Assessment of autophagy. (A) Confocal images of RPMI8226 cell line treated with bortezomib, tipifarnib, or the combination for 24 hours. Images were acquired using a Zeiss LSM 510 confocal mounted on a Zeiss Axioplan 2 microscope. A 63× oil objective (NA 1.4) was used for image acquisition using Zeiss LSM software Version 4.2. All images were acquired with equivalent acquisition settings to allow for intensity measurements between samples. Images were processed in Adobe Photoshop (CS3) with equal contrast expansion for image display. Cells were stained with antibodies against ubiquitin and p62 SQSTM1antibody as well as 4,6-diamidino-2-phenylindole. Colocalization of ubiquitin and p62SQSTM1 in the merged image is visualized in yellow. (B) Confocal images of RPMI8226 cells that were treated with bortezomib and tipifarnib combination for 24 hours. Cells were stained for ubiquitin and then separately stained with vimentin and γ-tubulin. Colocalization of ubiquitin and vimentin in the merged image is visualized in yellow. γ-Tubulin is visualized in the periphery of ubiquitin aggregates distinct from the ubiquitin aggregates. (C) Western blotting for p62SQSTM1 and LC3BII inMM.1S cells and also in RPMI8226 cells in the presence and absence of bafilomycin A1. Relative intensity of LC3BII was calculated by normalizing the untreated control to a relative intensity of 1.0 and then dividing subsequent LC3BII band intensity by actin band intensity in the same treatment group using densitometry.
