flag tag  (Proteintech)

Journal: Genes & Diseases

Article Title: Blockage of SUMO E1 enzyme inhibits ocular lens fibrosis by mediating SMAD4 SUMOylation

doi: 10.1016/j.gendis.2025.101827

Figure Lengend Snippet: ML792 disrupts SMAD4 SUMOylation-dependent nuclear translocation in TGFβ 2 -stimulated lens epithelial cells (LECs). (A – F) FHL124 LECs were treated with or without TGFβ2 (10 ng/mL, 2 h). Triple immunofluorescence staining of SMAD4 (green), SUMO1 (red)/SUMO2/3 (red), and DAPI (nuclei, blue) shows spatiotemporal dynamics of SMAD4-SUMO colocalization. (A, D) SMAD4-SUMO1/SUMO2/3 immunofluorescence staining and colocalization scatterplot. (B, E) Pearson's r analysis of colocalization performed by Image J. n = 9 replicates per group. (C, F) Quantification of nuclear SMAD4 intensity. n = 30 cells in (C) and n = 44 cells in (F). Unpaired Student's t -test; ∗ P < 0.05 and ∗∗∗ P < 0.001. (G, H) Flag-SMAD4 immunoprecipitation in engineered FHL124 LECs overexpressing Flag-SMAD4. Treatments were 0.1% DMSO, TGFβ 2 (10 ng/mL), ML792 (10 μM), or their combination for 2 h. (G, H) Whole-cell lysates were blotted with anti-Flag and anti-SMAD4 (INPUT). Cell lysates were immunoprecipitated with anti-Flag, followed by SUMO1 immunoblotting (G) and SUMO2/3 immunoblotting (H). (I, J) Subcellular fractionation analysis. (I) Immunoblots of cytoplasmic/nuclear SMAD4 after 8 h treatments in FHL12.4 LECs. (J) Quantification was normalized to GAPDH (cytoplasm) and lamin A/C (nucleus). One-way ANOVA with Bonferroni correction; ns, not significant; ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (K, L) SMAD4 nuclear translocation analysis. (K) Triple immunofluorescence staining SMAD4 (red), F-actin (Phalloidin, green), and DAPI (nuclei, blue) in LECs treated as indicated in (I). Scar bar: 20 μm. (L) Nuclear SMAD4 fluorescence intensity quantification. n = 30 cells per group. One-way ANOVA with Bonferroni post-hoc test; ∗ P < 0.05 and ∗∗∗ P < 0.001.

Article Snippet: Cells were lysed in 0.5% NP-40 buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 10% glycerol) containing protease inhibitors (#P2714, Sigma–Aldrich, Missouri, USA) on ice for 5 min. Lysate (2 mg) was precleared with control IgG (#2729, #53484, CST) at 4 °C for 2 h. Immunoprecipitation was performed at 4 °C overnight using anti-Flag antibody/Nano-Agarose beads (#FNM-25-500, NuoyiBio, Tianjin, China), anti-SUMO1 or anti-HA antibody with protein A/G Magnetic beads (#HY-K0202, MedChemExpress, New Jersey, USA).

Techniques: Translocation Assay, Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Fractionation, Fluorescence

Journal: Genes & Diseases

Article Title: Blockage of SUMO E1 enzyme inhibits ocular lens fibrosis by mediating SMAD4 SUMOylation

