Fig. 3
WNT activation and signaling following priming in 3D microwell culture. a, b Presence of WNT3A and β-catenin siRNA suppressed expression of WNT-β-catenin-related and stem cell-related proteins. c, d Recombinant WNT3A and β-catenin plasmid upregulated WNT3A-β-catenin-related and stem cell-related proteins. e Knockdown of WNT3A reduced differentiation capacity of SGSCs and decreased formation of acinus-like organoids. Scale bars represent 100 μm. f Diameter of acinus-like organoids normalized to total number of spheroids measured after knockdown of WNT3A. Differentiation capacity also determined by measuring average number of organoids per plate after plating same number of cells. Data from five independent experiments analyzed and presented as mean ± SEM (n = 5). One-way ANOVA; Tukey’s post hoc test. *Compared with control siRNA. ***P < 0.001. g Protein levels of SG acinar cell markers (α-amylase and AQP5), tight junction proteins (TJP1), and intercellular adherence protein (E-cadherin) determined by western blotting in acinus-like organoids after induction of differentiation in presence of WNT3A siRNA. SGSC salivary gland-resident stem cell, SGSCs2D monolayer-cultured SGSCs, SGSCs3D 3D spheroid-derived SGSCs, siRNA small interfering RNA, CON control