Fig. 4: HIF1α is stabilized by TET1 via the activation of AKT/GSK3β pathway.
From: Hypoxia switches TET1 from being tumor-suppressive to oncogenic
A Western blot analysis of TET1 and HIF1α in C643 cells with the indicated treatments under hypoxia. MG132 concentration: 10 μM. B Western blot analysis of TET1 and HIF1α in 8505C cells with the indicated treatments. The band intensity of HIF1α was normalized to that of β-actin, and subsequently normalized to that of cells treated without CHX. Data are shown as the mean ± SD. CHX cycloheximide at 200 μg/ml. Western blot analysis of TET1, HIF1α, p-AKTT308, t-AKT, p-GSK3β, GSK3α/β, p-β-catenin and β-catenin in 8505C (C) and C643 (D) cells with the indicated treatments. E The IHC staining for p-Akt and p-Gsk3β (left panel) and their quantification (right panel) in mice with different genotypes. F Western blot analysis of TET1, p-AKTT308, t-AKT, p-GSK3β, GSK3α/β and HIF1α in 8505C cells with the indicated treatments. LY PI3K inhibitor LY294002 at the indicated concentration. The densitometry ratio of the indicated proteins to β-actin (loading control) was shown below the corresponding band. G Western blot analysis of TET1, p-GSK3βS9, GSK3α/β, HIF1α and HIF2α in C643 cells with the indicated treatments. SB GSK3 inhibitor SB-216763 at the indicated concentration. The densitometry ratio of the indicated proteins to β-actin (loading control) was shown below the corresponding band. H Co-immunoprecipitation (Co-IP) assay showing the interaction of HIF1α with GSK3β, FBW7γ and VHL in 8505C cells with the indicated treatments. LY cells treated with 20 μM LY294002 for 24 h. I Co-IP assay showing the interaction of HIF1α with GSK3β, FBW7γ and VHL in C643 cells with the indicated treatments. In vitro protein ubiquitination analysis of the K48-linked ubiquitination of HIF1α in 8505C (J) and C643 (K) cells with the indicated treatments. Anti-His tag was used to show the K48-only His-tagged ubiquitin. L K48-linked ubiquitination of HIF1α in C643 cells with the indicated treatments. The IP shows K48-only His-tagged ubiquitin and HIF1α in the precipitants (left panel). Cells were treated with SB and MG132 at the indicated concentration for 24 h. M A schematic model summarizing the mechanism of TET1 stabilizing HIF1α via the activation the AKT/GSK3β pathway under hypoxia. Brafm/+ Braf heterozygous mutation, Tet1+/+ Tet1 wild-type, Tet1−/− Tet1 homozygous deletion. Scale bare in IHC pictures, 50 μm. Sections were rank scored [“negative” (−), “weak” (+), “moderate” (++) and “strong” (+++)]. Graph shows the proportion of each score for the indicated groups. Dox− negative control, Dox+ cells inducibly expressing TET1, sgCon control cells, sgTET1 TET1-knockout C643 cells, p-AKTT308 phosphorylated AKT at T308, p-GSK3β phosphorylated GSK3β at S9, p-β-catenin phosphorylated β-catenin at S33/37/T41, WCL whole cell lysates.
