FIG. 6.

NC cells transfected with TCOF1 siRNA impair regular migration of NC and MSC. (A) H9s-derived NC cells were transiently transfected with a siRNA to TCOF1. Left panel: Flow cytometry was performed for CD44 and P75 following 5 days of differentiation from NC to MSC, demonstrating that TCOF1 KD does not impair MSC differentiation. Right panel: Flow cytometric analysis of CD44 and P75 in NC transiently transfected with a siRNA to TCOF1. Red contour plots represent CD44+P75 double stained populations, and blue contour plots represent isotype control staining. (B) MTT cell proliferation assay was performed using TCOF1 KD NC cells (NC siRNA TCOF1) and siRNA scramble NC cells (NC siRNA SCRAMBLE) during 4 days. Results are presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01. Two-sided Student's t-test. (C) Representative images of scratch wound assays of HESC-derived NC cells transiently transfected with Scramble siRNA or TCOF1 siRNA. Images were collected 4 days following transfection, at the indicated time points. Scale bar: 100 μm. (D) Box plot depicting the quantification of chemotaxis potential and migration of NC cells transfected with TCOF1 siRNA or Scramble siRNA during a 6 h CytoSelect Cell Migration Assay. FGF8B was used as a NC chemoattractant. DMEM/F12 + 10% fetal bovine serum (SERUM) was used as a positive control for cell migration. Data are expressed relative to the NC cells transfected with TCOF1 siRNA, maintained in FSB medium. *P < 0.05; **P < 0.01. Two-sided Student's t-test. (E) Representative images of scratch wound assays of NC-derived MSC transiently transfected with Scramble siRNA or TCOF1 siRNA. Images were collected after 5 days of differentiation, at the indicated time points. Scale bar: 100 μm. KD, knockdown; MTT, 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; siRNA, small interfering RNA. Color images available online at www.liebertpub.com/scd