Fig. 4.

Src family kinase inhibitor Saracatinib suppresses FynY531F-driven dopaminergic neuron loss, Fyn activation, and il1b and il12a cytokine induction. (A) Time course of Saracatinib treatment beginning at 72 h post fertilization. (B) Live confocal imaging of vDC cluster eGFP dopaminergic neurons in 5 dpf control dat:eGFP and elavl3:Gal4; UAS:fynY531F larvae mock treated with DMSO or treated with 20 mM Saracatinib. (C) Quantification of vDC eGFP-positive neuronal cell bodies in 5 dpf control dat:eGFP and dat:eGFP; elavl3:Gal4; UAS:fynY531F larvae mock treated with DMSO or treated with 20 mM Saracatinib (n=12). Analysis was performed using two-way ANOVA with Tukey's multiple comparison. (D) Western blot of extracts from 5 dpf wild-type +/+ and dat:eGFP; elavl3:Gal4; UAS:fynY531F larvae treated with DMSO and increasing amounts of Saracatinib, and probed with anti-Src family kinase P-Y416-SFK. Anti-acetylated tubulin and anti-alpha-tubulin were used as loading controls. (E) RT-qPCR of il1b, il12a, tnfa and irg1 in RNA extracts from untreated and 20 µM Saracatinib-treated 5 dpf elavl3:Gal4; UAS:fynY531F larvae (n=3 biological replicates for all genotypes and conditions). This experiment was performed alongside the CAPE and Ac-YVAD-cmk experiments in Fig. 7C,D, using shared untreated controls. Control data in this panel are also shown in Fig. 7C,D. Statistical analysis was performed with two-tailed unpaired Student's t-test. Bars represent mean±s.e.m. ns, not significant; *P<0.05; ***P<0.001; ****P<0.0001. Scale bars: 50 µm.