Fig. 3

Neratinib inhibits cell proliferation and HER2 signalling in vitro. Proliferation of a TBCP-1 and b SKBR3 cells was measured over 3 days in the presence of TKIs in a standard 3-day SRB colorimetric assay. Dose-response curves were plotted to determine the half-maximal inhibitory concentrations (IC₅₀) for each compound using Hill’s equation in the GraphPad Prism software. Each point on the curves represents the mean ± SD of three independent experiments performed in triplicate (n = 3). c Summary of TKI receptor selectivity and IC₅₀ in TBCP-1 and SKBR3 cells. d Changes in levels of p-EGFR, total EGFR, p-HER2, total HER2, p-MAPK, total MAPK, p-AKT and total AKT proteins in TBCP-1 or SKBR3 cells following 1 h of neratinib treatment (300 nM for TBCP-1 or 5 nM for SKBR3) were measured by western blotting. Representative blots are shown on the left. GAPDH was used as a loading control. Densitometric quantitation of phospho/total protein ratio (right panels) was completed using ImageJ software. Data show mean ± SD of three independent experiments (n = 3). *p < 0.05, **p < 0.01, ****p < 0.0001