Fig. 4
A) Design of the siRNA screen to elucidate target space. siRNAs against 98 proteins involved in ATM regulation were screened for ability to prevent inhibition of Wnt3a/β-catenin firefly luciferase activity by PAWI-2 (100 nM), measured in the TCF-responsive pBARLRen reporter cells and normalized to Renilla luciferase as in the primary screen.
B) Screen results expressed as a Z-score relative to control (inert sequence) siRNA (blue).
C) Plot of % inhibition of Wnt3a-dependent luciferase activity for siRNAs with Z-score ≥ 2 (red). n=4; error bars = s.e.m.
D,E) Western blots showing the effect of siRNA knockdown of Aurora kinases and BUB3 on the ability of PAWI-2 (200 nM, 4 hours) to induce S1981 phosphorylation of ATM in SW480 cells (D). Quantification of phospho-S1981-ATM normalized to total ATM protein levels (E). Note siRNA directed against AURKB and BUB3 prevented phosphorylation of ATM in response to PAWI-2. n=4; * indicates p-value <0.05 (T-test).
F) Western blot showing effect of PAWI-2 (0–200 nM, 4 hours) on S15 phosphorylation and total p53 in HEK293 cells, and quantification of phospho-S15 and total p53 normalized to β-actin levels.
G) PAWI-2 (100 nM, 24 hours) induction of p53 target genes and simultaneous suppression of Wnt/β-catenin target genes in HEK293T cells treated with recombinant Wnt3a (100 ng/ml) by TaqMan qRT-PCR. Error bars = s.e.m. (n=4). *,**, *** indicates p-value <0.05, <0.01, <0.005 (T-test).
H) Model that PAWI-1, 2 mimics stress signaling to activate p53 and modulate the ability of a cell to respond to Wnt. PAWI compounds bind tubulin and activate ATM and HIPK2 at lower concentrations than needed to destabilize microtubules. ATM and HIPK2 phosphorylate and activate p53. HIPK2 also phosphorylates TCF/LEF proteins modifying their availability for Wnt/β-catenin transcriptional activity. Points of siRNA block that were used to substantiate the model in panels B,C,D and Fig. 3F,,GG are indicated.