Caki 2

Human kidney cancer cell lines 786‐O and Caki‐2 and the non‐malignant human kidney cell line HEK293T were purchased from ATCC (Manassas, VA, USA) and maintained in basal media (RPMI for 786‐O and Caki‐2, DMEM for HEK293T) supplemented with 10% FBS and 1% gentamicin–tyrosine solution (Invitrogen, Eugene, OR, USA) in a humidified incubator set to 37°C and 5% CO₂. For coculture experiments, the three cell lines were split into 35‐mm dishes (Becton Dickinson) and incubated for 2 days to reach 80–90% confluence. Transwell inserts (0.4‐μm pore size; Corning, Corning, NY, USA) were then placed into the wells and 5 × 10⁶ PBMCs from healthy volunteers were added to each Transwell insert. After 18 h, PBMCs were collected and lysed with Buffer RLT Plus with beta‐ME (RNeasy Plus Mini Kit; Qiagen, Venlo, Netherlands) and subjected to RNA extraction and qRT‐PCR.

RCC tissue and adjacent normal tissue were obtained from 40 RCC patients (median age 55 years, male:female 3:2) receiving nephrectomy at our hospital. The resected tumors were snap-frozen in liquid nitrogen and then stored at -80°C. Tumor stage was defined following the TNM classification standard of American Joint Committee on Cancer. Experimental protocols were approved by the ethics committees of Huaihe Hospital of Henan University. Written informed consent was obtained from all RCC patients prior to tissue use.
HK2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in keratinocyte serum-free medium containing 0.05 mg/mL bovine pituitary extract and 5 ng/mL human recombinant EGF (Thermo Fisher Scientific, Waltham, MA, USA). Human RCC cells (769P, 786O, ACHN, A498, Caki2) were purchased from the China Center for Type Culture Collection (Wuhan, China). 769P and 786O cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Thermo Fisher Scientific). ACHN cells were maintained in MEM-EBSS (HyClone, Logan, UT, USA) containing 10% FBS. A498 cells were maintained in the MEM-EBSS supplemented with 10% FBS and 1% inessential amino acids (Thermo Fisher Scientific). Caki2 cells were grown in modified McCoy’s 5A medium (Thermo Fisher Scientific) containing 10% FBS. All cultures were kept in a humidified atmosphere with 5% CO₂ at 37°C.

The human normal renal tubular epithelial HK-2 and RCC 786-O, A498, ACHN, and CAKI-2 cell lines were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (CCCAS, Shanghai, China). 786-O cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA). A498, ACHN, and CAKI-2 cells were cultured in RMPI 1640 (Thermo Fisher Scientific), while HK-2 cells were cultured in Keratinocyte Medium (KM, ScienCell, Carlsbad, CA, USA). All cell culture media were supplemented with 10% Fetal Bovine Serum (FBS, Hyclone, Logan, UT, USA), and cells were cultured at 37°C with 5% CO₂.
The lentivirus expressing shRNA against OGT was produced by Jiman Co. (Shanghai, China). 786-O and A498 cells were infected with LV-sh-OGT or LV-sh-NC and selected by puromycin (Sigma–Aldrich, St. Louis, MO, USA). The expression levels of OGT and O-GlcNAc were examined at the RNA and protein levels.

Human RCC cell lines (A498, ACHN, CAKI-2, OS-RC-2, 769P and 786-O) and the normal kidney cell line HK-2 were obtained from the Shanghai Institute of Life Sciences Cell Resource Center (Shanghai, China). OS-RC-2, 769P and 786-O cell lines were cultured in RPMI modified medium (GE Healthcare Life Sciences, Logan, UT, USA), and A498 and ACHN cell lines were cultured in minimum essential medium (Eagle; Corning Inc., Corning, NY, USA). The CAKI-2 cell line was cultured in McCoy's 5A medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the HK-2 cell line was cultured in DMEM (GE Healthcare Life Sciences). All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.), according to the American Type Culture Collection. All cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂. According to the expression of Pyk2 in RCC cell lines, we selected ACHN cells to generate Pyk2 knockdown cells and A498 cells to generate stable Pyk2 overexpression cells.