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. 2022 Jan 26;11:808300. doi: 10.3389/fonc.2021.808300

Figure 3.

Figure 3

Involvement of PI3K/AKT/GSK-3β signaling in NRF2 activation in HCT116 spheres. (A) Protein levels of p-AKT (S473), total AKT, p-GSK-3β (S9), and total GSK-3β were determined in monolayer and sphere HCT116 cells. Bar graph represents quantified protein levels from at least three experiments. aP < 0.05 compared with the monolayer group. (B) HCT116 cells were transfected with the non-specific siRNA (siCtrl) or CD133-specific siRNA (siCD133) and grown in either monolayer or sphere culture system. Protein levels for p-AKT (S473), AKT, p-GSK-3β (S9), and GSK-3β were assessed using western blotting. Bar graph represents quantified protein levels from at least three experiments. aP < 0.05 compared with the monolayer group. (C) LY294002 (10 μM), a pharmacological inhibitor of PI3K, was incubated in monolayer and sphere HCT116 cells for 24 h, and protein levels of NRF2, p-AKT (S473), AKT, p-GSK-3β (S9), and GSK-3β were determined. Bar graph represents quantified protein levels from at least three experiments. aP < 0.05 compared with the monolayer vehicle group. bP < 0.05 compared with the sphere vehicle group. (D) Transcript levels of GCLC AKR1C1, and NQO1 were measured using a relative qRT-PCR analysis in vehicle- or LY294002-treated cells. aP < 0.05 compared with the vehicle-treated sphere group. Quantification results of western blotting were relative values to the loading control GAPDH. Values represent the mean ± SEM of more than three experiments.