Fig. 2: AREG induces signaling and proliferation of NSCLC cell lines.
From: Amivantamab efficacy in wild-type EGFR NSCLC tumors correlates with levels of ligand expression
A NSCLC H292 cells were stimulated with 100 ng/mL of rAREG in the presence of 10 μg/mL of amivantamab or IgG1 isotype control (Isotype). Cells were harvested and lysed before stimulation (time 0) and at 5-, 15-, and 30 min post AREG treatment. Lysates were subjected to capillary-based electrophoresis to quantitate the levels of pEGFRY1173/T669/Y1086, EGFR, pErk1/2T202/Y204, Erk1/2, pAktS473, Akt, and actin (as loading control). Densitometry calculations are included in the Supplementary Fig. 2B. B Serum-starved H292 cells were stimulated with increasing concentrations of rAREG. Luminescence compared to T0, determined by ATP-dependent CellTiter-Glo (CTG) assay, were plotted over a time-course (24, 48, and 72 h post-stimulation). Dotted line represents T0. P-value was determined by two-way ANOVA with Dunnet’s multiple comparison tests; **** P < 0.0001. C AREG-low H1703 and H838 NSCLC cell lines were transduced at multiplicity of infection (MOI) of 1 or 10 with an AREG-containing lentiviruses or an empty vector control (EV). Equal amount of whole cell protein lysates was subjected to western blotting for AREG and loading control α-tubulin. The densitometry calculations are included in the Supplementary Fig. 2G. D Cell-free supernatants from H1703 and H838 cells stably expressing AREG were collected and AREG secretion measured by ELISA. Gray areas = above/below the limit of detection. E Stably expressing AREG H1703 and H838 cells were cultured overnight in low-serum conditions (1%) and whole cell lysates were collected and subjected to capillary-based electrophoresis for pEGFRY1086, EGFR, pErk1/2T202/4, Erk1/2, pAktS473, Akt, and β-actin detection and quantification. The densitometry calculations are included in the Supplementary Fig. 2G. F H1703 (i) and H838 (ii) cells were cultured for up to 6 days (144 h) in low serum (1%) and cell number measured by CTG to determine cell proliferation over time (0, 72, and 144 h). Dotted line represents T0. P-value was determined by two-way ANOVAs with Dunnet’s multiple comparison tests, ****P < 0.0001.
