Journal: Biomedicines

Article Title: Norisoboldine, a Natural Isoquinoline Alkaloid, Inhibits Diaphyseal Fracture Healing in Mice by Alleviating Cartilage Formation

doi: 10.3390/biomedicines11072031

Figure Lengend Snippet: NOR suppresses cartilage formation and chondrogenic differentiation of BMSCs. ( A ) Safranin O staining of the fracture site at 7 and 14 days post-fracture. (Scale bar, 500 μm). ( B ) Relative cartilage area in the fracture callus at 7, 14, and 21 days after fracture. ( C ) Relative bone mineral area in the fracture callus at 7 and 14 days after fracture. * p < 0.05.

Article Snippet: After the BMSCs reached 80% confluence, they were incubated from basic medium to chondrogenic differentiation medium [high-glucose DMEM (Corning, USA) with the addition of 10% FBS, 1% P/S, 40 mg/mL proline (Solarbio, Beijing, China), 100 mg/mL sodium pyruvate (Solarbio, China), 50 mg/mL vitamin C (Sigma Aldrich, St. Louis, MO, USA), 50 mg/mL insulin–transferrin–selenium (ThermoFisher Scientific, Waltham, MA, USA), and 10 ng/mL transforming growth factor (Abbkine, Wuhan, China)] [ ].

Techniques: Staining

Journal: Biomedicines

Article Title: Norisoboldine, a Natural Isoquinoline Alkaloid, Inhibits Diaphyseal Fracture Healing in Mice by Alleviating Cartilage Formation

doi: 10.3390/biomedicines11072031

Figure Lengend Snippet: NOR suppresses the ability of BMSCs differentiation into chondrocytes. ( A ) Wound healing assay representing cell migration ability in control and NOR group. (Scale bar, 100 μm). ( B ) Alcian blue staining of the BMSCs incubated in chondrogenic medium for 14 days. (Scale bar, 200 μm). ( C – H ) Bmp2 , Aggrecan , Col2a1 , Alk2 , IL-1β and IL-6 mRNA levels in 7 and 14 days differentiated BMSCs. * p < 0.05.

Article Snippet: After the BMSCs reached 80% confluence, they were incubated from basic medium to chondrogenic differentiation medium [high-glucose DMEM (Corning, USA) with the addition of 10% FBS, 1% P/S, 40 mg/mL proline (Solarbio, Beijing, China), 100 mg/mL sodium pyruvate (Solarbio, China), 50 mg/mL vitamin C (Sigma Aldrich, St. Louis, MO, USA), 50 mg/mL insulin–transferrin–selenium (ThermoFisher Scientific, Waltham, MA, USA), and 10 ng/mL transforming growth factor (Abbkine, Wuhan, China)] [ ].

Techniques: Wound Healing Assay, Migration, Control, Staining, Incubation

Journal: bioRxiv

Article Title: BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells (MSCs)

doi: 10.1101/104927

Figure Lengend Snippet: (A-D) LacZ staining (blue) of sagittal sections of mandibular molars from Gli1-LacZ mice at P0.5, P3.5, P7.5, and P21.5. Arrowheads indicate Gli1+ cells in the apical region of the mesenchyme. (E-H) Visualization of sagittal sections of mandibular molars from Gli1-CreER;tdTomato mice at P4.5 and P18.5 after induction at P3.5. The progeny of the Gli1 lineageappear red. Boxes in E and G are shown magnified in F and H, respectively. (I-L) Colony forming assay and osteogenic, chondrogenic, and adipogenic differentiation assays of cells from the Gli1+ region in the apical mesenchyme of molars from P5.5 Gli1-CreER;tdTomato mice induced at P3.5. Toluidine blue staining was used to visualize colony formation after culture for two weeks (I). Insert shows colonies are derived from Gli1+ cells (red). Alizarin Red (J), Alcian Blue (K), and Oil Red O (L) staining to detect osteogenic, chondrogenic, and adipogenic differentiation after three weeks. Scale bars, 100μm.

Article Snippet: For the differentiation assays, cells from the colonies were cultured until confluent and then induced in osteogenic, adipogenic or chondrogenic differentiation medium (05504, 05503, 05455, Stemcell Technologies, Vancouver, Canada) for 2-3 weeks according to the manufacturer’s instructions.