doi: 10.1016/j.gendis.2025.101827

Figure Lengend Snippet: SUMOylation site mutagenesis abolishes SMAD4-mediated epithelial–mesenchymal transition (EMT) in TGFβ 2 -stimulated lens epithelial cells (LECs). (A) Sanger sequencing validation of SMAD4 mutants. WT, wild-type; K113R, Lys113→Arg; K159R, Lys159→Arg. The black frames indicate WT and mutated codons. (B, C) SUMOylation capacity analysis in SMAD4 mutants. (B) FHL124 LECs stably overexpressed empty vector and flag-SMAD4 variants treated with TGFβ 2 (10 ng/mL, 2 h). Whole-cell lysates were immunoblotted with anti-Flag and anti-SMAD4. β-Tubulin served as the loading control. The cell lysates were immunoprecipitated with anti-Flag nano beads, followed by immunoblotting for SUMO1, SUMO2/3, and Flag antibody. (C) Quantification of SMAD4 expression (Input lysates). One-way ANOVA with Bonferroni correction; ns, not significant; ∗∗∗ P < 0.001. (D, E) SMAD4 nuclear translocation analysis. (D) Triple fluorescence imaging of Flag (SMAD4, red), F-actin (phalloidin, green), and DAPI (nuclei, blue) in engineered LECs treated with TGFβ 2 (10 ng/mL, 2 h). (E) Nuclear SMAD4 intensity quantification ( n = 15–18 cells/group). One-way ANOVA with Bonferroni post-hoc test; ∗∗∗ P < 0.001. (F, G) Functional consequence of double site mutant (K113 plus 159R) SMAD4 protein. (F) EMT marker immunoblotting 24 h after TGFβ 2 treatment in human LECs overexpressing empty vector, WT Flag-tagged SMAD4, or double site mutant Flag-tagged SMAD4. (G) Densitometric analysis from (F). β-Tubulin served as the loading control. One-way ANOVA followed by Bonferroni correction; ns, not significant; ∗ P < 0.05. ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Cells were lysed in 0.5% NP-40 buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 10% glycerol) containing protease inhibitors (#P2714, Sigma–Aldrich, Missouri, USA) on ice for 5 min. Lysate (2 mg) was precleared with control IgG (#2729, #53484, CST) at 4 °C for 2 h. Immunoprecipitation was performed at 4 °C overnight using anti-Flag antibody/Nano-Agarose beads (#FNM-25-500, NuoyiBio, Tianjin, China), anti-SUMO1 or anti-HA antibody with protein A/G Magnetic beads (#HY-K0202, MedChemExpress, New Jersey, USA).

Techniques: Mutagenesis, Sequencing, Biomarker Discovery, Stable Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Expressing, Translocation Assay, Fluorescence, Imaging, Functional Assay, Marker

