Figure 4
- ID
- ZDB-FIG-220912-43
- Publication
- Routledge et al., 2022 - The scaffolding protein Flot2 promotes cytoneme-based transport of Wnt3 in gastric cancer
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< (A) Immunohistochemistry (IHC) analysis showing endogenous localisation of Flot2 (green) in AGS cells. TRITC phalloidin was used to visualise actin. Arrows indicate the localisation of Flot2 to filopodia. Scale bars 5 µm. High-magnification images indicate an example of a Flot2-bearing cytonemes. Scale bars 2.5 µm. (B), IHC analysis shows that Flot2 co-localises with Wnt3 on cytonemes. (C) Confocal images showing the subcellular localisation of Flot2-GFP in AGS cells. Arrows indicate the localisation of Flot2-GFP on cytonemes. (D) Confocal images highlighting co-localisation of Flot2-GFP and Wnt3-mCh on cytonemes in AGS cells (arrows). Flot2-GFP and Wnt3-mCherry also cluster and co-localising at a cytoneme contact point (arrow). (E) Representative images of SuperTOPFlash (STF) reporter fluorescence for indicated conditions. Scale bar 100 uM. (F) Relative quantification of STF mCherry reporter fluorescence in HFE cells co-cultivated with AGS cells expressing indicated constructs. Quantifications are relative to AGS control. (n per condition = 322, 443, 403, 258, 336, 306, and 297; n=number of nuclei measured). (G) Relative number of HFE cells per image after co-cultivation with AGS cells expressing indicated constructs. Significance calculated by Student’s t-test with Bonferroni correction for multiple comparisons. (n per condition = 28, 27, 26, 27, 22, 24, and 15; n=number of images). (H) Quantification of BrdU-stained cells as a percentage of the population, after co-cultivation of AGS and HFE cells, as described in Figure 2a. significance calculated by Student’s t-test with Bonferroni correction for multiple comparisons. (n per condition = 20; n=number of images). (I), Colony-forming assay of AGS cells. AGS cells were transfected with the indicated constructs and co-cultivated with AGS-RFP cells for 2 days. After sorting, AGS-RFP expressing cells were plated at clonal density; (J), Quantification of colonies after 10–12 days. Significance is calculated by Student’s t-test (n=9).
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