Fig. 2.
Proteomic analysis reveals alterations in cellular iron homeostasis in sapatinib-treated tumors. Index tumors from MMTV-NIC-PTEN+/− mice treated with vehicle (n = 4) or sapatinib (n = 5) were subject to liquid chromatography-tandem mass spectrometry (LC–MS/MS). Protein identification and quantitation were performed using MaxQuant. a Volcano plot of proteins identified by MaxQuant. Highlighted are proteins associated with cellular iron homeostasis, ferritin heavy chain (FTH), ferritin light chain (FTL), ceruloplasmin (CP) and heme oxygenase-1 (HO-1). Dotted lines represent p = 0.05 and twofold ratio. b Bar chart showing significantly upregulated gene ontology (GO) terms related to proteins that were differentially regulated between vehicle-treated and sapatinib-treated tumors. Dashed bars represent GO terms upregulated in sapatinib-treated tumors, whereas solid bars represent those downregulated in sapatinib-treated tumors. Differentially regulated genes were selected as those which showed > twofold change and p < 0.05, as determined by MaxQuant, Wilcoxon–Mann–Whitney test. Benjamini–Hochberg corrected p-values and GO terms obtained from DAVID. c Representative immunohistochemical staining of HO-1 (vehicle: n = 23; sapatinib: n = 9). Scale bar: 50 µm. Quantification of immunohistochemical staining by Definiens Architect (bottom panels). Results presented as mean ± standard deviation. Kruskal–Wallis test, Dunn’s post hoc test, not significant = NS, p < 0.05*, p < 0.01**. d Representative immunohistochemical staining of NRF2 (vehicle: n = 20; sapatinib: n = 9). Scale bar: 50 µm. Quantification of immunohistochemical staining by Definiens Archtect (bottom panel). Results presented as mean ± standard deviation. Two-tailed Mann–Whitney test*, p < 0.01**. e G6PDH kinetic assay with measurements at 5 and 30 min in vehicle (n = 4) and sapatinib-treated (n = 5) tumors. Linear regression, not significant = NS. f GSH/GSSG ratio determined in vehicle (n = 4) and sapatinib-treated (n = 5) tumors. Results presented as scatter plot with median ±interquartile range. Two-tailed Mann–Whitney test, not significant = NS