AP-4-mediated axonal transport controls endocannabinoid production in neurons - Imaging data 1

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.