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 is highly expressed in T PEX cells and decreased after anti–PD-1 treatment. (A) UMAP plot showing the clustering of OT-I cells infiltrating the MC38-OVA tumor. (B and C) UMAP (B) and bubble (C) plots showing the marker genes expression in each cluster. (D) Volcano plot displaying the fold change expression of Mettl genes in T PEX cells compared with T EX cells. Genes were considered significant if the adjusted P value (FDR) was <0.01. (E and F) Bubble (E) and UMAP (F) plots showing the expression of Mettl8 in each cluster. (G and H) Bubble plots showing the expression of HAVCR2 , TOX , TCF7 (G), and METTL8 (H) in the CD8 + T cells of cancer patients before (treatment-naïve) and after (response) anti–PD-1 treatment. (I) Relative expression of METTL8 in tumor-infiltrating T cells from anti–PD-1/CTLA-4–treated melanoma (left) and anti-CD19 CAR-T–treated chronic lymphocytic leukemia (CLL) (right) responders (R) and nonresponders (NR) and in TILs from nivolumab treated NSCLC (middle) R and NR assessed by Tres ( https://resilience.ccr.cancer.gov/ ). Each dot represents a tumor with the average value among all cells on the y axis. The thick line represents the median value. The bottom and top of the boxes are the min and max, respectively. (Melanoma: R = 9, NR = 10; NSCLC: R = 4, NR = 12; CLL: R = 3, NR = 11.) (J) Schematic diagram of the tumor model: Mettl8-tdTomato-Flag mice were subcutaneously injected with 2 × 10 5 B16F10 cells. Mice were harvested at 13 dpi. (K) Representative cytometry plots showing the Ly108 + Tim3 − T PEX and Tim3 + Ly108 − T EX cells (left). Cells are gated on tumor-infiltrating CD8 + CD44 + T cells. Representative cytometry plots (middle) and statistic diagram (right) showing the expression of Mettl8-tdTomato in these cells. n = 6–7 per group. (L) Schematic diagram of the tumor model: CD45.1 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 2 × 10 6 CTV-labeled Mettl8-tdTomato-Flag OT-I cells transfer at 9 dpi. Mice were harvested at 12 dpi. (M) Representative cytometry plots showing the divisions of CD44 + OT-I cells at day 3 after transfer (left). Representative cytometry plots (middle) and statistic diagram (right) showing the expression of Mettl8-tdTomato in each division. The mean fluorescence intensity (MFI) of tdTomato in division 1, 2, and 3 are all compared with that in division 0. n = 5 per group. Data are representative of two independent experiments. P values were calculated two-tailed Student’s t test, **P < 0.01; ****P < 0.0001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Marker, Expressing, Injection, Cytometry, Labeling, Fluorescence, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 expression in CD8 + T cell subsets and generation of Mettl8-tdTomato-Flag mice. (A) UMAP plot showing the clustering of 9,557 OT-I cells infiltrating in the B16-OVA tumor. (B and C) Bubble (B) and UMAP (C) plots showing the character gene expression in each cluster. (D and E) UMAP (D) and bubble (E) plots showing the expression of Mettl8 in each cluster. (F) UMAP plot showing the clustering of CD8 + cells infiltrating in the mouse melanoma and colon adenocarcinoma tumor model. (G and H) Bubble plot showing signature gene (G) and Mettl8 (H) expression in effector/exhausted and stem-like clusters. (I) RNA-seq analysis was performed on tumor-infiltrating OT-I cells sorted into Ly108 + Tim3 − T PEX and Tim3 + Ly108 − T EX populations. Heatmaps depict signature genes associated with T PEX and T EX cells (Log 2 FC > 1, FDR < 0.05). (J) Targeting strategy of Mettl8 allele. (K) Expression of tdTomato in T cells of the spleens from Mettl8-tdTomato-Flag mice. n = 5–8 per group. Data are representative of two independent experiments. P value was calculated by one-way ANOVA; ****P < 0.0001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Expressing, Gene Expression, RNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: T cell maturation in the thymuses and spleens of Mettl8 conditional KO mice. (A) Schematic diagram of “conditional ready” (floxed) Mettl8 allele. Two loxP sites (red triangle) flanking the Mettl8 exon (blue square) within the 5′ and -3′ homology regions (∼1.5 kb, respectively) are indicated. (B) Relative expression of Mettl8 in CD3 + T cells from the spleens of Mettl8 fl/fl (WT) and Mettl8 fl/fl Cd4 cre (KO) mice, detected by qPCR. (C−E) Representative plots (C) and cumulative data (D and E) show different states of T cell development in the thymus of WT and KO mice as tested by flow cytometry. (F) Representative flow cytometry plots and cumulative data show the frequency and absolute number of CD44 hi CD62L lo effector memory cells and CD44 lo CD62L hi naïve cells gated on TCRβ + CD24 − CD69 − mature CD4 + T cells (top) and mature CD8 + T cells (upper middle) from the thymuses, CD44 hi CD62L lo active cells, CD44 lo CD62L hi naïve cells, and CD44 hi CD62L hi central memory cells gated on CD4 + T cells (lower middle) and mature CD8 + T cells (bottom) from the spleens of Mettl8 fl/fl Cd4 cre mice and littermate controls. (G) Representative flow cytometry plots and cumulative data show the frequency and absolute number of Foxp3 + CD4 + T cells the spleens (top) and the thymuses (bottom) of Mettl8 fl/fl Cd4 cre mice and littermate controls. (H) Body weight of enteritis mice induced by transfer of CD4 + T cell to Rag1 −/− mice and suppression by Mettl8 −/− or WT T reg cells. n = 4–7 mice per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Expressing, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 deficiency restricts tumor progression by promoting T PEX cell transition. (A) Schematic diagram of the adoptive transferred tumor model: CD45.1 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 2 × 10 6 CD45.2 WT or Mettl8 −/− OT-I cells transfer at 9 dpi. Mice were harvested at 21 dpi. (B) Tumor growth in each group of the mice in A. n = 8 per group. (C) Survival curve in each group of the mice in A. n = 8 per group. (D) The absolute number of tumor infiltrating OT-I cells from the mice in A. n = 8 per group. (E and F) Representative flow cytometry plots and cumulative data show the frequency and absolute number of Tcf1 + Tim3 − T PEX , Tim3 + Tcf1 − T EX (E), and CX3CR1 + Tcf1 − Int-T EX cells (F) gated on tumor-infiltrating OT-I cells. n = 6–8 per group. (G) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of GzmB + , IFN-γ + , and perforin + cells gated on tumor-infiltrating OT-I cells. n = 7 per group. (H) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and MFI of GzmB, IFN-γ, and perforin gated on tumor-infiltrating Tcf1 + Tim3 − T PEX , CX3CR1 + Tcf1 − Int-T EX, and CX3CR1 − Tcf1 − T EX subsets. n = 5–6 per group. Data are representative of three independent experiments. P value was calculated by two-way ANOVA (B), Log-rank test (C), and two-tailed Student’s t test (D−H); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Injection, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 promotes T PEX differentiation without affecting their proliferation and apoptosis. (A and B) Representative flow cytometry plots and cumulative data show the frequency of CD44 (A) and PD-1 (B) OT-I cells infiltrating in tumors. (C) Representative flow cytometry plots and cumulative data show the frequency of caspase and Ki67 in tumor-infiltrating OT-I cells. (D) Cumulative data show the frequency of caspase and Ki67 in Tcf1 + Tim3 − T PEX , CX3CR1 + Tcf1 − Int-T EX , and CX3CR1 − Tcf1 − T EX subsets. (E) Cumulative data show the frequency of GzmB, IFN-γ, and perforin in tumor-infiltrating OT-I subsets mentioned above. (F) Schematic diagram of the classic CRC liver metastases model: Mettl8 fl/fl Cd4 cre and Mettl8 fl/fl mice were intrasplenically injected with 2 × 10 5 MC38 cells (left), and imaging of livers on day 21 after injection (right). (G) Representative flow cytometry plots and cumulative data show the ratio of Tcf1 + Tim3 − T PEX to Tim3 + Tcf1 − T EX cells gated on CD44 hi CD62L lo CD8 + T cells of the livers from mice in F. (H) Representative flow cytometry plots and cumulative data show the frequency of GzmB, IFN-γ, and TNF-α gated on CD44 hi CD62L lo CD8 + T cells from the livers of mice in F. (I) Schematic diagram of the classic melanoma lung metastases model: Mettl8 fl/fl Gzmb cre and Mettl8 fl/fl mice were i.v. injected with 2 × 10 5 B16F10 cells (top) and the survival curve (bottom). (J) Schematic diagram of adoptive transfer model: CD45.1.2 + Mettl8 −/− or WT P14 CD8 + T cells were adoptively transferred into CD45.2 + WT recipients, followed by LCMV-clone 13 (LCMV-C13) infection 24 h later and then analyzed on 30 dpi. (K) Statistical analysis show the absolute number of P14 cells from the spleens of mice in J. (L) Representative flow cytometry plots and cumulative data show Tcf1 + Tim3 − T PEX , Tim3 + Tcf1 − T EX , and CX3CR1 + Tcf1 − Int-T EX cells gated on P14 cells from the spleens of mice in J. n = 4–8 mice per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test (A–H, K, and L) or Log-rank test (I); *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Flow Cytometry, Injection, Imaging, Adoptive Transfer Assay, Infection, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Reconstitution of Mettl8 expression in Mettl8 −/− OT-I cells restored their phenotype to that of WT OT-I cells. (A) Schematic diagram of the rescue experiment: CD45.2 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells. Mettl8 overexpression (OE) or empty vector (EV) retrovirus were transduced to CD45.1.2 WT or Mettl8 −/− OT-I cells. 