Zhongguo shi yan xue ye xue za zhi · 2026
[Regulation of Mitochondrial Autophagy by FADD in Jurkat Cells].
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Lin Cun-Zhe, Zhu Zhan-Xing, Zhong Qi, Zhang Xue-Rui, Liu Yuan-Yuan
Аннотация
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.
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.