Fig 2: APP upregulates JNK3 expression and function through AICD Representative western blots show JNK3 expression in a pcDNA3-FLAG-APP695 transfected or pcDNA3 transfected SH-SY5Y cells, e retinae from wild-type or APP-null mice, or i pcDNA4/V5-His-AICD59 or pcDNA4/V5-His transfected SH-SY5Y cells. The samples were lysed, resolved in SDS-page gel, and immunoblotted using antibodies against JNK3 or GAPDH. b, f, j The bar charts show quantitative analysis of relative JNK3 expression (n = 3 for each experiment). Representative western blots show phosphorylative activation of JNK3 in c APP695-overexpressing or normal SH-SY5Y cells, g retinae from wild-type or APP-null mice, or k pcDNA4/V5-His-AICD59 or pcDNA4/V5-His transfected SH-SY5Y cells. JNK3 was immunoprecipitated with anti-JNK3 antibody and then detected for kinase activity with anti-phospho-JNK (pJNK) or anti-JNK3 antibody. Whole-cell lysate western blots show the downstream substrate pc-Jun73 or c-Jun level. d, h, l The bar charts show quantitative analysis of activated pJNK/mg lyate proteins or pc-Jun73/c-Jun ratio (n = 3 for each experiment). Student’s t-test: *p < 0.05; **p < 0.01. Error bars represent SEM

Fig 3: APP deficiency suppresses the upregulation of JNK3 expression and JNK function in mouse retina after ONA a Representative western blots show JNK3 expression and overall phosphorylative activation of JNK in retinae from wild-type or APP-null mice 1 day (1 d), 1 week (1 w) and 2 weeks (2 w) after ONA (n = 3 for each time point). Anti-JNK3, anti-pJNK and anti-GAPDH antibodies were used to detect the respective protein expression. b The bar charts show quantitative analysis of relative JNK3 expression or overall phosphorylative activation of JNK in the ipsilateral-injured retinae vs. the contralateral-uninjured retina. One-way ANOVA followed by post-hoc Bonferroni test: **p < 0.01. Error bars represent SEM

Fig 4: JNK3 is upregulated and involved in RGC death after ONA in mice a Quantitative analysis of relative mRNA levels of Jnks in intact retinae or retinae 1 day (1 d), 1 week (1 w) or 2 weeks (2 w) after ONA (n = 3 for each time point). b Representative western blots from at least three biological repeats showing JNK expression after ONA. Intact retina or retinae 1 d, 1 w, 2 w after ONA were harvested, lysed and immunoblotted using antibodies against JNK1, JNK2, JNK3, pJNK or GAPDH. The location of molecular weight markers is shown in kDa on the right. c Representative fluorescence microscope images show immunostaining of mouse retina flat-mounts from wild-type or JNK1, JNK2, or JNK3-null mice (n = 6 for each genotype) 14 days after ONA using an anti-Tuj1 antibody. d The bar chart shows quantitative analysis of Tuj1+ RGC numbers 14 days after ONA. Scale bar, 50 µm. One-way ANOVA followed by post-hoc Bonferroni test: **p < 0.01. Error bars represent SEM