Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of AS-IV on proliferation and osteogenic differentiation of PDLSCs. ( A ) CCK-8 analysis of AS-IV effects on PDLSCs on day 1, 3, and 5. ( B and C ) ALP activity ( B ) and extracellular matrix mineralization ( C ) quantification of PDLSCs affected by AS-IV. ( D ) ALP staining. Scale bar: 500 μm. ( E ) Alizarin Red staining. Scale bar: 500 μm. ( F – H ) qRT-PCR analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV. ( I – L ) Western blot and semi-quantitative analysis of COL-1 (J), ALP(K), and RUNX-2 ( L ) in PDLSCs cultured with AS-IV. 20 μM AS-IV group had a significant promoting effect. Data are presented as mean ± SD. ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: CCK-8 Assay, Activity Assay, Staining, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of LY294002 on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and D ) ALP staining( A ) and ALP activity quantification( D ) of PDLSCs affected by LY294002. Scale bar: 500 μm. ( B and E ) Immunofluorescence staining( B ) of COL-1 (green) and DAPI staining (blue) and quantification(E). Scale bar: 50 μm. ( C and F ) Immunofluorescence staining( C ) of eNOS (green) and DAPI staining (blue) and quantification(F). Scale bar: 50 μm. ( G ) NO2- concentration represented the level of NO in cell supernatants. ( H–K ) Western blot and semi-quantitative analysis of COL-1(I), ALP(J), and RUNX-2 ( K ) in PDLSCs cultured with AS-IV and LY294002. ( L–O ) Western blot and semi-quantitative analysis of eNOS(M), iNOS( N ) and p-AKT/AKT ( O ) after addition of AS-IV at different time points. ( P–S ) Western blot and semi-quantitative analysis of eNOS(Q), iNOS( R ) and p-AKT/AKT ( S ) in PDLSCs cultured with AS-IV and LY294002. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Concentration Assay, Western Blot, Cell Culture

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of L-NAME on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and B ) ALP staining( A ) and ALP activity quantification( B ) of PDLSCs affected by L-NAME. Scale bar: 500 μm. ( C and D ) Immunofluorescence staining( C ) of COL-1 (green) and DAPI staining (blue) and quantification(D). Scale bar: 50 μm. ( E–H ) Western blot and semi-quantitative analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV and L-NAME. ( I–K ) Western blot and semi-quantitative analysis of eNOS( J ) and p-AKT/AKT ( K ) in PDLSCs cultured with AS-IV and L-NAME. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ns: P> 0.05. Each experiment was repeated five times.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Western Blot, Cell Culture

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Hematoxylin and eosin staining and immunohistochemical staining. ( A ) HE staining of the tension-side periodontal tissues of the control group (A-a) and AS-IV group (A-b) after OTM for 14 days. Scale bar: 50 μm. ( B ) Expression of ALP of the control group (B-a) and AS-IV group (B-b) after OTM for 14 days and quantitative analysis (B-c). Scale bar: 20 μm. ( C ) Expression of COL-1 of the control group (C-a) and AS-IV group (C-b) after OTM for 14 days and quantitative analysis (C-c). Scale bar: 20 μm. ( D ) Expression of eNOS of the control group (D-a) and AS-IV group (D-b) after OTM for 14 days and quantitative analysis (D-c). Scale bar: 20 μm. Data are presented as mean ± SD (n=8 rat/group). * P <0.05, ** P <0.01.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: Staining, Immunohistochemical staining, Control, Expressing

Journal: Cancer Research

Article Title: ROCK Inhibition Induces Terminal Adipocyte Differentiation and Suppresses Tumorigenesis in Chemoresistant Osteosarcoma Cells

