[Regulation of Mitochondrial Autophagy by FADD in Jurkat Cells].

OBJECTIVE

To investigate the regulatory effect of Fas-associated death domain protein (FADD) on the number of mitochondria in Jurkat cells.

METHODS

The FADD gene in Jurkat cell line was knocked out by lentivirus packaging CRISPR/Cas9-FADD plasmid. The protein levels of FADD and PGC1-α in virus-transfected Jurkat cells and the expression of LC3B/β-actin protein after rapamycin-induced autophagy were detected by Western blot. And the effect of FADD on autophagy was reversely validated through a rescue experiment. qPCR was used for relative quantification of mtCO1/β-globin in rapamycin induced or chloroquine inhibited WT Jurkat and FADD-/- Jurkat cells. The changes of mitochondria in WT Jurkat and FADD-/- Jurkat cells induced or inhibited autophagy under different conditions were analyzed by laser scanning confocal microscope and transmission electron microscope.

RESULTS

FADD gene was deleted in FADD-/- Jurkat cell line, and there was no significant change in PGC1-α protein level. Mitochondria increased significantly in FADD-/- Jurkat cells compared with WT Jurkat cells. After rapamycin-induced autophagy in WT Jurkat cells and FADD-/- Jurkat cells, mitochondrial fluorescence was significantly weakened and mtCO1/β-globin mRNA levels were also significantly down-regulated. The mitochondrial fluorescence was significantly enhanced and the mtCO1/β-globin mRNA level was significantly up-regulated after chloroquine inhibited autophagy in WT Jurkat cells. The number of mitochondria in WT Jurkat cells and FADD-/- Jurkat cells showed the same trend by transmission electron microscopy.

CONCLUSION

FADD promotes autophagy in Jurkat cells. After FADD is knocked out, mitochondria accumulate, and induction of autophagy can reduce the number of mitochondria.