Induction of axin puncta by TNKSi is proteasome-dependent.

<p><b>A.</b> SW480 cells were treated with single or combined doses of tankyrase (5 μM G007LK) and proteasome (20 μM MG132) inhibitors. The MG132 was added for 6 h, either simultaneously with G007-LK for a 6 h treatment, or during the last 6 h of a 24 h G007-LK treatment. As shown in the immunofluorescence images, the addition of MG132 caused a reduction in the % of cells with visible TNKSi-induced axin puncta after 6 h treatment. The 6 h MG132 treatment also caused a modest increase in nuclear staining of axin. The later addition of MG132 (at the end of a 24 h G007-LK treatment) caused the induced axin puncta to relocate to the perinuclear region. <b>B.</b> The number of axin puncta per cell were scored by microscopy and categorized (<5, 5–15 or >15 puncta per cell). As shown, TNKSi induced a high number of puncta per cell (more than 80% of cells scored >15 puncta per cell) and this was blocked at 6 h or reduced after 24 h by MG132 treatment. <b>C.</b> A diagram summarizing the effect of G007-LK +/- MG132 on axin pattern and cellular distribution (shown in green).