Cyclosporin a

Naïve CD44^low CD8⁺ T cells were purified from the spleen by negative selection using the BD-IMag system (BD Biosciences, San Jose, California, U.S.A.). To measure G0S2 expression, CD8⁺ T cells were activated in 96-well plates coated with 10 μg/ml anti-CD3 (BD Biosciences, San Jose, California, U.S.A.) in the absence or presence of rapamycin (Sigma-Aldrich), LY294002 (Sigma-Aldrich, St Louis, Missouri, U.S.A), cyclosporin A (Millipore, Billerica, Massachusetts, U.S.A.), or PD98059 Millipore, Billerica, Massachusetts, U.S.A.) for 24 hours.
For in vitro stimulation, CD44^low CD8⁺ T cells labeled with 4 μM CFSE (LifeTechnologies, Grand Island, New York, U.S.A.) or 5 μM eFluor 670 (eBioscience, San Diego, California, U.S.A.) were cultured for 2 or 3 days at a density of 1×10^{5 (link)} cells per well in RPMI 1640 medium containing 10% (vol/vol) FBS in 96-well plates coated with 10 μg/ml anti-CD3 (BD Biosciences, San Jose, California, U.S.A.) in the presence of anti-CD28 (2 μg/ml; BD Biosciences). For homeostatic proliferation, CFSE-labeled CD44^low CD8⁺ T cells from CD45.2⁺ WT or G0s2^−/− mice were transferred into sub-lethally irradiated B6.SJL (CD45.1⁺) mice. Dilution of CFSE dye was analyzed by flow cytometry and the proliferation index (PI) was calculated using FlowJo software (TreeStar, Ashland, Oregon, U.S.A.).

Calcein AM and cyclosporin A were obtained from Sigma-Aldrich (St. Louis, MO). Rhodamine 123, paclitaxel, vinblastine, doxorubicin, and cisplatin were purchased from Wako (Tokyo, Japan). Tariquidar was purchased from AdooQ BioScience (Irvine, CA). CellTiter 96 AQueous One Solution Reagent was purchased from Promega (Madison, WI). [¹²⁵I]-Iodoarylazidoprazosin (IAAP) was obtained from Perkin-Elmer Life Science (Wellesley, MA).