Fig 1: 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 2: 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 Human IL-18 enzyme-linked immunoassay kit