Effects of bortezomib on ubiquitin cellular distribution and HR molecules at the posttranscriptional level. (A) Immunoblot analysis for FANCD2 and RAD51 protein expression in MM cell lines treated with 2.5nM bortezomib for 12 and 24 hours. (B-C) Protein extracts from MM1S and OPM2 cells treated for the indicated times with 2.5nM bortezomib were subjected to Western blot analysis. As shown, treatment with bortezomib results in the accumulation of polyubiquitin (B) and decreases the ubiquitylation of BRCA1 (C). (D-G) Immunofluorescent microscopy was used to analyze free ubiquitin and polyubiquitin cellular distribution and to detect γ-H2AX, BRCA1, and RAD 51 foci formation induced by bortezomib and/or ABT-888 treatment in OPM2 and MM1S (images for OPM2 cells are included in the supplemental materials). Cells were co-treated with bortezomib (2.5nM) and/or ABT-888 (5μM) for 24 hours. Shown in panel D are the effects of bortezomib on ubiquitin cellular distribution in MM with rapid depletion of the nuclear ubiquitin pool in bortezomib-treated cells. Cy5-labeled Ab was used to detect free ubiquitin and DAPI was selected for nuclear staining. (E) Effects of ABT-888 and/or bortezomib on γH2AX foci formation and polyubiquitin (FK2). Bortezomib did not affect ABT-888 γH2AX foci formation, but did inhibit the polyubiquitylation of γH2AX. (F-G) Effects of ABT-888 and/or bortezomib on BRCA1 (H) and RAD51 (G) foci formation. BRCA1 and RAD1 foci induced by ABT-888 were completely resolved in cells cotreated with bortezomib. Image acquisition was performed with an epifluorescence microscope (BX51; Olympus) and multispectral color camera (Nuance FX; CRi) with a 60× or 100× magnification lens and oil immersion.