Journal: Advanced Science

Article Title: METTL7A Downregulation Drives SLC1A5‐Mediated Glutamine Competition to Promote Tumor Proliferation and Suppress CD8 + T Cell Immunity in Gastric Cancer

doi: 10.1002/advs.76963

Figure Lengend Snippet: High SLC1A5 expression is associated with poor prognosis and promotes proliferation via glutamine accumulation in GC. (A) Venn diagram representing the overlap between DEGs (transcriptomic) and DEPs (proteomics). (B, C) Heatmaps displaying the expression patterns of the 30 hub genes at the transcriptomic (B) and proteomic (C) levels. (D, E) qRT‐PCR analysis (D) and Western blot analysis (E) of SLC1A5 expression in paired GC and adjacent noncancerous tissues. (F, G) qRT‐PCR analysis (F) and Western blot analysis (G) of SLC1A5 expression in GES‐1 and GC cells. (H) Representative immunohistochemistry (IHC) images and quantification of SLC1A5 in GC tissues and paired adjacent noncancerous tissues from the in‐house cohort. Scale bar: 50 µm. (I) Kaplan–Meier survival curves according to SLC1A5 expression in the in‐house GC cohort. (J, K) Colony formation (J) and EdU staining (K) assays in SLC1A5 knockdown AGS and MKN‐45 cells. n = 3. Scale bars: 100 µm. (L, M) Intracellular glutamine concentration (L) and glutamine concentration of the cell culture supernatant (M) in AGS and MKN‐45 cells after SLC1A5 knockdown. n = 3. (N) Viability effect of glutamine in GES‐1, AGS, and MKN‐45 cells. Half‐maximal effective concentration (EC 50 ) values are indicated. n = 3. (O, P) Colony formation (O) and EdU staining (P) assays of SLC1A5‐overexpressing cells under normal (2 mM) or reduced (1 mM) glutamine conditions. n = 3. Scale bars: 100 µm.

Article Snippet: The stability of SLC1A5 mRNA was assessed by treating cells with actinomycin D (Act‐D; 10 μg/mL; MedChemExpress, HY‐17559) for the indicated durations (0, 2, 4, and 6 h), after which RNA was extracted and analyzed via qRT‐PCR.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining, Knockdown, Concentration Assay, Cell Culture

Journal: Advanced Science

Article Title: METTL7A Downregulation Drives SLC1A5‐Mediated Glutamine Competition to Promote Tumor Proliferation and Suppress CD8 + T Cell Immunity in Gastric Cancer

doi: 10.1002/advs.76963

Figure Lengend Snippet: GC cells inhibit CD8 + T cell antitumor immunity through SLC1A5‐mediated glutamine competition. (A) t‐SNE plot showing cell clustering of GC single‐cell RNA‐seq data ( GSE163558 ; GSE184198 ). (B) t‐SNE plot showing SLC1A5 expression across all cell types. (C) Network plot of interaction weights/strength among different cell types. (D) Heatmap of interaction strength between epithelial cells and other cell types. (E) Representative immunofluorescence staining images of CD8 + T cells and GZMB + CD8 + T cells in human GC tissues with high or low SLC1A5 expression. Scale bars: 50 µm. (F–H) Flow cytometric analysis of human CD8 + T cells positive for GZMB (F), IFN‐γ (G), or TNF‐α (H) across different treatment groups. n = 3. (I, J) Viability of MKN‐45 cells assessed by Calcein‑AM/PI staining (I) and flow cytometry (J) across different treatment groups. n = 3. (K–M) Representative images (K), tumor growth curves (L), and tumor weights (M) of sh‐NC or SLC1A5 knockdown MFC tumors in BALB/c mice with or without intratumoral glutamine treatment. n = 5. (N, O) Flow cytometry analysis of tumor‐infiltrating CD8 + T cells (N) and GZMB + CD8 + T cells (O) from sh‐NC or SLC1A5 knockdown MFC tumors in BALB/c mice with or without intratumoral glutamine treatment. n = 5. (P) Representative IHC images showing CD8 and GZMB staining from sh‐NC or SLC1A5 knockdown MFC tumors in BALB/c mice with or without intratumoral glutamine treatment. Scale bars: 100 µm.

