Fig. 4. TaPin1 activates the oncogenic c-Jun pathway via FBW7 ubiquitination.

a. Endogenous TaPin1 interacts with FBW7α isoform. Protein extracts from TBL3 expressing Flag-hFBW7 isoforms or Flag-Control “Con” were immunoprecipitated and immunoblotted with TaPin1 or Flag antibodies.

b. Inhibition of TaPin1 by Bup, Jug or DTM increased FBW7α protein levels and decreased c-Jun expression in TBL3 cells. Actin/Tubulin were loading controls.

c. Inhibition of FBW7 increases c-Jun protein levels in TBL3 cells, whereas ectopic FBW7α expression reduced c-Jun protein levels. Bovine actin/Tubulin were loading controls. Con = empty vector.

d. Inhibition of Jun reporter activity (BIC promoter-Luciferase, an AP-1 target gene) in TBL3 cells transfected with siRNA against FBW7 or siControl (siCon).

e. Jun reporter activity (BIC promoter-Luciferase) in TBL3 cells treated with Buparvaquone (Bup) or Juglone (Jug) was rescued by siRNA against FBW7 but not siControl.

f. siFBW7 inhibition rescued drug effects on c-Jun ubiquitination in parasitized TBL3 cells, incubated with MG132, followed by immunoprecipitation of endogenous c-Jun and immunoblotting with c-Jun and ubiquitin antibodies. “Ig” = non-specific control immunoglobulin.

g. siFBW7 depletion increased the half-life of endogenous c-Jun protein. TBL3 cells treated with Buparvaquone (Bup) or Juglone (Jug) were incubated with cycloheximide, followed by immunoblotting with c-Jun or Tubulin antibodies. Relative c-Jun protein levels at T0 were set at 1.

h. Colony formation of TBL3 cells was markedly reduced by ectopic expression of FBW7α isoform or c-Jun siRNA. Efficiency of two independent si-c-Jun is shown. Bovine Tubulin loading control.

Data are representative of 3 independent experiments (average ± sd, n=3). The p-values with the Bonferroni method based on the number of pairwise comparisons were calculated for Figure 4e. The statistics in Figure 4h used the Dunnett procedure. *p<0.05, **p<0.01, ***p<0.001.