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Flow cytometry assay For cell cycle assay, A498, 786-O, and ACHN cells were collected and fixed with ice-cold ethanol overnight.

Then the cells were washed three times with phosphate-buffered saline (PBS) and responded in PBS containing 500 ng/μL RNase A and incubated at 37°C for 30 min.

Accepted for publication 2 November 2016 Published 20 February 2017 Volume 20 Pages 711—724 DOI https://doi.org/10.2147/OTT.

S117933 Checked for plagiarism Yes Review by Single-blind Peer reviewers approved by Dr Lucy Goodman Peer reviewer comments 2 Editor who approved publication: Dr Ingrid Espinoza contributes to most cancers.

The occurrence of RCC has increased in recent years; worldwide, ~100,000 patients die of disease every year.

Similar procedure of cell invasion was also performed for cell migration transwell assay, except that the transwell membranes were not precoated with Matrigel. 5174; 1:1,500); mouse polyclonal antibody anti-ICAM-1 (cat. ab20; 0); rabbit polyclonal antibody anti-VCAM-1 (cat. Blots were then incubated for 1 h at 37°C with goat anti-mouse or anti-rabbit secondary antibody (cat. A0208 and A0216; 1:1,000; Beyotime Institute of Biotechnology), and intensities were measured using enhanced chemiluminescence (Thermo Fisher Scientific). The blotting bands were quantified with Image J software.

Reverse transcription and real-time polymerase chain reaction (PCR) Total RNA was extracted from normal kidney cells and four kidney cancer cell lines using Trizol reagent (Life Technologies) and detected by agarose gel electrophoresis according to the manufacturer’s instructions. In vivo experiments For tumor growth assay, A-498 cells infected with NC-sh RNA or CTNNB1-sh RNA were trypsinized, washed, and resuspended in DMEM without FBS.

The lower chamber was filled with 750 μL DMEM containing 10% fetal bovine serum (FBS). Later, the cells that were able to pass through the filter were fixed with 4% paraformaldehyde and stained by crystal violet solution for 30 min.

Cells on the top surface of the insert were removed with a cotton swab and counted under a microscope in five fields (×200). All the primary antibodies were purchased from Abcam (Cambridge, MA, USA) expect for anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA, USA).

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