Newborn calf serum

For cultured cells, the purified total RNA was obtained from GFP-negative cells as described above. For RNA isolation from murine pancreas, pancreas was cut into small pieces using scissors and incubated in 2 mg ml⁻¹ collagenase (Wako) in the RPMI1640 medium (Sigma-Aldrich) containing 2% (vol/vol) new-born calf serum (Equitech-Bio) for 30 min at 37 °C with stirring. The cell suspensions were filtered through cell strainers (pore size, 70 µm; BD Biosciences) and used for gene expression analysis. The total RNA was extracted from the isolated cells using TRIzol and an RNeasy Mini Kit (QIAGEN) and reverse-transcribed using a QuantiTect Reverse Transcription Kit (QIAGEN). GeneAce SYBR qPCR Mix (NIPPON GENE) was used to perform qPCR using the StepOne system (Thermo Fisher Scientific). The primer sequences used are listed in Supplementary Table 1. We used GAPDH or RPL13A as a reference gene to normalize data.

The present study was performed using five human bladder cancer cell lines: T24 (grade 3), KK47 (grade 1), 5637 (grade 2), 1376 (grade 3) and RT4 (grade 1). The T24, 5637, 1376 and RT4 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), and KK47 was generously provided by Dr Naito (Kyushu University, Fukuoka, Japan). The T24 and KK47 cells were cultured in minimum essential medium (MEM) with Earle’s salts, L-glutamine (Invitrogen, Carlsbad, CA, USA), 10% newborn calf serum (Equitech-Bio, Inc., Kerrville, TX, USA) and 1% penicillin (PC) streptomycin (SM; Gibco, Grand Island, NY, USA). The 5637 and 1376 cells were incubated in RPMI-1640 medium supplemented with 1% L-glutamine (Invitrogen), 10% fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan), 1% HEPES (Gibco) and 1% PCSM. The RT4 cells were grown in modified McCoy’s 5A medium (Gibco), supplemented with 10% FBS and 1% PCSM. All cell lines were maintained in a humidified incubator at 37°C and 5% CO₂.