Ference experiments. NHERF1 was stably knocked down by shNHERF1 in HeLa (Hela-NHERF1-KD) cells and transiently knocked down by siRNA in CaSki (CaSki-NHERF1-KD) cells. pCMV-HA and pCMV-HA-NHERF1 plasmids had been kindly offered by Dr. Randy Hall (EmoryUniversity, Atlanta, GA). pSuper.puro luciferase handle and pSuper. puro-shNHERF1 plasmids have been sort gifts of Dr. Margaret J. Wheelock (University of Nebraska Medical Center, Omaha, NE).Western blotting and reagentsMaterials and methodsCell culture and transfectionCells had been purchased from the National Infrastructure of Cell Line Resource (Beijing, China). HeLa and CaSki cervical cancer cells have been cultured in DMEM, RPMI 1640 medium (Gibico, Cleveland, TN), respectively, with ten FBS (Gibico, Cleveland, TN) at 37 in an atmosphere of 5 CO2. The steady transfection of HeLa cells was performed as previously described25.RNA interference and plasmid constructionsWestern blotting assay was performed as previously described46. The anti-NHERF1 was bought from Sigma-Aldrich (HPA009672, St. Louis, MO) and Becton Dickinson Labware (#611161, Billerica, MA), respectively. Anti-HA (#561) was purchased from Medical Biological Laboratories (Nagoya, Japan). Anti-c-Myc (#ab32072) and anti–catenin (#ab22656) have been bought from Abcam (Cambridge, UK). Anti-GAPDH (#5174), anti–catenin (#9581), and anti-TCF-1 (#2206) have been purchased from Cell Signaling Technologies (Danvers, MA). Anti-ACTN4 was purchased from Enzo Life Sciences (#ALX-210-356, Shanghai, China) and Santa Cruz Biotechnology (#sc134236, Santa Cruz, CA), respectively. Anti-Ki67 (#zm0166) and horse radish peroxidase-conjugated secondary antibodies had been bought from ZSGB-BIO (Beijing, China). Infrared fluorescent dyes-conjugated secondary antibodies had been bought from LI-COR Biosciences (Lincoln, NE). IWR-1-endo (#S7086) was bought from Selleck (Houston, TX).Cell proliferation assaysiRNAs have been purchased from Invitrogen (Carlsbad, CA) along with the sequences have been shown as follows: NHERF1 siRNA1#: 5-GCUAUGGCUUCAACCUGCA TT-3. NHERF1 siRNA2#: 5-GUCGACCACCAGCAGGCGC ACGGCGUUG-3.Official journal with the Cell Death Differentiation AssociationFor CCK-8 assay, cells were seeded in 96-well plates at a density of 3000 per well and cultured for 1? days, and CCK-8 (Dojindo, Kumamoto, Japan) was added in accordance with the manufacturer’s guidelines and absorbance was measured at 450 nm with an EnSpire label microplate reader (PerkinElmer, Waltham, MA). For CFSE (carboxy fluorescein succinimidyl ester) assay, single-cell Trifloxystrobin Purity suspension at a density of 1 ?106 cells per ml was stained with CFSE Cell Proliferation Kit (#C34554, Life Technologies, Carlsbad, CA) at day 1 and continued the culture for 3 days. The labeling cells were analyzed by flow cytometry at day 1 and day 3, respectively. TheWang et al. Cell Death and Disease (2018)9:Web page 12 ofproliferation index of each group was analyzed by comparing the fluorescence value of day 1 and day 3 by Modfit LT (Verity Software Flavonol Cancer program Property, Topsham, ME). For RTCA (real-time cell evaluation) assay, cells were cultured in 16-well plates (3000 cells per nicely, E-Plate 16, ACEA Biosciences Inc) and proliferation index was monitored by the xCELLigence technique (ACEA Biosciences Inc, San Diego, CA) for 72 h. For colony formation assay, cells had been cultured in 6-well plates (1000 cells per nicely) for 7 days. The amount of colonies ( 50 cells) had been counted after staining with 0.5 crystal violet.In vivo xenograft formation assayThis study was performed fo.