Journal: The FASEB Journal

Article Title: Matrix Metalloproteinase‐8 Deficiency Alters the Balance Between Viral Clearance and Immunopathology in H1N1 Infection

doi: 10.1096/fj.202601719RRR

Figure Lengend Snippet: H1N1 IAV infection induces greater increases in Type I (but not Type II) IFN levels in Mmp‐8 −/− versus WT mice. WT and Mmp‐8 −/− mice were infected with 10 3 PFU H1N1 via the intranasal route. Uninfected mice were used as controls. At intervals post‐infection, lung levels of IFN‐α (A, n = 4–10 mice per group), IFN‐β (B, n = 4–15 mice per group), and IFN‐γ (C, n = 4–10 mice per group) were measured using ELISAs and normalized to lung total protein levels. (D–H) Primary airway epithelial cells isolated from WT and Mmp‐8 −/− mice were treated with 20 ng/mL rhIFN‐β for 24 h. Eif2ak2 ( Pkr ) mRNA expression was measured by RT‐qPCR (D). Representative immunofluorescence images are shown in E and G, and the corresponding p‐RIP3 and p‐MLKL signals are quantified in F and H, respectively. In A–C, boxes in the box‐plots show the medians and 25th and 75th percentiles, and the whiskers show the 10th and 90th percentiles. Data were analyzed using a Kruskal–Wallis One‐Way ANOVA followed by pair‐wise testing with Mann–Whitney U tests. In D–H, data are from 3 independent experiments and are presented as mean ± SD. * p ≤ 0.05; ** p ≤ 0.01 versus the indicated group.

Article Snippet: ELISA kits to quantify murine IFN‐β were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: Infection, Isolation, Expressing, Quantitative RT-PCR, Immunofluorescence, MANN-WHITNEY

Journal: The FASEB Journal

Article Title: Matrix Metalloproteinase‐8 Deficiency Alters the Balance Between Viral Clearance and Immunopathology in H1N1 Infection

doi: 10.1096/fj.202601719RRR

Figure Lengend Snippet: Active MMP‐8 cleaves IL‐1β and IL‐6 in vitro. Recombinant human (rh) pro‐MMP‐8 was activated, and its activity was confirmed in each experiment using a quenched fluorogenic substrate that is susceptible to cleavage by MMP‐8, as described in the . Recombinant human cytokines, including 1000 nM IFN‐α2 (A), 1000 nM IFN‐β (B), 1100 nM IL‐1β (C), and 900 nM IL‐6 (D), were incubated with or without 300 nM active rhMMP‐8 for 18 h, with or without a non‐selective metalloproteinase inhibitor (1,10‐phenanthroline; 1,10 phen), which inhibits MMP‐8. The reaction products were separated on Tris‐tricine gels and visualized with silver staining. The arrows indicate the bands corresponding to rhMMP‐8 and the intact cytokines. The long arrows indicate the cleavage products of IFN‐α2, IFN‐β, and IL‐6 (in A, B, and D). MMP‐8 completely degraded IL‐1β; no degradation products were detected (C). The addition of 1,10‐phenanthroline blocked the cleavage or degradation of the four cytokines that was mediated by active MMP‐8.

Article Snippet: ELISA kits to quantify murine IFN‐β were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: In Vitro, Recombinant, Activity Assay, Incubation, Silver Staining

Journal: mLife

Article Title: z‐VAD‐FMK triggers macrophage necroptosis during mycobacterial infection via a non‐canonical off‐target mechanism

doi: 10.1002/mlf2.70101

Figure Lengend Snippet: Mtb‐related necroptosis relies on Toll‐like receptor (TLR) – tumor necrosis factor (TNF) signaling as well as type I interferon (IFN) signaling. (A) Effects of various gene deficiencies on Mtb‐related macrophage necroptosis based on the cell viability assay in the presence of Mtb + z‐VAD. BMDMs from wild‐type (WT) and indicated knockout mice were infected with Mtb at an MOI of 20 in the presence of z‐VAD, as in Figure , and cell viability was assayed at 24 hpi by measuring the ATP level. Detailed data are shown in Figure . (B, C) Effects of genetic deficiency on TNFα secretion by Mtb‐infected macrophages. BMDMs were infected with Mtb at indicated MOI and the cell culture supernatant was harvested at 24 hpi and analyzed by Enzyme‐Linked Immunosorbent Assay (ELISA). (D) Effects of Ifnar1 deficiency on Mtb‐related necroptosis activation. BMDMs were treated as in (A) and cells were harvested at indicated time for Western blot. (E, F) Effects of blockade of type I IFN signaling on macrophage necroptosis. Immunoblots of cell lysates (E) and cell viability measured by the ATP assay (F) are shown. BMDMs were pretreated with the indicated antibody at 20 μg/ml and compounds of Z or Z and N 2 h before infection and the treatment was continued till harvest at 10 hpi (E) or at 24 hpi (F). Data are means ± SD of two (B) or three technical replicates (C, F) and representative of two experiments. D, DMSO; Z, z‐VAD; N, Nec‐1; ND, not detected; ns, not significant; * p < 0.05; and ** p < 0.01 (two‐tailed unpaired Student's t test).

