Journal: Journal of cell science

Article Title: Telomere-based proliferative lifespan barriers in Werner-syndrome fibroblasts involve both p53-dependent and p53-independent mechanisms.

doi: 10.1242/jcs.00331

Figure Lengend Snippet: Fig. 5. Microinjection of control IgG or anti-p53 antibodies into senescent AG05229 cells. The injected cells are marked by Texas Red, nuclei are detected by DAPI staining and BrdU incorporation is marked by FITC. The images are merged in the bottom panels. The DO-1-injected cells show two nuclei incorporating BrdU. Bar, 100 µm.

Article Snippet: Cells were fixed as described above and BrdU incorporation was detected by incubation in a 1:100 dilution of anti-BrdU (isotype IgG1κ) (Dako) with DNAase I (25 U ml–1) for 2 hours at 37°C, followed by incubation for 1 hour at room temperature in a 1:50 dilution of fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG1isotype specific (Southern Biotechnology Associates).

Techniques: Microinjection, Control, Injection, Staining, BrdU Incorporation Assay

Journal: Drug Design, Development and Therapy

Article Title: Regulation of bone formation by baicalein via the mTORC1 pathway

doi: 10.2147/dddt.s81578

Figure Lengend Snippet: Figure 2 Effects of BN on mRNA expression of ALP, RUNX2, OCN, and Col1α1. Notes: Total cytosolic RNA was prepared from MC3T3-E1 cells cultured in osteogenic differentiation medium and exposed to BN (0.1–10 μM) for 14 days, and used for quantitative RT polymerase chain reaction analysis of ALP (A), RUNX2 (B), OCN (C), and Col1α1 (D) as described in the Materials and methods section. Data are expressed as fold change versus the control group, taken as calibrator for comparative quantitation analysis of GAPDH mRNA levels. It was shown that BN could promote expression of ALP, RUNX2, OCN, and Col1α1 in a dose-dependent manner. Each sample was measured in triplicate and the experiment was repeated twice with similar results. *P,0.05 versus group without BN; +P,0.05 versus group with 0.1 μM BN; #P,0.05 versus group with 1 μM BN. The values shown are the mean ± standard error of data from independent experiments (A–D). Abbreviations: ALP, alkaline phosphatase; BN, baicalein; Col1α1, collagen Iα1; GADPH, glyceraldehyde-3-phosphate dehydrogenase; OCN, osteocalcin; RT, real-time; RUNX2, runt-related transcription factor 2.

Article Snippet: The paraffin-embedded longitudinal bone sections with 5 μm slice thickness were then blocked by 5% goat serum for 1 hour at room temperature, and incubated overnight with the mouse monoclonal osteocalcin antibodies (product code sc-376835, diluted 1:50, Santa Cruz Biotechnology).

Techniques: Expressing, Cell Culture, Polymerase Chain Reaction, Control, Quantitation Assay

Journal: Drug Design, Development and Therapy

Article Title: Regulation of bone formation by baicalein via the mTORC1 pathway

doi: 10.2147/dddt.s81578

Figure Lengend Snippet: Figure 3 Effects of BN on protein expression of RUNX2, OCN, P-4E/BP1, and P-S6K1. Notes: MC3T3-E1 cells were cultured in differentiation medium and exposed to BN (0.1–10 μM) for 2 weeks. MC3T3-E1 cell lysates (20 μg protein) were prepared, subjected to Western blot, and analyzed using antibodies specific for RUNX2 (A), OCN (A), P-4E/BP1 (B), and P-S6K1 (C) as described in the methods section. Expression of RUNX2 (A, D), OCN (A, E), and P-S6K1 (C, F) increased gradually with increasing concentrations of BN and peaked at 10 μM. Expression of P-4E/BP1 protein (B, F) was decreased gradually and reached a nadir at 10 μM. Each sample was measured in triplicate, and the experiment was repeated twice with similar results. *P,0.05 versus group without BN; +P,0.05 versus group with 0.1 μM BN; #P,0.05 versus group with 10 μM BN. The values shown are the mean ± standard error of the mean of data from independent experiments (D–G). Abbreviations: ALP, alkaline phosphatase; BN, baicalein; OCN, osteocalcin; OVX, ovariectomized; RUNX2, runt-related transcription factor 2.

Article Snippet: The paraffin-embedded longitudinal bone sections with 5 μm slice thickness were then blocked by 5% goat serum for 1 hour at room temperature, and incubated overnight with the mouse monoclonal osteocalcin antibodies (product code sc-376835, diluted 1:50, Santa Cruz Biotechnology).

Techniques: Expressing, Cell Culture, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Regulation of bone formation by baicalein via the mTORC1 pathway

doi: 10.2147/dddt.s81578

Figure Lengend Snippet: Figure 6 Histochemistry for ALP, immunohistochemistry for RUNX2, and immunofluorescence for OCN in paraffin sections from femur. Notes: (A) ALP expression was significantly increased in OVX + BN mice (D) that received BN (10 mg/kg per day) for 3 months when compared with the OVX group (D). Histological sections from the sham, OVX, and OVX + BN groups were incubated with RUNX2 (B) and OCN (C) antibody. Expression of RUNX2 (E) and OCN (F) around trabecular bone decreased after ovariectomy but was rescued with BN. Each group contained ten mice. The black arrows indicate ALP positive cells (A). *P,0.05 versus OVX group. The values shown are the mean ± standard error of the mean of data from independent experiments (D–F). Abbreviations: BN, baicalein; OCN, osteocalcin; RUNX2, runt-related transcription factor 2.

Article Snippet: The paraffin-embedded longitudinal bone sections with 5 μm slice thickness were then blocked by 5% goat serum for 1 hour at room temperature, and incubated overnight with the mouse monoclonal osteocalcin antibodies (product code sc-376835, diluted 1:50, Santa Cruz Biotechnology).

Techniques: Immunohistochemistry, Immunofluorescence, Expressing, Incubation

Journal: Drug Design, Development and Therapy

Article Title: Regulation of bone formation by baicalein via the mTORC1 pathway

doi: 10.2147/dddt.s81578

Figure Lengend Snippet: Figure 7 BN promotes differentiation of MC3T3-E1 cells into osteoblasts. mTORC1 signaling favors maturation and survival of osteoblasts, and promotes expression of OCN and RUNX2. Abbreviations: BN, baicalein; mTORC1, mammalian target of rapamycin complex 1; OCN, osteocalcin; RUNX2, runt-related transcription factor 2.

Article Snippet: The paraffin-embedded longitudinal bone sections with 5 μm slice thickness were then blocked by 5% goat serum for 1 hour at room temperature, and incubated overnight with the mouse monoclonal osteocalcin antibodies (product code sc-376835, diluted 1:50, Santa Cruz Biotechnology).

Techniques: Expressing