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. 2021 Dec 30;14(1):173. doi: 10.3390/cancers14010173

Figure 1.

Figure 1

CME of EGFR is essential for CTX-induced apoptosis. (A,B) Localization of EGFR after EGF stimulation. HSC3, Ca9-22, and TSU cells were stimulated with 100 ng/mL EGF for 60 min. (A) EGFR localization was analysed via immunofluorescence staining. Scale bars, 10 μm. (B) Cell-surface EGFR was analysed using flow cytometry. (C) Amount of total EGFR after EGF stimulation. Cells were stimulated with 100 ng/mL EGF for the indicated times and total EGFR expression was analysed by using Western blotting. (D) Cell-surface EGFR expression after CTX treatment. Cells were treated with 100 μg/mL CTX for 60 min, and then cell-surface EGFR was stained with an anti-EGFR antibody (clone LA1). (E) Co-localization of EGFR with endosomes and lysosomes after CTX treatment. The localizations of EGFR and the endosome and lysosome markers were analysed via immunofluorescence staining after 100 μg/mL CTX treatment for 30 min (Rab5 and Rab7) or 60 min (LAMP1). Scale bars, 10 μm. (F) Amount of total EGFR expression after EGF stimulation. Cells were stimulated with 100 μg/mL CTX for the indicated times and total EGFR expression was analysed by using Western blotting. (G) Effects of CPZ on EGFR internalization and degradation after EGF or CTX treatment. HSC3 cells were pretreated with 5 μM CPZ for 30 min and were then stimulated with 100 ng/mL of EGF or 100 μg/mL of CTX for 60 min. CHX was added before adding CPZ. The localization of EGFR was analysed via immunofluorescence staining (upper panels). Total EGFR expression was analysed via Western blotting (lower panels). Scale bars, 10 μm. (H) Apoptosis after CTX given with CPZ. HSC3 cells were cultured in the presence of 5 μM CPZ for 30 min, followed by incubation with 100 μg/mL of CTX for 12 h in serum-free medium. Cells were harvested and stained with Annexin V-APC and 7-AAD. NT, no treatment. Bars indicate the percentage of apoptotic cells. * p < 0.05. (I) Phosphorylation of EGFR and major downstream molecules after CTX given with CPZ. Cells were pretreated with 5 μM CPZ for 30 min, after which they were treated with 100 μg/mL of CTX for the indicated times.