Supplementary Figure 6

(A) Unmerged images of primary neurons (DIV3) overexpressing various SSNA1 variants shown in Fig. 6. The axons are labeled in red by Tau1 antibody (first row, red), while the dendrites are marked with the MAP2 antibody (second row, green). The expression of the SSNA1 proteins was confirmed by concomitant GFP expression (third row, cyan). In the merged view of the SSNA1 WT, axon is guided with a dotted line. (B-E) Neuron morphology analysis of various overexpression conditions B) Distribution of neurons based on total branch length/axon length and pie graphs showing the distribution of the number of processes (major branches plus minor protrusions along axon). Sample size: Control (n = 266 cells), wild type (n = 289 cells), 1–104 (n = 537 cells), 21-C (n = 274 cells), 5 A (n = 358 cells) pooled from 3 independent experiments and (C) Pie graphs showing the distribution of the total number of processes. Sample size: Control (n = 266 cells), wild type (n = 289 cells), 1–104 (n = 537 cells), 21-C (n = 274 cells), 5 A (n = 358 cells) pooled from 3 independent experiments. In (B) and (C), statistics of 5 A show significant difference (χ2 = 23.0, p < 0.001 and 36.3, p < 0.001 respectively) compared to control, indicating a negative effect of the 5 A mutant overexpression to neurons. In (C), wild type overexpressed neurons show the significant difference (χ2 = 12.83, p < 0.01). (D) Scatter dot plots of axon length under over-expression of various SSNA1 swap mutants. The promotion of axon development occurs in over-expression of swap-KK/EE, while slight dominant negative effect (shortening of axon) was observed in over-expression of swap-KK/EEE. Every cell is represented by a single point: Control (n = 1348 cells), swap-KK/EE (n = 789 cells), swap-KK/EEE (n = 1129 cells) and the overlaid box-and-whisker plots cover 50% (boxes) and 90% (whiskers) of the entire population, with median values indicated as lines within the boxes. (E) Pie graphs showing the distribution of the number of branches. Distributions of the branches in swap-mutants expressed neurons differ significantly from control (GFP over-expression) according to χ² two-sample test (χ² = 14.4, p < 0.005 and 29.1, p < 0.000005, respectively). Sample size: Control (n = 1348 cells), swap-KK/EE (n = 789 cells), swap-KK/EEE (n = 1127 cells). (F) Purification of chTOG and EB3. (G) Mixtures of tubulin with chTOG (upper) and EB3 (lower) were treated in the same way as SSNA1-tubulin mixture to test the induction of microtubule branches. No microtubule branching was observed in the tested conditions.