Figure 2. Endocytic and exocytic endogenous NOTCH1 receptor trafficking in MCF10A cells.

(A) Confocal sections of MCF10A cells treated and immunolabeled as indicated. Upon BafA1 treatment, plasma membrane (PM) localization of both NOTCH1 and EGFR is reduced, and both receptors accumulate in LAMP1-positive compartments (arrows) (A') Quantification of panel (A). (B) Confocal sections of MCF10A cells treated and immunolabeled as indicated. 4-h EGTA treatment depletes NOTCH1 from the PM and strongly accumulates it in a RTN3-positive compartment (yellow arrow), whereas EGFR remains on the PM (red arrow). In unstimulated cells, a pool of both NOTCH1 and EGFR is visible in the RTN3-positive compartment (white arrow). (B') Quantification of panel B. (C) Confocal sections of MCF10A cells treated and immunolabeled as indicated. An intracellular pool of NOTCH1 and EGFR colocalizes with the Golgi apparatus (GA) marker, GIANTIN (white arrow). Compared with non-EGTA–treated cells, 4-h EGTA treatment causes diffused NOTCH1 and EGFR accumulation in the cytosol, with some EGFR remaining on the PM (green arrows). The GIANTIN signal is also diffused consistent with an expected fragmentation of the GA. EGTA washout (w/o) for 1 h causes all NOTCH1 and most EGFR signal to localize in the GA (red arrows). EGTA w/o for 4 h or overnight (ON) restores normal intracellular distribution of NOTCH1 and EGFR. (C') Quantification of panel (C). *, **, ***, ****, ns indicate P < 0.05, P < 0.01, P < 0.001, P < 0.0001, and not significant, respectively).