Figure 3. Effect of bortezomib and metformin on autophagosome formation.

A. RPMI8226 cells were treated with either bortezomib (10nM), metformin (1mM) or both agents for 18 hours under standard growth conditions. GRP78 was detected by IHC and confocal microscopy. Aggresomes and autophagosomes were detected using the dye-based methods. Shown are representative images seen on in at least three different experiments. B. Relative level of fluorescent intensity of autophagosomes after treatment of RPMI8226 cells with the indicated drugs. C. RPMI8226 cells were transfected with scrambled (control) or HSPA5-specific shRNA, treated with drugs as indicated and GRP78, aggresomes and autophagosomes detected as in Figure 3A. Shown are representative images seen on in at least three different experiments. D. Relative level of fluorescent intensity of autophagosomes after treatment of RPMI8226 cells transfected with either scrambled control or HSPA5 shRNA and then treated with drugs as indicated. E. Co-localization of GRP78 with aggresomes as determined by IHC and confocal microscopy. RPMI8226 cells were treated with bortezomib (10nM), metformin (1mM) or both and stained using a GRP78-specirfic antibody, for aggresomes using dye-based reagent or both the GRP78 antibody and the dye-based reagent. Shown are representative images from multiple experiments. F. Co-localization of GRP78 with autophagosome as determined by IHC and confocal microscopy. RPMI8226 cells were treated with bortezomib (10nM), metformin (1mM) or both and stained using a GRP78-specirfic antibody, for autophagsomes using dye-based reagent or both the GRP78 antibody and the dye-based reagent. Shown are representative images from multiple experiments. G. Effect of bortezomib and metformin on aggresomes and autophagosomes in MM patient tumor cells. Patient bone marrow was obtained, CD138⁺ cells purified, treated with drugs as indicated and aggresomes and autophagosomes detected using the dye-based methods and confocal microscopy.