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. 2023 Feb 14;15(4):1216. doi: 10.3390/cancers15041216

Figure 5.

Figure 5

Integrin β3 modulates iron metabolism and antioxidant response in TKI-resistant cells. (A) Changes in the basal expression of iron metabolism effectors were analysed in the age-matched sensitive (PAR) or resistant (NR) TBCP-1 cell lysates by Western blot and GAPDH was used as loading control. The intensity ratio of each band relative to GAPDH is indicated in red. The data show representative blots and (B) normalised expression relative to GAPDH ± SD from three independent lysates (n = 3). Statistical significance was determined by paired t-test, p < 0.05 was considered significant, * p < 0.05, ns = not significant. (C) The impact of addition of exogenous iron (500 μM Ferric Ammonium Citrate, FAC) on neratinib sensitivity of TBCP-1 or TBCP-1NR cells was determined using a 3-day SRB assay. Data shows mean IC50 ± SD from three independent experiments (n = 3). Statistical significance was determined using a non-parametric Wilcoxon matched-pair t-test, ns = not significant, * p < 0.05. (D) Changes in the basal expression of ferroportin-1 and ferritin were analysed in TBCP-1NR control or integrin β3 KO or OE whole cell lysates by Western blot and GAPDH was used as loading control. The intensity ratio of each band relative to GAPDH is indicated in red. The data show representative blots from two independent experiments (n = 2). (E) Neratinib (0.3 μM) or Erastin (5 μM)-induced cell death in TBCP-1 cells was prevented by the addition of NAC (2 mM). Cell death induced by the combination of ABT263 (0.5 μM) and S63845 (0.5 μM) was not blocked in combination with NAC. Cells were treated with the indicated inhibitors and growth relative to control was determined after 72 h using a 3-day SRB assay. Data show mean percentage growth relative to control ± SD of triplicates from a representative experiment of three independent experiments (n = 3). Statistical significance was determined using two-way ANOVA Tukey’s multiple comparison test. p < 0.05 was considered significant. **** p < 0.0001 ns = not significant. (F) The impact of neratinib treatment on System Xc- activity was determined by quantitation of FITC-labelled cystine uptake (0–1000 μM) in control or neratinib (100 μM)-treated TBCP-1 cells. Data are expressed as uptake rate (nmol/(mg protein.min)) (left panel) or represented as double reciprocal Lineweaver Burk plot (right panel) indicating reduced Vmax but same Km in neratinib treated TBCP-1 cells. (G) Cell surface expression of SLC3A2/CD98 was analysed in TBCP-1, TBCP-1NR, integrin β3 KO, and OE cells using standard flow cytometry. Data is presented as a staggered plot to highlight subtle changes in expression profiles. (H) TKI resistance and integrin β3 overexpression were characterised by increased uptake of FITC-labelled cystine in mouse TBCP-1 cells. Data are expressed as uptake rate (nmol/(mg protein.min)) and statistical significance was determined using the Mann–Whitney t-test, p < 0.05 was considered significant, *** p < 0.001. The uncropped blots are shown in Supplementary File S1.