Accell non targeting pool

The following siRNAs were used in this study: Accell Mouse Stat5a (20850) siRNA-SMART pool (Dharmacon, Cambridge, UK) and Accell Non-targeting Pool (Dharmacon). Transfection was performed by following the manufacture’s instruction. Briefly, isolated cells were incubated with 1 μM Accell siRNA in Accell siRNA Delivery Media (Dharmacon) at 37 °C, 5% CO₂ for 48 hr. Knockdown was assessed by Western blotting.

For downregulation of Mcu, 1 × 10⁶ Tregs or Tcons were transfected with 1 μM siRNA-targeting Mcu (Accell Mouse Mcu siRNA-SMARTpool, E-062849-00-0010, Dharmacon, Lafayette, CO, USA) or non-silencing control RNA (Accell Non-targeting Pool, D-001910-10-20, Dharmacon, Lafayette, CO, USA) using P3 Primary Cell 4D-Nucleofactor^TM Kit (Lonza, Basel, Switzerland) according to manufacturer’s instructions. Tcons were cultured in medium containing 20 ng/mL IL-2 after transfection, and 24 h later, 1 mg/mL PHA-P was included within to improve cell survival. Tregs were cultured in maintenance medium containing 50 ng/mL IL-2, 5 ng/mL TGF-β, and mouse T activator CD3/CD28 Dynabeads at a ratio of 1:10 (beads:cells). Experiments were performed 72 h after transfection.
To monitor the effect of downregulation of Mcu on mitochondrial Ca²⁺ uptake, EG7 or EL4 cells were transfected with 1 µM siRNA-targeting Mcu using SF Cell Line 4D-Nucleofector^TM Kit (Lonza, Basel, Switzerland) according to manufacturer’s instructions. The mitochondrial Ca²⁺ indicator mtCEPIA3 was transduced as described in 4.9. Measurements were performed 48 h after transfection.