Fig. 5: Clathrin silencing preferentially impacts confinement of ligand-bound EGFR.

APRE-19 cells were treated with siRNAs targeting clathrin heavy chain (clathrin) or non-targeting siRNA (con.). Cells were subjected to SPT using either Fab-Cy3B to label total EGFR in the absence (A, D, G) or presence (B, E, H) of unlabelled EGF, or labelled using EGF-Cy3B (C, F, I). A–C are the mean ± SE of the fraction of EGFR tracks in each mobility category. Also shown are mean ± SE of diffusion coefficient (D–F) or confinement radius (G–I). Data is from 4 and 5 independent experiments for EGF-Cy3B (ligand-bound) Fab-Cy3B (total EGFR), respectively. Each experiment involved detection and tracking of >500 EGFR objects. ^*p < 0.05. J An additional analysis of the images from Fig. 2 involving detection of CD81 objects followed by intensity-based analysis of overlap with eGFP-clathrin, following subtraction of background (as described in Methods) n = 3 independent experiments each with >10 cells and >5000 EGFR objects quantified. Shown are the means of eGFP clathrin intensity within CD81 objects in individual experiment (dots), and the overall mean ± SE; ^*p < 0.05 relative to basal condition. K An additional analysis of the images shown in Fig. 2. Results of conditional, position-based analysis, as in Methods. Shown are the individual measurements per cell of clathrin colocalization with CD81 (regardless of EGFR context, left panels), clathrin colocalization with CD81 (conditional on additional co-localization of EGFR objects, middle panels, shaded grey), clathrin colocalization with CD81 (conditional on exclusion from co-localization of EGFR objects, right panels). Individual cellular values are grouped by experiments (circle, triangle, diamond), and experimental mean for actual image sets (blue) as well as that from randomized position of clathrin (black) and EGFR (green) channel objects relative to CD81 objects. ^*p < 0.05, ^†p < 0.5 relative to corresponding colocalization extent in (i) clathrin w/CD81 (irrespective of EGFR), (ii) clathrin w/ (CD81 w/EGFR) with randomized clathrin positions and (iii) clathrin w/ (CD81 w/EGFR) with randomized EGFR positions. Statistical analysis and p-values are indicated in Supplementary Table 1. Source data are provided as a Source Data file.