Fig. 4: Mitochondrial FoxO3A regulates mitochondrial gene expression in metabolically stressed cancer cells. | Cell Death & Disease

Fig. 4: Mitochondrial FoxO3A regulates mitochondrial gene expression in metabolically stressed cancer cells.

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

Fig. 4

a Scheme of plasmids used. b ChIP analysis of exogenous FoxO3A recruitment at FHRE #1–2 sites on mtDNA (FHRE #1: bp 14,963–15,110; FHRE #2: bp 15,400–15,469) upon LG (0.75 mM glucose, 24 h) in HCT116 cells transfected with HA-FoxO3A-WT-FLAG. c, f ChIP analysis of endogenous FoxO3A recruitment at FHRE #1–2 sites on mtDNA in HCT116 (c), and HT29 and Rho0 HT29 (negative control) (f) cells upon LG (24 h). d, g Mitochondrial gene regulation in HCT116 (d) and HT29 (g) cells upon LG (24 h) assessed by RT-PCR. Black bars: ATPase 6 and 8 genes; white bars: COX1, COX2, and COX3 genes; gray bars: ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 genes; light gray bar: CYTOCHROME B gene. The dotted line corresponds to the expression levels detected in cells cultured in HG. e Co-immunoprecipitation analysis with the indicated antibodies of PK-treated HCT116 mitochondrial fractions upon LG (24 h). h Upper panel: scheme of gRNA location in human FoxO3A locus. Targeting sites and proto-spacer adjacent motifs (PAMs) and the deleted region are indicated. Lower panel: immunoblot analysis of HCT116-FoxO3A+/+ and HCT116-FoxO3A−/− cells with different anti-FoxO3A antibodies upon 2-DG (1 mM, 6 h) treatment. i–p HCT116-FoxO3A−/− cells were transfected with the indicated FoxO3A plasmids for 48 h and treated with 2-DG (1 mM, 6 h). i Immunoblot analysis of total and mitochondrial proteins. l, m, o, p ChIP analysis of exogenous FoxO3A recruitment at FHRE p21–p27 sites on nuclear DNA (l, o) and FHRE #1–2 sites on mtDNA (m, p). n Immunoblot analysis of nuclear and mitochondrial proteins. b, c, f, l, m, o, p Anti-IgGs were used as controls. e, h, i, n β-actin, TFAM, and LAMIN B were used as total, mitochondrial and nuclear lysate controls, respectively, as appropriate. 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. Where applicable, data are presented as mean ± SEM and significance was calculated with Student’s t test; *p < 0.05, **p < 0.01, and ***p < 0.001

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