Fig. 3: Phosphorylation at S12 and S30 of FoxO3A N-terminus is required for mitochondrial accumulation in metabolically stressed cancer cells. | Cell Death & Disease

Fig. 3: Phosphorylation at S12 and S30 of FoxO3A N-terminus is required for mitochondrial accumulation in metabolically stressed cancer cells.

From: Uncoupling FoxO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy

Fig. 3

a Scheme of FoxO3A-WT-FLAG and mutated FoxO3A-FLAG plasmids obtained by site-directed mutagenesis. b, c HCT116 cells were transfected with the indicated FoxO3A-WT-FLAG and mutant plasmids for 48 h. Upon LG (0.75 mM glucose, 24 h), total and mitochondrial proteins were analyzed by immunoblot. d HCT116 cells were transfected with the indicated FoxO3A-WT-FLAG and mutant plasmids for 48 h. Upon LG (24 h), purified mitochondrial fractions were treated with PK and analyzed by immunoblot. BCL2 outer membrane control, TFAM mitochondrial matrix control. e HCT116 cells were transfected with FoxO3A-WT-FLAG or FoxO3A-Δ1-30-FLAG for 48 h, cultured in LG (24 h). f Total proteins were isolated from HCT116 cells grown in standard and LG (24 h) conditions and analyzed by immunoblot. g Pharmacological inhibition of MEK using PD98059 in HCT116 cells cultured in LG (24 h). Total and mitochondrial proteins were analyzed by immunoblot. h Pharmacological inhibition or activation of AMPK using compound C (CC, 5 μM) or AICAR (5 mM), respectively, in HCT116 cells cultured in LG (24 h). Total and mitochondrial proteins were analyzed by immunoblot. b, c, e–h β-actin and TFAM were used as total lysate and mitochondrial fraction controls, respectively. i Scheme of GST-FoxO3A recombinant proteins. l, m In vitro kinase assays performed with the indicated GST-FoxO3A(1–148) and mutant recombinant proteins as substrates, in the presence of recombinant ERK (l) or recombinant AMPK (m). In both assays, GST-FoxO3A(386–525) was used as a positive control. GST-empty protein was used as a negative control. Coomassie gel staining (lower panels) was used as a 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

Back to article page