doi: 10.1158/0008-5472.can-18-2693

Figure Lengend Snippet: Figure 2. The ROCK inhibitor fasudil induces terminal adipocyte differentiation and growth arrest in chemoresistant AO cells in vitro. A, Activation of RhoA–ROCK signaling induces actin polymerization and inhibits adipocyte differentiation, whereas inactivation of such signaling elicits actin depolymerization and induces adipogenesis. B, RT and real-time PCR analysis of Pparg, Slc2a4, and Plin1 mRNAs in AO cells incubated in the absence (Cont) or presence of various concentrations of fasudil in growth medium for 72 hours. Data are means þ SD for triplicate experiments. C, Immunoblot analysis of PPARg, lamin C (loading control for nuclear proteins), FABP4, PLIN1, and GAPDH (loading control for cytoplasmic proteins) in nuclear (Nuc) or cytoplasmic (Cyto) fractions prepared from cells treated as in B. D, Fluorescence microscopy of the actin cytoskeleton (stained with phalloidin) and of PLIN1 expression in AO cells incubated in the absence (control) or presence of 50 mmol/L fasudil for 72 hours. Nuclei were stained with Hoechst 33342. Scale bars, 50 mm. E, Assay of cell growth and viability for cells treated as in B. Data are means þ SD for triplicate experiments. F, Cell-cycle analysis by flow cytometry for AO cells incubated in the absence (control) or presence of 50 mmol/L fasudil for 72 hours and then stained with PI. Representative results as well as quantitative data (means SD from triplicate experiments) for the proportions of cells in each phase (G0–G1, S, or G2–M) of the cell cycle are shown. , P < 0.01 for G0-G1 (Student t test). G, RT and real-time PCR analysis of PPARG, SLC2A4, and PLIN1 mRNAs in human osteosarcoma cell lines (HOS, 143B, SJSA1) incubated in the absence (control) or presence of 50 mmol/L fasudil in growth medium for 72 hours. Data are means þ SD from triplicate experiments. , P < 0.05; , P < 0.01; NS, nonsignificant (Student t test).

Article Snippet: Frozen human osteosarcoma sections were obtained from OriGene.

Techniques: In Vitro, Activation Assay, Real-time Polymerase Chain Reaction, Incubation, Western Blot, Control, Fluorescence, Microscopy, Staining, Expressing, Cell Cycle Assay, Cytometry

Journal: Cancer Research

Article Title: ROCK Inhibition Induces Terminal Adipocyte Differentiation and Suppresses Tumorigenesis in Chemoresistant Osteosarcoma Cells

doi: 10.1158/0008-5472.can-18-2693

Figure Lengend Snippet: Figure 5. Effects of sequential treatment with doxorubicin and fasudil in heterogeneous osteosarcoma cells. A, Experimental protocol for sequential treatment of OSi cells with doxorubicin (Dox) and fasudil (Fas). The cells were thus incubated in the absence or presence of doxorubicin (50 ng/mL) for 48 hours and then in growth medium (GM) with or without fasudil (50 mmol/L) for an additional 48 hours. B, Immunoblot analysis of PPARg, lamin C (loading control for nuclear proteins), FABP4, PLIN1, and GAPDH (loading control for cytoplasmic proteins) in nuclear (Nuc) or cytoplasmic (Cyto) fractions prepared from cells treated as in A. C, Fluorescence microscopy of the actin cytoskeleton (stained with phalloidin) and PLIN1 expression in cells treated as in A. Nuclei were stained with Hoechst 33342. Scale bars, 50 mm. D, Experimental protocol for subcutaneous injection of OSi cells, intravenous treatment (or not) with doxorubicin (2 mg/kg), and intraperitoneal treatment with fasudil (50 mg/kg) or saline in C57BL/6 mice. E, Macroscopic images of all tumors removed at day 25 from mice treated as in D (scale bar, 1 cm) and tumor volume at day 25 presented as box-and-whisker plots for the 10 tumors in five mice per group. , P < 0.01 (Mann–Whitney U test). F, Serial sections of tumors at day 25 for mice treated as in D were subjected to hematoxylin and eosin (H&E) staining and to IHC staining of GFP (to identify injected cells) and PLIN1. The boxed regions in the left and middle images are shown at higher magnification in the images on the right. Scale bars, 100 mm (right) or 200 mm (left and middle).

Article Snippet: Frozen human osteosarcoma sections were obtained from OriGene.

Techniques: Incubation, Western Blot, Control, Fluorescence, Microscopy, Staining, Expressing, Injection, Saline, Whisker Assay, MANN-WHITNEY, Immunohistochemistry

Journal: Cancer Research

Article Title: ROCK Inhibition Induces Terminal Adipocyte Differentiation and Suppresses Tumorigenesis in Chemoresistant Osteosarcoma Cells

doi: 10.1158/0008-5472.can-18-2693

Figure Lengend Snippet: Figure 6. Therapeutic strategies for heterogeneous osteosarcoma cells. Treatment with the ROCK inhibitor fasudil induces terminal adipocyte differentiation as well as suppresses tumorigenesis in chemoresistant AO cells that maintain an adipogenic (Adipo) potential, whereas that with the chemotherapeutic agent doxorubicin (Dox) kills AX cells as well as AOT cells (AO cells that have acquired AX-like properties and lost their adipogenic potential). Administration of the combination of doxorubicin and fasudil thus provides a potential therapeutic approach to the targeting of heterogeneous osteosarcoma cells based on trans–terminal differentiation.

Article Snippet: Frozen human osteosarcoma sections were obtained from OriGene.

Techniques: