Figure 6
Figure 6. Loss of GSK-3β induces cell migration via HIF-1α. (A) GSK-3β+/+ and GSK-3β−/− cells were transfected with vectors allowing expression of scrambled control shRNA (shCtr) or shRNA against HIF-1α (shHIF-1α). In addition, GSK-3β+/+ cells were transfected with expression vectors for the V5-tagged full-length HIF-1α (WT) or either the hydroxylation resistant V5-tagged HIF-1α P402A/P564A/N803A (PPN) or HIF-1α containing additional mutations in the GSK-3 sites P402A/S551A/T555V/ P564A/S589A/N803A (PPNSTS) or the empty vector (Ctr). Cells were seeded into Transwell chambers and the number of migrated cells was counted and quantified using ImageJ software. Data represent the number of cells relative to the control, which was set to 100%. *Significant difference between GSK-3β+/+ or GSK-3β+/+ + shRNA control versus GSK-3β−/− or GSK-3β−/− + shRNA control, as well as between control vector versus HIF-1α WT, HIF-1α PPN, or HIF-1α PPNSTS. **Significant difference between shRNA control versus HIF-1α shRNA. (B) Photographs from a representative Transwell chamber experiment. (C) HCT116 Fbw7+/+ and HCT116 Fbw7−/− cells were cultured in Transwell chambers for 16 hours and the number of migrated cells was counted and quantified using ImageJ Version 1.457 software. Data represent the number of cells relative to the control, which was set to 100%. *Significant difference between HCT116 Fbw7+/+ versus HCT116 Fbw7−/−. (D) Photographs from a representative Transwell chamber experiment. (E) HMEC-1 cells were either transfected with empty control vector (Control), expression vectors for Fbw7α, Fbw7β, Fbw7γ, or with vectors allowing expression of scrambled control shRNA (shCtr) or shRNA1 or shRNA2 against Fbw7. Transfected cells were seeded onto Matrigel-coated wells for 2 hours, and then exposed to normoxia (N) or hypoxia (H) for 6 hours. Formation of capillary-like structures was assessed by counting the number of tubules using ImageJ software. Data represent the number of tubules relative to the control, which was set to 100%. *Significant difference between the control vector versus Fbw7 isoforms under normoxic conditions; **significant difference between the control vector versus Fbw7 isoforms under hypoxic conditions. (F) Photographs from representative examples of an in vitro angiogenesis experiment.

Loss of GSK-3β induces cell migration via HIF-1α. (A) GSK-3β+/+ and GSK-3β−/− cells were transfected with vectors allowing expression of scrambled control shRNA (shCtr) or shRNA against HIF-1α (shHIF-1α). In addition, GSK-3β+/+ cells were transfected with expression vectors for the V5-tagged full-length HIF-1α (WT) or either the hydroxylation resistant V5-tagged HIF-1α P402A/P564A/N803A (PPN) or HIF-1α containing additional mutations in the GSK-3 sites P402A/S551A/T555V/ P564A/S589A/N803A (PPNSTS) or the empty vector (Ctr). Cells were seeded into Transwell chambers and the number of migrated cells was counted and quantified using ImageJ software. Data represent the number of cells relative to the control, which was set to 100%. *Significant difference between GSK-3β+/+ or GSK-3β+/+ + shRNA control versus GSK-3β−/− or GSK-3β−/− + shRNA control, as well as between control vector versus HIF-1α WT, HIF-1α PPN, or HIF-1α PPNSTS. **Significant difference between shRNA control versus HIF-1α shRNA. (B) Photographs from a representative Transwell chamber experiment. (C) HCT116 Fbw7+/+ and HCT116 Fbw7−/− cells were cultured in Transwell chambers for 16 hours and the number of migrated cells was counted and quantified using ImageJ Version 1.457 software. Data represent the number of cells relative to the control, which was set to 100%. *Significant difference between HCT116 Fbw7+/+ versus HCT116 Fbw7−/−. (D) Photographs from a representative Transwell chamber experiment. (E) HMEC-1 cells were either transfected with empty control vector (Control), expression vectors for Fbw7α, Fbw7β, Fbw7γ, or with vectors allowing expression of scrambled control shRNA (shCtr) or shRNA1 or shRNA2 against Fbw7. Transfected cells were seeded onto Matrigel-coated wells for 2 hours, and then exposed to normoxia (N) or hypoxia (H) for 6 hours. Formation of capillary-like structures was assessed by counting the number of tubules using ImageJ software. Data represent the number of tubules relative to the control, which was set to 100%. *Significant difference between the control vector versus Fbw7 isoforms under normoxic conditions; **significant difference between the control vector versus Fbw7 isoforms under hypoxic conditions. (F) Photographs from representative examples of an in vitro angiogenesis experiment.

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