) BMSCs were transfected with pcDNA (oe)-PML and oe-HIF1AN, and PML and HIF1AN expressions were measured by Western blot. (G, H) Osteogenic differentiation of BMSCs was detected by Alizarin red S (ARS) and alkaline phosphatase (ALP) staining (scale bar=50 μm), ARS staining was used to check the calcium deposition in BMSCs, and the ALP staining was employed to detect the number of ALP-positive BMSCs. (I) The expressions of HIF1α, SOD3, RUNX2, OCN, and ALP were analyzed by using Western blot. Data were exhibited as mean±SD from three independent experiments. OM: osteogenic medium, NC: negative control. *p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: When cells grow to 80% confluence, they were cultivated in the osteogenic medium (OM) (MDADGM; iXCells) which encompassed 1% FBS, 50 mg/mL ascorbic acid, 10 mM b-glycerophosphorus sodium, and 0.1 mg/mL dexamethasone for further incubation for 2 weeks.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Over Expression, Western Blot, Immunofluorescence, Staining, Transfection, Negative Control

Journal: International Journal of Stem Cells

Article Title: PML Regulated HIF1AN Ubiquitination and Activated PI3K/AKT Pathway to Promote Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation

doi: 10.15283/ijsc24110

Figure Lengend Snippet: Promyelocytic leukemia protein (PML) promoted HIF1α to activate SOD3 and PI3K/AKT pathway to enhance bone marrow mesenchymal stem cells (BMSCs) osteoblast differentiation. BMSCs were transfected with pcDNA (oe)-SOD3 and/or short hairpin RNA (sh)-PML, and then induced with osteogenic medium (OM). (A) The potential HIF1α binding site on the SOD3 promoter region was predicted JASPAR database. (B, C) The relationship between HIF1α and SOD3 was confirmed by dual luciferase reporter and chromatin immunoprecipitation assays. BMSCs were transfected with oe-SOD3 and/or sh-PML, and then induced with LY294002 under OM. (D) The expressions of SOD3 and PML were measured by Western blot. (E, F) The osteogenic differentiation potential of BMSCs was measured by Alizarin red S (ARS) and alkaline phosphatase (ALP) stainings (scale bar=50 μm), ARS staining was used to check the calcium deposition in BMSCs, and the ALP staining was employed to detect the number of ALP-positive BMSCs. (G) HIF1AN, HIF1α, RUNX2, OCN, ALP, p-PI3K/PI3K, and p-AKT/AKT expressions were determined by Western blot. Data were exhibited as mean±SD from three independent experiments. WT: wild-type, MUT: mutant, NC: negative control. *p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: When cells grow to 80% confluence, they were cultivated in the osteogenic medium (OM) (MDADGM; iXCells) which encompassed 1% FBS, 50 mg/mL ascorbic acid, 10 mM b-glycerophosphorus sodium, and 0.1 mg/mL dexamethasone for further incubation for 2 weeks.

Techniques: Transfection, shRNA, Binding Assay, Luciferase, Chromatin Immunoprecipitation, Western Blot, Staining, Mutagenesis, Negative Control