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Fig. 4 | Breast Cancer Research

Fig. 4

From: Neoadjuvant neratinib promotes ferroptosis and inhibits brain metastasis in a novel syngeneic model of spontaneous HER2+ve breast cancer metastasis

Fig. 4

Neratinib induces cell death by ferroptosis. a Liproxstatin-1 prevents erastin- or RSL3-induced ferroptotic cell death in TBCP-1 (red bars) or SKBR3 cells (black bars). b Neratinib-induced cell death in TBCP-1 or SKBR3 is rescued by co-incubation with liproxstatin-1. c Erlotinib-, lapatinib-, afatinib- and tucatinib-induced TBCP-1 (red bars) or SKBR3 (black bars) cell death is not rescued by liproxstatin-1. Cells in a and b were incubated with erastin (5 μM), RSL3 (0.5 μM) or neratinib (300 nM, TBCP-1 or 5 nM, SKBR3) with or without liproxstatin-1 (2 μM) as indicated. TKIs in c were used at the following concentrations based on their relative activity determined in Fig. 3a and b for TBCP-1 and SKBR3, respectively; erlotinib (16 μM and 500 nM), lapatinib (500 nM and 100 nM), tucatinib (300 nM and 50 nM) and afatinib (500 nM and 100 nM). Viable proliferating cells were quantitated after 72 h by SRB staining. Data in ac are expressed as percentage of control vehicle-treated cells and show mean ± SD of three independent experiments performed in triplicates. ****p < 0.0001. n/s, not significant. d Neratinib does not induce caspase-dependent apoptosis. Cells were treated for 6 h with a combination of BH3 mimetic (ABT263, 0.5 μM) + MCL1 inhibitor (S63845, 0.5 μM) or with neratinib (300 nM) in the absence or presence of caspase inhibitor Q-VD (20 μM). e Whole-cell lysates from control (DMSO), BH3 mimetics or neratinib-treated TBCP-1 cells were analysed by western blotting for the expression of caspase-3. A representative blot from three independent experiments is shown on the left and quantitation of cleaved caspase-3 bands relative to α-tubulin is shown on the right (n = 3). **p < 0.01, ***p < 0.001. f, g Lysates from control, neratinib or neratinib + liproxstatin-treated cells were analysed for the expression of ACSL4, ferritin, ferroportin-1 and transferrin receptor-1. A representative blot from three experiments is shown in f and quantitation relative toGAPDH (n = 3) shown in g. *p < 0.05, **p < 0.01. h ICP-MS analysis of iron content in control and inhibitor-treated TBCP-1 cells. Data are expressed as ng/106 cells and show mean ± SD of six replicate samples. **p < 0.01

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