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Fig. 4
Role of PLC?1 nSH2-CSH2 domains and its interaction with SHP2 for cetuximab resistance. A and B, Western blotting of CACO-2 (shControl and shPLCG1) and SW48 (parental and pTriex-PLCG1 overexpressed) cells treated with cetuximab for 72 hr. EGFR downstream signaling, namely ERK and AKT are shown. ?-actin was used as the loading control. C, Cell viability of parental SW48 and PLCG1-overexpressing cells: full length WT; constitutively active mutant - D1019K; constitutively inactive mutant - H335A; deletion of both nSH2-CSH2 tandem domains - ?SH2; and deletions of SH3 domain - ?SH3 (n = 3). Analysis was performed using two-way ANOVA test (****P < 0.0001). D, Western blotting of EGFR downstream signaling of SW48 parental and overexpressing PLCG1 WT and PLCG1 ?SH2 mutant, after 72 hr of cetuximab treatment. ?-Actin was used as the loading control. E, Co-immunoprecipitation of PLC?1 with anti-Stag antibody and Western blot analysis of PLC?1 and SHP2 of 72 hours cetuximab treated SW48 cells. F, Co-immunoprecipitation of SHP2 with anti-SHP2 antibody and Western blot analysis of PLC?1 and SHP2 of 72 hours cetuximab treated SW48 cells. G, Proximity ligation assay of PLC?1 and SHP2 using anti-Stag and anti-SHP2 antibodies in SW48 parental, PLCG1 WT- and ?SH2-overexpressing cells and corresponding quantification (n = 4). H, Cell viability of SW48 parental and overexpressing PLCG1 WT and ?SH2 mutant, treated with cetuximab and cetuximab + SHP099 for 72 hours (n = 3). I, Western blotting of EGFR downstream signaling of SW48 parental and overexpressing PLCG1 WT and ?SH2 mutant, treated with cetuximab and cetuximab + SHP099. Results are presented as the mean ± SEM. Statistical analysis was performed using unpaired t test [not significant (ns), P > 0.05; **, P < 0.01].