5 × 10 5 GFP + cells were sorted 48 h after transduction and adoptively transferred into the tumor-bearing mice at 9 dpi. Mice were harvested at 19 dpi. (B) Tumor growth in each group of the mice in A. n = 6 per group. (C) Tumor growth in each group displayed in each replicate. n = 6 per group. (D) Tumor weight (left) and the absolute number of tumor infiltrating OT-I cells (right) from the mice in A. n = 6 per group. (E) Representative flow cytometry plots and cumulative data show the frequency of GFP in OT-I cells. n = 6 per group. (F) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of Tcf1 + Tim3 − T PEX and Tim3 + Tcf1 − T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (G) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of CX3CR1 + Tcf1 − Int-T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (H) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of GzmB + , IFN-γ + , and perforin + cells gated on tumor-infiltrating OT-I cells. n = 6 per group. Data are representative of two independent experiments. P value was calculated by two-way ANOVA (B) and two-tailed Student’s t test (D to H); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Expressing, Injection, Over Expression, Plasmid Preparation, Transduction, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 deficiency alters transcriptional profiles of CD8 + T cells. RNA-seq analysis were performed using WT or Mettl8 −/− (KO) OT-I cells from the adoptive transferred tumor model. (A) Volcano plot illustrates genes differentially expressed between OT-I cells from WT and KO mice. Lines indicate a twofold difference between OT-I cells from KO and WT mice (DESeq2, FDR < 0.05). (B) Gene Ontology (GO) enrichment (biological processes) analysis of DEGs obtained from the R package named clusterProfiler. (C) GSEA shows enrichment of Tcf1 + T PEX signature genes (top), GzmK + T EM signature genes (middle), and T EMRA signature genes (bottom) in the transcriptome of KO versus WT cells. (D) Heatmaps of DEGs related to stem-like, exhausted, or effector-like features (Log2FC > 1, FDR < 0.05). (E) Representative flow cytometry plots (left) and cumulative data (right) show the expression of the indicated molecules gated on tumor-infiltrating OT-I cells. n = 5–6 per group. (F) Representative flow cytometry plots (left) and cumulative data (right) show the expression of the indicated molecules gated on tumor-infiltrating Tcf1 + Tim3 − T PEX , CX3CR1 + Tcf1 − Int-T EX , and CX3CR1 − Tcf1 − T EX subsets. n = 5–6 per group. Data are representative of two independent experiments. P values were calculated by two-tailed Student’s t test; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: RNA Sequencing, Flow Cytometry, Expressing, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: The expression of effector/exhausted molecules across CD8 + T cell subsets. (A) Representative plots and cumulative data show the expression of PD-1 and Tox gated on tumor-infiltrating OT-I cells. n = 5 per group. (B) Representative plots and cumulative data show the expression of Tcf1 gated on OT-I cells in TdLNs. n = 4–8 per group. (C) Representative flow cytometry plots and cumulative data show the expression of the indicated molecules gated on tumor-infiltrating CD44 + CD8 + T cells in the B16F10 tumor model: Mettl8 fl/fl CD4 cre mice and littermate controls were subcutaneously injected with 2 × 10 5 B16F10 cells. Mice were analyzed at 13 dpi. (D) RNA-seq analysis of Ly108 + Tim3 − T PEX , Tim3 + CX3CR1 + Ly108 − Int-T EX , and Tim3 + CX3CR1 − Ly108 − T EX cells gated on tumor-infiltrating WT and Mettl8 −/− (KO) OT-I cells. Heatmaps depict stem-like, exhausted and effector-like gene signatures in WT and Mettl8 −/− (KO) OT-I cells. (E) Representative flow cytometry plots and cumulative data show the expression of PD-1 and Tox gated on tumor-infiltrating Tcf1 + Tim3 − T PEX , CX3CR1 + Tcf1 − Int-T EX , and CX3CR1 − Tcf1 − T EX subsets. n = 5 per group. (F) Diamond graphs show chromatin interactions in WT (left) and Tcf1 −/− Lef1 −/− dKO (right) CD8 + T cells, with gene structures on the left. (G) Single-cell transcription levels of representative genes illustrated in the UMAP plot. Transcription levels are color coded: gray, not expressed; blue, expressed. (H) Representative flow and cumulative data show the frequencies of CX3CR1, Tim3, and PD-1 in Tcf1 + Tox − , Tcf1 − Tox + , and Tcf1 + Tox + cells, which are gated from B16F10 tumor-infiltrating CD8 + CD44 + T cells. n = 9 per group. (I) Schematic diagram of GA treatment to B16F10 cells in vitro (left) and frequency and absolute number of GA-treated cells (right). n = 3 per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Expressing, Flow Cytometry, Injection, RNA Sequencing, Single Cell, In Vitro, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 promotes m 3 C modification of Tcf7 mRNA and its genome-specific loops of Tox in CD8 + T cells. (A) Venn plot illustrates the overlap of downregulated genes from RNA-seq, m 3 C-seq, and Mettl8-binding genes from RIP-seq. (B) Mettl8 occupancy at the Tcf7 gene loci is revealed through m 3 C-seq (WT and Mettl8 −/− ) of EG7-OVA tumor-infiltrating OT-I cells and RIP-seq (Mettl8-tdTomato-Flag) of B16F10 tumor-infiltrating CD44 + CD8 + T cells. The binding peaks on Tcf7 loci are depicted. The m 3 C tracks are all plotted on a consistent scale. (C) The RNA decay assay demonstrates the remaining Tcf7 mRNA of CD8 + T cells from the spleens of WT and Mettl8 −/− mice detected by qRT-PCR, normalized to t = 0. (D) Heatmaps display changes in total Tcf1-targeting genes between WT and Mettl8 −/− EG7-OVA tumor-infiltrating OT-I cells and Mettl8-targeting genes in B16F10 tumor-infiltrating CD44 + CD8 + T cells of Mettl8-tdTomato-Flag mice as detected by CUT&Tag. (E) Diamond graphs exhibit chromatin interactions in WT and Mettl8 −/− tumor-infiltrating OT-I cells at the Tox gene loci (top), with CUT&Tag and ATAC-seq tracks, and gene structures on the bottom. An enlarged view highlights the signal profiles across the Tox gene region. (F) co-IP of Tcf1 by anti-Flag magnetic beads in CD3 + T cells from the spleens of Mettl8-tdTomato-Flag (RPT) and WT mice. IB, immunoblot. (G) co-IP of Tcf1 by Flag-tagged Mettl8 protein with anti-Flag magnetic beads after co-transfection into HEK293T cells. (H) Single-cell transcription levels of representative genes illustrated in the UMAP plot. Transcription levels are color coded: gray, not expressed; blue, expressed. (I) Schematic diagram of the tumor model: Mettl8 fl/fl Cd4 cre mice were subcutaneously injected with 2 × 10 5 B16F10 cells and harvested after 13 days. (J) Representative flow cytometry plots and cumulative data show the frequency of Tcf1 + Tox + cells gated on tumor-infiltrating CD8 + CD44 + T cells (right). n = 6 per group. (K) Schematic diagram of the OT-I–transferred tumor model: CD45.1 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 2 × 10 6 WT or Mettl8 −/− OT-I cells transfer at 9 dpi. Mice were harvested at 21 dpi. Representative flow cytometry plots and cumulative data show the frequency of Tox + cells gated on Tcf1 + OT-I cells. n = 6 per group. (L) The MFI of Tox gated on Tcf1 + OT-I cells of the mice in K. n = 6 per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available for this figure: .

