Fig. 2. Chemical screen for the identification of enhancers of H_CT and α-p75^NTR accumulation in the soma of ES cell-derived motor neurons.

a The results of a small-molecule chemical screen are shown as an XY plot of the normalized mean staining intensity for α-p75^NTR versus AlexaFluor 555-conjugated H_CT. α-p75^NTR was detected using an AlexaFluor 647-conjugated donkey anti-rabbit secondary antibody. Active compounds (red dots) were defined as those able to increase accumulation of α-p75^NTR and H_CT by at least three times the standard deviation of the dataset (represented by the yellow rectangle). The negative control (EHNA) is shown in blue (n ≥ 25 cell bodies analysed per condition; n = 3 repeats). b, c Dose–response analysis of the top four active compounds identified by the screening displayed in A. The accumulation assay was performed at 0.4, 2 and 10 μM and the amount of H_CT (b) and α-p75^NTR (c) in the soma was quantified as the mean staining intensity per pixel ( ± SEM) (n ≥ 25 cell bodies per condition, n = 3 repeats). d Effects of 2 μM A1 and C3 on the accumulation of H_CT in the cell body of primary motor neurons isolated from E13 SOD1^G93A embryos (DIV6). The amount of H_CT was quantified as described in B, C and shown as mean ± SEM. Significance levels were determined using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. n = 3 repeats; *** p < 0.001; * p < 0.05