Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP in CRC cells inhibits neutrophil infiltration and NETs formation through IL‐8. (A) Human cytokine array of the CM collected from SW620 cells with vector control and MIIP overexpression. (B) ELISA quantification of secreted IL‐8 protein levels in CM derived from three CRC cell lines with differential MIIP expression ( n = 3). (C) Volcano plot of DEGs from RNA‐seq analysis of CRC cell lines with varying levels of MIIP expression ( n = 3 per group). (D) CXCL8 mRNA expression in human CRC cells with different levels of MIIP expression was tested by RT–qPCR ( n = 3). (E) Quantification of neutrophils that migrated to CRC CM with or without recombinant human IL‐8 protein (rhIL‐8). Quantification of neutrophils that migrated to the CRC CM with or without anti‐IL‐8 neutralizing antibody (N‐IL‐8) ( n = 3). (F–H) Representative images and statistical quantification of IF staining ( n = 5) and MPO‐DNA ELISA ( n = 3) for NETs formation after neutrophils were co‐cultured with the CM from SW620 cells treated with recombinant IL‐8 protein and with the CM from SW480 and HCT116 cells treated with an anti‐IL‐8 neutralizing antibody. Scale bar, 50 µm. All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using Student's t‐test and two‐way ANOVA in (B, D, E, G, and H).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Plasmid Preparation, Control, Over Expression, Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, RNA Sequencing, Analysis, Quantitative RT-PCR, Recombinant, Staining, Cell Culture

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP in CRC cells decreases neutrophil infiltration and NETs formation through NF‐κB p65/IL‐8 signaling. (A) Bubble plot of the enriched KEGG pathways showing that the NF‐κB signaling pathway was enriched in HCT116 cells (marked with a red border). (B) GSEA revealed that MIIP knockdown in HCT116 cells significantly activated the NF‐κB signaling pathway. (C) Western blotting analysis of key proteins involved in the NF‐κB p65 signaling pathway in human CRC cells with different levels of MIIP expression. (D) Representative images of H&E and IHC staining for MIIP, p‐p65, IL‐8, and Ly6G in tumors from the cecum of an orthotopic CRC mouse model between the MIIP‐overexpressing group and the control group (n = 5 per group). Scale bar, 50 µm. (E) Statistical analysis of Ly6G + neutrophils and the expression of p‐p65 and IL‐8 in different groups. (F) Representative images and statistical quantification of IF staining for MPO (red), Cit‐H3 (green), and DAPI (blue) in tumor tissue from the cecum and comparisons between the two groups ( n = 5). Scale bar, 25 µm. (G) Western blotting of NF‐κB p65 and NF‐κB p‐p65 in CRC cells treated with or not treated with an NF‐κB inhibitor (Bay 11–7082, 10 µM). (H) RT‐qPCR of CXCL8 mRNA in CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (I) ELISA of IL‐8 in CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (J) Quantification of neutrophils that migrated to the CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (K–M) ELISA of MPO‐DNA and representative images and statistical quantification of IF staining for NETs formation after neutrophils were cultured with CM from SW480 and HCT116 cells treated or not treated with an NF‐κB inhibitor ( n = 3–5). Scale bar, 50 µm. All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using Student's t‐test and the Mann‐Whitney U test in (E); the Mann‐Whitney U test in (F); and two‐way ANOVA in (H, I, J, K, and M).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Knockdown, Western Blot, Analysis, Expressing, Immunohistochemistry, Control, Staining, Tissue, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, MANN-WHITNEY

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP modulates the NF‐κB p65/IL‐8 signaling pathway ‐mediated neutrophil infiltration and NETs formation through interactions with PKR. (A) IP–MS revealed PKR as a MIIP‐interacting protein. (B) The binding structure of MIIP and PKR was predicted using HDOCK molecular docking software. (C) Endogenous interaction between MIIP and PKR were detected via Co‐IP in HCT116 cells. (D) 293T cells were cotransfected with MIIP‐Flag and PKR‐HA, and reverse and forward Co‐IP analyses were performed. (E) Co‐IP assays were used to determine the PKR‐binding region of MIIP. (F) Co‐IP assays revealed that the PKR binding site for MIIP encompasses its IKKβ‐binding domain. (G) The expression of PKR was determined by Western blotting in CRC cells with different levels of MIIP expression. (H) Co‐IP was performed to examine the effect of MIIP expression on the interaction between PKR and IKKβ. (I–M) Double‐stable transgenic CRC cell lines were used in a rescue assay to demonstrate that the regulatory effects of MIIP on the NF‐κB p65‐mediated IL‐8 signaling pathway (I–K) and NETs formation (L, M) were dependent on PKR ( n = 3–5). All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using two‐way ANOVA in (J–M).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Protein-Protein interactions, Binding Assay, Docking Assay, Software, Co-Immunoprecipitation Assay, Expressing, Western Blot, Transgenic Assay, Rescue Assay

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: Schematic illustration shows that MIIP inhibits colorectal cancer progression by decreasing TANs infiltration and NETs formation through the PKR/NF‐κB p65/IL‐8 axis, providing potential therapeutic strategies for colorectal cancer.

