Bortezomib (Velcade)

PS-341 impairs FPV replication in A549 cells. (A and B)A549 cells were either pretreated for 1 h with different concentrations of PS-341 or with solvent only or were left untreated. Then, cells were infected with FPV at an MOI of 0.001 (A) or 0.05 (B). After virus inoculation cells were posttreated with different concentrations of PS-341. (A) At 24 h p.i. supernatants were obtained and progeny virus titers were measured by standard plaque assay. (B)Proteasome activity and the ability of PS-341 to inhibit the proteasome was determined 24 h p.i. (C) A549 cells were pretreated with 50 nM PS-341 or solvent or left untreated for 1 h. Afterwards cells were infected with FPV at an MOI of 0.0005. Subsequent to virus inoculation cells were posttreated with 50 nM PS-341 or solvent or left untreated. After the indicated times p.i.supernatants were obtained and progeny virus titers were determined by standard plaque assay. Arrow bars in all experiments represent standard deviations of three independent experiments.

Early steps of viral replication within the first replication cycle are affected. (A) For time-of-addition kinetics analysis, A549 cells were either left untreated or were pretreated for 10 h or 1 h with 50 nM PS-341 before infection and additionally posttreated after infection. Cells were infected with FPV at an MOI of 0.005. After virus inoculation cells were posttreated with 50 nM PS-341. Then the proteasome inhibitor was added after virus inoculation (10 h, 1 h, and 30 min) or it was added at the different times p.i. as indicated (1 h, 2 h, 4 h, 6 h, and 8 h; cells were not pretreated before infection). At 9 h p.i. supernatants were obtained and progeny virus titers were determined by standard plaque assay. Shown is one representative experiment out of three independent experiments. (B) A549 cells were pretreated with 50 nM PS-341 or left untreated for 1 h. Afterwards cells were infected with avian FPV or human PR8 at an MOI of 1. Subsequent to virus inoculation cells were posttreated with 50 nM PS-341 or left untreated. After the indicated times p.i. cells were lysed and analyzed by Western blotting for accumulation of viral proteins polymerase PB1 and matrix protein M1. Cellular protein ERK2 served as a control to demonstrate equal amounts of protein loading. Shown is one representative blot out of three independent experiments.

A549 cells were treated with PS-341 at 50 nM for the indicated times or left untreated. Western blotting was performed with total cell lysates, using phospho-specific antibodies against JNK and the transcription factors c-Jun and ATF-2 or loading controls, respectively.

The stable cell line HepAD38 was incubated for 18 h in the presence of the indicated amount of Bortezomib. The medium was removed and replaced by medium containing Bortezomib dissolved in PBS. In case of the control cells the same amount of PBS was added to the medium. 4 h later this procedure was repeated and again 14 h later the supernatant was collected. The amount of viral particles was quantified by real time PCR. HBV-genome quantification was done using COBAS® AmpliPrep/COBAS® TaqMan® HBV test (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. The assay shows relative values (the value for untreated control cells was arbitrarily set as 1) that are based on three independent experiments. The cell viability was analyzed by MTT assays. For does up to 50 nM no significant effect on cell viability was observed within 18h, for 100 nM the proportion of metabolically active cells was reduced to 83%.

Western blot of extracts of infected cells treated with different proteasome inhibitors at different concentrations, reacted with the indicated antibodies. p53 was used to monitor proteasome inhibition, and actin was used as a loading control.

Time window treatment with proteasome inhibitors. (A) Scheme of the experiment performed with MA104 cells exposed to virus (OSU; MOI, 3) for 1 h and analyzed at the starting point and endpoint of the indicated time window treatments with DMSO, MG132, or bortezomib. (B) Western blot of cellular lysates derived from cells infected for the indicated time periods and treated with the proteasome inhibitors or DMSO. NI, noninfected cells. Blots were reacted with the indicated antibodies; p53 was used to monitor proteasome inhibition, and actin was used as a loading control.

Fluorescence analysis of viroplasm formation on NSP5-EGFP cells infected with rotavirus (OSU; MOI, 3) and treated or not treated with MG132 (10 M) or bortezomib (10 M) at different times p.i., as indicated. Cells were analyzed at the starting points (1 h, 3 h, 5 h, 7 h) and endpoints (9 h) of the inhibitor’s window treatment.

Quantification of the accumulation of viroplasms in infected NSP5 -EGFP/MA104 cells. At different times p.i., cells were treated for 4 h with DMSO or the indicated proteasome inhibitor and the number of viroplasms/cell was quantified at the starting (1 h, 3 h, 5 h; white bars) and endpoints (5 h, 7 h, 9 h) of treatment.

Control wild-type and Fmn2–/–oocytes observed at different stages of meiotic maturation [prophase I (Pro I), NEBD, 3 hours and 8 hours after NEBD] using anti-Fmn2. wt + Bortezo, wild-type oocytes treated with 0.1M Bortezomib for 90 minutes before fixation. All oocytes were observed using the same settings and the images treated the same way (three independent experiments). Red arrows indicate cortical labeling. Scale bar: 10m.

LNCaP-AI cells were starved in 1% stripped medium for 24 h. The cells were then treated with Erlotinib (20 lM), Gefitinib (20 lM), Lapatinib (20 lM), CI-1033 (8 lM), LY294002 (20 lM) and Bortezomib (20 lM) for 24 h. Cell culture medium was collected from each sample and subjected to ELISA for sPLA2-IIa. The condition medium samples were diluted 10 times for ELISA. Average of duplicate samples was converted to nanogram per milliliter against standard curve. The data represent one of five repeated experiments.

(B–C) LNCaP (B) and LNCaP-AI (C) cells were transiently transfected with sPLA2-IIa(-800)-Luc (0.5 lg). The cells were then treated with Erlotinib (20 lM), Gefitinib (20 lM), Lapatinib (20 lM), CI-1033 (8 lM), LY294002 (20 lM) and Bortezomib (20 lM) without or with EGF (100 ng/ml) for 24 h. Luciferase assay was performed according to a standard protocol with Renilla luciferase as an internal control. Data are presented as the mean (±SD) of duplicate values of a representative experiment that was independently repeated for five times.