Article Snippet: Mouse IFN‐αA (#12100‐1) and mouse IFN‐β (#12405‐1) were from PBL Assay Science.

Techniques: Cell Viability Assay, Knock-Out, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, ATP Assay, Two Tailed Test

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A interacts with IRF3 and is associated with colorectal cancer (A and B) Whole-cell lysates (WCL) from RKO and HCT116 cells were collected for IP using anti-IRF3 or anti-KMT5A antibodies, followed by IB analysis. (C) Cytoplasmic and nuclear proteins from RKO were collected for IP using anti-KMT5A antibodies, followed by IB analysis. (D) Boxplots were used to compare KMT5A mRNA expression in COAD and READ, with Student’s two-tailed t test, p < 0.0001. (E) Disease-specific survival analysis of TCGA-COAD and READ based on KMT5A expression levels. (F) Protein expression of KMT5A was analyzed by IB in colorectal tumor and paired adjacent normal tissues. (G) mRNA was extracted from colorectal tumors and paired adjacent normal tissues, and KMT5A mRNA levels were analyzed by qPCR. (H) The levels of IFN-β colorectal tumor and paired adjacent normal tissues were measured using ELISA. For (D)–(H) statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Data are presented as mean ± SD. ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. All immunoblotting experiments were performed independently three times with similar results.

Article Snippet: Mouse IFN-beta ELISA kit , ABclonal , RK00420.

Techniques: Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A affects immune infiltration in colorectal tumors (A) To establish tumor models, MC38 cells with either control or mKMT5A knockdown via shRNA were injected subcutaneously into C57BL/6 mice. (B and C) Tumor mass and volume derived from the experiments in (A) were quantified. (D and E) Flow cytometry was conducted to analyze CD8 + T cell infiltration in the tumors from (A). (F) The levels of IFN-β in the tumors from (A) were measured using ELISA. (G and H) Activated T cells co-cultured with pre-treated RKO cells. Representative images (G) and statistical analysis (H) are shown. (I) The levels of IFN-β in the cell culture supernatant from (G) were measured using ELISA. For (B), (E), (F), (H), and (I) statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. For (C), two-way ANOVA with Tukey’s post-hoc test was used. Data are presented as mean ± SD. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse IFN-beta ELISA kit , ABclonal , RK00420.

Techniques: Control, Knockdown, shRNA, Injection, Derivative Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A inhibits phosphorylation activation of IRF3 (A) RKO cells were treated with poly(I:C) and protein samples were collected at 0, 8, 16, and 24 h for Western blot (WB) analysis. (B) Quantification of p-IRF3, RIG-1, and MDA5 protein levels relative to control in the samples collected in (A). (C) IFN-β secretion levels at different time points from (A) were measured using ELISA. (D, E) RKO and HCT116 cells transfected with either an empty vector or a KMT5A overexpression plasmid were treated with poly(I:C), followed by WB analysis of whole-cell lysates. (F) RKO cells transduced with control shRNA (shNC) or KMT5A-specific shRNAs (#1 and #2) were treated with poly(I:C) and analyzed by WB using whole-cell lysates. (G) RKO cells from (F) were co-transfected with the IFN-β-Luc reporter plasmid and pRL-TK plasmid, and after 24 h, luciferase activity was measured using a dual-luciferase assay kit. (H) The relative mRNA levels of KMT5A and INF-β in RKO cells from F were quantified using qPCR. (I) IFN-β levels in the RKO cells from (F) were measured using ELISA. (J) RKO cells, either wild-type or stably expressing HA-KMT5A, were treated with poly(I:C) and whole-cell lysates were collected at 0, 8, 16, and 24 h for WB analysis. (K) Quantification of p-IRF3 protein levels relative to control in samples collected in (J). For (G)–(I), statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. For (K), two-way ANOVA with Tukey’s post-hoc test was used. Data are presented as mean ± SD. Statistical significance is indicated as ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. All western blot analyses were performed independently three times, yielding consistent results.

