The cells were incubated at 37C with 5% CO2 and fed by the same medium for 1C2 week, twice per week

The cells were incubated at 37C with 5% CO2 and fed by the same medium for 1C2 week, twice per week. declaring the anti-proliferation efficacy of MK2 inhibitor IV in MM cells and gained further insights to determine whether MK2 inhibitor is more effective than monotherapeutic agent (such as bortezomib, doxorubicin, or dexamethasone). Additionally, combination of MK2 inhibitor IV and bortezomib, doxorubicin or dexamethasone presented better inhibition effect on MM cell proliferation. Materials and Methods Cell Lines and Cell Culture Human MM cell lines, APR1, and OCI-MY5 cells were maintained Serping1 in RPMI 1640 (Gibco, Grand Island, NY) and supplemented with 10% fetal bovine serum (FCS) (Gibco, Grand Island, NY), penicillin and streptomycin solution (100 g/mL, Sigma, St. Louis, MO) under the condition of 37C in a humidified atmosphere of 5% CO2. Reagents MK2 antibody, MK2 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) lentiviral activation particles, and MK2-shRNA lentiviral particle were purchased from Santa Cruz Biotechnology (Dallas, Texas). Caspase-3, PARP, -actin, and pAKT antibodies were obtained from Cell Signaling Technology (Danvers, MA). Trypan Blue, dexamethasone and doxorubicin were from Sigma (St. Louis, MO). Bortezomib was purchased from Selleck Chemicals (Houston, TX). MK2 inhibitor IV was purchased from Cayman Chemical (Ann Arbor, MI). Cell Growth Assays Following cell transfection or 48 h after different drug treatment, MK2 inhibitor IV IC50 15 M; bortezomib IC50 80 nM; dexamethasone IC50 25 M; doxorubicin IC50 1.5 M; MK2 inhibitor IV IC25 4.75 M + bortezomib IC25 46.2 nM; MK2 inhibitor IV IC25 4.75 M + dexamethasone IC25 6.6 M; MK2 inhibitor IV IC25 4.75 M + doxorubicin IC25 0.3 M. Cell growth was evaluated using MTT assay according to the method described in the literature (13). In short, cells were seeded at 4,000~5,000 cells/well in 96-well plates, 10 L of MTT and 90 L of complete medium were added to each well according to the manufacturer’s instructions, and the culture plate was incubated at 37C for 4 h. Then, the culture medium was removed and 150 L dimethyl sulfoxide was added to each well. After vigorous shaking for 10 min, the absorbance was read by a fluorometer (CytoFluor; Applied Biosystems, Foster City, CA, USA) at 570 nm. Cell Proliferation Cells were enumerated using a hemocytometer. The fraction of dead cells was determined by trypan blue staining. Flow Cytometry A flow cytometry assay was applied to analyze the cell cycle distribution as previously reported (14). Briefly, samples were washed with PBS and stained with PI solution (Yeasen, Shanghai, China) for 30 min. All samples were E7449 analyzed using FlowSight (Merck Millipore, Darmstadt, Germany). Western Blot Analysis Western blots were utilized to measure the protein levels of MK2, pAKT and apoptotic markers in MM cells. In brief, around 20 g protein per sample was extracted and whole-cell lysate were electrophorezed by 4C12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. After incubation with primary and second antibodies, blots E7449 were subsequently stripped and re-probed for -actin as protein controls. The immunostaining band was quantified using Image J software. Soft Agar Clonogenic Assay Clonogenic formation was monitored by plating 10,000 MM cells in 0.5 mL 0.33% agar RPMI 1640 medium (Invitrogen, Carlsbad, CA) with 10% FBS in 12-well plate. The cells were incubated at 37C with 5% CO2 and fed by the same medium for 1C2 week, twice per week. The colonies were imaged and colony numbers were counted by Image J. Apoptosis and Dye-Efflux Multidrug-Resistance Assays Drug-affected apoptosis following E7449 exposure to bortezomib or doxorubicin was detected in MM cells using flow-cytometric Annexin V apoptosis detection kit APC (catalog number 88C8007) from eBioscience (San Diego, CA). In brief, one million cells were washed and suspended in 1 mL binding buffer, followed by adding the Annexin V-APC (5 L) to the cells suspension (100 L). Then, cells were incubated for 15 min at room temperature, washed and re-suspended in binding buffer. The labeled cells were analyzed via flow cytometry. The eFluxx-ID? Multidrug resistance assay was applied to measure drug.