Fig 1: N1-polarized neutrophils induced by cisplatin-mediated tumor ferroptosis exerted an anti-tumor effect. (A) Relative mRNA expression of CXCL1 and CXCL2 was tested by RT-qPCR assay in A549 cells treated with 5 µM DDP or rescued by 10 µM ferrostatin-1. (B) and (C) Concentration of CXCL1 (B) and CXCL2 (C) released by A549 cells in culture medium, which was tested by ELISA assay. A549 cell were treated with 5 µM DDP or rescued by 10 µM ferrostatin-1. (D) and (E) Relative mRNA expression of neutrophil cytotoxic markers (D) and pro-inflammatory markers (E) were tested by RT-qPCR assays in neutrophils co-cultured with A549 cells, which were pretreated with 5 µM DDP or rescued by ferrostatin-1. (F) Cell death ratio of A549 cells treated with DDP or rescued by ferrostatin-1 before and after being co-cultured with neutrophils. (G) Quantification of data in (F). DDP pretreatment remarkably increases the cytotoxic effect of neutrophils which would be rescued by ferrostatin-1. Bar graphs represent the mean ± SD of indicated samples. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: Chemokines released by colonoids at 20 or 2% O2. Conditioned medium from colonoids at 20 or 2% O2, followed by 24h treatment with TNF, IL17 or TNF/IL17, and untreated colonoids were analyzed for chemokine concentration (A) Mean protein concentration (pg.ml-1) of chemokines in conditioned medium from colonoids (n = 3 independent assays) analyzed by Human Chemokine Panel featuring 40 magnetic bead-based immunoassays. The 18 heatmaps show expression of significantly regulated proteins and are grouped according to 1) Highest protein expression by TNF/IL17 treatment at 20% O2. 2) Highest protein expression by TNF/IL17 treatment at 2% O2. 3) Highest protein expression by TNF treatment at 20% O2, and 4) Highest protein expression by TNF or TNF/IL17 treatment, similar at 20 and 2% O2 (B) CCL20, CXCL1, CXCL2, CXCL5, CXCL8, CXCL10 and CXCL11 protein concentrations (pg.ml-1) in conditioned medium from minimum six assays measured by ELISA. Left panels: paired data at 2 or 20% O2 across all treatments for the selected chemokines, analyzed with Wilcoxon matched-pairs signed rank test. Significance level is indicated as p values or not significant (ns). Right panels: concentrations for each chemokine in 2% (blue) or 20% (red) O2 plotted as individual values with mean and SD. Statistical analysis were performed on log2 transformed data (Supplementary Data Sheet 2). Alterations across different treatments within each oxygen concentrations were analyzed with one-way ANOVA followed by Tukey’s multiple comparisons or Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Alterations across different oxygen concentrations within same treatment were analyzed by two-way ANOVA followed by Holm-Šídák multiple comparisons test. *p < 0.05; red and blue asterisks show comparisons across different treatments within 20 and 2% O2 conditions, respectively. Black asterisks indicate significant comparisons between 2 and 20% oxygen concentrations within a specific treatment.
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