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