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. Author manuscript; available in PMC: 2025 Apr 16.
Published in final edited form as: Sci Signal. 2024 Apr 16;17(832):eadf4299. doi: 10.1126/scisignal.adf4299

Fig. 7. EPS15 is required for Wnt9a-Fzd9b endocytosis and signaling.

Fig. 7.

(A) Super TOP Flash assays in HEK293 cells stably expressing Fzd9b-mKate and the Super TOP Flash reporter, treated with the indicated siRNA, and induced with conditioned medium (CM). Data presented are from N=3 independent experiments conducted on different days, each with 3 biological replicates. (B) Representative confocal z-stacks showing mKate and Gamillus in Fzd9b-mKate cells, treated with siRNAs as indicated, and induced with CM containing Wnt9a-Gam for one minute. Scale bar, 10 μm. The percentage of cells showing endocytosis of Fzd9b-mKate was quantified on the right; N=3-6, biological replicates, as indicated by dots. (C) Zebrafish at 40 hpf stained for the hematopoietic stem and progenitor cell maker cmyb by in situ hybridization. Scale bar, 100 μm. The number of cmyb+ cells in the aorta was quantified on the right. Data presented are from N=15 biological replicates, with experiments conducted on 3 different days with similar results. (D) EPS15 construct schematics. (E) Super TOP Flash assays conducted in HEK293 cells stably expressing Fzd9b-mKate and Super TOP Flash, transfected with EPS15 constructs, and induced with Wnt9a-Gam CM. Data presented are from N=3 biological replicates, with different experiments conducted on different days a total of 3 times with similar results. ****P<0.0001 by Two-tailed student’s t-test in (C). *P<0.05, **P<0.01, ***P<0.001 by ANOVA with Tukey post-hoc comparison for all other panels.