Techniques: Staining, Derivative Assay

Journal: Development (Cambridge, England)

Article Title: BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells in mice

doi: 10.1242/dev.150136

Figure Lengend Snippet: Gli1 is an in vivo marker for MSCs in the developing molar apical mesenchyme. (A-D) X-gal staining (blue) of sagittal sections of mandibular molars from Gli1-LacZ mice at P0.5, P3.5, P7.5 and P21.5. Arrowheads indicate Gli1+ cells in the apical region of the mesenchyme. (E-H) Visualization of sagittal sections of mandibular molars from Gli1-CreER;tdTomato mice at P4.5 and P18.5 after induction at P3.5. The progeny of the Gli1 lineage appear red. Boxes in E and G are shown at higher magnification in F and H, respectively. (I-L) Schematic drawing of Gli1+ cells (dark blue, at the base of the developing root) and their progeny (light blue) contribute to root mesenchyme growth during root initiation (I,J), elongation (K) and eruption (L) stages. (M-P) Colony-forming assay and osteogenic, chondrogenic and adipogenic differentiation assays of cells from the Gli1+ region in the apical mesenchyme of molars from P5.5 Gli1-CreER;tdTomato mice induced at P3.5. Toluidine Blue staining was used to visualize colony formation after culture for 2 weeks (M). Inset shows that colonies are derived from Gli1+ cells (red). Alizarin Red (N), Alcian Blue (O) and Oil Red O (P) staining to detect osteogenic, chondrogenic and adipogenic differentiation after 3 weeks. Scale bars: 100 μm.

Article Snippet: For the differentiation assays, cells from the colonies were cultured until confluent and then induced in osteogenic, adipogenic or chondrogenic differentiation medium (05504, 05503 and 05455, Stemcell Technologies) for 2-3 weeks according to the manufacturer's instructions.

Techniques: In Vivo, Marker, Staining, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: SETD7 regulates chondrocyte differentiation and glycolysis via the Hippo signaling pathway and HIF-1α

doi: 10.3892/ijmm.2021.5043

Figure Lengend Snippet: mRNA expression of SETD7 decreases during chondrogenic differentiation. (A) Alcian blue staining of differentiated ATDC5 cells cultured in micromass at 1 and 20% O 2 . Scale bar, 100 µ m. mRNA expression of (B) aggrecan, (C) SOX9, (D) COL2A1 and (E) SETD7 during chondrogenic differentiation at 1% O 2 . Results are representative of ≥3 independent experiments. * P<0.05, *** P<0.001 vs. 0 days. SETD7, SET domain containing 7; HIF-1α, hypoxia inducible factor-1α; COL2A1, collagen II, α1; SOX9, SRY-related box gene 9.

Article Snippet: At 70-90% confluence, growth medium was replaced with chondrogenic differentiation medium supplemented with 1x insulin-transferrin-selenium (Corning, Inc.).

Techniques: Expressing, Staining, Cell Culture

Journal: International Journal of Molecular Medicine

Article Title: SETD7 regulates chondrocyte differentiation and glycolysis via the Hippo signaling pathway and HIF-1α

doi: 10.3892/ijmm.2021.5043

Figure Lengend Snippet: Knocking down SETD7 activates chondrogenic differentiation. (A) Immunofluorescence staining of ATDC5 cells at 1% O 2 incubated with secondary antibodies and DAPI. (B) Immunofluorescence staining with SETD7 primary antibody, secondary antibodies and DAPI in ATDC5 cells at 1% O 2 . (C) mRNA expression and (D) protein levels of SETD7 after knocking down SETD7 at 1% O 2 . (E) Alcian blue staining after knocking down SETD7 in cells cultured in micromass at 1% O 2 . mRNA expression of (F) aggrecan, (G) SOX9 and (H) COL2A1 after knocking down SETD7 at 1% O 2 . * P<0.05, ** P<0.01, *** P<0.001 vs. con. SETD7, SET domain containing 7; HIF-1α, hypoxia inducible factor-1α; COL2A1, collagen II, α1; SOX9, SRY-related box gene 9; con, control; sh, short hairpin.

Article Snippet: At 70-90% confluence, growth medium was replaced with chondrogenic differentiation medium supplemented with 1x insulin-transferrin-selenium (Corning, Inc.).

Techniques: Immunofluorescence, Staining, Incubation, Expressing, Cell Culture, Control

Journal: International Journal of Molecular Medicine

Article Title: SETD7 regulates chondrocyte differentiation and glycolysis via the Hippo signaling pathway and HIF-1α

doi: 10.3892/ijmm.2021.5043

Figure Lengend Snippet: SETD7 regulates chondrocyte differentiation and glycolysis via the Hippo and HIF-1α. In normal conditions, SETD7 activates the Hippo signaling pathway, which phosphorylates YAP and retains it in the cytoplasm. In hypoxic conditions, expression of SETD7 is inhibited, resulting in increased YAP and HIF-1α in the cytoplasm. The accumulated YAP and HIF-1α translocate into the nucleus and combine to form a complex, which further promotes expression of glycolysis-associated genes and chondrogenic differentiation. SETD7, SET domain containing 7; YAP, Yes-associated protein; HIF-1α, hypoxia inducible factor-1α; GLUT1, glucose transporter 1; LDHA, lactate dehydrogenase A; PGK1, phosphoglycerate kinase 1; PKM2, pyruvate kinase isoenzyme 2; LATS1, large tumor suppressor 1; SOX9, SRY-related box gene 9; Me, methylation; P, phosphorylation.

Article Snippet: At 70-90% confluence, growth medium was replaced with chondrogenic differentiation medium supplemented with 1x insulin-transferrin-selenium (Corning, Inc.).

Techniques: Expressing, Methylation, Phospho-proteomics