Fig 2: MIR503HG inhibits NETs-triggered NSCLC cells metastasis capacity and NLRP3 inflammasome activation dependently on NF-?B. (A) Western blotting was used to detect the changes of p-p50, p50, p-p65 and p65 in MIR503HG overexpression A549 and SK-MES-1 cells after NETs treatment. (B) qRT-PCR and (C) Western blotting analyses of up-regulating p50 in A549 and SK-MES-1 cells. (D) Analysis of the NLRP3 and Caspase1 protein levels in MIR503HG-overexpressed NSCLC cells transfected with p50-pcDNA3.1 and pcDNA3.1 vector by western blot with NETs stimulated. Transwell invasion (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F) were performed to identify the effects of NETs on MIR503HG-overexpressed NSCLC cells invasion and migration transfected with p50-pcDNA3.1 and pcDNA3.1 vector. (*P < 0.05, **P < 0.01).

Fig 3: Differential expression of pi-p50 and pi-p65 in lung tissues from the IKKe-/- and WT groups at 24 or 48 h following IRI. (A) Representative western blotting results of each group. (B) Relative protein expression levels were normalized to GAPDH/total protein expression (n=4). *P<0.5, **P<0.01 vs. WT group. NF-?B, nuclear factor-?B; IRI, ischemia reperfusion injury; WT, wild-type; IKKe-/-, inhibitor of ?B kinase e knockout; pi, phosphorylated.

Fig 4: Cumulus-cell cytoplasmic Src-homology-2-containing phosphotyrosine phosphatase (SHP2) inhibition deteriorates oocyte maturation and embryo development. (A) SHP2/ERa (GFP-labeled SHP2 and red fluorescent protein-labeled ERa) proteins analyzed via immunofluorescence in cumulus cells during germinal vesicle breakdown (GVBD) and MII stages of control cumulus–oocyte complexes (COCs) and phenylhydrazonopyrazolone sulfonate 1 (PHPS1)-treated COCs. (B) Representative images of p-ERK1/2 and p-AKT in GVBD- and MII-stage oocytes in the control and PHPS1-exposed groups. 4',6'-Diamidino-2-phenylindole was used to stain the nucleus. (C) Immunofluorescence images of Caspase-3 and phosphorylated NF-Kb in control and PHPS1-treated bovine day 8 blastocysts. Qualitative analysis showed that PHPS1 treatment enhances p-NF-kB nuclear localization and enhances Caspase-3 expression. All experiments were repeated thrice, and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: NLRP3 inflammasome induced by NETs promotes NSCLC progression is associated with the activation of NF-?B. (A) The protein expression level of p-p50, p50, p-p65, p65 in NSCLC cells treated with NETs was detected by Western blotting. (B) Nuclear translocation of NF-?B in A549 and SK-MES-1 cells treated with NETs or combined with DNase I were detected by con-focal microscopy (magnification, 3000×; scale bar, 5µm). Immunofluorescence assays (C) and Western blot (D) were used to detect the effect of NETs on NLRP3 inflammasome in A549 and SK-MES-1 cells after p50 knockdown (magnification, 200×; scale bar, 50 µm). (E) Downregulation of p50 attenuated the effect on promoting EMT of NETs in NSCLC cells by Western blot. (F, G) Downregulated p50 reverses NETs-induced promotion of NSCLC cells metastasis using transwell assay (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F).