Journal: Stem Cells International

Article Title: Matricellular Protein SMOC2 Potentiates BMP9-Induced Osteogenic Differentiation in Mesenchymal Stem Cells through the Enhancement of FAK/PI3K/AKT Signaling

doi: 10.1155/2023/5915988

Figure Lengend Snippet: SMOC2 potentiates BMP9-induced ectopic bone formation in MEF implantation in vivo, while knockdown of FAK inhibits bone formation. (a) Macrographic images of ectopic bone mass. MEFs were implanted subcutaneously after infection with the designed adenoviruses. Ectopic osseous masses were retrieved at 4 weeks. Representative images are shown. (b) Masson's trichrome staining results showed the effect of SMOC2 and FAK knockdown on BMP9-induced ectopic bone masses in MEFs (scale bar is 50 μ m for the panel). (c) Hematoxylin-eosin (H&E) staining results showed that the osteogenesis ability was affected by SMOC2 and/or FAK knockdown in MEFs (scale bar is 200 μ m for upper panel and 50 μ m for lower panel). (d) Immunohistochemical staining results showed the expression relationship between p-FAK and p-AKT1/2/3. Ectopic osseous masses were retrieved at 4 weeks. The expression of p-FAK and p-AKT1/2/3 was assessed by immunohistochemical staining analysis. Representative images are shown (scale bar is 50 μ m for the panel).

Article Snippet: Primary antibodies against RUNX2 (12556S), p-AKT1/2/3 (4060S), and AKT1/2/3 (9272) were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: In Vivo, Infection, Staining, Immunohistochemical staining, Expressing

Journal: Diabetes

Article Title: A Novel Strategy to Prevent Advanced Atherosclerosis and Lower Blood Glucose in a Mouse Model of Metabolic Syndrome

doi: 10.2337/db17-0744

Figure Lengend Snippet: S597 has no detected direct effects on isolated macrophages and endothelial cells. All in vitro studies were performed with vehicle (Veh), insulin (Ins; 100 nmol/L), or S597 (200 nmol/L). A and B: Thioglycollate-elicited macrophages (n = 3) were harvested and adhesion purified for 1 h, followed by stimulation for 18 h. Cytokine mRNA levels, Il6 (A) and Tnfa (B), were measured by real-time PCR. C–G: Bone marrow–derived macrophages (BMDM; n = 3) were differentiated for 7 days in the presence of L-cell conditioned medium, followed by M1 (5 ng/mL LPS + 12 ng/mL IFN-γ) or M2 (4 ng/mL IL-4) stimulation, or no additional stimulation (Basal) for 24 h in the presence of vehicle, insulin, or S597. C: Ym1 mRNA, an M2 marker. D: Arg1 mRNA, an M2 marker. E: Il1b mRNA, an M1 marker. F: Nos2 mRNA, an M1 marker. G: Il6 mRNA, an M1 marker. H and I: Thioglycollate-elicited macrophages (n = 3) were harvested and stimulated with vehicle or 100 μg/mL AcLDL (H) or AcLDL together with 10 μg/mL ACAT inhibitor 58035 for 24 h (I) in the presence or absence of insulin or S597. Free cholesterol (FC) and cholesteryl esters (CE) were extracted and measured. Macrophages were stimulated with 100 μg/mL AcLDL or loaded with free cholesterol, using 100 μg/mL AcLDL during ACAT inhibition (compound 58035, 10 μg/mL; Sigma-Aldrich). J: Assays of cell death, as measured by TUNEL staining (TiterTACS; R&D Systems), were performed 24 h after stimulation. abs 450, absorbance 450 nm; FC, free cholesterol loading; Tun, tunicamycin (5 μg/mL); TG, thapsigargin (5 μmol/L). K: Aortic Vcam1 mRNA. Data were normalized to Rn18s values and presented using the ΔΔCT method with the chow group as controls (chow: n = 5; DDC groups: vehicle, n = 14; insulin, n = 13; S597, n = 13). Similar results were obtained when the data were normalized to Rpn32 or Gapdh. **P < 0.01; ***P < 0.001. L–N: Primary mouse aortic endothelial cells (AoEC) or heart endothelial cells (N) were stimulated with 20 ng/mL TNF-α in the presence and absence of insulin or S597. L: Endothelial cell Vcam1 mRNA. M: Primary mouse bone marrow monocytes were added to a monolayer of endothelial cells, and adhesion of monocytes to the endothelium was determined. AU, arbitrary units. N: Endothelin 1 (ET-1) release 20 min after insulin or S597 stimulation. O: Isolated thioglycollate-elicited macrophages (eMPM) were stimulated with vehicle (PBS), 100 nmol/L insulin, 200 nmol/L S597, or a combination of 100 nmol/L insulin and 200 nmol/L S597 for 15 min. Samples were analyzed using p-Akt1–specific ELISAs (n = 6–9). P: Aortic endothelial cells were stimulated with vehicle, 100 nmol/L insulin, 200 nmol/L S597, or a combination of 100 nmol/L insulin and 200 nmol/L S597 for 15 min and analyzed for p-Akt1. Data are expressed as mean ± SEM (n = 5). Symbol definitions in panel O also apply to P. Q: Hepa1-6 cells, AoECs, and eMPMs were harvested and analyzed for expression of the IR and IGF-1 receptor (n = 3). None of the cell lines used in this study is included in the database of commonly misidentified cell lines (International Cell Line Authentication Committee). Cells were free of mycoplasma contamination, as determined by the MycoProbe Mycoplasma Detection Kit (R&D Systems).

Article Snippet: The total Erk1/2 antibody was a rabbit polyclonal ( 20 ). p-Akt1/2/3 ELISAs were from Cell Signaling.

Techniques: Isolation, In Vitro, Purification, Real-time Polymerase Chain Reaction, Derivative Assay, Marker, Inhibition, TUNEL Assay, Staining, Expressing