X 001 programme

For transfection to HEK-293T cells, plasmids were transfected to 60–80% confluent HEK-293T cells by polyethylenimine ‘Max' (Polysciences, Inc.) according the manufacturer's protocol and cultured 2 days in culture media. For ICER-overexpressing experiments in murine primary T cells, cells were collected 1 day after starting culture and empty vector, ICER-overexpressing plasmid, or ICERγ-overexpressing plasmid were transfected using the Amaxa Mouse T Cell Nucleofector Kit with the X-001 programme (Amaxa) according to the manufacturer's protocol. The supernatant of each culture was saved in 4 °C during transfection and recovery. After 4 h recovery at 37 °C, cells were again cultured in those supernatant for 2 days. The efficacy of the transfection always exceeds 10%.

Luciferase reporter plasmid was transfected using the Amaxa Mouse T Cell Nucleofector Kit with the X-001 programme (Amaxa) on day 2 of culture. Each reporter experiment included 200 ng renilla luciferase construct as an internal control. Luciferase activity was quantified using the Promega Dual Luciferase Assay System (Promega) on day 3 of culture according to the manufacturer's instructions.