Were generated by selection with the appropriate drug. The short hairpin
Were generated by selection with the appropriate drug. The short hairpin RNA (shRNA) constructs were purchased from SigmaAldrich (St Louis, MO, USA) (SHCLND-NM_198095) and lentiviral particles were generated at the Gene Transfer Vector Core at the University of Iowa.Flow cytometryFive-week-old C57BL/6NCr and BALB/cAnNCr female mice were used. Mice were sacrificed when they became moribund. Tumor volume (TV) was calculated as: TV = 0.5(length*width2) [22]. Tumor latency was calculated as the number of tumor-free injected mice/number of injected mice ?100. To assess morbidity, the following clinical score ranking was used: (0) no abnormal clinical signs, (1) ruffled fur but lively, (2) ruffled fur, activity levelCell monolayers were washed with PBS and treated with Versene (Life Technologies). Single cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse BST-2 (eBioscience, San Diego, CA, USA), allophycocyanin (APC)-conjugated anti-human BST-2 (BioLegend, San Diego, CA, USA), and appropriate immunoglobulinMahauad-Fernandez et al. Breast Cancer Research (2014) 16:Page 3 ofGs (IgGs) [16,17] at 4 for 1 hour. After washing, cells were incubated with a fluorescent intercalator – 7aminoactinomycin D (7-AAD) (BioLegend) at 4 for 30 minutes to assess cell viability. Stained cells were quantified using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). At least 10,000 events were collected per sample. Fluorescence-activated cell sorting (FACS) data were analyzed by Flowjo software (TreeStar, Ashland, OR, USA).Reverse transcriptase quantitative real-time PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27597769 PCR (RT-qPCR)2 hours. Nonspecific sites were blocked with 40 l of 2 mg/ml bovine serum albumin (BSA) in PBS. Wells were washed once with PBS. Cancer cells were labeled with PKH67 Green fluorescent cell linker, following the manufacturer’s instructions (Sigma-Aldrich). Labelled cells were added to pre-coated wells (20,000 cells/well) and allowed to adhere for 4 hours. Non-adhered cells were washed off with PBS and plates were read at 485 nm/535 nm (excitation/emission) wavelengths using a Tecan Infinite M200 Pro plate reader (Tecan, Maennedorf, Switzerland). Values are represented as relative fluorescence unit.Scratch assayIsolation of RNA was accomplished using the RNeasy mini kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. Equivalent amounts of DNase I (Qiagen)-treated RNA were reverse-transcribed with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA). cDNA was amplified with target-specific primers (GAPDH-Forward: 5-CCCCTTCA TTGACCTCAACTACA-3, Reverse: 5-CGCTCCTGGA GGATGGTGAT-3; mouse BST-2-Forward: TCAGGAG TCCCTGGAGAAGA, Reverse: ATGGAGCTGCCAGAG TTCAC; human BST-2 RT2 qPCR Primer Assays (SABiosciences, Frederick, MD, USA). RT-qPCR was carried out with an ABI 7500 FAST thermal PD-148515 manufacturer cycler (Applied Biosystems) as previously described [24].Western blotConfluent monolayers of cancer cells plated in 12-well plates were scratched using a pipette tip. Fresh medium was added to the wells. Cells were allowed to migrate for 0, 6 or 24 hours before fixation (4 paraformaldehyde (PFA) for 45 minutes). Fixed cells were washed (1 ?PBS) and imaged with a Nikon Eclipse Ti microscope adjusted with a Nikon digital sight camera (Nikon, Tokyo, Japan). Images were processed and migrated cells counted using Image J software.Boyden chamber assayWestern blots were performed as previously described [24]. Blots were p.