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques:

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP in CRC cells inhibits neutrophil infiltration and NETs formation through IL‐8. (A) Human cytokine array of the CM collected from SW620 cells with vector control and MIIP overexpression. (B) ELISA quantification of secreted IL‐8 protein levels in CM derived from three CRC cell lines with differential MIIP expression ( n = 3). (C) Volcano plot of DEGs from RNA‐seq analysis of CRC cell lines with varying levels of MIIP expression ( n = 3 per group). (D) CXCL8 mRNA expression in human CRC cells with different levels of MIIP expression was tested by RT–qPCR ( n = 3). (E) Quantification of neutrophils that migrated to CRC CM with or without recombinant human IL‐8 protein (rhIL‐8). Quantification of neutrophils that migrated to the CRC CM with or without anti‐IL‐8 neutralizing antibody (N‐IL‐8) ( n = 3). (F–H) Representative images and statistical quantification of IF staining ( n = 5) and MPO‐DNA ELISA ( n = 3) for NETs formation after neutrophils were co‐cultured with the CM from SW620 cells treated with recombinant IL‐8 protein and with the CM from SW480 and HCT116 cells treated with an anti‐IL‐8 neutralizing antibody. Scale bar, 50 µm. All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using Student's t‐test and two‐way ANOVA in (B, D, E, G, and H).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Plasmid Preparation, Control, Over Expression, Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, RNA Sequencing, Analysis, Quantitative RT-PCR, Recombinant, Staining, Cell Culture

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP in CRC cells decreases neutrophil infiltration and NETs formation through NF‐κB p65/IL‐8 signaling. (A) Bubble plot of the enriched KEGG pathways showing that the NF‐κB signaling pathway was enriched in HCT116 cells (marked with a red border). (B) GSEA revealed that MIIP knockdown in HCT116 cells significantly activated the NF‐κB signaling pathway. (C) Western blotting analysis of key proteins involved in the NF‐κB p65 signaling pathway in human CRC cells with different levels of MIIP expression. (D) Representative images of H&E and IHC staining for MIIP, p‐p65, IL‐8, and Ly6G in tumors from the cecum of an orthotopic CRC mouse model between the MIIP‐overexpressing group and the control group (n = 5 per group). Scale bar, 50 µm. (E) Statistical analysis of Ly6G + neutrophils and the expression of p‐p65 and IL‐8 in different groups. (F) Representative images and statistical quantification of IF staining for MPO (red), Cit‐H3 (green), and DAPI (blue) in tumor tissue from the cecum and comparisons between the two groups ( n = 5). Scale bar, 25 µm. (G) Western blotting of NF‐κB p65 and NF‐κB p‐p65 in CRC cells treated with or not treated with an NF‐κB inhibitor (Bay 11–7082, 10 µM). (H) RT‐qPCR of CXCL8 mRNA in CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (I) ELISA of IL‐8 in CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (J) Quantification of neutrophils that migrated to the CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (K–M) ELISA of MPO‐DNA and representative images and statistical quantification of IF staining for NETs formation after neutrophils were cultured with CM from SW480 and HCT116 cells treated or not treated with an NF‐κB inhibitor ( n = 3–5). Scale bar, 50 µm. All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using Student's t‐test and the Mann‐Whitney U test in (E); the Mann‐Whitney U test in (F); and two‐way ANOVA in (H, I, J, K, and M).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Knockdown, Western Blot, Analysis, Expressing, Immunohistochemistry, Control, Staining, Tissue, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, MANN-WHITNEY

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP modulates the NF‐κB p65/IL‐8 signaling pathway ‐mediated neutrophil infiltration and NETs formation through interactions with PKR. (A) IP–MS revealed PKR as a MIIP‐interacting protein. (B) The binding structure of MIIP and PKR was predicted using HDOCK molecular docking software. (C) Endogenous interaction between MIIP and PKR were detected via Co‐IP in HCT116 cells. (D) 293T cells were cotransfected with MIIP‐Flag and PKR‐HA, and reverse and forward Co‐IP analyses were performed. (E) Co‐IP assays were used to determine the PKR‐binding region of MIIP. (F) Co‐IP assays revealed that the PKR binding site for MIIP encompasses its IKKβ‐binding domain. (G) The expression of PKR was determined by Western blotting in CRC cells with different levels of MIIP expression. (H) Co‐IP was performed to examine the effect of MIIP expression on the interaction between PKR and IKKβ. (I–M) Double‐stable transgenic CRC cell lines were used in a rescue assay to demonstrate that the regulatory effects of MIIP on the NF‐κB p65‐mediated IL‐8 signaling pathway (I–K) and NETs formation (L, M) were dependent on PKR ( n = 3–5). All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using two‐way ANOVA in (J–M).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Protein-Protein interactions, Binding Assay, Docking Assay, Software, Co-Immunoprecipitation Assay, Expressing, Western Blot, Transgenic Assay, Rescue Assay

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: Schematic illustration shows that MIIP inhibits colorectal cancer progression by decreasing TANs infiltration and NETs formation through the PKR/NF‐κB p65/IL‐8 axis, providing potential therapeutic strategies for colorectal cancer.

