cat 21829 1 ap  (Proteintech)

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: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

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: NSUN5 confers cisplatin resistance in a GLUT1-dependent manner in vitro and in vivo . (A) Effect of GLUT1 knockdown on cisplatin sensitivity in NSUN5-overexpressing cells. Cellular viability and cisplatin IC 50 values were determined by CCK-8 assay in NSUN5-overexpressing A549 cells following GLUT1 knockdown. (B) Effect of GLUT1 knockdown on cisplatin-induced apoptosis in NSUN5-overexpressing cells. Apoptosis was assessed by flow cytometry in NSUN5-upregulated A549 cells after GLUT1 knockdown and cisplatin exposure. (C) Western blot analysis of indicated proteins in NSUN5-overexpressing A549 (left panel) and PC9 (right panel) cells, with or without cisplatin exposure and with or without GLUT1 knockdown. (D) Representative comet assay images (left panel) and quantitative tail moment analysis (right panel) in NSUN5-overexpressing A549 cells following GLUT1 knockdown. (E) Immunofluorescence showing nuclear γ-H2AX foci density in designated treatment groups. Scale bars, 10 μm. (F) Bioluminescence images of xenograft tumors across groups. (G) Tumor volume measurements in nude mice under indicated conditions. (H) Terminal tumor weights across groups. (I) H&E staining and IHC for NSUN5, GLUT1, p -RPA2, γ-H2AX, and Cleaved Caspase 3 (Cleaved C3) in mice tumor sections. Scale bars (the upper panel), 200 μm. Scale bars (the lower panel), 50 μm. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (A, D, E, H). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: In Vitro, In Vivo, Knockdown, CCK-8 Assay, Flow Cytometry, Western Blot, Single Cell Gel Electrophoresis, Immunofluorescence, Staining

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: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

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: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

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

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 promotes glycolysis and HR through GLUT1. (A) The glucose uptake was measured in NSUN5-overexpressing A549 cells with shNC or shGLUT1 transfection by fluorescently labeled glucose analogue 2-NBDG. The nucleus (blue) was stained with Hoechst. Scale bars, 100 μm. (B) Glycolytic flux analysis by extracellular acidification rate (ECAR). Real-time ECAR tracing in A549 cells sequentially treated with glucose, oligomycin (oligo), and 2-DG across experimental groups (left panel). Quantification of glycolytic parameters, including the basal glycolytic rate, maximal glycolytic capacity, and spare glycolytic capacity (right panel). (C) Mitochondrial respiration analysis by oxygen consumption rate (OCR). Real-time OCR tracing in A549 cells sequentially treated with oligomycin, FCCP, and rotenone/antimycin A across groups (left panel). Quantification of mitochondrial parameters, including basal respiration, ATP production, maximal respiration, and spare respiratory capacity (right panel). (D) Relative lactate production in designated A549 cell groups. (E) Schematic representation of the HR reporter. (F) The HR levels of the indicated HEK293T cells were detected by flow cytometry. (G-J) Representative immunofluorescence images of MRE11 (G), p -RPA2 (H), BrdU (I), and RAD51 (J) foci in A549 cells under indicated treatments. Scale bars, 10 μm. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (B-D, F-J), ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: Transfection, Labeling, Staining, Flow Cytometry, Immunofluorescence