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. 2021 Jan 19;11(7):3392–3416. doi: 10.7150/thno.52435

Figure 2.

Figure 2

Activated ERK and AKT signaling mediated EGFR-induced ILT4 expression. (A) MAPK was among the most significantly altered signaling pathways upon treatment with osimertinib in H1975 cells with osimertinib (0.2 µM) for 24 h. mRNA was extracted and microarray was performed for cluster analysis of altered signaling pathways. (B) EGFR expression in NSCLC tissues was positively correlated with EKR (r = 0.44), NF-κB1 (r = 0.38), P65 (r = 0.37), and AKT1(r = 0.32) in the TCGA database. The online tool GEPIA was used to analyze the correlation of EGFR with key modulators of MAPK, NF-κB, and AKT signaling pathways. A correlation coefficient (r) of > 0.3 was considered significant. (C) pEGFR expression in lung adenocarcinoma and squamous cell carcinoma was positively correlated with activation of MAPK1/3, AKT1/2/3, and NF-κB1 in the RPPA database. The online tool cBioportal and Spearman correlation coefficient were used to evaluate the relevance. (D-E) Treatment with a gradient concentration of gefitinib or osimertinib inhibited ERK1/2, P65, and AKT phosphorylation in PC9 (D) or H1975 (E) cells. PC9 or H1975 cells were treated with different concentrations of gefitinib or osimertinib for 24 h and Western blotting was performed to determine the phosphorylation of signaling molecules. (F) EGF stimulation activated ERK1/2, P65, and AKT phosphorylation in H1299 cells treated with 100 ng/mL EGF for 24 h before Western blotting. (G-I) Treatment with ERK (U0126) or AKT (MK2206) inhibitor rather than NF-κB (PDTC) inhibitor decreased ILT4 expression in both PC9 and H1975 cells treated with different concentrations of specific inhibitors for 72 h and Western blotting was performed. The average results from 3 independent experiments are shown. Gef: gefitinib; Osi: osimertinib.