Addition of ceramides (CER) mimics ELOVL6-dependent effects in BTZ-resistant MM cells. (A) Parental (P) and BTZ-resistant (R) MM cells expressing or not empty vector or ELOVL6 were treated or not with the indicated amounts of BTZ for 16 hours followed by lipidomic analysis in independent triplicates. Raw values of each CER species (each row) were transformed as z scores and plotted in the heatmap, with red representing the highest value and blue representing the lowest value. (B) Mean ratios of the levels of individual CER species between designated BTZ-R MM cells were calculated and plotted as heatmaps. Each row represents an individual CER ratio. (C) BTZ-resistant MM.1S-R and RPMI8226-R cells were incubated with d18:1_16:0 CER (20 μM) and/or indicated amounts of BTZ for 16 hours, followed by immunoblotting with indicated antibodies. Shown immunoblots are representative images of at least 2 independent experiments with no tangible experimental variability. (D) BTZ-R MM.1S-R and RPMI8226-R cells were treated or not with the indicated amounts of d18:1_16:0 CER and/or BTZ for 24 hours and probed in trypan blue cell viability assay. (E) BTZ-R MM.1S-R and RPMI8226-R cells were treated for 24 hours with indicated amounts of VLDL and/or BTZ and probed in trypan blue cell viability assay. All viability data are presented as the mean values of triplicates ± standard errors of the mean. P values were determined by Student t test. *P < .05, **P < .01, ***P < .001. VLDL, very low density lipoprotein.