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Fig. 5 | Journal of Hematology & Oncology

Fig. 5

From: Blocking of Transient Receptor Potential Vanilloid 1 (TRPV1) promotes terminal mitophagy in multiple myeloma, disturbing calcium homeostasis and targeting ubiquitin pathway and bortezomib-induced unfolded protein response

Fig. 5

AMG9810 in combination with bortezomib elevates mitochondrial ROS and calcium levels, induces mitochondrial and lysosomal destabilization and promotes ER stress. RPMI8226 and OPM1 cells were exposed to low-dose bortezomib (BTZ) (4 nM) in the absence or presence of AMG9810 (10 µM) for 24 h. a Mitochondrial ROS levels and apoptosis were measured using MitoSOX and Annexin V staining. b Mitochondrial calcium levels were assessed using Rhod-2 staining. c Mitochondrial depolarization (ΔΨm loss) was detected by DiOC6 staining and flow cytometry analysis. d Lysosomal membrane destabilization was assessed using LysoTracker staining. Representative images with RPMI8226 cells are shown in upper panels. Bars showing mean of triplicates ± SD (**p < 0.01) in RPMI8226 and OPM-1 cells are presented in lower panels. e RPMI8226, CAG and OPM-1 cells were treated with bortezomib (5 nM), AMG9810 (10 µM) or their combination for 24 h. RNA was extracted and was subjected to qRT-PCR analysis. CHOP, GADD34 and HO-1 mRNA levels were evaluated, using β2-microglobulin as endogenous control gene. f Western blot analysis of ER stress response chaperons HSP70 and HSP40 in RPMI826, CAG and OPM-1 cells before and after 24-h treatment with bortezomib (5 nM), AMG9810 (10 µM) or their combination. β-Actin was used as internal control. Representative data from at least two independent experiments are shown

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