Figure 3.

Effect of metformin on GRP78 and bortezomib-induced autophagy. (a) Western blot to determine the effect of bortezomib treatment on GRP78 levels in RPMI8226 cell lysates. Cells were treated with bortezomib at indicated concentrations for 24 h, lysates prepared and probed using a GRP78-specific antibody. (b) Western blot to determine the effect of metformin co-treatment with bortezomib on GRP78 levels in RPMI8226 cells. Cells were treated with bortezomib and metformin at indicated concentrations for 24 h; lysates prepared and probed using an antibody specific to GRP78. (c) Western blot to determine the effect of metformin co-treatment with bortezomib on GRP78 levels in SP and MP cells isolated from RPMI8226. Cells were treated with bortezomib and metformin at indicated concentrations for 24 h; lysates prepared and probed using an antibody specific to GRP78. (d) Effect of metformin co-treatment with bortezomib on the conversion of LC3B-I to LC3B-II in RPMI8226 cells. Cells were treated with bortezomib (5 nM), metformin (500 μM) for 24 h. Bafilomycin A1 (100 nM), a specific inhibitor of the vacuolar type H⁺-ATPase was then added for the final 4 h of incubation to inhibit autophagy flux and to prevent autophagosome recycling and lead to LC3B-II accumulation. Lysates were prepared and probed by western blot to detect the relative levels of LC3B-I and LC3B-II. (e) Effect of bortezomib (5 nM) and metformin (500 μM) alone and combined on the levels of GRP78 and autophagosomes in RPMI8226 cells. Cells were treated with drugs for 16 h and GRP78 was then visualized by immunohistochemistry staining and confocal microscopy. Autophagosomes were detected using the cytologic identification (cyto-ID) autophagy detection kit (Enzo Life Sciences, Farmingdale, NY, USA) and visualized using a Zeiss LSM710 confocal microscope (Zeiss Microscopy, Thornwood, NY, USA). Settings for fluorescein isothiocyanate detection were EX_488nm and EM_550nm. (f) Effect of HSPA5-specific shRNA on autophagosome formation. RPMI8226 cells were transfected with control or HSPA5-specific shRNA and transfectants treated as indicated. Autophagosomes were detected by dye-based staining and confocal microscopy. (g) Autophagosomes were detected by the dye-based method and the relative amount of green fluorescence was determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Statistically significant differences in fluorescence are indicated by *. (h) Effect of bortezomib (5 nM) and metformin (500 μM) alone and combined on autophagosome formation in SP cells. SP were treated with drugs as indicated for 16 h and autophagosome formation visualized by dye-based staining and confocal microscopy. Shown are representative images observed in multiple experiments.