Mem medium

HCT-116 cells (DSMZ, Braunschweig, Germany) were grown in McCoy’s 5A medium (Biowest) containing 10% fetal bovine serum (FBS) (PAA Laboratories, Pasching, Austria). Caco-2 cells (ECACC, Salisbury, UK) were grown in MEM medium (Biowest, Nuaillé, France) containing 15% FBS (PAA Laboratories) and MEM non-essential amino acids (Biowest). Culture media were supplemented with 2 mM glutamine (Biowest) and Zell Shield antibiotics (Minerva Biolabs, Berlin, Germany). Cells were maintained in a humidified atmosphere at 37 °C and 5 °C CO₂.

Before RNA extraction, 0.5 × 10⁶ fibroblasts were seeded in a 25 cm² flask and maintained in a MEM growth medium (Biowest) for 24 h. Afterward, cells were starved for 48 h in a starvation medium (MEM medium [Biowest] supplemented with 1.75% L-glutamine [Biowest] and 1.25% antibiotic [Biowest]). RNA isolation was performed by the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. For cDNA synthesis, 500 ng RNA was transcribed using random primers and SuperScript III Reverse Transcriptase (Invitrogen). RT–PCR was performed to analyze the RPGR transcript (primers and conditions are listed in Table S3). Vinculin transcripts were used as an internal control.

RPMI 2650 cells were cultivated in MEM medium (BioWest, Nuaillé, France) containing 10% FBS, 2 mM Gln and 10U penicillin, 10 µg streptomycin at 37 °C, 5% CO₂. 95% rH. Cells were split on a regular basis at 80–90% confluency by trypsination (Trypsin/EDTA, Biochrom, Stockelsdorf, Germany).