Fig. 1

inhibition effects of MAP4Ks in the development of synapses and neurites. A The diagram of a hypothesized MAP4Ks-JNK-GAP43 cascade. According to ref. 20, brain-specific MAP2K for JNK is thought to be MKK7. B The workflow of the study methods. C Cell imaging: Synaptic analysis of neurons (2 weeks in culture) treated with DMSO (control) and with 160 μM PF06260933, immunostained by SHANK2 and synaptophysin. D Synaptic analysis of two-weeks old neurons treated with DMSO, and 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μM PF06260933. E Cell imaging: Analysis of neurite lengths (day 3 in culture) treated with DMSO and with 160 μM PF06260933, immunostained by MAP2. F Neurite analysis of day 3 old MAP2-immunostained neurons treated with DMSO, and 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM PF06260933 (eight different concentrations for neurite analysis). G Cell imaging: Neurite length analysis treated with DMSO or 5 μM PF06260933, immunostained using anti-phosphorylated JNK (pJNK) antibody. H Analysis of neurites on three-days old using pJNK-immunostained neurons, treated with DMSO or PF06260933 (the same concentrations as in G). I Cell imaging: Neurite length analysis treated with DMSO and 5 μM PF06260933, immunostained using phosphorylated GAP43 (pGAP43) antibody. J Neurite analysis of day 3 old pGAP43-immunostained neurons treated with DMSO and PF06260933 (same concentrations as in G). K Cell imaging: Neurite length analysis treated with DMSO and 10 μM PF06260933 enhanced by JNK immunostaining. L Neurite analysis of three-days old JNK-immunostained neurons treated with DMSO and PF06260933 (same concentrations as in G). M Cell imaging: Neurite length analysis treated with DMSO and 10 μM PF06260933 enhanced by GAP43 immunostaining. N Neurite analysis of day 3 old GAP43-immunostained neurons treated with DMSO and PF06260933 (same concentration as in G). O–V Protein expression in cells treated with control, and 2.5, 5, and 10 μM PF06260933 in gross protein visualization of pJNK (O), pGAP43 (Q), JNK (S), and GAP43 (U); and signal intensity quantification of pJNK (P), pGAP43 (R), JNK (T), and GAP43 (V). Significant comparison is marked with (*) at p < 0.05/8 (D, F, H, J, L, N; n = 6 each concentration) and at p < 0.05/3 (P, R, T, V; n = 3 each concentration), and error bars represent standard deviation