mouse ifn-β elisa kit  (Multi Sciences (Lianke) Biotech Co Ltd)

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: Molecular Therapy Oncology

Article Title: Endothelial injury is a central driver of systemic IFN-β toxicity and is reversible through Jak inhibition

doi: 10.1016/j.omton.2026.201226

Figure Lengend Snippet: Elevated transaminases and toxicity from high plasma mIFNβ levels after hydrodynamic injection of plasmid DNA encoding mIFNβ in Balb/c mice (A) Schematic representation of the sleeping beauty transposon system with a transposon plasmid pKT2/mIFNβ-CLP PGK-Luc encoding mIFNβ, and sleeping beauty SB100X transposase. (B) Plasma IFN-β levels from Balb/c mice [0.0037 μg DNA ( n = 5), 0.011 μg DNA ( n = 3), 0.033 μg DNA ( n = 3), 0.05 μg DNA ( n = 3), 0.1 μg DNA ( n = 4), 0.2 μg DNA ( n = 3), 0.4 μg DNA ( n = 6), 0.8 μg DNA ( n = 3), and 1.6 μg DNA ( n = 1)] at 6–10 days after hydrodynamic injection of increasing amounts of plasmid DNA. Mean ± SEM. (C) AST and (D) ALT levels of individual mice with the corresponding plasma mIFNβ are plotted. The respective Pearson correlation factor and p values are shown. High systemic IFN-β is associated with elevated AST and ALT.

Article Snippet: Murine IFN-β was analyzed using VeriKine Mouse IFNβ ELISA kit (42400, PBL Assay Science, Piscataway, NJ).

Techniques: Clinical Proteomics, Injection, Plasmid Preparation

Journal: Molecular Therapy Oncology

Article Title: Endothelial injury is a central driver of systemic IFN-β toxicity and is reversible through Jak inhibition

doi: 10.1016/j.omton.2026.201226

Figure Lengend Snippet: Lack of toxicity in the absence of IFNR signaling and toxicity is reversible after IFN-β production is terminated Transgenic mice knockout for type I IFN receptor (IFNRko) and C57/B6 mice received one intramuscular injection of saline or 2 × 10 11 vg AAV1-mIFNβ and were monitored over time. (A) Plasma mIFNβ, (B) Kaplan-Meier survival curves, (C) ALT, (D) AST from saline (black, n = 6), or 2 × 10 11 vg AAV1-mIFNβ-treated IFNRko mice (red, n = 10), and C57B/6 mice (blue, n = 7) were plotted at regular intervals. Line denotes mean. Half of the mice were bled for each time point for blood analysis. C57/B6 mice ( n = 25) were given one intramuscular injection of AAV-mIFNβ-iCas9 and 14 days later, given one intraperitoneal dose of AP1903 a prodrug to activate iCas9 to initiate apoptosis of AAV-transduced muscle cells expressing IFN-β. (E) Plasma mIFNβ, (F) ALT, (G) AST, and (H) platelet counts in C57B/6 mice before (day 0) and after termination of mIFNβ production using AP1903. Mean ± SEM.

Article Snippet: Murine IFN-β was analyzed using VeriKine Mouse IFNβ ELISA kit (42400, PBL Assay Science, Piscataway, NJ).

Techniques: Transgenic Assay, Knock-Out, Injection, Saline, Clinical Proteomics, Expressing

Journal: Molecular Therapy Oncology

Article Title: Endothelial injury is a central driver of systemic IFN-β toxicity and is reversible through Jak inhibition

doi: 10.1016/j.omton.2026.201226

Figure Lengend Snippet: Concomitant treatment with ruxolitinib protects VSV-IFNβ-NIS treated MPC-11 tumor-bearing Balb/c mice from toxicity (A) Kaplan-Meier survival, (B) individual tumor volumes, (C) predicted tumor volumes using mixed effects modeling, (D) plasma IFNβ, (E) ALT, (F) AST, and (G) platelet count of mice at scheduled bleeds on day 3 and day 14, or at necropsy (at various time points). Treatment groups are color coded, saline (black, n = 5), VSV alone (red, n = 10), ruxolitinib (gray, n = 5), and VSV plus ruxolitinib (blue, n = 10). One intravenous dose of VSV at 10 7 TCID 50 was given per mouse, and/or ruxolitinib at 2 mg twice a day by oral gavage for 10 days, starting on the same day as VSV therapy.