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques:

Journal: International Dental Journal

Article Title: NLRP12 Alleviated Periodontal Destruction via Suppressing Nuclear Factor Kappa-B Signalling Pathway

doi: 10.1016/j.identj.2026.109417

Figure Lengend Snippet: LPS triggered an inflammatory response and suppressed the osteogenic differentiation potential of PDLSCs. (A) The proliferative capacity of PDLSCs exposed to varying LPS concentrations over 2, 4, and 6 days was measured with CCK-8 assay. (B) The qRT-PCR analysis revealed elevated mRNA expression of inflammatory mediators IL-6, IL-8 , and IL-10 in PDLSCs following treatment with 10 µg/mL LPS. The qRT-PCR (C) and WB (D) analysis revealed significantly lower expression of osteogenic markers (COL1 and RUNX2) in PDLSCs exposed to 10 µg/mL LPS. The ALP (E) and alizarin red (F) staining assays showed that 10 µg/mL LPS significantly inhibited the osteogenic differentiation capacity of PDLSCs compared to the control group. Data were presented as mean ± SD ( n = 3). *** P < .001, **** P < .0001.

Article Snippet: After blocking with 10% normal goat serum (Beyotime), sections were covered overnight at 4°C with primary antibodies against IL-6 (#21865-1-AP; Proteintech), IL-8 (#94407; CST), IL-10 (#ab290735; Abcam), COL1 (#12256; CST) and RUNX2 (#8486; CST), then covered with secondary antibodies Goat Anti-Rabbit IgG (Zsbio), and then stained with DAB (Zsbio) for 20 to 60 seconds and counterstained with haematoxylin.

Techniques: CCK-8 Assay, Quantitative RT-PCR, Expressing, Staining, Control

Journal: International Dental Journal

Article Title: NLRP12 Alleviated Periodontal Destruction via Suppressing Nuclear Factor Kappa-B Signalling Pathway

doi: 10.1016/j.identj.2026.109417

Figure Lengend Snippet: NLRP12 overexpression reduced LPS-induced inflammatory response and reversed LPS-induced suppression of osteogenic differentiation in PDLSCs. (A and B) The qRT-PCR and WB data revealed that expression level of NLRP12 in oeNLRP12 group was increased significantly compared to oeNC group. (C) The qRT-PCR data revealed that mRNA expression of IL-6 and IL-8 was suppressed, while the mRNA expression of IL-10 was increased in oeNLRP12 + LPS group compared to oeNC + LPS group. (D) The expression of COL1 and RUNX2 was increased significantly detected by WB assays in oeNLRP12 + LPS group compared to oeNC + LPS group. (E) Representative pictures of ALP staining showed lower staining in oeNC + LPS group compared to oeNC group and higher staining in oeNLRP12 + LPS group compared to oeNC + LPS group (scale bar = 500 μm). (F) Representative pictures of alizarin red staining showed fewer mineralized nodules in oeNC + LPS group compared to oeNC group and more mineralized nodules in oeNLRP12 + LPS group compared to oeNC + LPS group (scale bar = 500 μm). oeNC: PDLSCs transfected via negative control lentiviral. oeNLRP12: PDLSCs transfected via lentiviral with overexpression- NLRP12 . oeNC + LPS: PDLSCs transfected via negative control lentiviral and subsequently cultured under 10 µg/mL LPS induction. oeNLRP12 + LPS: PDLSCs transfected via lentiviral with overexpression- NLRP12 and subsequently cultured under 10 µg/mL LPS induction. Data were presented as mean ± SD ( n = 3). ns, no significant difference, *** P < .001, **** P < .0001.

