All the primer sequences are listed in Additional?file?1: Table S1

All the primer sequences are listed in Additional?file?1: Table S1. Western blot Western blotting was performed as we previously described [5]. JTK12 S.D. (value ?0.05 were chosen as the cut-off criteria. All microarray data were uploaded to Gene Expression Omnibus (accession number, “type”:”entrez-geo”,”attrs”:”text”:”GSE109007″,”term_id”:”109007″GSE109007 and “type”:”entrez-geo”,”attrs”:”text”:”GSE109008″,”term_id”:”109008″GSE109008). siRNA, plasmids, and virus infection siRNAs used to knockdown target lncRNAs or mRNAs were designed by the online tool (BLOCK-iT? RNAi Designer) and synthesized by GenePharma (Suzhou, China). For overexpression, the full length of LincK was inserted into lentivirus expression vector PCDH-CMV-MCS-EF1-puro (termed as LV-Control thereafter). For knockdown, lentivirus shRNA expression vectors targeting the same sequences as siRNAs were constructed and packaged by GenePharma. In vitro migration and invasion assay For MCF-7 and MDA-MB-453 (with LincK overexpression, knockdown, or their corresponding controls), tumor cells were co-cultured with hAD-MSCs for 2?weeks before being subjected to the migration and invasion assay. Then, tumor cells were resuspended in 200-l serum-free 1-Naphthyl PP1 hydrochloride medium at a density of 1 1??106/ml and seeded into the upper chamber of 24-well Transwell chambers (8-m pore, costar) coated without (migration) and with (invasion) Matrigel (BD Biosciences). The lower chambers were filled with 600?l of medium containing 20% FBS. After 24?h (migration) or 36?h (invasion), cells on the lower surface of the inserts were stained with 0.1% Crystal Violet. For MDA-MB-231 cells, 5??104 cells were added into the top chamber and permitted to migrate for 8?h; 2??105 cells were seeded into the top chamber coated with Matrigel and permitted to invade for 24?h. Three randomly selected fields per filter were counted. Colony formation assay For MCF-7 and MDA-MB-231 cell lines, 2000 cells were suspended in 5?ml complete medium and seeded in a 60-mm dish. For 1-Naphthyl PP1 hydrochloride MDA-MB-453 cell line, 2??104 cells were cultured in a 6-mm dish. After 2?weeks, colonies were stained by 0.1% crystal violet. Photos of colonies were taken by Cannon EOS 600D and number of colonies were analyzed by ImageJ software. RNA extraction and quantitative reverse transcription-polymerase chain reaction Total RNA was extracted using the Trizol reagent (Invitrogen), and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of mRNA and miRNAs was performed as we previously described [5]. All the primer sequences are listed in Additional?file?1: Table S1. Western blot Western blotting was performed as we previously described [5]. Antibodies against the following proteins were obtained as indicated: ZEB1, E-cadherin, N-cadherin, ZO-1, Vimentin, and GAPDH (Proteintech, China); PBK, p38 MAPK, and phosphorylated p38 MAPK 1-Naphthyl PP1 hydrochloride (Cell Signaling Technology?). Cell proliferation assay Cells were plated in 96-well plates (2000 cells/well). Cell proliferation was determined every 24?h for 5?days according to the manufacturers instructions. Briefly, 10?l of MTS (#G3582, Promega) was added to each well. After incubation at 37?C for 1?h, the absorbance at 490?nm was detected. BrdU proliferation assay Cell proliferation was monitored using the BrdU-ELISA kit (#11647229001, Roche) according to the manufacturers instructions. Briefly, 1??104 cells were plated in 96-well plates for 48?h and then labeled with BrdU for 2?h. After incubation with BrdU antibody-peroxidase (POD), photometric detection was performed at 370?nm wavelength. Northern blot Northern blots were performed using the DIG Northern Starter Kit (#12039672910, Roche) as we described previously [20]. Digoxigenin (DIG)-labeled LNA probes were designed using online software (Stellaris probe designer) and synthesized by Exiqon. 5 and 3 rapid amplification of cDNA ends The transcriptional initiation and termination sites of LincK were detected using the FirstChoice RLM-RACE Kit (#AM1700, Ambion) according to the manufacturers instructions. The primer sequences are listed in Additional file?1: Table S1. Subcellular fractionation The separation of the nuclear and cytosolic fractions was performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (#78833, Thermo Scientific) according to the manufacturers instructions. RNA was extracted, and qRT-PCR was performed to assess the relative proportion in the nuclear and cytoplasmic fractions. Dual luciferase reporter assay Wild types of full-length LincK and 3UTR of ZEB1 and PBK were obtained by PCR or RT-PCR and cloned into the luciferase reporter vector psiCHECK2 (Promega). Mutants were prepared by deleting of 16 base-pair binding sequences of miR-200b. The cells were harvested 24?h after transfection, and Renilla and firefly luciferase activity were analyzed using the Dual-Luciferase? Reporter Assay System (#E1910, Promega). RNA immunoprecipitation assay The Ago2-LincK RNA immunoprecipitation (RIP) assay was performed with the EZ-Magna RIP Kit (Millipore). The AGO2 antibody 1-Naphthyl PP1 hydrochloride was purchased from Millipore (#03-110, Merck). The Flag-MS2bp-MS2bs-based RIP assay was conducted according to previous reports [21]. Briefly, 293?T cells were co-transfected with pcDNA3-Flag-MS2bp and LincK-MS2bs or mutant-MS2bs, and the cell lysates were harvested.