Article Snippet: Mouse IFN-beta ELISA kit , ABclonal , RK00420.

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Over Expression, Transduction, shRNA, Luciferase, Activity Assay, Stable Transfection, Expressing

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A induces mono-methylation of lysine 193 on IRF3 (A) HEK293T cells were transfected with FLAG-IRF3 and/or HA-KMT5A plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads. Subsequent analysis was conducted via IB. (B) Whole-cell lysates were extracted from RKO cells with either control (shNC) or KMT5A shRNA (#1 and #2) silencing. IP was performed using anti-IRF3 antibodies, followed by WB analysis. (C) RKO cells were transfected with HA-KMT5A WT or HA-KMT5A D338A, IP was performed using anti-IRF3 antibodies, followed by WB analysis. (D) RKO cells were treated with either DMSO or UNC0379. IP was performed using anti-IRF3 antibodies, followed by IB analysis. (E) RKO cells were treated with either DMSO or varying concentrations of UNC0379. IP was conducted using anti-IRF3 antibodies, followed by IB analysis. (F) The levels of IFN-β in RKO cells from experiment (E) were quantified using ELISA. Data were analyzed using one-way ANOVA with Tukey’s post-hoc test, presented as mean ± SD. Statistical significance was defined as ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. (G) In vitro methylation assays were conducted by incubating purified His-IRF3 with KMT5A in the presence of S-adenosyl-L-methionine, followed by IB analysis. (H) Secondary mass spectrometry results of IRF3 K193 methylation were obtained. (I) HEK293T cells were transfected with FLAG-IRF3 wild-type or mutant plasmids, followed by transfection with either a vector or HA-KMT5A. Whole-cell lysates were collected, and IP was performed using anti-FLAG magnetic beads, followed by IB analysis. (J) Amino acid sequences at the K193 site of IRF3 were compared across different species. All immunoblotting experiments were conducted independently in triplicate, yielding consistent results.

Article Snippet: Mouse IFN-beta ELISA kit , ABclonal , RK00420.

Techniques: Methylation, Transfection, Magnetic Beads, Control, shRNA, Enzyme-linked Immunosorbent Assay, In Vitro, Purification, Mass Spectrometry, Mutagenesis, Plasmid Preparation, Western Blot

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: UNC0379 could be used as an adjuvant therapy for colorectal tumors (A) The treatment protocol of anti-PD-1 treatment combined UNC0379 in CRC. (B) C57BL/6 mice were subcutaneously injected with MC38 cells and subsequently treated with PBS, anti-mPD1, UNC0379, or a combination of anti-mPD1 and UNC0379 to establish a tumor model. (C and D) Tumor weight and volume were quantified from the samples obtained in experiment (A). (E) The concentration of IFN-β in the tumors from experiment (A) was assessed using an ELISA assay. (F–H) Flow cytometric analysis was conducted to evaluate the populations of CD8 + T cells and NK1.1+ cells in the tumors from experiment (A). For (C), (E), (G), and (H), data were analyzed using one-way ANOVA with Tukey’s post-hoc test. (D) was analyzed using two-way ANOVA with Tukey’s post-hoc test. Data are presented as mean ± SD, with statistical significance indicated as ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse IFN-beta ELISA kit , ABclonal , RK00420.

Techniques: Adjuvant, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Interferon-λ drives renal fibrosis by coordinating epithelial–fibroblast crosstalk

