The ccRCC cell lines 786-O and ACHN were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The cells were cultured in 1640 medium supplemented with 10% fetal bovine serum. In order to obtain ccRCC cells stably over-expressing KDF1, the cells were transduced with a recombinant lentivirus Lenti-KDF1. Lenti-KDF1 was made by inserting KDF1 coding sequence (152-1348 of NM_152365.3) into the NotI/XbaI site of the lentivirus expression vector pCDH-CMV-MCS-EF1. Infection of ccRCC cells was performed when the cells reached about 50% confluence. Stably transduced cells were obtained by screening the cells with 5μg/ml of puromycin, and the overexpression of KDF1 in the cells was verified by RT-PCR and Western blot.

Lentivirus-mediated short hairpin (sh) RNA was employed to inhibit the expression of KDF1 in KDF1-overexpression cells. To produce the KDF1 shRNA expression Lentivirus Lenti-KDF1shRNA, a shRNA targeting KDF1 was synthesized and cloned into the BamH I and EcoR I site of pLVshRNA-EGFP(2A)puro. Following is the shRNA sequence: 5’-GAGGAGTACTATTCTTTCCATCTCGAGATGGAAAGAATAGTACTCCTCTTTTTT-3’.

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