Fig. 2: Fluorescence turn-on response and molecular pharmacology of HX103 on EGFR.

a Fluorescence spectra of HX103 (5 µM) in the absence and presence of EGFR (5 µM) in PBS buffer (0.1% DMSO) and after the addition of EGFR-TKI (50 µM, e.g., gefitinib, afatinib, and compound 4). b Quantification of the fluorescence intensity at 570 nm is shown in (a). Data represent average values ± SEM, n = 3 independent experiments per group. The excitation wavelength for emission fluorescence spectra was 440 nm. c Fluorescence spectra of HX103 (5 µM) in the presence of distinct forms of EGFR (wild-type, L858R, 19del, and T790M) in PBS buffer (0.1% DMSO). d Quantification of the fluorescence intensity at 570 nm is shown in (c). Data represent average values ± SEM, n = 3 independent experiments per group. The excitation wavelength for emission fluorescence spectra was 440 nm. e Kinase inhibitory activity of HX103 on recombinant human EGFR wild-type, EGFR L858R, EGFR 19del, and EGFR T790M determined by a fluorescent mobility-shift assay. Data represent average values ± SEM, n = 3 independent experiments per group. f Western blots were used to evaluate the impacts of HX103 (0 to 10 µM) on EGFR activation with EGF (25 ng/mL) and its downstream AKT and ERK1/2 phosphorylation levels in HCC827. Uncropped blots are in Source Data. g–k Quantification of protein bands shown in (F and Supplementary Fig. 3). Data represent average values ± SEM, n = 3 independent experiments per group. Source data are provided as a Source Data file.