The antibodies to Caspase3 (#9662), Rad51 (#8875), MLH1 (#3515), Chk1 (#2360), Gamma H2A.X (#9718), MSH2 (#2850), Topoisomerase II (D10G9) (#12286) were purchased from Cell Signaling; PMS2 (#2251.00.02), MSH6 (#2203.00.02), were purchased from Sdix, Alpha tubulin – Abcam (ab15246); Lamin A/C -Cell Signaling (#2032) and beta-Actin (#sc-81178) was purchased from Santa Cruz Biotechnology. were obtained from the Ohio State University Leukemia Tissue Bank after getting informed consent approved by the cancer Institution Review Board. Primary cells were thawed and death cells were removed using dead cell removal kit (Miltenyi Biotec) according to manufacturers instructions. The cells were allowed to recover overnight (16C18 hrs) after which drug treatment studies were carried out. Primary cells were cultured in StemSpan SFEM (STEMCELL Technologies) supplemented with StemSpan CC100 cytokine cocktail (STEMCELL Technologies) and 20% FBS. Compounds Selinexor was obtained from Karyopharm Therapeutics. Idarubicin, mitoxantrone and etoposide were purchased from Selleckchem. Daunorubicin was purchased from Sigma. LY2228820 (Ralimetinib) Taqman Gene Assays and Antibodies All the real time PCR Taqman gene assays were purchased from Life Technologies (MSH2: Hs00953523_m1; MLH1: Hs00179866_m1; MSH6: Hs00264721_m1; PMS2: HS00241053_m1; Rad51: Hs00153418_m1; Chk1: Hs00967506_m1). The antibodies to Caspase3 (#9662), Rad51 (#8875), MLH1 (#3515), Chk1 (#2360), Gamma H2A.X (#9718), MSH2 (#2850), Topoisomerase II Rabbit Polyclonal to HEXIM1 (D10G9) (#12286) were purchased from Cell Signaling; PMS2 (#2251.00.02), MSH6 (#2203.00.02), were purchased from Sdix, Alpha tubulin – Abcam (ab15246); Lamin A/C -Cell Signaling (#2032) and beta-Actin (#sc-81178) was purchased from Santa Cruz Biotechnology. The secondary antibodies for Western Blotting were purchased form LI-COR and for immunofluorescence were purchased from Invitrogen (#A11008). Real-time quantitative reverse transcription-PCR Cells were treated with the indicated selinexor concentrations and cells were collected at different time points. RNA was extracted from cells using RNeasy Kit (#74106, Qiagen) and reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit (#4368813, Applied Biosystems). mRNA for the indicated genes was quantified using ViiA7 Real-Time PCR system and analyzed by the V1.2 software (Life Technologies). Trizol/chloroform extraction step was performed for the primary AML samples prior to the actual RNA extraction step. Immunofluorescence Cells were exposed to the indicated treatment regimen. 100C200uL of the cell suspension from each treatment condition was loaded onto a cytospin cuvette with a coverslip and was spun at 800 rpm for 5mins using a Cytospin. The adhered cells were fixed using ice-cold 100% methanol for 15mins, washed with 1XPBS and then blocked/permeabilized using a solution containing 0.1% Tween-20, 0.3M Glycine, 1%BSA in 1XPBS. The cells were incubated with the primary antibody overnight at 4C. The cells were washed 3 times with 1XPBS, incubated with 1:2000 of the secondary antibody for 1hr. The cells were washed with 1XPBS, treated with 1:1000 1ug/mL Dapi for 5mins and then mounted to a glass slide using Vectashield mounting medium (# H-1400, Vector Laboratories). Western Blotting Cells were washed with 1X PBS and then lysed with RIPA LY2228820 (Ralimetinib) buffer (#89901, Thermo Scientific) supplemented with protease inhibitor (# 05892791001, Roche) and phosphatase inhibitor (# 04906837001, Roche). The nuclear and cytoplasmic fractions were isolated using the NE-PER? Nuclear and Cytoplasmic Extraction Reagents (#78833, Thermo Scientific) according to manufacturers protocol. The protein level of each sample was quantified and normalized using BCA assay (#23225, Thermo Scientific). 20ug of each sample were run in 4C12% Bis-Tris Gel (Life Technologies) and later transferred to nitrocellulose membrane using iBlot Gel Transfer Kit (Life Technologies). The membranes were blocked using LI-COR blocking buffer (#927-40000, LI-COR), probed with the indicated antibodies and analyzed using Licor Odyssey. WST-1 assay and Calculation of Combination Index Cells were seeded into 96-well plates (50,000 cells per well) and treated for 48 hrs with individual drug- selinexor, idarubicin, daunorubicin, mitoxantrone, LY2228820 (Ralimetinib) etoposide, or the combination of selinexor with one other individual Topo II inhibitor drug. Cell viability was evaluated using the cell proliferation reagent WST-1 (Roche, Germany) according to manufacturers protocol. The absorbance of wells at 450 nm (reference wavelength 650 nm) was measured with a micro-plate reader (SoftMax Pro, Molecular Devices). The doses for each drug was chosen according to their individual IC50 (2 fold dilutions) that was determined previously by WST-1 assay. For sequential treatments, the second drug was added 24 hrs after the first drug treatment without washing. Plates were read 48 hrs after second drug was added. The effects of the combinations were calculated using CalcuSyn software, where CI 1 indicates synergy, CI=1 is additive and CI 1 is antagonistic. HR Assay HR was assessed using a direct repeat green fluorescent protein (DR-GFP) assay essentially as previously described (36). The HeLA-DR cells possess an integrated DR-GFP construct, whose.
The antibodies to Caspase3 (#9662), Rad51 (#8875), MLH1 (#3515), Chk1 (#2360), Gamma H2A