Journal: Stem Cells (Dayton, Ohio)

Article Title: TAFA2 Induces Skeletal (Stromal) Stem Cell Migration Through Activation of Rac1‐p38 Signaling

doi: 10.1002/stem.2955

Figure Lengend Snippet: TAFA2 levels are regulated during the process of fracture healing. ( A): Immunohistochemical analysis of TAFA2 protein expression in a fractured femur, 48 hours after induction of fracture. Scale bar: 1 mm. (B): Analysis of TAFA2 immunoreactivity at the fracture site (boxed area from A ) at different time points (2, 24, and 48 hours, 4 days, and 1 week) after induction of femoral fracture (upper panel) and nonfractured femur (lower panel). Scale bar: 100 μm. (C): Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analysis of TAFA2 mRNA expression in nonfractured and fractured mouse femur at different time points after fracture induction (2 hours, day 1, day 4, and day 14). (D): Flow cytometry analysis of TAFA2 protein expression in bone marrow cells obtained from fractured femur at different time points. Data represent MFI ± SEM from three independent experiments. **, p ≤ .01; two‐tailed unpaired Student t test. (E): qRT‐PCR analysis of IL‐1β mRNA expression in mouse femur before fracture and at different time points after fracture induction (2 hours, day 1, day 4, and day 14). (F): qRT‐PCR analysis of TAFA2 mRNA expression in human skeletal (mesenchymal, stromal) stem cell (hMSC) cultures in the presence of different doses of IL‐1β (1, 10, and 50 ng/ml) for 48 hours. Data represent three independent experiments ± SEM; **, p ≤ .01, two‐tailed paired Student t test. (G, H): Western blot analysis and quantification of TAFA2 protein levels in hMSC cultures in the presence of different doses of IL‐1β. Data represent mean ± SEM from four independent experiments. *, p ≤ .05, two‐tailed unpaired Student t test. (I, J): Flow cytometry analysis of TAFA2 protein expression and percentage of leukocytes (CD45 + ), monocytes (CD14 + ), T cells (CD3 + ), and B cells (CD19 + ) at 1 and 3 days postfracture. Data are obtained from pooled bone marrow cells isolated from fractured femurs of three mice. (K): Enzyme‐linked immunosorbent assay measurement of TAFA2 levels in consecutive serum samples from patients with hip fracture. **, p ≤ .01, two‐tailed paired Student t test. (L): Proposed model for role of TAFA2 in recruiting MSC to the site of fracture. Abbreviations: IL, interleukins; MFI, mean fluorescence intensity; MSC, skeletal (mesenchymal) stem cell.

Article Snippet: ELISA measurements of TAFA2 concentrations in human serum samples were performed using an established ELISA procedure (Mybiosource, San Diego, USA) according to the manufacturer's instructions.

Techniques: Immunohistochemical staining, Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Flow Cytometry, Two Tailed Test, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay, Fluorescence