Article Snippet: The stability of SLC1A5 mRNA was assessed by treating cells with actinomycin D (Act‐D; 10 μg/mL; MedChemExpress, HY‐17559) for the indicated durations (0, 2, 4, and 6 h), after which RNA was extracted and analyzed via qRT‐PCR.

Techniques: Single Cell, RNA Sequencing, Expressing, Immunofluorescence, Staining, Flow Cytometry, Knockdown

Journal: Advanced Science

Article Title: METTL7A Downregulation Drives SLC1A5‐Mediated Glutamine Competition to Promote Tumor Proliferation and Suppress CD8 + T Cell Immunity in Gastric Cancer

doi: 10.1002/advs.76963

Figure Lengend Snippet: METTL7A suppresses SLC1A5 expression via m6A modification to inhibit GC cell proliferation and restore CD8 + T cell antitumor immunity. (A) Predicted m6A modification sites on SLC1A5 mRNA from the SRAMP database. (B) Venn diagram identifying METTL7A as the sole overlapping gene between the m6A2Target database and multi‐omics analysis. (C, D) qRT‐PCR analysis (C) and Western blot analysis (D) of METTL7A expression in paired GC and adjacent noncancerous tissues. (E, F) qRT‐PCR analysis (E) and Western blot analysis (F) of METTL7A expression in GES‐1 and GC cells. n = 3. (G) Representative IHC images and quantitative analysis of METTL7A protein expression. Scale bar: 50 µm. (H) Kaplan–Meier survival curves according to METTL7A expression in the in‐house GC cohort. (I, J) Colony formation (I) and EdU staining (J) assays were conducted in AGS and MKN‐45 cells as indicated. Scale bars: 100 µm. n = 3. (K–M) Flow cytometric analysis of human CD8 + T cells positive for GZMB (K), IFN‐γ (L), or TNF‐α (M) across different treatment groups. n = 3. (N, O) Viability of MKN‐45 cells assessed by Calcein‑AM/PI staining (N) and flow cytometry (O) across different treatment groups. n = 3. (P) Dot blot assay was conducted in METTL7A overexpression AGS and MKN‐45 cells. (Q) RNA stability assay was conducted in METTL7A overexpression AGS and MKN‐45 cells. n = 3. (R) MeRIP‐qPCR analysis of m6A enrichment across 3′ UTR of SLC1A5 mRNA in oe‐NC and METTL7A‐overexpressing cells. n = 3. (S) Schematic of the luciferase reporter constructs containing the wild‐type (WT) or mutant (Mut1–4) SLC1A5 3′ UTR. (T) Luciferase activity of reporters containing the WT or mutant (Mut1–4) SLC1A5 3′ UTR in oe‐NC versus METTL7A‐overexpressing cells. n = 3.

Article Snippet: The stability of SLC1A5 mRNA was assessed by treating cells with actinomycin D (Act‐D; 10 μg/mL; MedChemExpress, HY‐17559) for the indicated durations (0, 2, 4, and 6 h), after which RNA was extracted and analyzed via qRT‐PCR.

Techniques: Expressing, Modification, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Staining, Flow Cytometry, Dot Blot, Over Expression, Stability Assay, Luciferase, Construct, Mutagenesis, Activity Assay

