Fig 1: RelB‐upregulated S100A4 in promoting PCa aggressiveness. (A, B) The correlation of S100A4 with Gleason score (GS) of PCa patients (n = 6). (C–E) TNF‐α increased the levels of nuclear RelB and cytosolic S100A4 in LNCaP cells. (F) The effect of S100A4 on LNCaP cell migration (n = 4). (G, H) The effect of S100A4 on LNCaP cell invasion (n = 4). (I, J) The S100A4‐overexpressed LNCaP cells were orthotopically implanted into nude mice prostates to examine the effect of S100A4 on tumour formation (n = 4). (K) The S100A4 enhancer was linked to the luciferase reporter. (L‐N) The reporter responses in RelB‐ and IL‐8‐overexpressed LNCaP cells (L), RelB‐ and IL‐8‐silenced PC‐3 cells (M), and TNF‐α‐ and PTL‐treated PC‐3 cells (N) (n = 4). (O, P) The RelB‐binding site was mutated to eliminate the reporter response in PC‐3 cells (n = 4). Data are shown as the mean ± SD. **p < .01, ***p < .001, ns, no significance; t‐test (E, J), one‐way ANOVA (B, F, H, L–N, P). Scale bar, 50 µm in (A) and 20 µm in (D).
Fig 2: Effects of CTLA4+ T cells-secreted S100A4 on the stemness phenotype of TNBC cells. A Heatmap of DEGs in CTLA4+ T cells. Red represents upregulated genes, and green represents downregulated genes. B and C S100A4 expression change in T cells and the culture supernatants of T cells after co-culture with MDA-MB-231 or BCSCs-231 cells as detected by RT-qPCR and ELISA. D Flow cytometry to determine the effect of co-culture of MDA-MB-231-T or BCSCs-231-T with T cells on the proportion of CD44+ and CD24− MDA-MB-453 cells. E Flow cytometry to determine the effect of exogenous hrS100A4 on the proportion of CD44+ and CD24− MDA-MB-231 and MDA-MB-453 cells. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. All cell experiments were repeated three times
Fig 3: Effects of DAMPs and MCP1 in macrophage migration. In the CytoSlectTM 24-well cell migration assay, THP1 was differentiated for 48 h. (A) Next, macrophages were exposed to S100A4 (2 ng/mL [pink]), S100A8 (3 ng/mL [purple]), S100A9 (2 ng/mL [blue]), HMGB1 (100 ng/mL [green]), or MCP1 (100 ng/mL [yellow]). (B) Macrophages were exposed to the same DAMPs with or without RAGE inhibitors (RAP [angled hatches]) and TLR4 (TAK [horizontal hatches]). The experiment was repeated three times (n = 3). The quantification of cells in the top and bottom of the insert was measured via fluorescence. Results are expressed as a fold change between treated and non-treated (NT) conditions and were analyzed via ANOVA statistical test (Kruskal–Wallis), followed by Dunn’s post hoc test. * p = 0.05. The horizontal line corresponds to the value of the non-treated cells reported as 1.
Fig 4: The correlations of PSA, IL‐8, and S100A4 to PCa patients’ malignancy. (A–C) Concentrations of PSA (A), IL‐8 (B), and S100A4 (C) in serum specimens from PCa patients (n = 70) vs. normal healthy donors (n = 20). (D–F) The corrections of PSA (D), IL‐8 (E), and S100A4 (F) with patients’ survival. Samples from PCa patients included 25 non‐metastasis, 22 lymph‐metastasis, and 23 bone‐metastasis). Data are shown as the mean ± SD. *p < .05, **p < .01, ***p < .001; one‐way ANOVA.
Fig 5: The mRNA expression profiles in RelB‐silenced PC‐3 cells. (A, B) After silencing RelB in PC‐3 cells (A), the mRNA expression profiles were examined by RNA sequencing. IL‐8 (CXCL8) and S100A4 are indicated by green arrows (B). (C–E) The mRNA profiles were analyzed by KEGG pathway enrichment, assembling in cytokine (C), cell adhesion (D), and calcium signalling (E). (F) The correlation between S100A4 and RelB or RelA in PCa was analyzed using the Oncomine database. (G, H) S100A4 mRNA and protein levels in PCa cell lines vs. a normal cell line (PZ‐HPV‐7). (I, J) S100A4 mRNA and protein levels in RelB‐ or IL‐8‐overexpressed LNCaP cells. Data are shown as the mean ± SD. n = 4, *p < .05, ***p < .001, ns, no significance; one‐way ANOVA).
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