Journal: The Journal of Biological Chemistry

Article Title: A Novel Suppressive Effect of Alcohol Dehydrogenase 5 in Neuronal Differentiation *

doi: 10.1074/jbc.C114.561860

Figure Lengend Snippet: ADH5 counteracted differentiation of human neural stem cells toward neurons. A, immunostaining of the neural progenitor markers Nestin, Sox2, Pax6, and Ki67 in hNSCs. Scale bar, 50 μm. B, scheme of differentiating hNSCs toward neurons. Briefly, hNSCs were transduced with lentiviral vectors (ADH5, ADH5(R115D), or empty vector) at day 0. At day 4, the transduced hNSCs subcultured in a 24-well microplate were induced to differentiation toward neurons. Immunofluorescence and qPCR were performed at day 40. C, qPCR analysis of ADH5 expression in hNSCs and their neuron derivatives. D, qPCR analysis of SOX2 and Nestin in hNSCs transduced with lentivirus expressing either EGFP (Vector) or ADH5. E, Transwell assay to analyze the relative number of migrated hNSCs transfected with lenti-EGFP or lenti-ADH5 (wt). F, immunofluorescence staining of differentiated neurons indicated in B in the absence or presence of ADH5 inhibitor C3. Scale bar, 50 μm. G, qPCR analysis of ADH5, MAP2, and GAD67, in the differentiated neurons indicated in B and F. H, qPCR analysis of synapsin as a marker of neuron maturation in the differentiated neurons indicated in B and F. I, qPCR analysis of glial fibrillary acidic protein (GFAP), α-smooth muscle actin (SMA), and α-fetoprotein (AFP) lineage-specific markers in the differentiated neurons indicated in B and F. J, scheme of differentiating hNSCs toward neurons in the presence of ADH5 inhibitor C3. Briefly, hNSCs subcultured in a 24-well microplate at day 0 were induced to differentiation toward neurons at day 3 by differentiation medium together with C3. Immunofluorescence and qPCR were performed at day 9. K, qPCR analysis of neuron-specific markers, MAP2 and GAD67, in the differentiated hNSCs treated with C3 for 6 days. All the values are shown as means ± S.E. n = 3. Single asterisk, p < 0.05; triple asterisk, p < 0.001. NS, not significant.

Article Snippet: The following antibodies were used: β-actin (Santa Cruz Biotechnology), HDAC2 (Santa Cruz Biotechnology), ADH5 (Proteintech), Sox2 (Abcam), Pax6 (Vector Laboratories), Nestin (Millipore), Ki67 (Covance), MAP2 (Millipore), and Tuj1 (Sigma-Aldrich).

Techniques: Immunostaining, Transduction, Plasmid Preparation, Immunofluorescence, Expressing, Transwell Assay, Transfection, Staining, Marker