Figure S3. Colocalization analysis of... | Rockefeller University Press

Figure S3.

$Colocalization analysis of endocytic Crb in ingressed NBs. (A–N) Single ingressed NBs co-stained for Crb, Dlg (lateral membrane marker), and the indicated markers in green. A and B are positive and negative co-localization controls, respectively: NBs from endo-Crb::GFP expressing embryo were co-stained for Crb and GFP. The GFP channel was rotated 90° clockwise in B. Scale bars, 2.5 μm. (O) Mander’s colocalization coefficients (orange bars, mean ± SD) between intracellular Crb and markers indicated in A–N and in Fig. 3 B. Blue bars indicate the fraction of NBs with PCostes >0.95. Mander’s coefficients were determined when >80% of NBs had PCostes >0.95 (markers to the left of dashed line). +/− control: 90 NBs, seven embryos; Rab5: 128 NBs, seven embryos; P(I)3P: 238 NBs, six embryos; Rab7: 159 NBs, six embryos; Sdt: 177 NBs, seven embryos; Lamp-1: 119 NBs, four embryos; Rab11: 89 NBs, four embryos; Par6: 99 NBs, four embryos; aPKC: 119 NBs, six embryos; Par3/Baz: 145 NBs, six embryos; PIP2: 42 NBs, four embryos; βH-Spectrin: 101 NBs, five embryos; Phospho-Moesin: 95 NBs, four embryos. (P) Pearson’s colocalization coefficients between intracellular Crb and the indicated markers in ingressed NBs. Bars indicate mean ± SD. N values as in O. (Q and R) Representative scatter plots of individual embryos displaying the relationship between apical surface and intracellular Crb levels in ingressing NBs (blue dots). (Q) Late stage 8 embryo (ingression is underway), 33 NBs. (R) Stage 9 embryo (ingression is complete in most NBs), 16 NBs. Note an increase in intracellular Crb as NBs reduce their apical surface. Fully ingressed cells (with apical surface ∼0 μm2) may display variable levels of intracellular Crb likely reflecting post-ingression Crb protein degradation. Purple lines represent the cubit fit of experimental data.$

Colocalization analysis of endocytic Crb in ingressed NBs. (A–N) Single ingressed NBs co-stained for Crb, Dlg (lateral membrane marker), and the indicated markers in green. A and B are positive and negative co-localization controls, respectively: NBs from endo-Crb::GFP expressing embryo were co-stained for Crb and GFP. The GFP channel was rotated 90° clockwise in B. Scale bars, 2.5 μm. (O) Mander’s colocalization coefficients (orange bars, mean ± SD) between intracellular Crb and markers indicated in A–N and in Fig. 3 B. Blue bars indicate the fraction of NBs with PCostes >0.95. Mander’s coefficients were determined when >80% of NBs had PCostes >0.95 (markers to the left of dashed line). +/− control: 90 NBs, seven embryos; Rab5: 128 NBs, seven embryos; P(I)3P: 238 NBs, six embryos; Rab7: 159 NBs, six embryos; Sdt: 177 NBs, seven embryos; Lamp-1: 119 NBs, four embryos; Rab11: 89 NBs, four embryos; Par6: 99 NBs, four embryos; aPKC: 119 NBs, six embryos; Par3/Baz: 145 NBs, six embryos; PIP2: 42 NBs, four embryos; β_H-Spectrin: 101 NBs, five embryos; Phospho-Moesin: 95 NBs, four embryos. (P) Pearson’s colocalization coefficients between intracellular Crb and the indicated markers in ingressed NBs. Bars indicate mean ± SD. N values as in O. (Q and R) Representative scatter plots of individual embryos displaying the relationship between apical surface and intracellular Crb levels in ingressing NBs (blue dots). (Q) Late stage 8 embryo (ingression is underway), 33 NBs. (R) Stage 9 embryo (ingression is complete in most NBs), 16 NBs. Note an increase in intracellular Crb as NBs reduce their apical surface. Fully ingressed cells (with apical surface ∼0 μm²) may display variable levels of intracellular Crb likely reflecting post-ingression Crb protein degradation. Purple lines represent the cubit fit of experimental data.