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. 2021 Oct 20;12:670338. doi: 10.3389/fimmu.2021.670338

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Copyright © 2021 Liu, Gokhale, Jung, Zhu, Luo, Saha, Guo, Zhang, Kyin, Zong, White and Xie

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Overexpression of MFF induced mitochondria-dependent apoptosis in human MM 8226 cells. Cells were transduced with individual lentiviral expression vector of MFF (pUB-Myc-MFF), MFF3 (pUB-Myc-MFF3), or an empty vector (pUB-Thy1.1). (A) Transduction efficiency of 8226 cells analyzed by Thy1.1 immunofluorescence staining and FACS. Cells were analyzed at day 3 post transduction. Gated population (Thy1.1+) indicates the cells that were successfully transduced with the lentiviral expression vector. (B) Ectopic overexpression of MFF and MFF3 in transduced 8226 cells. Total cellular proteins were prepared at day 4 post transduction, and then immunoblotted for the Myc tag, followed by actin. Bands of Myc-MFF and Myc-MFF3 are indicated in the figure. (C–H) Cell apoptosis, DNA fragmentation and mitochondrial membrane permeabilization were analyzed at day 6 post transduction. (C) Representative FACS profiles of annexin V staining. Gated populations indicate the annexin V+ apoptotic cells. (D) Graphical results of the percentage of annexin V+ apoptotic cells. (E) Representative FACS profiles of cell cycle analysis by PI staining. Gated populations indicate apoptotic cells with DNA fragmentation (DNA content < 2n) and proliferating cells (2n < DNA content ≤ 4n). (F) Graphical results of the percentage of apoptotic cells with DNA fragmentation. (G) Representative FACS profiles of mitochondrial membrane permeabilization measured by MitoProbe JC-1 staining. Gated populations in H show cells with healthy mitochondria and gated populations in P indicate cells with permeabilized mitochondria. (H) Graphical results of the percentage of cells with permeabilized mitochondria. (D, F, H) The graphs depict the results of 3 independent experiments with duplicate samples in each experiment (mean ± SD). ***p < 0.001 by one-way ANOVA.