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Modification, RNA Sequencing, Binding Assay, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Cotransfection, Single Cell, Injection, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: GA enhances the antitumor response of CD8 + T cells. (A) Schematic diagram of detecting Mettl8 expression in vitro : HEK293T cells were transfected with pMigR1-Mettl8-Flag plasmid, and Flag-tagged Mettl8 was detected by western blotting. (B) The detection included GA-treated HEK293T cells after transfection (top) or treated protein extracted from transfected HEK293T cells (bottom). (C) Schematic diagram of GA treatment to B16F10 tumor–bearing Mettl8 -tdTomato-Flag mice: Mice were subcutaneously injected with 2 × 10 5 B16F10 cells, followed by GA treatment every 2 days from day 6 to day 12. Mice were harvested at day 13. (D) Tumor growth of the mice in C. n = 7 per group. (E) Tumor growth of the mice in C displayed in each replicate. (F and G) Tumor weight (F) and the absolute number of tumor-infiltrating CD44 + CD8 + T cells (G). n = 7 per group. (H) Representative flow cytometry plots (left) and cumulative data (right) show the MFI of tdTomato and Ly108 in tumor-infiltrating CD8 + T cells. n = 5–6 per group. (I and J) Representative flow cytometry plots and cumulative data show the frequency and absolute number of CX3CR1 + Tcf1 − Int-T EX (I), Tcf1 + Tim3 − T PEX , and Tim3 + Tcf1 − T EX cells (J) gated on tumor-infiltrating CD44 + CD8 + T cells. n = 7 per group. (K) Representative flow cytometry plots (left) and cumulative data (right) show the frequency of GzmB, IFN-γ, and perforin gated on tumor-infiltrating CD44 + CD8 + T cells. n = 6–8 per group. (L and M) Cumulative data show the absolute number (L) and MFI (M) of IFN-γ, GzmB, and perforin gated on tumor-infiltrating CD44 + CD8 + T cells. n = 6–8 per group. Data are representative of three independent experiments. P value was calculated by two-way ANOVA (D) and two-tailed Student’s t test (F–M); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: .

