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. 2017 Aug 3;8(47):82872–82884. doi: 10.18632/oncotarget.19958

Figure 3. Induction of HER1/HER2 degradation, inhibition of downstream cascades signalling and reduction of H292 cells viability.

Figure 3

(A) H292 cells were incubated during 8 hours with pooled immune sera (PAbs) from extraction day 56 (diluted 1:100), and further stimulated with EGF (10 min, 100ng/mL). STAT3, Akt and ERK1/2 expression and phosphorylation state was analysed by Western blot, and compared with cells incubated with pre-immune sera (PI) used as negative control. AG1478 (TKI) (10μM) was included as a positive control of receptor inhibition. (B) HER1 and HER2 expression was assessed by Western blot at different end points (from 30 minutes to 24 hours) in H292 cells treated with PAbs from day 35, diluted 1: 100. Cells treated with PI were considered as negative control. (C) Cells viability was determined by MTT assay after 24, 48 and 120 hours of incubation with PAbs (pattern box) or PI (white box) diluted 1: 20 and heated at 56°C for 30 minutes to inactivate the complement. Mitomycin C (MitoC, 25μg/mL) was considered as positive control of cell viability reduction (black box). The 100% of viability was referred to PI incubation. Mean ± S.D. of six experiments are represented. Statistical analysis was performed by Kruskall-Wallis test, followed by Games Howell pos-test. a vs b (p<0.05), a vs c (p< 0.01), a vs d (p<0.001).