Supplementary Methods, Supplementary Figures 1-17, Supplementary Tables 1-19 from Complex Rab4-Mediated Regulation of Endosomal Size and EGFR Activation
Authors: Tubbesing Kate , Ward Jamie , Abini-Agbomson Raymond , Malhotra Aditi , Rudkouskaya Alena , Warren Janine , Lamar John , Martino Nina , Adam Alejandro P. , Barroso Margarida 
Published: April 2023

<p>Figure S1. Comparison of colocalization analysis coefficients, to support the colocalization approach and use of Pearson's coefficient as detailed in Supplementary Methods section entitled Colocalization analysis. Figure S2. Human mammary epithelial cells (HMEC) were internalized with AF555-Tf (shown as red) at 50 μg/mL continuously for 1 h, fixed and subjected to immunostaining with anti-EEA1 (shown as green). Figure S3. MCF10A, MDAMB231 and T47D cells were internalized with fluorescently labeled Tf for 1 h at 37oC to identify early and recycling endocytic pathway. Figure S4. Rab5-mRFP expression in MDAMB231 results in heterogenous EEA1 staining, not unlike the endogenous EEA1. Figure S5. dSTORM microscopy analysis of Tf and EEA1 endocytic compartments. Figure S6. Whole-cell morphometric quantification of early endosomes using 3D rendering by Imaris Cell Modules. Figure S7. Cells grown on glass-bottom dishes and pre-cleared with imaging medium for 30 minutes were washed, fixed, permeabilized, immunostained with anti-TfR and anti-EEA1, and labeled with DAPI. Figure S8. Immunoblotting analysis of TfR, LDLR and EGFR in whole-cell lysates of MCF10A, MDAMB231 and T47D. Figure S9. Additional time-point specific data corresponding to Figure 3D-E. Figure S10. (A-D) MCF10A and MDAMB231 cells were stimulated with unlabeled EGF for 0 or 5 min and chased for different periods of time. Figure S11. Example of quantitative analysis and visualization assays of EGF-induced EGFR-p1068 activation according to endosomal size. Figure S12. (A) EGF and DAPI merged panels associated with Figure 4D-E. Figure S13. RT-qPCR of Rab4A and Rab4B mRNA in MCF10A and MDAMB231 cells. Figure S14. MCF10A (A-D) and MDAMB231 (E-G) cells were subjected to shRNA Rab4A (shRab4A-KD) or empty vector (EV) with GFP reporter lentiviral infection. Figure S15. MCF10A over-expressing empty vector (EV) or Rab4A-myc constructs were stimulated with unlabeled EGF for 0 or 5 min, and either fixed immediately or chased for different periods of time. Figure S16. (A) EGF and DAPI merged panels associated with Figure 6B-C. Figure S17. Alterations in Rab4 expression can result in enlargement of early endosomes (EEs). Table S1. Solutions and Kits Table S2. Plasmids Table S3. List of primary and secondary antibodies Table S4. Statistical analysis for the quantification of Tf, EEA1 and LC3 colocalization from Figure 1E. Table S5. Statistical analysis (T-test, two-tail: p-value) corresponding to Figure 1G-1 (Rab5 and Rab7), Figure5AC (Rab4 and Rab11). Table S6. Statistical analysis (T-test, two-tailed) of vesicle diameter from super-resolution STORM imaging of Figure 2A, panel q. Table S7. Statistical analysis for the quantification of 3D whole-cell endocytic morphology in Figure 2B-F and Figure S7. Table S8. Statistical analysis for colocalization of EGF vs. EEA1 in Figure 3B. Table S9. Statistical analysis for Tf recycling assay in Figure 3C. t-test, two-tailed. Table S10. Statistical analysis (T-test, two-tail: p-value) corresponding to FigureS8A and S8C. Table S11. Statistical analysis for Figure 3D, for each ligand population with EGF 3D objects (Figure 3D-E and Figure S9A), LDL 3D objects (Figure S9B) and Tf 3D objects (Figure S9C-D). Table S12. Statistical analysis of quantitative immunoblotting analysis from Figure 4B-D. Table S13. Statistical analysis (t-test two-tail: p-value) of data in Figure 4E(panel i-iii, and iv). Table S14. Statistical analysis of endocytic volume data in Figure 5F and Figure 5H. Table S15. Statistical analysis of colocalization analysis in Figure 5G and Figure 5I. Table S16. Statistical analysis of endocytic volume data in Figure 5M and Figure 5O. Table S17. Statistical analysis of quantitative immunoblotting analysis in Figure 6A and Figure S15. Table S18. Statistical analysis of quantitative immunoblotting analysis from Figure 6 and Figure S14. Table S19. Statistical analysis of data in Figure 6B-C.</p>