Fig. 5

Overactivation of RANK signaling in HER2-positive cell lines increased NF-κB activation and lapatinib resistance. a Expression levels of RANK mRNA in HER2-positive SKBR3, BT474, and HCC1954 cells stably transduced with control (empty) or RANK-overexpressing (RANK) vectors. RANK expression values were quantified by RT-qPCR relative to PP1A gene expression. Experiments were performed in triplicates and standard error is depicted. b Expression levels of RANK/NF-κB downstream gene targets BIRC3, ICAM1, TNFα, and IL8 relative to PP1A gene expression in cells described in a, with and without RANKL treatment (24 h). Experiments were performed in triplicates and standard error is depicted. c Relative number of living (relative survival) SKBR3, BT474, and HCC1954 cells stably transduced with control (empty) or RANK-overexpressing (RANK) vectors incubated for 4 days with the indicated concentrations of lapatinib and/or stimulated with RANKL. Cells were seeded in growth media with/without 100 ng/ml RANKL; 24 h later, lapatinib was added and cells were analyzed with CCK8 after 4 days as detailed in the “Methods” section. A representative experiment out of three independent experiments is shown. For each experiment, data was obtained from triplicates and SD, and a two-way ANOVA p value is included. d. Western blot analyses of NF-κB (p-p65) and HER2 (p-HER2, p-ERK1/2, and p-AKT) pathway activation in cells depicted in c. Before collecting the cells, they were cultured in media without FBS for 12 h and pretreated with/without lapatinib for 2 h followed by 10 min stimulation with RANKL. Representative blots from three independent experiments are shown. Tubulin was used as a loading control (see Fig. S6B for total protein levels, Fig. S6C for quantifications and Fig. S7 for EGF/HRG stimulations)