Article Snippet: Murine IFN-β was analyzed using VeriKine Mouse IFNβ ELISA kit (42400, PBL Assay Science, Piscataway, NJ).

Techniques: Clinical Proteomics, Saline

Journal: Advanced Science

Article Title: RNA‐Binding Protein RBM25 Targets the mRNA Stability of GTPase Rab22a to Restrict Viral Entry and Infection

doi: 10.1002/advs.76160

Figure Lengend Snippet: RBM25 mediates antiviral protection independent of the IFN‐I signaling pathway. (A) A scheme of RNA‐seq analysis of WT and Rbm25 ‐cKO peritoneal macrophages infected with VSV for 4 h. (B) Heatmap of differentially expressed genes (DEGs) in Rbm25 ‐deficient and WT peritoneal macrophages infected with VSV for 4 h. (C) GSEA showing enrichment of DEGs in the pathways involving in the response to IFN‐α, IFN‐β production, JAK‐STAT3, IFN‐I receptor binding, toll like receptor, and cGAS target gene signaling pathways. (D) RT‐qPCR analysis of IFN‐I ( Ifna4, Ifnb1 ) mRNA in the lung tissues from WT and Rbm25 ‐cKO mice infected with PR8 influenza virus with a sublethal dose (50 PFU., n = 5 per group) at 7 dpi. (E) RT‐qPCR analysis of Cxcl10, Mx1, Isg15 , and Mx2 mRNA in the lung tissues from WT and Rbm25 ‐cKO mice treated as in (D). (F) ELISA of IFN‐β in serum from Rbm25 ‐cKO and WT mice infected with PR8 influenza virus, as in (D). (G) RT‐qPCR analysis of IFN‐I ( Ifna4, Ifnb1 ) mRNA in peritoneal macrophages from Rbm25 ‐cKO and WT mice infected with PR8 influenza virus for 12 h. (H) RT‐qPCR analysis of Cxcl10, Isg15 , and Rsad2 mRNA in peritoneal macrophages from WT and Rbm25 ‐cKO mice treated as in (G). (I) Immunoblots analysis of RBM25, RIG‐I, MAVS, p‐TBK1, TBK1, p‐IRF3, and IRF3 in peritoneal macrophages from WT and Rbm25 ‐cKO mice infected with PR8 influenza virus for the indicated times. β‐ACTIN was used as a loading control. (J) A scheme of experiment analyzing the infection of viruses in peritoneal macrophages from Irf3 −/− mice transfected with control siRNA or siRNA targeting Rbm25 . (K) RT‐qPCR analysis of PR8 HA and IAV M1, HSV‐1 TK and ICP0, VSV‐G mRNA in peritoneal macrophages from Irf3 −/− mice transfected with control siRNA or siRNA targeting Rbm25 followed by infection with PR8 influenza virus for 12 h, HSV‐1 for 12 h, or VSV for 8 h, respectively. (L, M) RT‐qPCR analysis of IFN‐I ( Ifna4, Ifnb1 ) mRNA in Rbm25 ‐cKO and WT peritoneal macrophages transfected with poly (I:C) for 4 h (L) or stimulated with UV‐VSV for 18 h (M). (N) Immunoblots analysis of p‐STAT1 and STAT1 in peritoneal macrophages from WT and Rbm25 ‐cKO mice infected with PR8 influenza virus for the indicated times. β‐ACTIN was used as a loading control. Data are presented as the mean ± SD. Unpaired two‐tailed Student's t ‐test (D–H, K–M). ns, not significant.

