(A) Immunohistochemistry (IHC) analysis of AGS cells stained for Ror2 (red) and Flot2 (green). Flot2 and Ror2 show co-localisation with a Pearson’s correlation coefficient (PCC) of 0.65 (n=10), highlighted at the membrane by arrows. Scale bars represent 10 µm, and in high-magnification images, 2.5 µm (right). (B–D), Confocal live-cell imaging of AGS cells expressing Ror2-BFP with Flot2-GFP (B), Flot2 siRNA (c) or ∆N-Flot2-GFP (D) and memCherry. Arrows highlight subcellular regions of co-localisations. (E) Live confocal images of AGS cells expressing Ror2-mCherry and indicated organelle markers +/- Flot2 siRNA. Arrows highlight the co-localisation of Ror2-mCherry and mTurq2-Golgi. Scale bar 10 µm F, Quantification of co-localisation of Ror2-mCherry with indicated markers, assessed by PCC. Significance is calculated by Student’s t-test. (n per condition [WT]=7, 10, 8, and 10) (n per condition [Flot2 siRNA]=7, 6, 7, 8). (G) Representative images of AGS cells stably expressing the JNK-KTR-mCherry reporter and indicated constructs after 48 hr. Blue dotted line encircles the cytoplasm and yellow dotted line the nucleus of a representative cell. Scale bar 20 µm. (H) Quantification of the JNK-KTR-mCherry reporter. Nuclear and cytoplasmic fluorescence of cells were measured, and the cytoplasmic: nuclear ratio was calculated. Significance is calculated by one-way ANOVA with Bonferroni correction for multiple comparisons. (n per condition = 136, 109, 74, 109, 82, and 79). (I) Representative confocal images of AGS cells expressing memCherry and indicated constructs for 48 hr. Scale bars 10 µm. (J, K) Quantification of cytoneme length and number of AGS cells transfected with constructs indicated in (i). Significance calculated by Student’s t-test with Bonferroni correction for multiple comparisons. (n per condition = 25, 22, 21, 23, 25, and 21; n=number of cells).