(A) H-NF and β-III tubulin expression in CD99-silenced cells that were maintained in standard culture conditions. Neural differentiation was reversed when CD99 was reexpressed in knockdown/rescue cells. Representative photomicrographs are shown for TC-71–derived cells. Scale bars: 120 μm. (B) Western blot analysis of H-NF and β-III tubulin expression in CD99-silenced cells that were maintained in standard culture conditions. Expression of H-NF was observed only in CD99-deprived cells, while β-III tubulin expression was induced. (C) Western blot analysis of H-NF TC-71 and TC-CD99-shRNA#1 cells after exposure to low-serum medium (1% IMDM) or NGF (20 ng/ml) for 72 hours. Again, expression of H-NF was observed only in CD99-silenced cells. (D) Downregulation of p21 in TC-CD99-shRNA#2 by transient silencing of the molecule, as detected by Western blotting, resulted in inhibition of the neural differentiation, as shown by H-NF expression. Cells were exposed to shRNA against p21 for 24 hours and then evaluated for H-NF expression. Representative photomicrographs are shown for TC-71–derived cells. Scale bars: 120 μm. (E) MSCs were cultured in neural differentiation medium, and terminal differentiation was monitored by H-NF and β-III tubulin staining that visualized neural development. The expression of CD99 is lost during neural development, as shown by avidin-biotin immunostaining. All digital images were taken under identical conditions, at the same time, and using the same image analysis software (Quips-XL genetic workstation). Scale bars: 600 μm; enlarged sections, 300 μm.