Fig. 7: The EGFR-NCE multiorganelle platform mediates EGF-induced migration in keratinocytes.

a CD147 internalization in HaCaT cells subjected to the indicated KDs or mock transfection (Ctrl). Internalized CD147 was measured by IF after stimulation with high dose Alexa647-EGF for 12 min at 37 °C, as described in Fig. 4a. Mean integrated fluorescence intensity is reported as a percentage of the control cells. N = number of cells: Control/N = 301/n = 6, RTN3-KD/N = 97/n = 3, RTN4-KD/N = 179/n = 5, MCU-KD/N = 221/n = 4, IP3R-KD/N = 210/n = 4. b HaCaT cells expressing the Ca²⁺ sensor PM-GCaMP6f were stimulated with high or low EGF. Left, Representative single-cell Ca²⁺ response curves presented as the ratio of fluorescence emission at 490/406 nm. Right, AUC of the Ca²⁺ response. N=number of cells: High EGF/N = 17, Low EGF/N = 20 (n = 2). c Ca²⁺ response in HaCaT cells subjected to RTN3 or RTN4 KD or to mock transfection (Ctrl) and stimulated with high EGF dose as in b. AUC is shown. N = number of cells: Control/N = 18, RTN3-KD/N = 25, RTN4-KD/N = 14. A representative experiment of three independent replicates is shown. d ΔΨm in HaCaT cells stimulated or not with low or high EGF, measured using TMRM. Total peak area is reported. N=number of cells: -EGF/N = 206, Low EGF/N = 253, High EGF/N = 327. A representative experiment of n = 2 is shown. e Wound healing in sub-confluent HaCaT cells stimulated or not with low or high EGF was monitored by time-lapse video microscopy. Left, representative images at the indicated time points. Middle, distance covered by cells in the different conditions corrected to the baseline at t = 0. Right, wound closure velocity (μm/h) at t = 24 h is shown. N=number of cells: -EGF/N = 15, Low EGF/N = 23, HighEGF/N = 34 (n = 3). f Wound closure velocity (μm/h) in sub-confluent HaCaT cells stimulated with high EGF dose and subjected to RTN3 KD or the indicated treatments for 24 h. N = number of cells: Control/N = 98/n = 6, DMSO/N = 24/n = ), RTN3-KD/N = 40/n = 2, OMY/N = 44/n = 3, MCUi11/N = 22/n = 2, CK666/N = 28/n = 2, XeC/N = 30/n = 2). g Schematic showing the crosstalk between EGFR-NCE, ER, and mitochondria in controlling EGFR endocytosis, receptor fate and cell migration. Box plots in all panels are defined as in Fig. 2f. Exact p-values (each pair Student’s t-test, two-tailed) are shown; ns not significant, n biological replicates. Source data are provided as a Source Data file.