Fig. 2: sFRP2 specifically promotes attachment of Wnt3a on cell surfaces.

a Western blot detection of GFP-Wnt3a in lysates of MDCK cells collected at different times after treatment of the CS of co-culture of GFP-Wnt3a/L cells with sFRP2-expressing or control HEK293 cells. Experiments were carried out at 4 °C or 37 °C. β-tubulin was detected as an internal control. b–f Visualization of GFP-Wnt3a in sFRP2-, sFRP3-, exFzd8-expressing, or control cells. To examine Wnt3a distribution in the presence of sFRP2 sFRP3, or exFzd8, GFP-Wnt3a-producing L cells were co-cultured with sFRP2-, sFRP3-, or exFzd8-expressing or control HEK293 cells for 2 days before observation. All images were processed by maximum intensity projection. Three independent sets were observed for each co-culture combination. GFP fluorescence images (b–e), bright field images (b’–e’), and their merged images (b”–e”) are shown. Images in the area where sFRP, exFzd8-expressing or control cells were surrounded by GFP-Wnt3a-expressing cells are shown. In addition, FRP2-expressing cells and surrounding GFP-Wnt3a/L cells were stained with FM 4–64 Fx, a lipophilic probe that fluoresces upon binding to cell membranes and quickly endocytosed (GFP-Wnt3a image in (f), FM 4–64 image shown in magenta in (f’), and merged image in (f”)). Note that many GFP-Wnt3a puncta are localized along the cell surface. Analysis of XZ planes also revealed that some green puncta in f” are incorporated into cells (Supplementary Fig. 11). Scale bar; 50 μm. g Time-course images of GFP-Wnt3a co-stained with FM 4–64 at indicated time-point from Supplementary Movie 3. The movement of GFP fluorescent puncta with FM 4–64 in sFRP2-expressing HEK293 cells was tracked. In (f, f’, f”), cells were fixed 1 min after treatment with FM 4–64. Many green puncta overlap with FM 4–64 signals following a short treatment. White and orange arrowheads in (g) indicate movement of GFP-Wnt3a puncta incorporated into cells. Scale bar; 10 μm.