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: Dose- and time-dependent effects of fucoidan on PI3K and Akt in HT-29 cells. (A) HT-29 cells were incubated for 6 h with various concentrations of fucoidan (0–200 µg/ml) and the phosphorylation of PI3K and Akt was detected by western blotting. (B) HT-29 cells were treated with fucoidan (200 µg/ml) for 0, 2, 4, 6, 8 and 10 h and the phosphorylation of PI3K and Akt was detected by western blotting. The protein expression levels were quantified and vallues are expressed as the mean ± standard error of the mean of five experiments, as determined by densitometry against ß-actin (*P<0.05, vs. non-treated cells). ROD, relative optical density; p-PI3K, phosphorylated phosphoinositide-3 kinase.

Fig 4: AKIP1 mediates NF-?B regulation via the Akt/IKKß pathway during the EMT. A, Serum-starved HeLa cells were pretreated with PI3K inhibitor Ly294002 (40 µM) or IKK inhibitor Bay117082 (15 µM) for 4 hours, followed by stimulation with AKIP1 protein (80 ng/mL) for 4 hours. Cell lysates were harvested and the levels of indicated proteins were measured using western blot analysis. B, pGL4.32 (luc2NF-?B-RE/Hygro) vector and pRL4-TK Renilla luciferase constructs were used for co-transfection of HeLa cells. The cells were further transfected 24 hours later with empty vector (EV) or AKIP1 expression plasmid. Some of the AKIP1-transfected cells were pretreated with Ly294002 (40 µM) or Bay117082 (15 µM) for 4 hours. The dual luciferase reporter assay was performed 24 hours following transfection of AKIP1. EV-transfected cells treated with TNFa (40 ng/mL) for 1 hour after transfection were used as the positive control. ** indicates P < .01. C, The HeLa cells were transfected with AKIP1 siRNA to knock down AKIP1. After 72 hours transfection, AKIP1 protein (80 ng/mL) was used to stimulate HeLa cells for 4 hours and the cell lysates were analysed using western blot

Fig 5: AKIP1 Suppresses PTEN during the EMT. A, HeLa and CaSki cervical cancer cells were transfected with a control siRNA (siNC) or AKIP1 siRNA. The cell lysates were collected and the protein expression of PTEN, AKIP1, p-Akt, total Akt, and ß-Actin was detected using western blot. B, Stable AKIP1-expressing cells were transfected with EV, wild-type PTEN, or G129E-mutant PTEN plasmids. At 48 hours after transfection, cell lysates were harvested after cell fractionation. Levels of indicated proteins were determined using western blot analysis