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. 2022 Mar 15;132(6):e154074. doi: 10.1172/JCI154074

Figure 3. CGA functions by binding to EGFR and activating EGFR downstream signaling in GC cells.

Figure 3

(A) Human phosphorylated RTK antibody array in SGC7901 cells serum starved for 12 hours and then treated with rCGA for 30 minutes. (B) Immunoblotting of CGA, EGFR, and p-EGFR in indicated serum-starved cells. (C) Immunoblotting of p-EGFR in serum-starved SGC7901 cells treated with indicated concentrations of rCGA (top) or treated with rCGA (20 μg/mL) at different time points (bottom). (D) Immunoblotting with indicated antibodies of lysates from CGA–/– SGC7901ADR cells that were pretreated with cetuximab (10 μg/mL) followed by rCGA treatment. (E) Immunoblotting of lysates from SGC7901 cells that were incubated with rCGA and immunoprecipitated with anti-EGFR antibody or normal IgG. (F) Immunoblotting of lysates from SGC7901 cells transfected with Flag-tagged RFP or EGFR containing FL, ECD, or ICD plasmids, treated with purified His-tagged rCGA, and subjected to anti-Flag and anti-His immunoprecipitation. (G) Molecular docking analysis of CGA to the ECD of EGFR. (H) SPR analysis of the interaction between CGA and the ECD of EGFR. Raw response (RU) curves (top) from a representative experiment were fitted to a 1-site-specific kinetic model (bottom) to derive on and off rates and a Kd value for the interaction. (I) IF staining of CGA, EGFR, and early endosome marker EEA1 in SGC7901 cells treated with rCGA at 37°C for a 30-minute time course. Scale bar: 10 μm. (J) Viability of SGC7901 and NCI-N87 cells stably expressing CGA, treated with fluorouracil, cetuximab, erlotinib (20 nM), or their combination. (K and L) SGC7901 cells stably expressing CGA and control SGC7901 cells (K) or SGC7901ADR cells (L) were injected subcutaneously into nude mice (n = 6–8). When the tumor size reached 100 mm3, mice received indicated treatment every 3 days (fluorouracil, 20 mg/kg, i.p. injection; cetuximab, 1 mg/mouse, i.p. injection). Tumor volume and tumor weight were measured. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01 by 1-way ANOVA with Bonferroni’s post hoc test (J–L) or by repeated-measures ANOVA with Bonferroni’s post hoc test (K and L).