Fig. 3

RANK expression increased in HER2-positive breast cancer cell lines after treatment with anti-HER2 therapies as well as in anti-HER2-resistant cells. a RANK gene expression levels determined by RT-qPCR in the indicated HER2-positive cell lines after short-term treatment with lapatinib (Lapa), trastuzumab (Trastu), or the combination of both, relative to corresponding untreated cells (Ctr). Quantifications were performed in triplicates from two independent experiments. b RANK gene expression levels determined by RT-qPCR in SKBR3 cell lines resistant to trastuzumab (SKTR), lapatinib (SKLR), or both drugs (SKTLR, SKLTR) compared to sensitive SKBR3 parental cells. Quantifications were performed in triplicates from at least three independent experiments. c RANK protein expression in parental SKBR3 cells (SK) or resistant to trastuzumab (SKTR), lapatinib (SKLR), or both (SKLTR). β-Actin was used as a loading control. Blots shown are representative of those obtained from 3 independent experiments. d RANK gene expression levels in BT474 cells, either control or resistant to lapatinib (LR, according to public datasets [36]). a.u., arbitrary units. e RANK and RANK/NF-κB downstream gene targets BIRC3, ICAM1, TNFα, and IL8 mRNA levels relative to housekeeping gene PP1A in parental (SKBR3, BT474) and lapatinib-resistant (SKLR, BTLR) HER2-positive cell lines with or without RANKL treatment (24 h). Gene expression levels were quantified by RT-qPCR. PP1A expression was used as an internal reference gene (a, b, e). Determinations were done in triplicates, and the mean values are depicted from n ≥ 2 independent analyses. p values were calculated by ordinary one-way ANOVA (a, b) and by unpaired t tests (d) (*≤ 0.05; **≤ 0.01, ***≤ 0.001; n.s., non-significant)