Journal: Advanced Science

Article Title: METTL7A Downregulation Drives SLC1A5‐Mediated Glutamine Competition to Promote Tumor Proliferation and Suppress CD8 + T Cell Immunity in Gastric Cancer

doi: 10.1002/advs.76963

Figure Lengend Snippet: METTL7A inhibits N‐glycosylation of SLC1A5 via downregulation of B4GALT5. (A) GSEA of the GSE84437 dataset based on SLC1A5 expression. (B) Western blot analysis of SLC1A5 glycosylation status in AGS and MKN‐45 cells treated with PNGase F, TM, or OSMI‐1. (C) Western blot analysis of SLC1A5 N‐glycosylation from four human GC tissues treated with or without PNGase F. (D) Western blot analysis of SLC1A5 N‐glycosylation in AGS and MKN‐45 cells with or without METTL7A overexpression, alongside TM treatment. (E) Venn diagram identifying the overlap between SLC1A5‐interacting proteins (by IP‐MS) and glycosyltransferases from the GlycoGene (GGDB) database. (F) qRT‐PCR was used to compare the expression of 17 candidate N‐glycosyltransferases in GC cells with and without METTL7A overexpression. n = 3. (G) Western blot analysis of SLC1A5 N‐glycosylation in AGS and MKN‐45 cells following knockdown of the six indicated N‐glycosyltransferases. (H) Western blot analysis of B4GALT5 protein levels in GC cells with or without METTL7A overexpression. (I) Western blot analysis of SLC1A5 N‐glycosylation under the indicated conditions. (J, K) Co‐immunoprecipitation (Co‐IP) assay probing the interaction between endogenous B4GALT5 and SLC1A5 in AGS cells. (L) Immunofluorescence staining showing the subcellular localization and co‐localization of B4GALT5 and SLC1A5 in AGS and MKN‐45 cells. Scale bars: 5 µm. (M) Schematic diagram of full‐length (FL) and truncated mutants of the B4GALT5 protein. (N) Co‐IP assays assessing the interaction between SLC1A5 and the series of B4GALT5 mutants.

Article Snippet: The stability of SLC1A5 mRNA was assessed by treating cells with actinomycin D (Act‐D; 10 μg/mL; MedChemExpress, HY‐17559) for the indicated durations (0, 2, 4, and 6 h), after which RNA was extracted and analyzed via qRT‐PCR.

Techniques: Glycoproteomics, Expressing, Western Blot, Over Expression, Protein-Protein interactions, Quantitative RT-PCR, Knockdown, Co-Immunoprecipitation Assay, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: METTL7A Downregulation Drives SLC1A5‐Mediated Glutamine Competition to Promote Tumor Proliferation and Suppress CD8 + T Cell Immunity in Gastric Cancer

doi: 10.1002/advs.76963

Figure Lengend Snippet: B4GALT5 prevents K48‐linked polyubiquitination of SLC1A5 by promoting its N‐glycosylation. (A) Western blot analysis of SLC1A5 expression in AGS and MKN‐45 cells treated with TM at different doses or for different amounts of time. (B) Western blot analysis of SLC1A5 expression in GC cells treated with TM in combination with the proteasome inhibitor MG132 or the lysosome inhibitor chloroquine (CQ). (C) Cycloheximide (CHX) chase assay assessed by Western blot to determine SLC1A5 protein half‐life in GC cells with or without B4GALT5 knockdown. (D) Evaluation of endogenous SLC1A5 ubiquitination in AGS cells under the indicated conditions. (E) Immunofluorescence staining of SLC1A5 protein levels and localization in AGS and MKN‐45 cells under the indicated conditions. Scale bars: 5 µm. (F) A set of Myc‐tagged ubiquitin mutants (WT, K6, K11, K27, K29, K33, K48, or K63) was co‐expressed with HA‐SLC1A5 and Flag‐B4GALT5 in HEK293T cells, followed by anti‐HA immunoblot to determine the chain linkage pattern on SLC1A5. (G) IP‐MS analysis identified N‐glycosylation at the N212 site of SLC1A5. (H) Western blot analysis of SLC1A5 N‐glycosylation in HEK293T cells co‐transfected with WT or N212Q mutant SLC1A5 and either full‐length (FL) or truncated (ΔB) B4GALT5. (I) CHX chase assay assessed by Western blot comparing the degradation rate of WT and N212Q mutant SLC1A5 protein with or without B4GALT5 overexpression. (J) HEK293T cells were co‐transfected with HA‐SLC1A5 (WT or N212Q), Flag‐B4GALT5 (FL or ΔB mutant), and Myc‐K48 ubiquitin. Immunoprecipitation of HA‐tagged SLC1A5 to analyze its K48‐linked polyubiquitination.

