Loss of HS mitigates the potentiating effect of R-spondin on Wnt signaling. (A) Western blot analysis of nuclear and cytoplasmic distribution of β-catenin in control-transduced (EXT1 wild-type [WT]) or EXT1 KO HMCLs after treatment with recombinant Wnt3a, R-spondin, or both. Tubulin (cytoplasm) and TBP (nucleus) served as fractionation and loading controls. For quantification, the expression level in Wnt3a and R-spondin–treated WT cells was normalized to an arbitrary level of 100 units. (B) Western blot analysis of β-catenin accumulation in control (buffer)– or heparitinase-treated primary MM cells stimulated with recombinant Wnt3a, R-spondin, or both. Actin served as a loading control. For quantification, the signal in Wnt3a and R-spondin–treated control 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 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 of 3 independent experiments in triplicate is shown. **P ≤ .01 using 1-way analysis of variance with Bonferroni correction.