Article Snippet: After blocking with 10% normal goat serum (Beyotime), sections were covered overnight at 4°C with primary antibodies against IL-6 (#21865-1-AP; Proteintech), IL-8 (#94407; CST), IL-10 (#ab290735; Abcam), COL1 (#12256; CST) and RUNX2 (#8486; CST), then covered with secondary antibodies Goat Anti-Rabbit IgG (Zsbio), and then stained with DAB (Zsbio) for 20 to 60 seconds and counterstained with haematoxylin.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Staining, Transfection, Negative Control, Cell Culture

Journal: International Dental Journal

Article Title: NLRP12 Alleviated Periodontal Destruction via Suppressing Nuclear Factor Kappa-B Signalling Pathway

doi: 10.1016/j.identj.2026.109417

Figure Lengend Snippet: Overexpression of NLRP12 alleviated the inflammatory responses and osteogenic differentiation inhibition of PDLSCs by suppressing the NF-κB pathway. (A) WB results showing the changes of protein expression levels of p-p65, p65, p-IκBα, and IκBα in PDLSCs after NLRP12 overexpression. (B) WB results showing the changes of protein expression levels of p-p65 and p65 in PDLSCs overexpressing NLRP12 after PMA treatment. (C)The qRT-PCR results demonstrating the transcriptional expression levels of IL-6, IL-8 , and IL-10 in PDLSCs overexpressing NLRP12 after PMA treatment. (D)WB results demonstrating alterations in the protein expression levels of COL1 and RUNX2 in PDLSCs overexpressing NLRP12 after PMA treatment. (E) Representative pictures showing ALP staining (scale bar = 500 μm) (F) Representative pictures showing alizarin red staining (scale bar = 500 μm). oeNC: PDLSCs transfected via negative control lentiviral. oeNLRP12: PDLSCs transfected via lentiviral with overexpression- NLRP12 . oeNC + LPS: PDLSCs transfected via negative control lentiviral and subsequently cultured under 10 µg/mL LPS. oeNLRP12 + LPS: PDLSCs transfected via lentiviral with overexpression- NLRP12 and subsequently cultured under 10 µg/mL LPS. Data were presented as mean ± SD ( n = 3). ns, no significant difference, * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: After blocking with 10% normal goat serum (Beyotime), sections were covered overnight at 4°C with primary antibodies against IL-6 (#21865-1-AP; Proteintech), IL-8 (#94407; CST), IL-10 (#ab290735; Abcam), COL1 (#12256; CST) and RUNX2 (#8486; CST), then covered with secondary antibodies Goat Anti-Rabbit IgG (Zsbio), and then stained with DAB (Zsbio) for 20 to 60 seconds and counterstained with haematoxylin.

Techniques: Over Expression, Inhibition, Expressing, Quantitative RT-PCR, Staining, Transfection, Negative Control, Cell Culture

Journal: International Dental Journal

Article Title: NLRP12 Alleviated Periodontal Destruction via Suppressing Nuclear Factor Kappa-B Signalling Pathway

doi: 10.1016/j.identj.2026.109417

Figure Lengend Snippet: Overexpression of NLRP12 in PDLSCs alleviated the inflammatory responses and promoted periodontal regeneration in periodontitis rats. (A) The timeline demonstrated the experimental procedures. (B) The micro-CT scanning results. (C) Representative images of H&E staining (the black scale bars and white scale bars are 200 and 100 μm, respectively). (D) Representative images of Masson trichrome staining (the black scale bars and white scale bars are 200 and 100 μm, respectively). (E) Cementoenamel junction (CEJ)–alveolar bone crest (ABC) distance. (F) bone volume/total volume (BV/TV). (G) Loss of attachment in H&E staining. (H) Semiquantitative analysis of Masson trichrome staining. (I) Representative immunohistochemical images showing the expression level of inflammatory factor IL-6, IL-8, and IL-10, osteogenic factor COL1, and RUNX2, and TRAP staining showing the number of osteoclasts. Scale bar = 100 μm. (J) Quantitative analysis of IL-6-positive cells, IL-8-positive cells, IL-10-positive cells, COL1-positive cells, and RUNX2-positive cells. (K) Quantitative result of TRAP+ cells. Control group: without treatment. Periodontitis group: ligature-induced experimental periodontitis model. oeNC group: periodontitis group treated with oeNC PDLSCs. oeNLRP12 group: periodontitis group treated with oeNLRP12 PDLSCs. IHC: immunohistochemistry. IOD, integrated option density. The data are presented as mean ± SD ( n = 6 rats per group). *P < .1 , **P < .01 , ***P < .001 , ****P < .0001 .

Article Snippet: After blocking with 10% normal goat serum (Beyotime), sections were covered overnight at 4°C with primary antibodies against IL-6 (#21865-1-AP; Proteintech), IL-8 (#94407; CST), IL-10 (#ab290735; Abcam), COL1 (#12256; CST) and RUNX2 (#8486; CST), then covered with secondary antibodies Goat Anti-Rabbit IgG (Zsbio), and then stained with DAB (Zsbio) for 20 to 60 seconds and counterstained with haematoxylin.

Techniques: Over Expression, Micro-CT, Staining, Immunohistochemical staining, Expressing, Control, Immunohistochemistry