cells were stained Biochanin A using an annexin V/FITC kit and analysed by flow cytometry following the manufacturer’s instructions. Cells were washed once with PBS. After re suspending the cell pellet in the binding buffer, 5 ll of annexin V/FITC was added to the cell suspension and incubated at room temperature for 10 min. Before adding 10 ll of PI , cells were washed once with PBS and re suspended in binding buffer. The samples were analysed by flowcytometry using Beckman Coulter EpicsXL FlowCytometry and software Expo 32 . Caspase activity assay Caspase Streptozotocin molecular weight 3 activity was determined by measuring enzymatic cleavage of the fluorescent substrate Acetyl Asp Glu Val Asp 7 amido 4 trifluoromethyl coumarin , using 15 lg of proteins from cell lysates and 20 lmol/l of substrates .
The fluorescence signals were detected by a fluorometer at excitation Agomelatine price and emission wavelengths. Caspase 3 proteolytic activity was expressed as the increase of fluorescence units. Measurement of ROS production Superoxide anions were detected as previously described by Chauhan et al . Briefly, serum starved MM cells were treated with bortezomib , PXD101 , or both for 15 h, harvested and stained with membrane permeable dye dihydroethidium for 20 min at 37 C. Dihydroethidium is converted to a red fluorescent product, ethidium, in the presence of cellular oxidants, particularly superoxide radicals. The cells were washed and resuspended in PBS for FACScan flow cytometry analysis using Beckman Coulter Epics XL Flow Cytometry and Expo 32 software. Fluorescent signals were displayed as histograms.
To determine chemical library the protection of traditional antioxidant N acetyl l cysteine against bortezomib/PXD101 mediated oxidative stress, the cells were pretreated with 5 mmol/l NAC for 3 h, and then exposed to combined drugs with NAC in the cultures. Western blot analysis Western blotting was performed as previously described . The treated cells were harvested, washed twice with cold PBS, and lysed with radioimmunoprecipitation assay buffer containing phosphatase and protease inhibitors . Cell lysates were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane . The blots were probed with antibodies against caspase 3, 8, 9, phospho p53 , phospho H2A.X, phospho p38 mitogen activated protein kinase , Acetyl Tubulin, Bcl 2, Bim, Bax, IkB a and phospho IkB a , p21, and Mcl 1 .
The stripped membrane was re probed with nausea b actin antibody to confirm equal loading. Immune complexes were detected using Amersham enhanced chemiluminescence Western blotting detection reagents . Osteoclast formation assay Non adherent mononuclear bone marrow cells were isolated from the healthy donors by overnight plastic adherence and seeded in 96 well multi plates at 105 cells/100 ll/well in aMEM containing 20% horse serum, 10 ng/ml M CSF and 50 ng/ml RANKL . For dose dependent inhibition assays, the following doses were tested: PXD101 10, 25, 50, 75, 100, 1000 nmol/l, and bortezomib 01, 1, 10, 100 nmol/l, alone or in combination. The drugs were added into appropriate wells every time half media changes were performed . The cultures were incubated for a total of 3 weeks at 37 C/5% CO2. OCL formation was assessed by staining with monoclonal antibody.