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: IRF3 Rabbit pAb , ABclonal , CAT#A2172SP; RRID: N/A.

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 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: IRF3 Rabbit pAb , ABclonal , CAT#A2172SP; RRID: N/A.

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: IRF3 Rabbit pAb , ABclonal , CAT#A2172SP; RRID: N/A.

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: KMT5A hindering production of IFN-β depends on IRF3 K193 methylation (A) HEK293T cells were transfected with vector, FLAG-IRF3 wild-type, or K193R mutant plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads, and subsequent analysis was performed via IB. (B) KMT5A-knockout RKO cells were transfected with FLAG-IRF3 wild-type or K193R mutant plasmids. The cells were treated with poly(I) or UNC0379, then whole-cell lysates were collected, and IP was conducted using anti-FLAG magnetic beads, followed by IB analysis. (C) RKO cells from experiment (B) were co-transfected with reporter plasmids IFN-β-Luc and pRL-TK. After 24 h, the cells were harvested, and luciferase activity was measured using a dual-luciferase reporter assay kit. (D) The relative mRNA levels of INF-β in RKO cells from experiment (B) were quantified using qPCR. (E) KMT5A knockout RKO cells were transfected with HA-KMT5A and FLAG-IRF3 wild-type or K193R mutant plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads, and subsequent IB analysis was performed. (F) Relative quantification of phosphorylated IRF3 (p-IRF3) protein was conducted in experiment (E). (G) The relative mRNA levels of INF-β in RKO cells from experiment F were quantified using qPCR. (C) and (D) were analyzed using one-way ANOVA with Tukey’s post-hoc test, with data presented as mean ± SD. (F) and (G) were analyzed using two-way ANOVA with Tukey’s post-hoc test. Statistical significance was defined as ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, while ns indicates no statistical difference. All immunoblotting experiments were performed independently in triplicate, yielding consistent results.

Article Snippet: IRF3 Rabbit pAb , ABclonal , CAT#A2172SP; RRID: N/A.

Techniques: Methylation, Transfection, Plasmid Preparation, Mutagenesis, Magnetic Beads, Knock-Out, Luciferase, Activity Assay, Reporter Assay, Quantitative Proteomics, 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 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: Phospho-IRF3-S386 Rabbit mAb , ABclonal , CAT#AP0623; RRID: AB_2771210.

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 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: Phospho-IRF3-S386 Rabbit mAb , ABclonal , CAT#AP0623; RRID: AB_2771210.

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: Phospho-IRF3-S386 Rabbit mAb , ABclonal , CAT#AP0623; RRID: AB_2771210.

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: KMT5A hindering production of IFN-β depends on IRF3 K193 methylation (A) HEK293T cells were transfected with vector, FLAG-IRF3 wild-type, or K193R mutant plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads, and subsequent analysis was performed via IB. (B) KMT5A-knockout RKO cells were transfected with FLAG-IRF3 wild-type or K193R mutant plasmids. The cells were treated with poly(I) or UNC0379, then whole-cell lysates were collected, and IP was conducted using anti-FLAG magnetic beads, followed by IB analysis. (C) RKO cells from experiment (B) were co-transfected with reporter plasmids IFN-β-Luc and pRL-TK. After 24 h, the cells were harvested, and luciferase activity was measured using a dual-luciferase reporter assay kit. (D) The relative mRNA levels of INF-β in RKO cells from experiment (B) were quantified using qPCR. (E) KMT5A knockout RKO cells were transfected with HA-KMT5A and FLAG-IRF3 wild-type or K193R mutant plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads, and subsequent IB analysis was performed. (F) Relative quantification of phosphorylated IRF3 (p-IRF3) protein was conducted in experiment (E). (G) The relative mRNA levels of INF-β in RKO cells from experiment F were quantified using qPCR. (C) and (D) were analyzed using one-way ANOVA with Tukey’s post-hoc test, with data presented as mean ± SD. (F) and (G) were analyzed using two-way ANOVA with Tukey’s post-hoc test. Statistical significance was defined as ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, while ns indicates no statistical difference. All immunoblotting experiments were performed independently in triplicate, yielding consistent results.

Article Snippet: Phospho-IRF3-S386 Rabbit mAb , ABclonal , CAT#AP0623; RRID: AB_2771210.

Techniques: Methylation, Transfection, Plasmid Preparation, Mutagenesis, Magnetic Beads, Knock-Out, Luciferase, Activity Assay, Reporter Assay, Quantitative Proteomics, Western Blot