Fig 1.
Proteasome inhibition reduces formation of degradasomes during the initial 6 h of TNKSi treatment.
(A) Confocal sections through SW480 GFP-TNKS1 cells treated with DMSO (upper panel) or G007-LK (lower panel) for 6 h and immunostained with antibodies against AXIN2, white and β-catenin, red. Hoechst, blue. Scale bar: 5 μm. (B) SW480 GFP-TNKS1 cells were examined by live microscopy after adding G007-LK, either alone (B’) or in combination with MG132 (B”). Images were captured every 10 minutes during a time frame of 6 h. One stack was acquired before addition of the inhibitors (timepoint "0 h"). Still frames of representative cells are shown. Scale bar: 5 μm. (C) Quantification of the number of GFP-TNKS1 puncta per cell. Shown are values +/- SEM of two independent experiments with 24 (G007-LK) and 36 (G007-LK + MG132) cells in total. (D) SW480 cells expressing GFP-TNKS1 were incubated with G007-LK and MG132 for 6 h, either alone or in combination. Cells were then lysed and whole cell lysate was applied for Western blotting. Membranes were incubated with antibodies against AXIN1, AXIN2 and Actin (loading control). One representative blot is shown.
Fig 2.
G007-LK-induced formation of degradasomes requires sustained protein synthesis.
(A) SW480 cells expressing GFP-TNKS1 were treated with CHX and G007-LK for 6 h, either alone or in combination. Images were taken with the Olympus ScanR microscope. GFP-TNKS1 spots, white; nucleus, blue. Scale bar: 10 μm. (B) The average number of GFP-TNKS1 puncta per cell was quantified using the ScanR analysis software. Shown are values +/- SEM of three independent experiments. At least 2000 cells were analyzed per condition in each experiment. (C) SW480 cells expressing GFP-TNKS1 exposed to the same treatment as in (A) were lysed and whole cell lysate was applied for Western blotting. Membranes were incubated with antibodies against AXIN1, AXIN2, total β-catenin and Actin (loading control). One representative blot is shown. (D) Graphs show quantification of Western blots in (C), from three independent experiments, +/- SEM.
Fig 3.
Late-phase upregulation of AXIN1 after prolonged TNKSi treatment is not required for degradasome formation.
(A) Western blots of whole cell lysates from SW480 cells treated with G007-LK for 0, 2, 4, 6 and 24 h, respectively. Membranes were incubated with antibodies against AXIN1, AXIN2 and Actin. (B) SW480 cells stably expressing GFP-TNKS1 were treated with G007-LK for 24 h after siRNA-mediated depletion of either AXIN1 or AXIN2, or both proteins for 48 h. Non-targeting siRNA (Scr) was used as a negative control. (C) Images taken with the Olympus ScanR microscope show the formation of GFP-TNKS1 puncta upon G007-LK treatment for 24 h in control (Scr siRNA) cells or cells depleted of AXIN1 and AXIN2, either alone or in combination. Scale bar: 10 μm. (D) The number of GFP-TNKS1 puncta was quantified using the ScanR analysis software. Bars show values +/- SEM from three individual experiments. (E) GFP-TNKS1 puncta from Fig 3D were sorted by size into small (2–20 pixels), medium (20–60 pixels) and large (60–500 pixels) puncta and the average number of puncta per cell in each size category is shown.
Fig 4.
AXIN2 plays a predominant role in degradasome-mediated degradation of β-catenin.
(A) SW480 cells stably expressing GFP-TNKS1 were treated with G007-LK for 24 h after depletion of either AXIN1 or AXIN2, or both proteins. The remaining total β-catenin levels were measured by Western blot analysis. Actin was used as a loading control. Quantification of Western blots from three independent experiments, +/- SEM. (B) Lysates from the samples described in (A) were applied for Western blotting. Membranes were incubated with an antibody specifically detecting non-phosphorylated (active) β-catenin. Quantification of Western blots from three independent experiments, +/- SEM.