doi: 10.1084/jem.20251858

Figure Lengend Snippet: Type I and II IFNs differ from type III IFN in their regulation of TGF-β expression and the ERK–JNK pathway in renal fibroblasts during kidney fibrosis. (A–C) Primary renal fibroblasts from WT mice were treated for 24 h with 100 ng/ml of various IFNs subtypes (IFN-α, IFN-β, IFN-γ, or IFN-λ2) or PBS. (A and B) Isg15 , Mx1 , Ifit1 , and Tgf-β mRNA levels were assessed by RT-qPCR ( n = 6), and (C) TGF-β protein in the culture supernatants was quantified by ELISA ( n = 4). (D–F) Primary skin fibroblasts from WT mice were treated for 24 h with 100 ng/ml of various IFNs subtypes (IFN-α, IFN-β, IFN-γ, or IFN-λ2) or PBS. (D and E) Isg15 , Mx1 , Ifit1 , and Tgf-β mRNA levels were detected by RT-qPCR ( n = 6), and (F) TGF-β protein in the culture supernatants was measured by ELISA ( n = 4). (G–J) WT, Ifnar –/– , and Ifngr1 −/− mice were subjected to sham or UUO surgery, and kidneys were collected on day 7. n = 6 per group. (G) Representative images and quantitative analysis of fibrotic areas with Masson’s trichrome and PSR staining (scale bars = 50 μm). (H) RT-qPCR analysis of Acta2 , fibronectin, and vimentin mRNA levels in kidneys. TGF-β mRNA and protein levels in kidneys were measured by RT-qPCR (I) and western blot (J). (K and L) Primary kidney fibroblasts were treated with 100 ng/ml IFN-α (K) or IFN-β (L) for the indicated times. (K and L) Western blot analysis of phosphorylated and total ERK and JNK protein levels. Data in A–I are pooled from two independent experiments. Data in J–L are representative of three independent experiments. Data are presented as mean ± SEM. *P < 0.05, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparison test (A–I). ns, no significant difference. Source data are available for this figure: .

Article Snippet: Primary renal fibroblasts isolated from WT and Ifnlr1 −/− mice were stimulated with or without 100 ng/ml IFN-λ2 (250-33; PeproTech) for 1 h or 24 h. In separate experiments, primary renal and skin fibroblasts from WT mice were treated with 100 ng/ml of IFN-λ2 (250-33; PeproTech), IFN-α (CK83; Novoprotein), IFN-β (HY- P73130 ; MedChemExpress), or IFN-γ (315-05; PeproTech) for different times.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Comparison

Journal: EMBO Reports

Article Title: IFN-α and IFN-β inhibit NLRP1-driven PANoptosis

doi: 10.1038/s44319-026-00888-0

Figure Lengend Snippet: ( A , B ) Representative images ( A ) and quantification ( B ) of cell death in wild type (WT), Irf3 –/– , and Irf7 –/– mouse bone marrow-derived macrophages (BMDMs) at 16 h after treatment with Val-boroPro (VBP) or VBP plus lipopolysaccharide (LPS). ( C ) Ifnb gene expression in WT and Irf3 –/– BMDMs at 2 h and 4 h after treatment with VBP or VBP plus LPS, expressed in fold change relative to the media control for each genotype, using Actb as the housekeeping gene. ( D , E ) Representative images ( D ) and quantification ( E ) of cell death in WT and Irf3 –/– BMDMs at 16 h after treatment with VBP or VBP plus IFN-β. ( A – E ) Data were representative of three independent biological replicates, n = 4 ( B , E ) or 2 ( C ) technical replicates. Scale bar: 50 μm ( A , D ). ( B ) WT VBP vs VBP + LPS, *** P = 0.0001; Irf3 –/– VBP vs VBP + LPS, P = 0.9895 (ns); Irf7 –/– VBP vs VBP + LPS, *** P = 0.0003. ( E ) WT VBP vs VBP + IFN-β, ** P = 0.0011; Irf3 –/– VBP vs VBP + IFN-β, ** P = 0.0017. Two-way ANOVA with Sidak’s multiple comparison test was used ( B , E ). The data were represented as mean ± SEM in ( B , E ) and as mean in ( C ). .

Article Snippet: Recombinant mouse IFN-β , PBL Assay , 12400-1.

Techniques: Derivative Assay, Gene Expression, Control, Comparison

Journal: EMBO Reports

Article Title: IFN-α and IFN-β inhibit NLRP1-driven PANoptosis

doi: 10.1038/s44319-026-00888-0

Figure Lengend Snippet: ( A ) Cell death kinetics in wild-type (WT) mouse bone marrow-derived macrophages (BMDMs) following treatment with Val-boroPro (VBP) or VBP plus lipopolysaccharide (LPS). ( B ) Quantification of IFN-β in the supernatant of WT BMDMs at 3 h after treatment with VBP or VBP + LPS. ( A , B ) Data were representative of three independent biological replicates. n = 4 ( A ) or 2 ( B ) technical replicates. The data were represented as mean ± SEM in ( A ) and mean in ( B ). .

