Fig. 5: Eliglustat inhibits OC formation in a TRAF3-dependent manner. | Nature Communications

Fig. 5: Eliglustat inhibits OC formation in a TRAF3-dependent manner.

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

Fig. 5

a RAW264.7 cells were differentiated into OCs with 50 ng/ml M-CSF and 75 ng/ml RANKL. Different doses of eliglustat (0.1, 1, 10, 25, 50 μM) were present throughout the culture period and OCs were identified by TRAP staining on day 5. Scale bar represents 200 μm. Result is representative of four independent experiments. b–e Number of OCs per well, number of nuclei per TRAP-positive cell, % OC area over total area and Alamar blue viability assay were quantified (n = 3 independent experiments). f Schematic graph illustrating binding of RANKL to its receptor activates the TRAF6-dependent canonical NFκB pathway, leading to proteasome-mediated IκBα degradation and nuclear translocation of p65 and p50. In the non-canonical NFκB pathway, binding downregulates TRAF3 via autophagy/lysosome-mediated degradation, induces nuclear translocation of p52 and RelB. Panel f is adapted from “NF-KB Signaling Pathway” and “Autophagy Process”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates. g–i RAW264.7 cells treated with M-CSF, RANKL, and eliglustat for 1 day. TRAF3 protein levels quantified by western blot and mRNA levels quantified by qRT-PCR. Treatment with BafA1 for the last 2 h (n = 6 independent samples). j–m Primary murine BM cells treated with RANKL together with BafA1 or MG132 or eliglustat for 2 h. TRAF6 (j, k) and IκBα (l, m) levels quantified by western blot (n = 8 biologically independent samples). n Lethally irradiated 8-week-old female CD45.1+ B6.SJL mice were reconstituted with littermate (WT) or myeloid specific TRAF3 knockout (LysM-Cre + , Traf3 fl/fl) BM cells. After 19 days of treatment with eliglustat, bone volume of tibiae was quantified by micro-CT (WT, n = 4; WT + Elig, n = 4; traf3 KO, n = 5; traf3 KO + Elig, n = 5 biologically independent animals). The experiment is representative of two independent experiments. Results from g, j, and i 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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