Fig. 7: Notch3 downregulation induces EGFR internalization and intracellular arrest. | Oncogenesis

Fig. 7: Notch3 downregulation induces EGFR internalization and intracellular arrest.

From: NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest

Fig. 7

a Upper panel: FACS analysis of the EGFR surface expression (EGFREC) in control (siCTR) and Notch3-silenced (siN3) cells after EGF stimulation for the time indicated, shown as percentage of the mean fluorescence intensity (MFI) respect to the EGF-untreated cells (t = 0). lower panel: Western blot analysis of the total extracts from the same cells probed with anti-Notch3 (N3IC) antibody, to test the efficiency of Notch3 silencing, and with anti-EGFR and anti-pEGFR(Y1173) antibodies, to evaluate the EGFR expression. The β-actin expression was used as loading control. b MDA-MB-468 cells were Notch3-silenced for 48 h and EGF-treated for 2 h: Immunofluorescence assay (IF) was performed by using anti-Notch3 green) or anti-EGFR (red) antibodies to test the efficacy of Notch3 silencing and to reveal the endogenous EGFR intracellular localization, respectively. Nuclei were DAPI labeled (blue). EGFR/DAPI merge is shown. White arrows indicate peri-nuclear EGFR localization in Notch3-silenced cells. The * indicate the higher magnification of a single EGF-stimulated control cell (left) and Notch3-silenced (right) cell. All the panels are representative single plane confocal IF images captured using a × 60 oil objective. Scale bar: 10 μm. The results are representative of three independent experiments

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