Article Snippet: IFN‐β levels in the supernatants or sera were measured using a mouse IFN‐β ELISA kit (PBL Biomedical Laboratories) according to the manufacturer's instructions.

Techniques: RNA Sequencing, Infection, Binding Assay, Protein-Protein interactions, Quantitative RT-PCR, Virus, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Transfection, Two Tailed Test

Journal: Cancer Research

Article Title: METTL3 Methylation Induces Decay of Endogenous Retroelement Transcripts to Promote Tumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2893

Figure Lengend Snippet: METTL3 K513 methylation enhances the interaction between METTL3 and SAM. A, RNA m 6 A levels in METTL3 WT and K513R LoVo cells analyzed by RNA dot blot and ELISA. B, RNA m 6 A levels in LoVo cells transduced with negative control or shSETD1A constructs (left), and in LoVo cells expressing vector control, SETD1A WT, or SETD1A R1495A (right), as measured by ELISA. C, The last frame of two complex samples obtained from molecular simulations, visualized using PyMOL. D–F, FEL analysis, DCCM analysis, and pocket volume analysis of METTL3 WT–METTL14 and METTL3 K513me2–METTL14 complexes. G and H, SPR analysis of protein complex-SAM affinity differences in Supplementary Fig. S3P and S3Q. I, Representative images (top) and quantification (bottom) of Cry2-mCherry-METTL3 WT or K513R after blue light stimulation. Scale bar, 10 μm. J, Schematic of METTL3 K513 methylation enhancing the interaction between METTL3 and SAM. Data information: All immunoblots were performed independently three times with similar results. Data are presented as mean ± SD. In I , statistical analysis was performed using the Student two-tailed t test. ns, not significant; ***, P < 0.001. RU, response unit. J, Created with Figdraw.com .

Article Snippet: IFNβ and CXCL10 levels in conditioned media were quantified using the Mouse IFNβ Enzyme-Linked Immunosorbent Assay (ELISA) Kit (RK00420, ABclonal) and the Mouse CXCL10 ELISA Kit (RK00056, ABclonal) according to the manufacturer’s protocol.

Techniques: Methylation, Dot Blot, Enzyme-linked Immunosorbent Assay, Transduction, Negative Control, Construct, Expressing, Plasmid Preparation, Control, Western Blot, Two Tailed Test

Journal: Cancer Research

Article Title: METTL3 Methylation Induces Decay of Endogenous Retroelement Transcripts to Promote Tumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2893

Figure Lengend Snippet: METTL3 K513 dimethylation suppresses type I IFN response and antitumor immune responses. A, GSEA of differentially expressed genes between METTL3 WT and METTL3 K513R cells based on prior sequencing data. NES, normalized enrichment score. B, IF staining using the J2 antibody to detect dsRNA levels in METTL3 WT and METTL3 K513R LoVo cells. C, IB analysis of DNA and RNA sensors, along with signaling proteins involved in the type I IFN pathway. D, Heatmap showing differential expression of ISGs in METTL3 WT and METTL3 K513R LoVo cells. E, ELISA analysis of IFNβ and CXCL10 secretion in METTL3 KO, METTL3 WT, and METTL3 K513R CT26 cells. F, Flow cytometry analysis of MHC-I expression in METTL3 KO, METTL3 WT, and METTL3 K513R CT26 cells. MFI, mean fluorescence intensity. G, Subcutaneous implantation of METTL3 KO, METTL3 WT, and METTL3 K513R CT26 cells into BALB/c mice ( n = 5). Representative bioluminescent images and tumor bioluminescence intensity. H, Flow cytometric analysis of the proportions of CD8 + T cells, NK cells, and M1 and M2 macrophages in tumor-infiltrating lymphocytes (TIL) from subcutaneous tumors ( G ). I, Subcutaneous implantation of METTL3 WT and METTL3 K513R CT26 cells into NOD/SCID/IL2Rγ null (NCG) mice ( n = 5). Tumor volumes were monitored. Data information: All immunoblots were performed independently three times with similar results. Data are presented as mean ± SD. In E , F , and G , statistical analysis was performed using one-way ANOVA with the Tukey test. In H , statistical analysis was performed using two-way ANOVA with Tukey test. In I , statistical analysis was performed using the Student two-tailed t test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: IFNβ and CXCL10 levels in conditioned media were quantified using the Mouse IFNβ Enzyme-Linked Immunosorbent Assay (ELISA) Kit (RK00420, ABclonal) and the Mouse CXCL10 ELISA Kit (RK00056, ABclonal) according to the manufacturer’s protocol.

