The glucosylceramide synthase inhibitor PDMP causes lyso-somal lipid accumulation and mTOR inactivation

We investigated subcellular sphingolipid distribution using a functionalized sphingosine analogue (pacSph) by confocal microscopy in control, PDMP and NB-DNJ (Miglustat) treated WT and SGPL1 knock-out cells (experimentA; channel 1: pacSph, channel 2: Lamp1). We studied lysosomal sphingolipid (pacSph) export using pulse-chase experiments in control and PDMP treated SGPL1 knock-out cells (experimentB; channel 1: pacSph, channel 2: Lamp1). We analyzed other lipids such as LBPA (immunofluorescence staining) and cholesterol (filipin staining) at the lysosome in control, PDMP and NB-DNJ (Miglustat) treated WT cells by microscopy (experimentC). We investigated a timeline of lipid accumulating effects arising from PDMP treatment by confocal microscopy. Therefore, we investigated LBPA (immunofluorescence staining in WT cells; channel 1: LBPA, channel 2: Lamp1), sphingolipids (using pacSph in SGPL1 knock-out cells; channel 1: pacSph, channel 2: Lamp1) and cholesterol (filipin staining in WT cells; channel 1: Filipin, channel 2: Lamp1) (experimentD). We analysed the timeline of mTOR (channel 1: mTOR, channel 2: Lamp1) and TFEB (channel 1: DAPI, channel 2: TFEB) subcellular translocation arising from PDMP treatment in WT cells by immunofluorescence microscopy (experimentE).