Cell-Type Resolved Protein Atlas of Brain Lysosomes Identifies SLC45A1-Associated Disease as a Lysosomal Disorder

Description

Methods

U2OS cells expressing stably mRFP-KDEL were seeded in 8-well LabTeks with 20x10³ cells per well. Transient transfection was performed by pre-mixing 1 µg DNA and 3 µg PEI (polyethylenimine, MW 25 kDa) in 100 µl OptiMEM (without serum and antibiotics). The transfection mix was incubated for 15 min at RT and 20µl added to each Labtek well. Incubation time was 16 h for transfection and replaced transfection media to DMEM high glucose and incubated further for 48 h (exception for ENPP5, 12h transfection, without further incubation).

Live-cell imaging of mRFP-KDEL U2OS was performed changing the medium (phenol-free, complete DMEM media) prior to imaging. Airyscan images were acquired in the SR (super resolution) mode on a Zeiss LSM 980 microscope equipped with an Airyscan2 detector using a Plan-Apochromat 63x/1.4NA oil DIC M27 objective. Images were deconvolved with the Airyscan processing tool of the ZEN Blue software. Subsequent post-processing, contrast adjustment, and image export were performed in ImageJ/Fiji.