Techniques: Sequencing, Staining, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Fluorescence, Western Blot, Two Tailed Test

Journal: Cancer Research

Article Title: METTL3 Methylation Induces Decay of Endogenous Retroelement Transcripts to Promote Tumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2893

Figure Lengend Snippet: SETD1A mediates colorectal cancer immune evasion via METTL3 K513 dimethylation. A, IF staining using J2 antibody to detect dsRNA levels in control and SETD1A knockdown LoVo cells. B, IB analysis of DNA and RNA sensors, along with signaling proteins involved in the type I IFN pathway. C, qRT-PCR analysis of ISGs in control and SETD1A knockdown LoVo cells. D, ELISA analysis of IFNβ and CXCL10 secretion in control and SETD1A knockdown CT26 cells. E, Flow cytometry analysis of MHC-I expression in control and SETD1A knockdown CT26 cells. F, Subcutaneous implantation of control and SETD1A knockdown CT26 cells into BALB/c mice ( n = 5). Tumor volumes were monitored. G, Flow cytometric analysis of the proportions of CD8 + T cells, NK cells, and M1 and M2 macrophages in tumor-infiltrating lymphocytes (TIL) from subcutaneous tumors ( F ). H, IB analysis of DNA and RNA sensors, along with signaling proteins involved in the type I IFN pathway. I, qRT-PCR analysis of ISGs in METTL3 WT and METTL3 K513R LoVo cells with or without SETD1A knockdown. J, Subcutaneous implantation of METTL3 WT and METTL3 K513R CT26 cells with or without SETD1A knockdown into BALB/c mice ( n = 5). Tumor volumes were monitored. K, Flow cytometric analysis of the proportions of CD8 + T cells, NK cells, and M1 and M2 macrophages in TILs from subcutaneous tumors ( J ). Data information: All immunoblots were performed independently three times with similar results. Data are presented as mean ± SD. In C–E , G , I , and K , statistical analysis was performed using one-way ANOVA with the Tukey test. In F and J , statistical analysis was performed using two-way ANOVA with Tukey test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. NC, negative control.

Article Snippet: IFNβ and CXCL10 levels in conditioned media were quantified using the Mouse IFNβ Enzyme-Linked Immunosorbent Assay (ELISA) Kit (RK00420, ABclonal) and the Mouse CXCL10 ELISA Kit (RK00056, ABclonal) according to the manufacturer’s protocol.

Techniques: Staining, Control, Knockdown, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Western Blot, Negative Control

Journal: Cancer Research

Article Title: METTL3 Methylation Induces Decay of Endogenous Retroelement Transcripts to Promote Tumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2893

