Data Availability StatementThe datasets used and/or analyzed during the current study
Data Availability StatementThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. of methyl–cyclodextrin, an agent to disrupt caveolae and lipid rafts led to a downregulation of caveolin-1 that reduced the expression of KCNA5. Furthermore, following caveolin-1 knockdown, the expression of KCNA5 was decreased in MDA-MB-231 human breast cancer and MCF-10A-neoT non-tumorigenic epithelial cell lines. In subsequent experiments, the MTT assay showed that increased caveolin-1 and KCNA5 expression promoted the survival of MCF-7 human breast cancer CP-724714 kinase activity assay cells, but cell survival was not affected following KCNA5 overexpression alone. Using small interfering RNA technology, KCNA5-silenced MCF-10A-neoT cells were established and a decreased level of phosphorylated-AKT serine/threonine kinase (AKT) was observed in the cells compared with the parental cells. Overall, these results suggested that caveolin-1 facilitated KCNA5 expression and may be associated with AKT activation. showed that KCNA5 can localize to caveolae microdomains, and KCNA5 was associated with caveolae (27). However, we are unknown for the mechanisms controlling their interactions and the physiological functions of this localization. Recent research has found a role of Cav-1 in transporting proteins to the cell membrane (30). And according to some recent studies, Cav-1 regulates proteins that co-localize with it, such as estrogen receptor (ER), KCNA5, and desmoglein 2 (Dsg2) (31C33). However, the role of Cav-1 in mediating the membrane localization of KCNA5 channel has not been elucidated. Our previous study demonstrated that Kv channels were required for the viability of the normal MCF-10A-neoT cells (7). In this study, we described that KCNA5 and Cav-1 co-localize in the cytoplasm of MCF-7 human breast cancer cells. The study also found that the knockdown KCNA5 inhibited the PI3K/AKT signaling pathway in MCF-10A-neoT cells, and cells upregulated CP-724714 kinase activity assay with Cav-1 and KCNA5 promoted survival in MCF-7 cells through PI3K/AKT signaling. In addition, it was showed that the downregulation of Cav-1 decreased the expression of KCNA5, indicating that Cav-1 was involved in the KCNA5-promoted survival of human mammary cells. Materials and methods Plasmids and antibodies The KCNA5 plasmid was from Dr Jie Zheng (University of California, Davis). The Cav-1 plasmid and siRNA plasmid specific for Cav-1 (target sequence Oligo 1, 5-ACCTCATTAAGAGCTTCCTGATTGAGTCAAGAGCTCAATCAGGAAGCTCTTAATTT-3, Oligo Rabbit polyclonal to CD14 2, 5-CAAAAAATTAAGAGCTTCCTGATTGAGCTCTTGACTCAATCAGGAAGCTCTTAATG-3) were obtained from the Cancer Center at Creighton University. Anti-KCNA5 (rabbit polyclonal, 1:500; EMD Millipore, Billerica, MA, USA), anti-Cav-1 (mouse monoclonal, 1:1,000, Santa cruz biotechnology), anti-p-MAPK (mouse monoclonal, 1:1,000), anti-MAPK (rabbit polyclonal, 1:1,000), anti-p-AKT (rabbit monoclonal, 1:1,000) (all from Cell Signaling Technology, Danvers, MA, USA), anti-AKT (goat polyclonal, 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), 1:500), anti-PCNA (mouse monoclonal, 1:500) and anti–actin (mouse monoclonal, 1:1,000) (all from Wuhan Boster Biological Technology, Ltd., Wuhan, China). HRP-conjugated goat anti-rabbit, anti-mouse or anti-goat specific secondary antibody (1:6,000; Zhongshan Golden Bridge Biotechnology, Beijing, China). Patients A total of 23 breast cancer tissues were obtained from patients in the First Affiliated Hospital of Dalian Medical University. All the patients were females aged 29C83 with infiltrative non-specific breast cancer. The selected tissue samples express both ER and ER-36 under immunofluorescence observation, and without any radiation, chemotherapy, or endocrinotherapy treatment before surgical resection. We got the patients’ relatives written informed consent for the procedures, which were also approved by the Ethics Committee on the Use of Human Subjects (the First Affiliated Hospital of Dalian Medical University). Cell culture and transfection The MCF-10A-neoT, MCF-7 and MDA-MB-231 cells were purchased from ATCC (Rockville, MD, USA). Stable clones (designated as MCF-10A-neoTCE) were established as described in our previous study (34,35). The MCF-10A-neoT and MCF-10A-neoTCE cells were cultured in DMEM/F12 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 5% horse serum (HyClone, Logan, UT, USA), penicillin (100 U/ml), streptomycin (100 g/ml) (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), hydrocortisone (1.410-6 M; HyClone), insulin (10 g/ml), cholera toxin (100 ng/ml) and EGF (20 ng/ml) (both from Sigma-Aldrich, Merck KGaA). MCF7, MDA-MB-231 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml), and streptomycin (100 g/ml). All cells were maintained in a humidified atmosphere at 37C in 5% CO2. Lipofectamine 2000TM (Invitrogen; Thermo Fisher Scientific, Inc.) was used CP-724714 kinase activity assay for cell transfection according to the manufacturer’s instructions. After 24C48 h of transfection and subsequent culture in 1 M wortmannin or 50 M “type”:”entrez-nucleotide”,”attrs”:”text”:”Ly294002″,”term_id”:”1257998346″,”term_text”:”LY294002″Ly294002 (Sigma-Aldrich, Tokyo, Japan), cells were harvested for western blot analysis or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Methyl–cyclodextrin (MCD) was used to disrupt caveolae. MCF-10A-neoT cells pretreated with MCD (2 mM) for 90 min were used for immunofluorescent microscopy analysis. Western blotting Cells were harvested and then lysed in a cold lysis buffer (20 mmol L?1 Tris-HCl, pH 7.5, 70 mmol L?1 NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100 and 1% PMSF) to extract protein (35). The concentration of total protein was determined by the Bradford method. CP-724714 kinase activity assay The protein samples were.