Citation

  • Authors: Yokoi K. et al.
  • Year: 2022
  • Journal: Biochemistry 61 639-655
  • Applications: in vitro / siRNA / INTERFERin
  • Cell type: Jurkat
    Description: Human acute T cell leukemia line

Method

Knockdown (KD) of Mitofusins in Jurkat Cells by Small Interfering RNA (siRNA) To a solution of transfection reagent (INTERFERin, 5 μL) in OPTI-MEM (0.3 mL) in a 1.5 mL Eppendorf tube was added siRNA (10 μM, 1 or 2 μL) in H2O, and the resulting solution was allowed to stand for 15 min at room temperature. The resulting solution was then added to Jurkat cells (2.0 × 105 cells/mL, 1.5 mL) in 10% FBS/RPMI 1640 medium that had been seeded on a 12-well plate and incubated overnight at 37 °C under 5% CO2 (the final concentration of siRNA was 5 or 10 nM), and the resulting solution was incubated for 48 or 72 h at 37 °C under 5% CO2.

Abstract

We previously reported that a cyclometalated iridium (Ir) complex-peptide hybrid (IPH) 4 functionalized with a cationic KKKGG peptide unit on the 2-phenylpyridine ligand induces paraptosis, a relatively newly found programmed cell death, in cancer cells (Jurkat cells) via the direct transport of calcium (Ca2+) from the endoplasmic reticulum (ER) to mitochondria. Here, we describe that CGP37157, an inhibitor of a mitochondrial sodium (Na+)/Ca2+ exchanger, induces paraptosis in Jurkat cells via intracellular pathways similar to those induced by 4. The findings allow us to suggest that the induction of paraptosis by 4 and CGP37157 is associated with membrane fusion between mitochondria and the ER, subsequent Ca2+ influx from the ER to mitochondria, and a decrease in the mitochondrial membrane potential (ΔΨm). On the contrary, celastrol, a naturally occurring triterpenoid that had been reported as a paraptosis inducer in cancer cells, negligibly induces mitochondria-ER membrane fusion. Consequently, we conclude that the paraptosis induced by 4 and CGP37157 (termed paraptosis II herein) proceeds via a signaling pathway different from that of the previously known paraptosis induced by celastrol, a process that negligibly involves membrane fusion between mitochondria and the ER (termed paraptosis I herein).

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