Fig 1: Chronic SF exacerbated the inflammatory response and promoted the formation of NETs in MI/RI mice. (A to C) Plasma concentrations of IL-1β, IL-6, and IL-18 in mice were measured by ELISA (n = 8 per group). (D to F) qRT-PCR assays were performed to determine mRNA levels of IL-1β, IL-6, and IL-18 in the hearts of MI/RI mice with SF (n = 6 per group). (G) Representative images of immunofluorescence staining of NETs in myocardial tissues from Sham, SF + Sham, MI/RI, and SF + MI/RI groups (green: MPO, red: citH3, blue: DAPI) (n = 6 per group). (H) Cell-free DNA (cfDNA) of each group (n = 6 per group). (I) The MPO–DNA complex relative levels were assessed in mice (n = 6 per group). Data are presented as mean ± SD. ###P < 0.001 versus Sham group; ***P < 0.001 versus SF + Sham group; $$P < 0.01 versus MI/RI group; $$$P < 0.001 versus MI/RI group. (J and K) Western blotting was used to analyze the expression of CXCR2 in NEs after treatment with EPI for 6 h (n = 6 per group). Data are presented as mean ± SD. ###P < 0.001 versus Control group. C + EPI, Control + EPI. (L) Schematic diagram of coculture of EPI-induced NEs and CMECs exposed to H/R. CMECs were treated with hypoxia for 12 h and reoxygenation for 24 h and then cocultured with NEs for 6 h. (M) Statistical analysis of the number of migrated NEs after coculture with H/R-induced CMECs (n = 6 independent experiments). (N and O) The cfDNA and MPO–DNA complex levels were assessed (n = 5 independent experiments). (P) After coculture, the ultrastructure of NEs was observed via the electron microscope. Data are presented as mean ± SD. ###P < 0.001 versus Control + NEs group; ***P < 0.001 versus H/R + NEs group. Scale bars, 50 μm (G) and 2.5 μm (P). NEs, neutrophils; EPI, epinephrine.
Fig 2: MCC950 attenuates the macrophage-modulating effects of ZNF831-overexpressing TNBC cells. A, B Representative images and quantification of RAW264.7 transwell migration induced by conditioned medium from ZNF831-overexpressing MDA-MB-231 or 4T1-luc cells, with or without MCC950 treatment. C, D Representative flow cytometry plots and quantification of F4/80⁺CD86⁺ macrophages after stimulation with conditioned medium from the indicated tumor cell groups. E, F RT-qPCR and western blot analyses of M1-associated markers (CD86, CD80, iNOS, and IL-6) in RAW264.7 cells treated with conditioned medium from the indicated groups. G, H Representative flow cytometry plots and quantification of F4/80⁺CD206⁺ macrophages after stimulation with conditioned medium from the indicated tumor cell groups. I, J RT-qPCR and western blot analyses of M2-associated markers (CD206, ARG1, CD163, and IL-10) in RAW264.7 cells treated with conditioned medium from the indicated groups. K LDH release assays showing that MCC950 attenuated the increase in lytic cell death associated with ZNF831 overexpression in MDA-MB-231 and 4T1-luc cells. Asterisks indicate comparisons with the corresponding Vector groups, and number signs indicate comparisons with the corresponding ZNF831 groups at the same time point. L Western blot analysis of cleaved caspase-1 (p20) and GSDMD-NT in MDA-MB-231 and 4T1-luc cells under Vector, ZNF831, or ZNF831 + MCC950 conditions. M Western blot analysis of mature IL-1β and mature IL-18 in concentrated culture supernatants from MDA-MB-231 and 4T1-luc cells under the indicated conditions. N ELISA quantification of secreted IL-1β and IL-18 in culture supernatants from MDA-MB-231 and 4T1-luc cells under the indicated conditions. Data are presented as mean ± SD from three independent experiments. For panel K, statistical comparisons were performed at each time point by one-way ANOVA with Tukey’s multiple-comparisons test. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 versus Vector; #p < 0.05, ##p < 0.01, ###p < 0.001 versus ZNF831
Fig 3: Preparation of CRPPR@Sch and assessment of the targeting ability of CRPPR@Sch. (A) Schematic illustration of CRPPR@Sch preparation. (B) TEM image of CRPPR@Sch. (C) Size of DSP@Sch and CRPPR@Sch (n = 3). (D) Fluorescence spectrum analysis of CRPPR@Sch and DSP@Sch (n = 3). (E) Zeta potential of DSP@Sch and CRPPR@Sch (n = 3). (F) Vivo fluorescence imaging of nanoparticles in the mice heart and other organs following DSP@Sch and CRPPR@Sch administration (n = 3). (G–J) Quantification of fluorescence intensity in the heart, liver, spleen and kidneys (n = 3). (K and L) Representative fluorescence images of CD31 (red), CRPPR@Sch (green) and DSP@Sch (green) in the heart of each group (n=6 per group). Scale bars: 10 μm. (M) Histology characteristics of major organs of CRPPR@Sch or DSP@Sch treated healthy mice were detected by HE staining (n = 6). Scale bars: 50 μm. (N–P) Quantification of serum IL-1β, IL-6 and IL-18 concentrations measured by ELISA in the indicated mouse groups (n = 6 per group). Data are presented as mean ± SD. ###P < 0.001 vs. Blank group; ***P < 0.001 vs. CRPPR@Sch group. ns, not significant.
Fig 4: ZNF831 promotes NLRP3-associated pyroptotic signaling and lytic cell death in TNBC cells. A Phase-contrast microscopy of MDA-MB-231 and 4T1-luc cells showing cell swelling and bubble-like membrane protrusions in the ZNF831-overexpression group. Scale bar, 50 μm. B, C LDH release assays showing that ZNF831 overexpression increased, whereas ZNF831 knockdown decreased, lytic cell death over time in MDA-MB-231 and 4T1-luc cells. In the overexpression panels, asterisks indicate comparisons between the ZNF831 and Vector groups at the corresponding time points. In the knockdown panels, asterisks indicate comparisons between the shZNF831 #1 and shNC groups, whereas number signs indicate comparisons between the shZNF831 #2 and shNC groups at the corresponding time points. D, E RT-qPCR analysis of NLRP3, ASC, caspase-1, GSDMD, IL-1β, and IL-18 mRNA levels in MDA-MB-231 and 4T1-luc cells with ZNF831 overexpression or knockdown. F, G Western blot analysis of NLRP3, ASC, caspase-1, cleaved caspase-1 (p20), GSDMD, and GSDMD-NT in MDA-MB-231 and 4T1-luc cells with ZNF831 overexpression or knockdown. H Western blot analysis of mature IL-1β and mature IL-18 in concentrated culture supernatants from MDA-MB-231 and 4T1-luc cells under the indicated conditions. I, J ELISA quantification of secreted IL-1β and IL-18 in culture supernatants from MDA-MB-231 and 4T1-luc cells under the indicated conditions. Corresponding intracellular pro-IL-1β and pro-IL-18 protein levels are shown in Fig. S3. Data are presented as mean ± SD from three independent experiments. For LDH assays, statistical comparisons were performed among groups at each individual time point. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001; #p < 0.05, ##p < 0.01, ###p < 0.001
Supplier Page from HUABIO for Mouse IL-18 enzyme-linked immunoassay kit