Figure Lengend Snippet: METTL3 dimethylation promotes colorectal cancer progression through type I IFN–mediated tumor immune microenvironment remodeling. A, Model of EREs-induced type I IFN production. B, qRT-PCR analysis of ISGs in indicated cells. C and D, Subcutaneous implantation of indicated CT26 cells into BALB/c mice ( n = 5; C ). Tumor volumes were monitored ( D ). E, Flow cytometric analysis of the proportions of CD8 + T cells, NK cells, and M1 and M2 macrophages in tumor-infiltrating lymphocytes (TIL) from subcutaneous tumors ( D ). F and G, Subcutaneous implantation of the indicated CT26 cells into BALB/c mice, with or without anti-IFNAR1 antibody treatment ( n = 5; F ). Tumor volumes were monitored ( G ). H, Flow cytometric analysis of the proportions of CD8 + T cells and M1 and M2 macrophages in TILs from subcutaneous tumors ( G ). I and J, Subcutaneous implantation of METTL3 WT and METTL3 K513R CT26 cells into BALB/c mice, with or without anti–PD-1 antibody treatment ( n = 10; I ). Tumor volumes were monitored ( J ). K, Flow cytometric analysis of the proportions of CD8 + T cells in tumor-infiltrating lymphocytes from subcutaneous tumors ( J ). Data information: All immunoblots were performed independently three times with similar results. Data are presented as mean ± SD. In B , E , H , and K , statistical analysis was performed using one-way ANOVA with the Tukey test. In D , G , and J , statistical analysis was performed using two-way ANOVA with Tukey test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. A, Created with Figdraw.com .

Article Snippet: IFNβ and CXCL10 levels in conditioned media were quantified using the Mouse IFNβ Enzyme-Linked Immunosorbent Assay (ELISA) Kit (RK00420, ABclonal) and the Mouse CXCL10 ELISA Kit (RK00056, ABclonal) according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Cancer Research

Article Title: METTL3 Methylation Induces Decay of Endogenous Retroelement Transcripts to Promote Tumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2893

Figure Lengend Snippet: Fluorouracil-induced E2F4 self-regulation activates SETD1A-mediated METTL3 methylation. A, TF prediction for SETD1A regulation using the JASPAR, ENCODE, ChIP-Atlas, CHEA3, and GTRD databases. B, IB analysis of SETD1A, E2F4, K513me2, and METTL3 expression in LoVo and HT29 cells transfected with Flag-E2F4 or control vector (left), and in cells transfected with negative control (NC) or siE2F4 (#1 and #2; right). C, Luciferase assay of full-length and truncated or mutated SETD1A promoter constructs cotransfected with control or Flag-E2F4 plasmids in HEK293T cells. D, ChIP assay detecting the abundance of E2F4 binding to the SETD1A promoter in LoVo cells. E, IB and RNA dot blot analysis of RNA m 6 A levels, SETD1A, METTL3, K513me2, and METTL3 expression in LoVo cells treated with different concentrations of HLM006474, and RNA m 6 A levels in indicated cells were quantified by ELISA. F, qRT-PCR analysis of E2F4 and SETD1A expression levels in LoVo cells treated with fluorouracil (0.5 mg/mL) for the indicated times. G, IB analysis of SETD1A, E2F4, K513me2, and METTL3 expression in LoVo cells treated with fluorouracil (0.5 mg/mL) for the indicated times. H, ChIP assay detecting the abundance of E2F4 binding to the E2F4 promoter in LoVo cells treated with or without fluorouracil (0.5 mg/mL). I and J, Subcutaneous implantation of CT26 cells into BALB/c mice, with control vehicle, E2F4 inhibitor (HLM006474), anti–PD-1 antibody, or combined treatment ( n = 10; I ). Representative bioluminescent images and tumor bioluminescence intensity at 16 days after cell injection ( J ). K, KM survival curves for BALB/c mice bearing CT26 tumors. L, Representative staining and quantification of CD8 in subcutaneous tumors ( n = 15; J ). Data are presented as mean ± SD. In C , D , F , and H , statistical analysis was performed using the Student two-tailed t test. In E , J , and L , statistical analysis was performed using one-way ANOVA with the Tukey test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: IFNβ and CXCL10 levels in conditioned media were quantified using the Mouse IFNβ Enzyme-Linked Immunosorbent Assay (ELISA) Kit (RK00420, ABclonal) and the Mouse CXCL10 ELISA Kit (RK00056, ABclonal) according to the manufacturer’s protocol.

Techniques: Methylation, Expressing, Transfection, Control, Plasmid Preparation, Negative Control, Luciferase, Construct, Binding Assay, Dot Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Injection, Staining, Two Tailed Test