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Expressing, In Vitro, Transfection, Plasmid Preparation, Western Blot, Injection, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: GA potentiates CD8 + T cell antitumor immunity via Mettl8 inhibition. (A) Schematic diagram of GA-treated OT-I–transferred model: CD45.2 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 5 × 10 5 CD45.1.2 WT or Mettl8 −/− OT-I cells transfer at 9 dpi. GA was administered i.p. every 2 days from 11 to 17 dpi. Mice were harvested at 19 dpi. (B–D) Tumor growth (B), tumor weight (C), and absolute number of tumor infiltrating OT-I cells (D) of the mice in A. n = 7–8 per group. (E and F) Representative flow cytometry plots (E) and cumulative data (F) show the frequency and absolute number of Tcf1 + Tim3 − T PEX and Tim3 + Tcf1 − T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (G and H) Representative flow cytometry plots (G) and cumulative data (H) show the frequency and absolute number of CX3CR1 + Tcf1 − Int-T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (I) Schematic diagram of Mettl8-mutated mouse model: CD45.2 Mettl8 fl/fl Cd4 cre mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells. Mettl8-WT or Mettl8-mutation (Mut) retrovirus were transduced to CD45.1.2 Mettl8 −/− OT-I cells. 5 × 10 5 GFP + cells were sorted 48 h after transduction and adoptively transferred into the tumor-bearing mice at 9 dpi. GA was administered i.p. every 2 days from 11 to 17 dpi. Mice were harvested at 18 dpi. (J) Tumor growth of the mice in I. n = 4–6 per group. (K and L) Representative flow cytometry plots (K) and cumulative data (L) show the frequency and absolute number of Tcf1 + Tim3 − T PEX , Tim3 + Tcf1 − T EX , and CX3CR1 + Tcf1 − Int-T EX cells gated on tumor-infiltrating OT-I cells. n = 4–6 per group. Data are representative of two independent experiments. P value was calculated by two-way ANOVA (B and J) and two-tailed Student’s t test (C, D, F, H, and L); *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Inhibition, Injection, Flow Cytometry, Mutagenesis, Transduction, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 inhibition promotes CD8 + T cell antitumor immunity and synergistically enhances PD-1 blockade. (A) Tumor growth of the mice in GA-treated adoptive-transferred model displayed in each replicate. n = 8 per group. (B) Western blot analysis of Mettl8-Flag and GAPDH in lysates from HEK293T cells transfected with pMIGR1-Empty, pMIGR1-Mettl8-WT, and pMIGR1-Mettl8-Mut plasmid. (C) Tumor growth curves for individual mice in the Mettl8-mutated mouse model. n = 4–6 per group. (D) Representative flow cytometry plots and cumulative data show the frequency of GzmB, IFN-γ, and perforin gated on tumor-infiltrating OT-I cells from the mice in C. n = 4–6 per group. (E) Tumor growth of the mice from the combined Mettl8 KO and anti–PD-1 treatment model displayed in each replicate. n = 8 per group. (F) Representative flow cytometry plots and cumulative data show the frequency of IFN-γ, GzmB, and perforin gated on tumor-infiltrating OT-I cells from the mice in E. n = 6 per group. (G) Tumor growth of the mice from GA and anti–PD-1 combined treatment model displayed in each replicate. n = 9 per group. (H) Cumulative data show the absolute number of tumor-infiltrating OT-I cells from the mice in G. (I) Representative flow cytometry plots cumulative data show the frequency and absolute number of CX3CR1 + Tcf1 − Int-T EX cells gated on tumor-infiltrating OT-I cells from the mice in G. n = 7 mice per group. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: .

