Fig 2: C9orf140 negatively regulates Wnt/β-catenin signaling at the destruction complex level by compromising Wnt3A-induced β-catenin accumulation. a Positioning C9orf140 in the Wnt pathway by epistatic analysis. Wnt/β-catenin signaling was induced by transfection of 293T cells with Wnt3A, LRP6ΔN, Dvl2, or pt.β-catenin. TOP luciferase reporter and TK-Renilla reporter, together with C9orf140, Axin1, or empty vector, were co-transfected as indicated. Data were normalized to empty vector-transfected cells and are presented as the mean + SD. b TOPFlash luciferase assays were performed in HepG2 cells transfected with C9orf140, Axin1, or empty vector. Luciferase activities were measured after treatment with L-CM or Wnt3A-CM for 18 h. Data are normalized to empty vector-transfected cells and presented as the mean + SD. c Overexpression of C9orf140 decreases β-catenin accumulation. RKO cells were first transfected with empty vector or C9orf140, treated with Wnt3A-CM, and finally harvested at the indicated times. Cell lysates were subjected to western blot analysis for the indicated proteins (upper panel). The lower panel shows quantitative analysis of active β-catenin levels normalized to GAPDH

Fig 3: ADP-ribosylation enhances interaction of Axin with phospho-LRP6 following Wnt stimulation.(a) Phospho-Arrow interacts with ADP-ribosylated Axin following Wg exposure. Immunoblot of S2R+ cells treated with dsRNA against Axin or white (negative control), then treated with Wg CM for 1 h, subjected to GST-WWE pull down, and analysed by immunoblotting with indicated antibodies. Treatment with Wg CM markedly increases the amount of ADP-ribosylated Axin pulled down with GST-WWE, but not the GST-WWER163A control. On treatment with Wg CM, phospho-Arrow is also pulled down with GST-WWE, but not the GST-WWER163A control. The pull down of phosphorylated Arrow by GST-WWE is prevented by treatment with Axin dsRNA. (a, input) Treatment with Wg CM induced a mobility shift in Axin. There is a small decrease in the level of phospho-LRP6 following Axin knockdown. These data suggest that in Drosophila, Axin is not essential for Wg-dependent phosphorylation of LRP6, but may facilitate this process. (b) ADP-ribosylation enhances the interaction of Axin with phospho-LRP6. HEK293T cells expressing Flag-Axin1 were treated with either the DMSO control or the small molecule Tnks inhibitor XAV939 for 24 h. XAV939 diminishes the Wnt3A-dependent interaction between Axin1 and phospho-LRP6. (c,d) The Tnks-binding domain enhances the interaction of Axin with phospho-LRP6 following Wnt exposure. HEK293T cells transfected with either Flag-Axin1 or Flag-Axin1ΔTBD, and subsequently treated with Wnt3A for 30 min. Lysates were immunoprecipitated by Flag antibody, followed by immunoblot using indicated antibodies. Both (c) phospho-LRP6 (S1490) and (d) phospho-LRP6 (T1572) antibodies demonstrate that Axin1 interacts with phospho-LRP6 following Wnt3A stimulation; however, deletion of the TBD of Axin1 diminishes this interaction. α-Tub was used as a loading control. These experiments were performed at least three times with representative results shown.

Fig 4: MAMDC4 is colocalized with the WNT signalosome.(A) SKCO cells were transfected with MAMDC4 and DVL2. 48 hours post transfection cultures were treated with vehicle or Wnt3a for 1hr. Cells were labeled with antibodies to MAMDC4 (green) and DVL2 (red) and counterstained with DAPI. MAMDC4 and DVL2 are colocalized in puncta regardless of Wnt3a stimulation Scale bar: 10μm. (B) Quantification of Pearson’s Correlation Coefficient (r). Wnt3a treatment does not influence MAMDC4 and DVL2 colocalization. (C) SKCO cells were transfected with MAMDC4 and Axin1. 48 hours post transfection cultures were treated with vehicle or Wnt3a for 5min. Cells were labeled with antibodies to MAMDC4 (green) and Axin1 (red) and counterstained with DAPI. MAMDC4 and Axin1 are colocalized in some puncta after Wnt3a stimulation (arrows). (D) Quantification of Pearson’s Correlation Coefficient (r). There is an increase in MAMDC4 and Axin1 colocalization that is dependent on Wnt3a stimulation. (E) Immunoblot of NeutrAvidin Pull down. (F) Quantification of LRP6 Pull down / Lysate ratio.

Fig 5: MAMDC4 promotes Wnt/β-catenin signaling.(A) Western blot analysis of HEK293 cells in the presence and absence of Wnt and with or without MAMDC4 overexpression. (B) Quantification of A shows that MAMDC4 overexpression and Wnt3a treatment results in increased non-phospho (active) β-catenin and p-LRP6. There is decreased active β-catenin in Wnt3a stimulated cells over-expressing MAMDC4 relative to Wnt3a treated vector control cells. The are no changes in total LRP6 or Axin1 levels with MAMDC4 overexpression or Wnt stimulation. Both endogenous and exogenous MAMDC4 levels are unchanged with Wnt stimulation (n = 3). (C) HEK293 cells expressing MAMDC4 or vector control were transfected with Super TOPFlash TCF/LEF reporter and renilla control vector. Cells were stimulated with Wnt3a conditioned media for 6 hours. There is an increase in luciferase with MAMDC4 overexpression. In control transfected cells, Wnt3a stimulates luciferase activity 4-fold, however when MAMDC4 is overexpressed, this increase is dampened. Error bars represent SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Statistical significance was determined using an Ordinary one-way ANOVA with Tukey’s Multiple Comparisons. (n = 3).