Fig. 3

Oxy186 and Oxy210 inhibit WNT signaling in A549 cells. A. Oxy186 and Oxy210 inhibit WNT reporter (TOPflash) luciferase activity in A549 cells. A549 cells transfected with TOPflash and pRL-TK were treated with the indicated compounds in the absence or presence of WNT CM. After 24 h, Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Oxy186 and Oxy210 inhibited WNT signaling, while the control Oxy189 did not. The WNT pathway inhibitor FH535 is shown as a positive control. Data from a representative experiment are reported as the mean of triplicate measurements ± SD. B. Oxy186 and Oxy210 inhibit WNT reporter luciferase activity independent of GLI activities. TopFlash luciferase assays were performed as above. Oxy186 and Oxy210 inhibit WNT CM-induced TOPflash luciferase activity in Gli1^−/−;Gli2^−/− MEFs. C. Oxy186 and Oxy210 inhibit WNT pathway target gene expression. A549 cells were cultured in the absence or presence of WNT CM for 24 h, RNA was extracted and analyzed by RT-qPCR for the expression of the indicated genes and normalized to RPS18 expression. Data reported as the mean (n = 5) ± SD (*** p < 0.001; ** p < 0.02). D. Oxy186 and Oxy210 inhibit TOPflash luciferase activity induced by expression of the WNT co-receptor LRP6, but not by expression of a combination of DVL2 and CSNK1E, or by CTNNB1. A549 cells were transfected with TOPflash and pRL-TK as well as the indicated plasmids, and treated with Oxy186, Oxy210 or control Oxy189 for 24 h before harvesting for luciferase assays. Data from a representative experiment are reported as the mean of triplicate measurements ± SD (***p < 0.001; **p < 0.02)