Combination of Wnt inhibition and Olaparib enhances senescence Protein levels of HR and FA pathway genes are reduced upon Wnt inhibition in both ETC-159 treated and combination groups.Figure 6Protein levels of HR and FA pathway genes are reduced upon Wnt inhibition in both ETC-159 treated and combination groups. Tumor lysates from mice treated with vehicle, ETC-159, olaparib or combination of ETC-159 and olaparib (as shown in Figures 1C-D) for 56 hours were resolved on a 10% SDS-PAGE gel. The indicated HR and FA pathway proteins were detected by immunoblotting. Each lane represents an individual tumor. Wnt inhibition alone or in combination with olaparib does not induce apoptosis. Tumor lysates from mice treated as above for 56 hours or 21 days (Figures 1C-D) were resolved on 12% SDS-PAGE gel and analyzed by immunoblotting with the indicated antibodies. Each lane represents an individual tumor. Co-treatment with olaparib and Wnt inhibitor regulates expression of senescence associated genes. (C Expression of senescence-associated genes was analyzed in the tumors from all four treatment groups (treated for 56 hours) by qRT-PCR. Each data point represents an individual tumor and horizontal lines represent mean. p-values for the significant changes as calculated by Mann-Whitney U test are shown. Each lane represents an individual tumor. Co-treatment with olaparib and Wnt inhibitor regulates expression of senescence associated genes. (D) Tumor lysates from indicated groups were immunoblotted for p21. Each lane represents an individual tumor. C-E. Co-treatment with olaparib and Wnt inhibitor regulates expression of senescence associated genes. E) Expression of senescence-associated genes was analyzed in the tumors from all four treatment groups (treated for 56 hours) by qRT-PCR. Each data point represents an individual tumor and horizontal lines represent mean. p-values for the significant changes as calculated by Mann-Whitney U test are shown. Each lane represents an individual tumor. F, Wnt inhibition induces senescence, which is further enhanced by co-treatment with olaparib. (F) Representative images of tumor sections from the four treatment groups (treated for 21 days) stained for SA-β-galactosidase, a senescence marker, and counterstained with nuclear fast red. Blue color indicates positive staining for senescent cells. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test. G. Wnt inhibition induces senescence, which is further enhanced by co-treatment with olaparib. (G) The percentage of SA-β-galactosidase positively stained area (blue) in the representative images from each of the groups is shown. Multiple areas were imaged from three tumor samples per group (indicated as mouse 1, 2 and 3). The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test. Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (H) HPAF-II cells were treated with DMSO, ETC-159 (50 nM), olaparib (20 µM) or both for 7 days. 53BP1 foci per cell were then assessed by immunofluorescence. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test. I. Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (I) Representative immunofluorescence images of 53BP1 foci (red) and nuclei counterstained with DAPI (blue) are shown for each group.