Imaging data from the article "AP-4-mediated axonal transport controls endocannabinoid production in neurons", published in Nature Communications by Davies et al., from Fig. 2, 3, 4, 6 and S2. Widefield images were captured on a Leica DMi8 inverted microscope equipped with an iTK LMT200 motorised stage, a 63x/1.47 oil objective (HC PL APO 63x/1.47 OIL) and a Leica DFC9000 GTC Camera, and controlled with LAS X (Leica Application Software X). HeLa_HADAGL_RUSC2_Fig_4: Widefield imaging of wild-type HeLa and HeLa cells stably expressing GFP-RUSC2, transiently expressing HA-DAGLB or HA-DAGLA, and labelled with anti-HA (Alexa Fluor 680), anti-ATG9A (Alexa Fluor 568) and DAPI. HeLa_KD_DAGLB_TGN_Fig_S2a,b: Widefield imaging of immunofluorescence double labelling of DAGLB (Alexa Fluor 568) and TGN46 (Alexa Fluor 680) in HeLa cells transfected with a non-targeting siRNA (control) or with siRNA to knock down AP-4. DAPI labelling of the nucleus is also shown. Note, the antibody used to label AP4E1 (Alexa Fluor 488) in replicate 1 had high non-specific background and so was not used in the analysis. HeLa_KO_DAGLB_TGN_Fig_2a,b: Widefield imaging of immunofluorescence double labelling of DAGLB (Alexa Fluor 568) and TGN46 (Alexa Fluor 680) in wild-type, AP4B1 knockout, and AP4B1 knockout HeLa cells stably expressing AP4B1 (functional rescue). DAPI labelling of the nucleus is also shown. HeLa_RUSC2_DAGLB_Fig_3: Widefield imaging of DAGLB (Alexa Fluor 568) labelling in wild-type and AP-4 knockout (AP4B1 KO or AP4E1 KO) HeLa cells, stably expressing GFP-tagged RUSC2. AP4B1 knockout HeLa expressing RUSC2-GFP were also transiently transfected with AP4B1 (rescue) or mock transfected without DNA as a negative control. DAPI labelling of the nucleus is also shown. iPSC_Neurons_DAGLB_TGN_Fig_6a,b: Widefield imaging of immunofluorescence triple labelling of DAGLB (Alexa Fluor 568), TGN46 (Alexa Fluor 680) and TUJ1 (Alexa Fluor 488; a marker to distinguish neurons from co-cultured astrocytes) in iPSC neurons from a patient with AP-4 deficiency syndrome (patient 1) and their matched control. SHSY5Y_DAGLB_TGN_Fig_2c,d_Fig_S2c,d: Widefield imaging of immunofluorescence double labelling of DAGLB (Alexa Fluor 568) and TGN46 (Alexa Fluor 680) in control (parental Cas9-expressing), AP4B1-depleted (BKO) and AP4E1-depleted (EKO) undifferentiated and neuronally-differentiated SH-SY5Y cells. DAPI labelling of the nucleus is also shown. See article for methods and further detail.

This work was funded by the German Research Foundation (DFG/Gottfried Wilhelm Leibniz Prize MA 1764/2-1) and the Max Planck Society for the Advancement of Science. A.K.D. received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no. 896725 and a Humboldt Research Fellowship. D.E.-F. had support from the CureAP4 Foundation, the Spastic Paraplegia Foundation, the National Institute of Health / National Institute of Neurological Disorders and Stroke (2R25NS070682 & 1K08NS123552-01) and the National Institutes of Health (BCH IDDRC, 1U54HD090255). M.Z. received scholarships from the DAAD (German National Exchange Service) and the German National Academic Foundation. J.E.A. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) 270949263/GRK2162, the DAAD (German National Exchange Service), the German National Academic Foundation and the Max Weber-Program of the State of Bavaria.