Loss of HS impairs Wnt pathway activation by paracrine Wnts. (A) Flow cytometry analysis of Wnt reporter activity in 6 TOP-GFP–transduced HMCLs treated with Wnt3a, R-spondin, or both. Wnt reporter activity is plotted as the percentage of mCherry/TOP-GFP double-positive live cells. The mean ± standard deviation (SD) of 3 independent experiments in triplicate is shown. (B) Western blot analysis of the nuclear and cytoplasmic distribution of β-catenin in control-transduced (wild-type [WT]) or EXT1 KO HMCLs after treatment with Wnt3a. Tubulin (cytoplasm) and TBP (nucleus) served as fractionation and loading controls. For quantification, the expression level in Wnt3a-treated WT cells was normalized to an arbitrary level of 100 units. (C) Flow cytometry analysis of Wnt reporter activity in control-transduced (EXT1 WT) or EXT1 KO HMCLs after treatment with Wnt3a. Left panel: representative picture of TOP-GFP reporter assay in HMCL OPM2 is shown. Right panel: Wnt reporter activity is plotted as the percentage of mCherry/TOP-GFP double-positive cells. The mean ± SD of 3 independent experiments in triplicate is shown. *P ≤ .05, **P ≤ .01 using 1-way analysis of variance with Bonferroni correction.