Article Snippet: The stability of SLC1A5 mRNA was assessed by treating cells with actinomycin D (Act‐D; 10 μg/mL; MedChemExpress, HY‐17559) for the indicated durations (0, 2, 4, and 6 h), after which RNA was extracted and analyzed via qRT‐PCR.

Techniques: Glycoproteomics, Western Blot, Expressing, Knockdown, Ubiquitin Proteomics, Immunofluorescence, Staining, Protein-Protein interactions, Transfection, Mutagenesis, Over Expression, Immunoprecipitation

Journal: Advanced Science

Article Title: METTL7A Downregulation Drives SLC1A5‐Mediated Glutamine Competition to Promote Tumor Proliferation and Suppress CD8 + T Cell Immunity in Gastric Cancer

doi: 10.1002/advs.76963

Figure Lengend Snippet: Luteolin inhibits GC progression by upregulating METTL7A and enhances the efficacy of anti‐PD‐1 therapy. (A) Chemical structure of the natural flavonoid Luteolin (Lut). (B) Molecular docking model predicting the binding pose of Lut within the METTL7A protein structure. (C) Cellular Thermal Shift Assay (CETSA) measuring the thermal stability of METTL7A protein in GC cells treated with DMSO or Lut. (D) Immunofluorescence staining of METTL7A, B4GALT5, and SLC1A5 protein levels in AGS and MKN‐45 cells under the indicated conditions. Scale bars: 5 µm. (E) The half‐maximal inhibitory concentration (IC 50 ) values of Lut in AGS and MKN‐45 cells. n = 3. (F, G) Colony formation (F) and EdU staining (G) assays in AGS and MKN‐45 cells under the indicated conditions. Scale bars: 100 µm. n = 3. (H–J) Representative images (H), tumor growth curves (I), and tumor weights (J) in MFC xenograft‐bearing mice across different treatment groups. n = 5. (K) Schematic of the combination therapy experiment timeline using MFC tumor‐bearing mice treated with Lut alone, anti‐PD‐1 antibody alone, or their combination. (L–N) Representative images (L), tumor growth curves (M), and tumor weights (N) in MFC xenograft‐bearing mice across different treatment groups. n = 5. (O, P) Flow cytometry analysis of tumor‐infiltrating CD8 + T cells (O) and GZMB + CD8 + T cells (P) across different treatment groups. n = 5. (Q) Representative IHC images showing CD8 and GZMB staining across different treatment groups. Scale bars: 100 µm.

Article Snippet: The stability of SLC1A5 mRNA was assessed by treating cells with actinomycin D (Act‐D; 10 μg/mL; MedChemExpress, HY‐17559) for the indicated durations (0, 2, 4, and 6 h), after which RNA was extracted and analyzed via qRT‐PCR.

Techniques: Binding Assay, Thermal Shift Assay, Immunofluorescence, Staining, Concentration Assay, Flow Cytometry