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Inhibition, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 inhibition synergizes with anti–PD-1 treatment. (A) Schematic diagram of the combined Mettl8 KO and anti–PD-1 treatment model: CD45.2 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 5 × 10 5 CD45.1.2 WT or Mettl8 −/− OT-I cells transfer at 9 dpi. Anti–PD-1 antibody was administered i.p. every 2 days from 11 to 17 dpi. Mice were harvested at 19 dpi. (B–D) Tumor growth (B), tumor weight (C), and absolute number of tumor infiltrating OT-I cells (D) of the mice in A. n = 6 per group. (E and F) Representative flow cytometry plots (E) and cumulative data (F) show the frequency and absolute number of Tcf1 + Tim3 − T PEX and Tim3 + Tcf1 − T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (G and H) Representative flow cytometry plots (G) and cumulative data (H) show the frequency and absolute number of CX3CR1 + Tcf1 − Int-T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (I) Schematic diagram of the combined GA and anti–PD-1 treatment model: CD45.2 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 2 × 10 6 CD45.1.2 OT-I cell transfer at 9 dpi. GA and anti–PD-1 antibody were administered i.p. in an alternating schedule from 11 to 19 dpi. Mice were harvested at 23 dpi. (J) Tumor growth (left) and survival curve (right) in each group of the mice in I. n = 8–9 per group. (K and L) Representative flow cytometry plots (K) and cumulative data (L) show the frequency and absolute number of Tcf1 + Tim3 − T PEX and Tim3 + Tcf1 − T EX cells gated on tumor-infiltrating OT-I cells. n = 6–7 per group. Data are representative of two independent experiments. P value was calculated by two-way ANOVA (B and J), two-tailed Student’s t test (C, D, F, H, and L), and Log-rank test (J); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Mettl8 -tdTomato-Flag mice were generated by Cyagen Biosciences, China.

