Loss of HS interferes with upstream, receptor-proximal Wnt pathway activation. (A) Flow cytometry analysis of Wnt reporter activity in control-transduced (EXT1 wild-type [WT]) or EXT1 KO HMCLs XG1 and LME1 treated with the GSK3 inhibitor CHIR99021 or DMSO. Wnt activity is plotted as the percentage of mCherry/TOP-GFP double-positive live cells. The mean ± standard deviation of 3 independent experiments in triplicate is shown. Not significant (ns) P > .05 using 1-way analysis of variance with Bonferroni correction. (B) Western blot analysis of phospho-LRP6 (pLRP6) and total LRP6 (tLRP6) in control-transduced (EXT1 WT) or EXT1 KO HMCLs XG1 and LME1 treated with Wnt3a, R-spondin, or both. Tubulin served as a loading control. For quantification, the phosphorylation level in Wnt3a and R-spondin–treated WT cells was normalized to an arbitrary level of 100 units.