Fig. 1

TRPV1 expression and the effect of TRPV1 inhibition on MM cell viability in MM BM samples and MM cell lines. a TRPV1 mRNA levels in bone marrow (BM) samples from healthy donors, MM patients and MM cell lines, measured by quantitative RT-PCR. b Effect of TRPV1 antagonist AMG9810 on viability of MM cell lines and primary CD138+ cells. Cells were incubated with increasing doses of AMG9810 for 48 h, and viability was measured by the XTT method. c–e Time course of the increase in AMG9810-induced vesicle acidification, mitochondrial depolarization and cell death. RPMI8226 and CAG MM cells were exposed to AMG9810 (10 µM) for indicated time points. c Vesicle acidification was measured by flow cytometry using acridine orange (AO) dye. Representative flow cytometry histograms from RPMI8226 cells (left side). Graphs showing percentages of AO-positive RPMI8226 and CAG cells obtained from representative experiment out of three repeats (right side). d Mitochondrial depolarization (ΔΨm loss) in AMG9810-treated cells detected by DiOC6 staining and flow cytometry analysis. Representative flow cytometry histograms from RPMI8226 cells (left side) and graphs showing percentages of MM cells with ΔΨm loss obtained from representative experiment out of three repeats (right side). e AMG9810-induced cell death detected by PI staining. Representative flow cytometry histograms from RPMI8226 cells (left side) and graphs showing the percentages of PI-positive dead RPMI8226 and CAG cells from representative experiment out of three repeats (right side). Data are presented as mean of triplicates ± SD (**p < 0.01). f Western blot analysis of anti-apoptotic proteins BCL-XL and MCL-1, ER stress marker CHOP and mTOR pathway target pS6 in RPMI826 and CAG MM cells before and after treatment with various doses of AMG9810 for 24 h. β-Actin was used as internal control. Representative data from at least two independent experiments are shown. g Caspase 3 activation in response to AMG9810 treatment (5–20 µM, 24-h exposure) was assessed using CaspGLOW Red staining and flow cytometry analysis. Representative histograms (upper panel) and graphs showing percentages of cells with activated Caspase 3 (lower panel). Data are presented as mean of triplicates ± SD (**p < 0.01)