Mycoplasma detection set

HEK293 and MCF7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA). Human neuroblastoma SH-SY5Y and SK-N-BE(2) cells were maintained in RPMI 1640 (Sigma) supplemented with 10% FBS. HEK293 cells were obtained from the JCRB Cell Bank and MCF7 and SH-SY5Y cells were from ATCC, while SK-N-BE(2) cells were purchased from the European Collection of Authenticated Cell Cultures. Mycoplasma contamination was tested by Mycoplasma Detection Set (Takara), and short tandem repeat analysis was performed for cell authentication (Promega). Transient transfection with C-terminal hemagglutinin (HA) or myc-tagged human NLRR1 plasmids was performed using Fugene HD (Roche) or Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. Seventy to eighty percent confluent monolayers of transfected cells were treated with growth factors for the indicated times after 16 h serum starvation, and cell lysates were subjected to western blot analyses. Cell proliferation was determined using a Cell Counting Kit-8 (Dojindo, Japan).

The human RCC cell lines, 786-O, Caki-1, 769-P, A498, ACHN and the immortalized proximal tubule epithelial cell line from normal adult human kidney (HK-2) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Mycoplasma detection was performed using a Mycoplasma Detection Set (Takara, Shiga, Japan) for all the cells. All the RCC cell lines were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml Penicillin, and 100 µg/ml streptomycin in the humidified atmosphere with 5% CO2 at 37°C.