H 3 bovine serum albumin in PBS for 30 minutes at room temperature. After washing with PBS, the cells were incubated with specific primary antibody at 4 overnight. The cells were then washed and incubated with Alexa Fluor 488- or 555-conjugated goat anti-rabbit IgG diluted in blocking solutions and incubated for 1 hour. The nuclei were stained with 4,6-diamidino2-phenylindole (DAPI). Sections were visualized by fluorescence microscopy. SP cells were cultured under differentiating conditions (DMEM supplemented with 10 FBS in the absence of growth factors) for 7 days to allow cells attachment and differentiation. In addition, SP cells were treated with 100 M sesamin for 7 days in DMEM/F12 medium supplemented with 20 ng/mL EGF and 10 ng/mL bFGF. The acquisition of epithelial markers (E-cadherin) and loss of mesenchymal markers (Vimentin) were evaluated by immunofluorescence as indicated above.Cell proliferation assaySorted SP cells and non-SP cells were cultured in 96-well plates at a density of 2 ?104 cells/mL in DMEM/F12 medium supplemented with 20 ng/mL EGF and 10 ng/mL bFGF. Conditioned medium was collected over 48 hours and the IL-6 concentrations were tested utilizing the human IL-6 ELISA Development Kit (Peprotech) according to the manufacturer’s instructions. Briefly, culture medium and IL-6 standards were incubated for 2 hours at room temperature in 96-well microplates, which were coated with IL-6 mAb. After washing, an antibody against IL-6 conjugated to alkaline phosphatase was added. Substrate and amplifier were added and the plates were read at 485 nm. Similar procedures were performed to study the effects of sesamin (0, 11, 33.3, 100 M) for 7 days on IL-6 production.Real-time PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26080418 reverse transcriptase PCR analysisFor non-SP cells, SP cells and SP cells exposed to sesamin (0, 11, 33.3, 100 M) for 7 days, total cellular RNA were collected from cells using 1 mL of TRIzol reagent (Invitrogen), then the samples were reverse-transcribed using random hexamers and reverse transcriptase (Invitrogen) to obtain cDNA. The expression levels of IL-6 mRNAs were determined by real-time reverse transcriptase-PCR. All reactions were carried out on 96-well PCR plates (ABI PRISM, Applied Biosystems) in an ABI PRISM 7000 sequence detection system. Standard thermal cycling conditions are a hot start at 50 for 5 min, 95 10 min, followed by up to 50 cycles of: 95 15 sec, 60 for 1 min. Data shown are normalized to GADPH expression. Primer sequences were as follows: IL-6, 5-GAGAAAGGAGACATGTAACAAGAGT-3 (forward), 5-GCGCAGAATGAGATGAGTTGT-3 (reverse); GADPH, 5-TGGGGAAGGTGAAGGTCGG-3 (forward), 5-CTGGAAGATGGTGATGGGA- 3 (reverse).Cell proliferation assays were conducted using the CCK-8 assay kits as described by the manufacturer. Sorted SPKong et al. BMC Complementary and Alternative Medicine 2014, 14:254 http://www.biomedcentral.com/1472-6882/14/Page 4 ofWestern blot analysisFor non-SP cells, SP cells and SP cells exposed to sesamin (0, 11, 33.3, 100 M) for 7 days, nuclear and cytosolic proteins were prepared using NE-PER Nuclear and Cytoplasmic Fractions Kit purchased from Thermo Scientific. CynarosideMedChemExpress Cynaroside Protein content was determined by Bradford assay. Equal amounts (30?0 g) of proteins were applied to an 8-12 SDSpolyacrylamide separating gel and transferred to a PVDF membrane (Millipore). The membrane was blocked with 5 skim milk or 1 BSA in TBST and then probed with indicated primary antibodies with gentle shaking at 4 overnight. Primary antibodies against N.
