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. 2018 Feb 14;9(2):231. doi: 10.1038/s41419-018-0336-0

Fig. 2. FoxO3A is cleaved at its N-terminus upon translocation into the mitochondria of normal and cancer cells.

Fig. 2

a Scheme of plasmids used. b–e Cells were transfected with the indicated FoxO3A plasmids for 48 h; upon LG (0.75 mM glucose, 24 h), purified mitochondria were treated with PK alone or with PK and Triton X-100. Total and mitochondrial proteins were analyzed by immunoblot. b HCT116 and HEK293 cells transfected with FoxO3A-WT-FLAG. c HCT116 cells transfected with HA-FoxO3A-WT-FLAG. d HCT116 cells transfected with FoxO3A-WT-FLAG or FoxO3A-Δ1–148-FLAG. e HCT116 cells transfected with FoxO3A-WT-FLAG or FoxO3A-Δ80-108-FLAG. β-actin and PDH were used as total lysate and mitochondria controls, respectively. f Immunoblots performed with two different anti-FoxO3A antibodies in mitochondria isolated from HCT116 and HEK293 cells cultured in LG (24 h). Mitochondrial fractions were subjected to PK treatment. β-actin and PDH were used as total lysate and mitochondrial fraction controls, respectively. g HCT116 cells were transfected with control (siNC) or FoxO3A-specific siRNAs for 48 h and FoxO3A mitochondrial levels were evaluated by immunoblot upon LG (24 h). PDH: loading control. fl. full-length FoxO3A, cl. cleaved FoxO3A, N-term. N-terminal domain, FKH-DBD forkhead DNA-binding domain, NLS nuclear localization signal, TAD transactivation domain, C-term. C-terminal domain. The presented results are representative of at least three independent experiments