Fig. 4

RANK knockdown slightly resensitizes SKLR cells to lapatinib. a The expression levels of RANK mRNA in lapatinib-resistant SKLR cells stably transduced with non-targeting (control) or two independent RANK knock-down (sh#3 and #4) vectors. RANK expression values were quantified by RT-qPCR relative to PP1A gene expression. Quantifications were performed in triplicates. b Relative number of living (relative survival) cells stably transduced with control (SKBR3 and SKLR), sh#3 or sh#4 (SKLR) and incubated for 4 days with the indicated concentrations of lapatinib. Cells were seeded in growth media; 24 h later lapatinib was added and cells were analyzed with CCK8 as detailed in the “Methods” section. The mean values and SD of four independent experiments are shown. For each experiment, data was obtained from quintuplicates. Paired t tests were done between the groups, and the two-tailed p value is depicted (**). In accordance with the lower expression of RANK achieved, sh#3 significantly reduced survival compared to SKLR control cells at 0.063 (p = 0.0097), 0.125 (p = 0.0055), and 0.25 (p = 0.0003) μM of lapatinib. For sh#4, a significant reduction in survival was observed at 0.125 μM of lapatinib (p = 0.014). The significance of relative survival was calculated for each concentration using two-tailed p values for one sample t test. c Western blot showing the levels of NF-κB (p-p65, p-IκB) and HER2 (p-HER2, p-ERK1/2, p-AKT) pathway activation in control SKBR3, lapatinib-resistant SKLR, and sh#3 SKLR cells treated with RANKL or lapatinib. Cells were serum-starved for 12 h and then treated with lapatinib (2 h) or RANKL (10 min) before processing them. Tubulin was used as a loading control (see Fig. S5B for total protein levels and Fig. S5C for relative quantifications)