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5 regulates the m5C modification and expression of its downstream target gene GLUT1. (A) Dot blot assay illustrating global m 5 C modification levels of total RNA in shNC or shNSUN5 A549/DDP cells. (B) Distribution profile of m 5 C modifications across diverse RNA regions (CDS, downstream, exon, intron, upstream, 3′UTR, and 5′UTR) from RNA Bis-seq in shNC- and shNSUN5-transfected A549/DDP cells. (C) Line chart depicting m 5 C site distribution by methylation level after NSUN5 knockdown. (D) Expression of differentially expressed genes (DEGs) from RNA-seq analysis of shNC- vs. shNSUN5-transfected A549/DDP cells. (E) Enriched pathways of those DEGs (D) in the RNA-seq. (F) Venn diagram of significantly m 5 C-modified genes (BiS-seq) and DEGs (RNA-seq). (G) Integrated volcano plot showing methylation (BiS-seq) and expression (RNA-seq) changes for 149 overlapping genes. GLUT1 exhibited the most pronounced methylation decrease in hypo-down group. (H) Correlation between NSUN5 and GLUT1 mRNA expression in TCGA-LUAD cohort. (I) IHC of NSUN5 and GLUT1 in serial sections from the same LUAD tumor tissue sample (left). Frequency of GLUT1 overexpression stratified by high/low NSUN5 expression. Scale bars (the upper panel), 200 μm. Scale bars (the lower panel), 50 μm. (J) Representative immunofluorescence staining showing the subcellular localization of GLUT1 (red) in shNC or shNSUN5 A549/DDP cells. Nuclei were stained with DAPI (blue). Scale bars, 15 μm. (K) Protein expression of GLUT1 in shNC and NSUN5-knockdown cells was assessed by Western blot assays. (L) m 5 C-MeRIP-qPCR analysis showing m 5 C modification of GLUT1 mRNA in shNC- or shNSUN5-transfected A549/DDP cells. (M) GLUT1 mRNA stability after actinomycin D (4 μg/mL) treatment. Half-life calculated from decay curves. (N) Western blot assays evaluating relative GLUT1 protein expression in NSUN5-overexpressing vs. control cells. (O) m 5 C-MeRIP-qPCR quantifying m 5 C modification levels of GLUT1 mRNA in NSUN5-overexpressing vs. control cells. (P) Actinomycin D assay determining GLUT1 mRNA half-life in NSUN5-overexpressing vs. control cells. Rep: Repeat. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (L, O), Pearson correlation test (H) or Chi-square test (I). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: To evaluate mRNA decay rates, cells were treated with actinomycin D (5 μg/mL; Cat# HY-12320, MedChem Express) to block transcription.

Techniques: Modification, Expressing, Dot Blot, Transfection, Methylation, Knockdown, RNA Sequencing, Over Expression, Immunofluorescence, Staining, Western Blot, Control

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: Cisplatin-induced ROS enhances methyltransferases activity of NSUN5 to promote m 5 C modification of GLUT1 mRNA. (A, B) NSUN5-bound m 5 C RNA detection by Co-IP. Western blot revealed m 5 C-modified RNA bound by HA-NSUN5 treated with cisplatin or Tempol. (C) Three-step catalytic mechanism of NSUN5-mediated m 5 C methylation. First, deprotonated Cys359 (motif VI, purple) initiated nucleophilic attack on cytosine C6, forming a covalent S-thioester intermediate (II) that polarizes C5. Second, Cys308 (motif IV, orange) abstractd the C5 proton, enabling methyl transfer from SAM to generate methylated intermediate (III). Finally, general base-catalyzed β-elimination released m 5 C-modified RNA and regenerates the enzyme. Top: Amino acid sequence alignment of regions forming the active sites of m 5 C methyltransferases NSUN5; The conserved motifs of NSUN5 (IV and VI) were boxed. Bottom: Reaction pathway of m 5 C formation. (D) Schematic of single-site (NSUN5 C308A , NSUN5 C359A ) and double mutant (NSUN5 DM ) constructs. Domains: N-terminal globular (green), RNA methyltransferase (blue), C-terminal (grey). Catalytic cysteines (C308/C359, orange) and SAM binding site (pink) were shown. Amino acid positions were numbered from the N-terminus. (E) Western blot revealed m 5 C-modified RNA bound by wild-type or mutant HA-NSUN5 treated with cisplatin or Tempol. (F) RNA pull-down assay coupled with Western blot validated NSUN5 as a binding protein for GLUT1 mRNA in resistant cells. (G) RNA immunoprecipitation (left panel) and agarose gel electrophoresis (right panel) assays confirmed direct binding between NSUN5 protein and GLUT1 mRNA in A549/DDP cells. (H) Western blot of GLUT1 expression after overexpression of NSUN5 WT , NSUN5 C308A , or NSUN5 C359A in A549 cells under cisplatin treatment. (I) RIP assay comparing the binding ability of NSUN5 with GLUT1 mRNA in overexpressed NSUN5 WT , NSUN5 C308A or NSUN5 C359A cells when treated with cisplatin or Tempol. (J) m 5 C-MeRIP-qPCR analysis of GLUT1 mRNA m 5 C modification levels in cells transfected with wild-type or single-point mutation constructs, following cisplatin or Tempol treatment. (K) GLUT1 mRNA half-life measured by actinomycin D assay after NSUN5 WT versus NSUN5 DM overexpression in A549 cells after cisplatin exposure. (L) Luciferase activity of wild-type and m 5 C-site-mutated GLUT1 reporters in A549 cells overexpressing NSUN5 WT or NSUN5 DM . Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (G, I, J, L). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: To evaluate mRNA decay rates, cells were treated with actinomycin D (5 μg/mL; Cat# HY-12320, MedChem Express) to block transcription.

