Fig 2: DLK1 cleavage influences p53 and Akt at hypoxia.a Graphical representation of Proteome profiler array results, showing variations of DLK-A vs. DLK-C kinase phosphorylation level ratio in relation to internal reference control (continuous line), in cells exposed to hypoxia for 24 h. Kinases with the most significant variations are highlighted in the figure. ELISA assay data showing p53 DNA binding activity in 3 different cell lines transiently transfected with DLKs, grown at 21% or 1% O2 for 24 (left, b) and 72 (right, c) h. d, e ELISA assay data showing Akt T308 phosphorylation in 3 different cell lines transiently transfected with DLKs, grown at 21% or 1% O2 for 24 (left, b) and 72 (right, c) h. Statistical analysis: all data are from three independent experiments and expressed as mean ± SEM, statistical significance was determined by one-way ANOVA, followed by Bonferroni post hoc test. In the whole figure significance is represented as *p < 0.05, **p < 0.01, and ***p < 0.001 as indicated by straight lines.

Fig 3: Protein levels of apoptosis biomarker. The level of Caspase-3 (A), p53 (B), Bax (C), and Bcl-2 (D) in HepG-2, MCF-7, Bj-1, and MCF-12F treated with or without the IC50 of MP, MP/NabM, and MP/IHM. MP: milk proteins concentrate; MP/NabM: milk proteins/Nabeq mucilage complex; MP/IHM: milk proteins/Isabgol husk mucilage complex. Data are average of triplicates. Measurements with different letters (a, b, c and d) are significantly different (p < 0.05).

Fig 4: Temporal modulation of Akt and p53 balance by DLK1 cleavage regulates cell metabolism at hypoxia.a ELISA time course experiments showing p53 DNA-binding activity and Akt T308 phosphorylation variations in U3084S stable cell lines grown in 1% O2 for up to 72 h. b Western blot time course experiments showing variations in Akt S473 phosphorylation and total Akt levels in 3084 stable cell lines grown in 1% O2 for up to 72 h. SDHA was used as loading control. c Colorimetric assay time course experiment showing different modulation of glucose consumption and lactate production in 3084 S stable cell lines grown in 1% O2 for up to 72 h. d Representative images and densitometric analysis of western blots showing phosphorylated and total AKT levels in U3084S stable cell lines pre-treated for 24 h with 10 µM PI3K inhibitor LY294002 and then grown in 21% or 1% O2 for 48 h. SDHA was used as loading control. e Colorimetric assay experiment showing glucose consumption and lactate production variations in U3084S stable cell lines pre-treated for 24 h with 10 µM PI3K inhibitor LY294002 and then grown in 21% or 1% O2 for 48 h. Statistical analysis: all data are from four independent experiments, with the exception of point c with n = 3, and expressed as mean ± SEM. Statistical significance was determined by two-way ANOVA (a, c) and one-way ANOVA (b, d, e), followed by Bonferroni post hoc test. In the whole figure significance is represented as *p < 0.05, **p < 0.01, and ***p < 0.001 vs. EMPTY control or as indicated by straight lines, ##p < 0.01 and ###p < 0.001 of DLK-A vs. DLK-C.

Fig 5: Graphical abstract. As mutant p53 has a mutation in its DNA binding domain, mutant p53 can be expected to have less transcriptional activity. When 4HR is coupled with mutant p53, its DNA binding activity may be increased by its conformational change. As a consequence, p53 transcriptional activity will be recovered. 4HR, 4-Hexylresorcinol.