Fig 1: Distribution of GnRH and c-FOS immunoreactive cells in the preoptic area and hypothalamus after administration of 3 doses of 15 mg human Kisspeptin-10 intravenously (at 1 hr intervals) during proestrous phase of estrous cycle in cows.A) GnRH immunoreactive neurons (arrow) in the medial preoptic area (mPOA) nucleus showing cytoplasmic DAB (brown) staining. B) GnRH immunoreactive neurons that co-express c-FOS immunoreactive reaction (Nickel-DAB black color). Note that c-FOS is localized in the nucleus (arrow). C-D) GnRH (brown) and c-FOS (black) immunoreaction cells and nerve fibers in the arcuate nucleus and median eminence.
Fig 2: Mechanistic of c-FOS promoting neutrophil recruitment and NETs formation in triple-negative breast cancer
Fig 3: p38-ROS regulates NETs formation through c-FOS. A Detection of protein expression levels of PAD4, p38, and p-p38 in neutrophils after stimulation with CM from various cell lines. B IF detection of ROS expression levels in neutrophils after stimulation with CM from various cell lines. C After simultaneously stimulating neutrophils with MDA-MB-231 or HCC1937 CM and inhibiting ROS (Acetylcysteine), inhibiting the p38 pathway (SB203580), activating the p38 pathway (Metformin HCl), or through combined interventions, detection of protein expression levels of PAD4 and c-FOS in neutrophils. D, E After simultaneously stimulating neutrophils with MDA-MB-231 or HCC1937 CM and inhibiting ROS (Acetylcysteine), inhibiting the p38 pathway (SB203580), activating the p38 pathway (Metformin HCl), or through combined interventions, IF detection of expression levels of c-FOS and SYTOX GREEN in neutrophils. Data are presented as means from three independent experiments ± S.D. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant
Fig 4: Inhibition of c-FOS can reduce the expression of inflammatory factors in neutrophils. A, B Luminex detects the expression levels of cytokines after c-FOS inhibition. C Luminex detects the expression levels of MIP-1α after c-FOS inhibition. D Luminex detects the expression levels of IL-1ra after c-FOS inhibition. E Heat map showing the correlation of inflammatory cytokines between the two groups. F Assessment of the effect of MIP-1α on neutrophil migratory ability
Fig 5: c-FOS is a key gene affecting NETs formation in breast cancer. A Detection of protein levels of c-FOS and PAD4 in neutrophils after stimulation with CM from different cell lines. B Detection of protein levels of c-FOS and PAD4 in neutrophils after stimulation with CM from different cell lines or CM pre-treated with T5224. C IF detection of expression levels of c-FOS and SYTOX GREEN in neutrophils after stimulation with CM from different cell lines. D IF detection of expression levels of c-FOS and SYTOX GREEN in neutrophils after stimulation with CM from different cell lines or conditioned media pre-treated with T5224. E SEM detection of NETs formation. F Detection of protein expression levels of c-FOS and PAD4 in neutrophils after inhibiting PAD4 (GSK484), inhibiting c-FOS (T5224), over-expressing c-FOS (recombinant c-FOS protein), or simultaneous interventions. G, H ChIP-qPCR detection of the binding ability of c-FOS and PAD4. I JASPAR predicts the c-FOS sequence that binds to the PAD4 promoter region. J Predicted sequences that bind to the PAD4 promoter region. K Detection of the binding region between the c-FOS and PAD4 promoters using a luciferase reporter assay. L, M T5224 reduced the enhanced tumor migratory ability induced by the neutrophil-conditioned medium (CM). Data are presented as means from three independent experiments ± S.D; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant
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