Techniques: Activity Assay, Modification, RNA Detection, Co-Immunoprecipitation Assay, Western Blot, Methylation, Sequencing, Mutagenesis, Construct, Binding Assay, Pull Down Assay, RNA Immunoprecipitation, Agarose Gel Electrophoresis, Expressing, Over Expression, Transfection, Luciferase

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5-catalyzed m 5 C modification of GLUT1 mRNA maintains its YBX1-mediated stability. (A) Silver staining of whole-cell extract, biotin-NC pull-down (Bio-NC), and biotin-GLUT1 mRNA (Bio-GLUT1) pull-down proteins from A549/DDP cells (left panel). HPLC-MS/MS results showing the sequence HT score and relative abundance of YBX1 (right panel). (B) Correlation between YBX1 and GLUT1 mRNA expression in TCGA-LUAD cohort. (C) IHC staining of serial sections from the same LUAD patients showing co-expression of YBX1 and GLUT1. Scale bars (the upper panel), 200 μm. Scale bars (the lower panel), 50 μm. (D, E) GLUT1 expression at mRNA and protein levels following YBX1 depletion (shRNA #1/#2) in cisplatin resistant cells. (F) GLUT1 mRNA half-life determined by actinomycin D chase assay after YBX1 knockdown in A549/DDP cells. (G, H) GLUT1 mRNA (G, qPCR) and protein (H, Western blot) expression upon YBX1 overexpression in cisplatin sensitive LUAD cells. (I) GLUT1 mRNA half-life was measured by actinomycin D assay after YBX1 overexpression. (J) RIP assay showing enrichment of GLUT1 mRNA by the YBX1 antibody compared with the negative control IgG. (K) RNA-pulldown assay demonstrating direct binding between GLUT1 mRNA and YBX1. (L) Western blotting showed that YBX1 depletion reversed the increase in GLUT1 protein levels induced by NSUN5 overexpression upon cisplatin exposure. (M) RIP analysis evaluating YBX1 binding to GLUT1 mRNA in A549 cells overexpressing NSUN5 WT or NSUN5 DM with cisplatin treatment. (N) Dual-luciferase reporter assay measuring YBX1-mediated activity of GLUT1-WT and GLUT1-MUT reporters. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (D, G, J, M, N), Pearson correlation test (B) or Chi-square test (C). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: To evaluate mRNA decay rates, cells were treated with actinomycin D (5 μg/mL; Cat# HY-12320, MedChem Express) to block transcription.

Techniques: Modification, Silver Staining, Tandem Mass Spectroscopy, Sequencing, Expressing, Immunohistochemistry, shRNA, Knockdown, Western Blot, Over Expression, Negative Control, Binding Assay, Luciferase, Reporter Assay, Activity Assay