Table 1. Presence of EGFR-NCE in various cell lines.

The presence of an EGFR-NCE pathway in the indicated cell lines was evaluated by comparing the internalization curves of WT versus clathrin-KD cells or dynamin-KD (see Supplementary Figure 9 for estimation of the KD level), or by comparing WT versus filipin-treated cells, and calculating the endocytic rate constants (Ke or Ke obs) as explained in detail in Supplementary Experimental Procedures (section—Definition of EGFR-NCE and experimental determination of CME and NCE of the EGFR—). Cells were also subjected to ¹²⁵I-EGF saturation binding assays to measure EGFR surface number/cell. Results are the average of triplicate points (s.e.m.<10%) and representative of at least two independent experiments. We would like to draw attention to the comparison between two different HeLa cell isolates, HeLa Milan and HeLa Oslo. Our previous studies (Sigismund et al, 2008; Sigismund et al, 2005) have shown that HeLa cells display EGFR-NCE. Another study, however, showed that the same type of cells only display EGFR-CME (Kazazic et al, 2006). To resolve the discrepancy, we compared the HeLa isolates used in both studies (HeLa Milan (our isolate)) and HeLa Oslo (kindly provided by IH Madshus, University of Oslo, Norway)) under the same experimental conditions. We were able to confirm the findings of both studies, with HeLa Milan, but not HeLa Oslo, displaying EGFR-NCE. In addition, since we observed that HeLa Milan displayed ∼four-fold more surface EGFRs than HeLa Oslo (∼3.0 × 10⁵ EGFRs/cell, versus ∼0.8 × 10⁵ EGFRs/cell, respectively), we investigated whether the differences in EGFR-NCE could be ascribed to the different levels of expression of the receptor. To this end, we attenuated the expression of EGFR in HeLa Milan by EGFR-KD (HeLa Milan EGFR-KD, 0.7 × 10⁵ EGFRs/cell) and tested the presence of EGFR-NCE. Also in this case, as shown in the panel, the cells displayed NCE of the EGFR.