Fig. 6: Eliglustat is an autophagy inhibitor. | Nature Communications

Fig. 6: Eliglustat is an autophagy inhibitor.

From: Modulating glycosphingolipid metabolism and autophagy improves outcomes in pre-clinical models of myeloma bone disease

Fig. 6: Eliglustat is an autophagy inhibitor.

a–d RAW264.7 cells were treated with RANKL and M-CSF with or without Elig for 24 h. 10 nM BafA1 added 2 h before protein harvest. LC3II (a, b) and p62 (c, d) levels were quantified by western blot. (n = 7 independent samples for DMSO control, Elig, BafA1, and Elig with BafA1). e, f BafA1, CQ and Elig increased LC3 puncta in U2OS cell transfected with GFP-LC3 plasmid after 2 h treatment (n = 43 cells for DMSO control, n = 17 cells for BafA1, n = 15 cells for CQ, n = 46 cells for Elig group). g–j Elig increased GFP/RFP ratio (h; n = 46 cells for DMSO control, n = 42 cells for BafA1, n = 38 cells for CQ, and n = 77 cells for Elig group) and GFP + RFP + puncta number (j; n = 31 cells for DMSO control, n = 42 cells for BafA1, n = 39 cells for CQ, and n = 20 cells for Elig group) in RFP-GFP-LC3 transfected U2OS cell after 6 h treatment. k, l Electron microscope images of Elig induced autolysosome (red arrow) formation in RAW264.7 cells after 2 h treatment (25,000×) (n = 23 cells for DMSO control, n = 13 cells for BafA1, n = 19 cells for CQ, and n = 15 cells for Elig group) (l). m, n TRAF3 and LAMP1 western blot for lysosome-enriched layer from the control group (DMSO), BafA1 group (10 nM, 2 h treatment), CQ group (20 μM, 12 h treatment), and Elig group (50 μM, 12 h treatment) (m); TRAF3 protein level was quantified based on the lysosome marker LAMP1, n = 12 independent samples (n). o, p Confocal images of TRAF3 and LAMP2 in Pre-OCs derived from RAW264.7 cells were treated with BafA1, CQ, and Elig for 60 min, Scale bar represents 10 μm, n ≥ 4 (o); the percentage of TRAF3 and LAMP2 co-localization is quantified (n = 6 cells for DMSO control, n = 9 cells for BafA1, n = 9 cells for CQ, and n = 7 cells for Elig group) (p). Results from a, c, e, g, k, m, and o are representative of three independent experiments. Data are presented as mean values ± SEM. Exact p-values are depicted in the figure. Statistical analysis was performed using one-way ANOVA. Source data are provided as a Source Data file.

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