Fig. 3
ATG9A accumulates at the trans-Golgi network (TGN) in AP-4 knockout HeLa and SH-SY5Y cells. a Widefield imaging of immunofluorescence double labelling of ATG9A and TGN46 in wild-type (WT), AP4B1 KO, and AP4B1 KO HeLa cells stably expressing AP4B1 (rescue). Scale bar: 20 μm. b Quantification of the ratio of ATG9A labelling intensity between the TGN and the rest of the cell using an automated microscope. Ratios were normalised to the mean wild-type ratio. The experiment was performed in biological triplicate (mean indicated, n = 3), and >1400 cells were scored per cell line in each replicate. Data were subjected to one-way ANOVA with Dunnett’s Multiple Comparison Test for significant differences from the wild-type: ***p ≤ 0.001; ns p > 0.05. c SH-SY5Y (neuroblastoma) cells stably expressing Cas9 were transduced with sgRNAs to AP4B1 or AP4E1. Mixed populations were selected for sgRNA expression and parental Cas9-expressing SH-SY5Y cells were used as a control. Western blot of whole cell lysates; α-Tubulin, loading control. Representative of two independent experiments. d Widefield imaging of immunofluorescence double labelling of ATG9A and TGN46 in control, AP4B1 depleted and AP4E1 depleted SH-SY5Y cells. Scale bar: 20 μm
