FIG 1.

DVL-deficient cells are unable to respond to Wnt3a. Both wild-type (WT) and DVL1/DVL2/DVL3 triple knockout (DVL KO) HEK293 cells were treated with Porcupine inhibitor (LGK974) for 24 h to reduce autocrine signaling, and then the cells were treated for 2 h (for Western blotting [WB]) or for 14 h (for the TopFlash reporter assay) by control (ø) or Wnt3a (3a) conditioned medium (CM). (A) In DVL KO HEK293 cells, S1490-LRP6 phosphorylation and active β-catenin were not induced by Wnt3a; total LRP6 (tLRP6 ) levels were not changed. A lack of DVL2 and DVL3 in DVL KO HEK293 cells served as a control for cell identity. (i) Western blots; (ii) quantification of density of pS1490-LRP6 compared to that of actin (n = 3). (B) DVL1 antibody testing. DVL KO HEK293 cells were transfected according to the loading scheme and analyzed by WB. None of the anti-DVL1 antibodies was able to detect DVL1 specifically. All DVL constructs were expressed to a comparable level, as demonstrated by antitag (GFP, FLAG, and HA) staining. (C) TCF/LEF-dependent transcription is not induced in DVL KO HEK293 cells, as analyzed by the TopFlash reporter assay (n = 4). (D) DVL KO HEK293 cells are not able to induce the expression of AXIN2 after Wnt3a treatment, as quantified by RT-PCR (n = 4). Cells were treated with mouse Wnt3a recombinant protein (rmWnt; 100 ng/ml). (E) (Top) Analysis of cells of the DVL KO HEK293 T-REx cell line. DVL KO T-REx cells are not able to phosphorylate S1490-LRP6 after Wnt3a induction. A lack of DVL2 and DVL3 in DVL KO HEK293 cells served as a control for cell identity. (Bottom) The guide RNA (gRNA) design and the sequences of verified deletions in the DVL KO T-REx cell line edited by the CRISPR/Cas9 system are shown. Analysis for statistically significant differences was performed by paired Student's t test (*, P < 0.05; **, P < 0.01).