Fig. 6

Combination of AMG810 with bortezomib suppresses ubiquitin signaling and induces mitophagy in MM cells. a RPMI8226 cells were exposed to bortezomib (5 nM), AMG9810 (10 µM) or their combination for 24 h and subjected to cell proteome analysis. a Heatmap of differentially expressed proteins associated with ubiquitin signaling pathway is presented (p < 0.05). b Western blot analysis of cytosolic and mitochondrial fractions of RPMI8226 cells that were exposed to bortezomib (5 nM), AMG9810 (10 µM) or their combination for 24 h. Blots were probed for poly-ubiquitin, HSP70, AIF, LAMP1, PINK1 and VDAC1. α-Tubulin and COX IV were used as markers for cytosolic and mitochondrial fractions, respectively. c, d Effect of bortezomib (5 nM), AMG9810 (10 µM) or their combination on mitochondrial mass in MM cells. Mitochondrial content was measured using MitoSpy staining and flow cytometry analysis. c Representative histograms showing the decrease in mitochondrial mass staining of RPMI8226 cells. d Graphs showing MitoSpy MFI levels in RPMI8226, CAG and OPM-1 cells presented as mean of triplicates ± SD (**p < 0.01)