10.1371/journal.pone.0187289.g005 Matilde Forcella Matilde Forcella Monica Oldani Monica Oldani Samantha Epistolio Samantha Epistolio Stefania Freguia Stefania Freguia Eugenio Monti Eugenio Monti Paola Fusi Paola Fusi Milo Frattini Milo Frattini EGFR phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment. Public Library of Science 2017 therapies sensitivity NSCLC cell viability NEU 3 EGFR point mutations TKI Non-Small Cell Lung Cancer Akt pathway NEU 3 overexpressing patients ERK pathway lung cancer cell lines plasma membrane-associated NEU 3 Adenocarcinoma sialidase NEU 3 EGFR pathways activation NEU 3 overexpression mucosa cell line Non-small cell lung cancer sialidase NEU 3 overexpression EGFR wild-type gene sequence 2017-10-31 17:47:15 Figure https://plos.figshare.com/articles/figure/EGFR_phosphorylation_levels_after_EGF_stimulation_sialidase_NEU3_overexpression_and_gefitinib_treatment_/5557243 <p>(A) Representative Western-blot analyses performed on a normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected either with the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-EGFR, anti-P-EGFR antibodies. GAPDH was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained in the absence of EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).</p>