Figure 1.

Individual and combined effects of 12 soluble differentiation factors on TUBB3 expression within microscale 3D hNPC cultures. (A) 3D hNPC differentiation screen schematic. (B) Representative immunofluorescence-based images used for high-content analysis of 3D hNPC differentiation outcomes. Chosen images depict antibody-based detection of TUBB3 expression (green) and Hoechst 33342-based detection of cell nuclei (blue) for the no primary antibody control, conditions which increased the relative TUBB3 expression (CHIR + RA and PURM + RA), and the spontaneous (no factors added) control. Scale bar = 300 μm. (C) The average Hoechst 33342 normalized TUBB3 expression from n = 12 collected from three independent screens is plotted for each condition tested. Mean ± SEM plotted. (D) Log₂ heat map of TUBB3 expression relative to spontaneous differentiation. Three replicate screens were performed and the log₂ relative fluorescence of each screened condition was normalized by that of the spontaneous differentiation. To elucidate the influence of individual and combination treatments, the log₂ values were used to generate a heat map by correlating values to a color gradient between the minimum (red) and maximum (green) values. (E) Select conditions from 3D hNPC neuronal differentiation screen. One-way ANOVA analysis of the Hoechst 33342 normalized TUBB3 expression identified several conditions that resulted in statistically significant different TUBB3 expression compared to the spontaneous differentiation control (*=p<0.05, **=p<0.01, ***=p<0.0001). Mean ± SEM plotted.