Mda mb 231 and mcf 7 cells

HEK293T cells, a non-cancerous human embryonic kidney epithelial cell line with a stable expression of the SV40 large T antigen, were obtained from the RIKEN BioResource Center (Tsukuba, Japan). Human pancreatic cancer PANC-1, AsPC-1, and MIA PaCa-2 cells, human breast cancer MDA-MB-231 and MCF-7 cells, human prostate cancer PC-3 and LNCaP cells, human squamous cancer A431 cells, human cervical cancer HeLa cells, human melanoma MeWo cells, human mesothelioma MSTO-211H cells, and human lung cancer NCI-H2170 cells were obtained from the American Type Culture Collection (Rockville, MD). All cell lines were cultivated in DMEM/F12 (D/F) medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS).
Human neutrophils were isolated from the blood of a healthy donor by the conventional method. In brief, the polymorphonuclear cell fraction was first collected from the whole blood by centrifugation at 2000 rpm for 30 min using the PolymorphPrep™ solution (Serumwerk Bernburg, Bernburg, Germany). The collected cells were then incubated with CD16 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and the unbound flow through the fraction was collected as the neutrophil-enriched fraction.

Sense and antisense oligonucleotides for shRNAs targeting human Bub1 (5′-GGAAGTGCCTCATGCTGAAGA-3′, 5′-GCTGCACAACTTGCGTCTACA-3′, 5′-GGGACTGTTGATGCTCCAAAC-3′) were generated, annealed, and cloned into the pSuper.puro vector, according to the manufacturer’s instructions (Oligoengine, Seattle, WA, USA). For Bub1-depleted stable clones, MDA-MB-231 or MCF7 cells were transfected with each shRNA, subject to puromycin selection 36 hrs after transfection. Bub1 expression of stably transfected clones was verified by western blot analysis. shRNAs targeting human RHAMM (TRCN0000061555 and TRCN0000333646) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MDA-MB-231 and MCF7 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell culture, transfection, and irradiation were performed as described previously50 (link)51 (link).