Article Snippet: Recombinant mouse IFN-β , PBL Assay , 12400-1.

Techniques: Derivative Assay

Journal: EMBO Reports

Article Title: IFN-α and IFN-β inhibit NLRP1-driven PANoptosis

doi: 10.1038/s44319-026-00888-0

Figure Lengend Snippet: ( A , B ) Representative images ( A ) and quantification ( B ) of cell death in wild type (WT), Ifnar1 –/– , and Tlr4 –/– mouse bone marrow-derived macrophages (BMDMs) at 16 h after treatment with Val-boroPro (VBP), VBP plus IFN-α, or VBP plus IFN-β. ( C , D ) Quantification of cell death in human macrophages at 24 h after treatment with VBP or VBP plus IFN-α ( C ), and human monocytes at 12 h after treatment with VBP or VBP plus IFN-α ( D ). ( E , F ) Quantification of cell death in human macrophages at 16 h after treatment with VBP or VBP plus IFN-β ( E ), and human monocytes at 12 h after treatment with VBP or VBP plus IFN-β ( F ). Donors in ( E , F ) are distinct from donors in ( C , D ). ( A – F ) Data were representative of three independent biological replicates, ( B – F ) n = 4 technical replicates. Scale bar: 50 μm ( A ). ( B ) WT VBP vs VBP + IFN-α, **** P < 0.0001; WT VBP vs VBP + IFN-β, **** P < 0.0001; Ifnar1 –/– VBP vs VBP + IFN-α, P > 0.9999 (ns); Ifnar1 –/– VBP vs VBP + IFN-β, P > 0.9999 (ns); Tlr4 –/– VBP vs VBP + IFN-α, **** P < 0.0001; Tlr4 –/– VBP vs VBP + IFN-β, **** P < 0.0001. ( C ) **** P < 0.0001. ( D ) ** P = 0.0050. ( E ) ** P = 0.0061. ( F ) * P = 0.0108. Two-way ANOVA with Sidak’s multiple comparison test ( B ) and Welch’s t -test ( C – F ) were used. The data were represented as mean ± SEM in ( B – F ). .

Article Snippet: Recombinant mouse IFN-β , PBL Assay , 12400-1.

Techniques: Derivative Assay, Comparison

Journal: EMBO Reports

Article Title: IFN-α and IFN-β inhibit NLRP1-driven PANoptosis

doi: 10.1038/s44319-026-00888-0

Figure Lengend Snippet: ( A ) Heatmap depicting NLR family gene expression from a single-cell RNA-Seq analysis of peripheral blood mononuclear cells (PBMCs) extracted from patients with systemic lupus erythematosus and treated with IFN-β as compared with untreated control PBMCs from the same patient set ( GSE96583 ). ( B ) Heatmap depicting NLR family gene expression from an RNA-Seq analysis of mouse bone marrow-derived macrophages (BMDMs) treated with IFN-β as compared with untreated controls ( GSE232827 ). ( C , D ) Expression of Nlrp1b in mouse BMDMs at 6 h after treatment with media, Val-boroPro (VBP), or VBP plus either IFN-β ( C ) or LPS ( D ). ( E , F ) Expression of NLRP1 in primary human macrophages at 6 h ( E ) and primary human monocytes at 4 h ( F ) after treatment with media, VBP, or VBP plus LPS. ( C – F ) Data were expressed as fold change relative to media controls, using 18s or 18S as the housekeeping gene. Data are representative of three independent biological replicates, n = 2 technical replicates combined from two biological replicates ( C ) or n = 3 technical replicates from one biological replicate ( D – F ). **** P < 0.0001. One-way ANOVA with Dunnett’s multiple comparison test ( C – F ) was used. The data were represented as mean ± SEM in ( C – F ). .

Article Snippet: Recombinant mouse IFN-β , PBL Assay , 12400-1.

Techniques: Gene Expression, Single Cell, RNA Sequencing, Analysis, Control, Derivative Assay, Expressing, Comparison

Journal: EMBO Reports

Article Title: IFN-α and IFN-β inhibit NLRP1-driven PANoptosis

doi: 10.1038/s44319-026-00888-0

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant mouse IFN-β , PBL Assay , 12400-1.

Techniques: Generated, Recombinant, Cell-Signaling, Sequencing, Control, Isolation, Transfection, Gene Expression, SYBR Green Assay, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Western Blot, Sensitivity, Software, Real-time Polymerase Chain Reaction