Deletion of heparan sulfate by EXT1 KO reduces Wnt signaling in HMCLs. (Ai) Flow cytometry analysis of cell-surface expression of HS in control-transduced cells (Cas9-empty control) or upon CRISPR/Cas9-mediated EXT1 KO (Cas9-sgEXT1) in HMCLs. (Aii) Confocal microscopy analysis of HS expression in control-transduced cells (EXT1 wild type [WT]) or upon CRISPR/Cas9-mediated EXT1 KO in HMCL LME1. Scale bars represent 2.5 μm. (B) Analysis of the nuclear and cytoplasmic distribution of β-catenin in HMCLs after EXT1 KO by western blot. Tubulin (cytoplasm) and TBP (nucleus) served as a fractionation and loading controls. For quantification, the expression level in WT cells was normalized to an arbitrary level of 100 units. (C) Flow cytometry analysis of Wnt reporter activity in LP1 cells transduced with Wnt signaling reporter TOP-GFP or control FOP-GFP. (D) Flow cytometry analysis of Wnt reporter activity in EXT1 WT or EXT1 KO LP1 cells. A representative plot is shown at left. Quantification of Wnt reporter activity is plotted as the percentage of mCherry/GFP double-positive live cells (right). The mean ± standard deviation of 3 independent experiments in triplicate is shown. **P ≤ .01 using 1-sample t test. (E) Analysis of c-Myc and cyclin D1 expression after EXT1 KO by western blot. Actin served as loading control. For quantification, the expression level in WT cells was normalized to an arbitrary level of 100 units.