Techniques: Inhibition, Injection, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 promotes m 3 C modification of Tcf7 mRNA and its genome-specific loops of Tox in CD8 + T cells. (A) Venn plot illustrates the overlap of downregulated genes from RNA-seq, m 3 C-seq, and Mettl8-binding genes from RIP-seq. (B) Mettl8 occupancy at the Tcf7 gene loci is revealed through m 3 C-seq (WT and Mettl8 −/− ) of EG7-OVA tumor-infiltrating OT-I cells and RIP-seq (Mettl8-tdTomato-Flag) of B16F10 tumor-infiltrating CD44 + CD8 + T cells. The binding peaks on Tcf7 loci are depicted. The m 3 C tracks are all plotted on a consistent scale. (C) The RNA decay assay demonstrates the remaining Tcf7 mRNA of CD8 + T cells from the spleens of WT and Mettl8 −/− mice detected by qRT-PCR, normalized to t = 0. (D) Heatmaps display changes in total Tcf1-targeting genes between WT and Mettl8 −/− EG7-OVA tumor-infiltrating OT-I cells and Mettl8-targeting genes in B16F10 tumor-infiltrating CD44 + CD8 + T cells of Mettl8-tdTomato-Flag mice as detected by CUT&Tag. (E) Diamond graphs exhibit chromatin interactions in WT and Mettl8 −/− tumor-infiltrating OT-I cells at the Tox gene loci (top), with CUT&Tag and ATAC-seq tracks, and gene structures on the bottom. An enlarged view highlights the signal profiles across the Tox gene region. (F) co-IP of Tcf1 by anti-Flag magnetic beads in CD3 + T cells from the spleens of Mettl8-tdTomato-Flag (RPT) and WT mice. IB, immunoblot. (G) co-IP of Tcf1 by Flag-tagged Mettl8 protein with anti-Flag magnetic beads after co-transfection into HEK293T cells. (H) Single-cell transcription levels of representative genes illustrated in the UMAP plot. Transcription levels are color coded: gray, not expressed; blue, expressed. (I) Schematic diagram of the tumor model: Mettl8 fl/fl Cd4 cre mice were subcutaneously injected with 2 × 10 5 B16F10 cells and harvested after 13 days. (J) Representative flow cytometry plots and cumulative data show the frequency of Tcf1 + Tox + cells gated on tumor-infiltrating CD8 + CD44 + T cells (right). n = 6 per group. (K) Schematic diagram of the OT-I–transferred tumor model: CD45.1 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 2 × 10 6 WT or Mettl8 −/− OT-I cells transfer at 9 dpi. Mice were harvested at 21 dpi. Representative flow cytometry plots and cumulative data show the frequency of Tox + cells gated on Tcf1 + OT-I cells. n = 6 per group. (L) The MFI of Tox gated on Tcf1 + OT-I cells of the mice in K. n = 6 per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available for this figure: .

Article Snippet: In briefly, cells were sorted enriched by ConA-magnetic beads and resuspended in wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCI, 0.5 mM spermidine; 1× protease inhibitor cocktail; 0.05% digitonin) and then incubated overnight with anti-Tcf1 (1:50, C63D9, cat. no. 2203; Cell Signaling Technology), anti-H3K27ac (1:50, cat. no. ab4729; Abcam), or anti-Flag (1:50, D6W5B, cat. no. 14793; Cell Signaling Technology).

Techniques: Modification, RNA Sequencing, Binding Assay, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Cotransfection, Single Cell, Injection, Flow Cytometry, Two Tailed Test