Fig 2: Expression of SH3GL2G276V in differentiated dopaminergic neurons recapitulates findings from Drosophila synapses(A) Schematic representation of gene editing strategy to knock in the G276V mutation in SH3GL2.(A') Sanger sequencing showing successful homozygous editing.(B) Representative maximum projection confocal images of terminally differentiated (60 days) SH3GL2WT ventral midbrain dopaminergic neurons (vmDAns) stained with the ventral midbrain marker FOXA2, dopaminergic marker TH, and neuronal marker MAP2. Scale bar: 100 µm.(B') Quantification of the number of dopaminergic neurons over the total number of neurons in the field of view expressed as percentage of TH+/MAP2+ neurons. Error bars: mean ± SEM. Statistical significance: one-way ANOVA with Tukey’s multiple comparison test: ns, not significant. Single data points representing single confocal images. Data from three independent vmDAn differentiations (circles and empty/filled triangles represent independent differentiations).(C and C') Western blot with anti-ENDOA1 from induced vmDAn. (C') Quantification of ENDOA1 intensity relative to ß-actin expression. Experiments performed with three biological replicates (neurons from three independent differentiations, displayed as circle and full/empty triangles). Error bars: mean ± SEM. Statistical significance: Student’s t test: ns not significant.(D–D'') Calcium imaging on induced vmDAns transfected with GCaMP6f. (D) Example traces of single SH3GL2WT (black) and SH3GL2G276V (blue) neurons. (D') Quantification of the average activity of neurons in one field of view (peak activity) and (D'') quantification of the average peak amplitude of the calcium responses. Error bars: mean ± SEM. Statistical significance: Student’s t test: ns, not significant(E) Representative maximum projection confocal images of induced vmDAn (60 days) SH3GL2WT vmDAn stained with anti-TH and anti-LC3B antibodies. Arrowheads: LC3B+ autophagosomes within TH+ neurites. Scale bar: 20 µm. Insert: zoom of the region indicated, where multiple LC3B+ puncta are visible.(E') Quantification of the number of LC3B+ autophagosomes within TH+ neurites normalized to the total TH+ neurite length in the field of view. Error bars: mean ± SEM. Statistical significance: Student’s t test. ***p < 0.001. Single data points represent single confocal images. Data from three independent vmDAn differentiations (circles and empty/filled triangles represent independent differentiations).Related to Figure S8.