Knockdown of mitofilin inhibits autophagy and facilitates starvation-induced apoptosis in HeLa cells

Document Type: Original Article

Authors

Zhejiang Provincial Key Laboratory of Medical Genetics, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China 325035

Abstract

Objective(s): Mitofilin contributes to the maintenance of mitochondrial structure and functions. This study was undertaken to determine the mechanisms underlying its regulation of apoptosis.  
Materials and Methods: Mitofilin was knockdowned by specific short hairpin RNA (shRNA) and the stable HeLa cell clone was selected. The autophagy activity were assessed with LC3-II conversion and puncta formation by western blot and fluorescence imaging in starved and normal cultured HeLa cells. Autophagy flux was measured in the presence of NH4Cl. Wortmannin was used to inhibit autophagy. Cell viability and apoptosis were detected with cell counting kit-8 (CCK-8) and fluorescence-activated cell sorting (FACS) assay, respectively.
Results: Mitofilin expression was down-regulated in starved HeLa cells. In established mitofilin stable knockdown cell lines, LC3-II conversion and puncta formation were detected, which are both hallmarks of autophagy, under both basal and starvation conditions. Mitofilin down-regulation decreased LC3-II conversion and puncta formation, which indicates that loss of mitofilin function inhibits both basal and starvation-induced autophagy activity. CCK-8 and FACS analysis confirmed mitofilin involvement in the regulation of cell survival since mitofilin down-regulation facilitated starvation-induced apoptosis in HeLa cells.
Conclusion: Taken together, mitofilin is a potent regulator of autophagy and it may modulate cell survival through regulation of autophagy.

Keywords

Main Subjects


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