anti nsun2  (Proteintech)

Journal: Frontiers in Pharmacology

Article Title: Targeting the NSUN2–DHODH axis reverses ferroptosis resistance and oxaliplatin resistance in colorectal cancer

doi: 10.3389/fphar.2026.1739981

Figure Lengend Snippet: NSUN2 is overexpressed in CRC and associated with poor prognosis. (A) Heatmap of differential mRNA expression of m5C-related genes between CRC tumor tissues (T) and adjacent normal tissues (N) across multiple bulk transcriptomic cohorts. Bar plot showing the rank order of each gene. (B) Volcano plot of differentially expressed genes (DEGs) between CRC tumor and adjacent normal tissues in GSE18105 . (C) Dot heatmap of m5C-related gene expression in malignant versus normal epithelial cells. Dot size indicates the fraction of expressing cells, color indicates average normalized expression (Epi, epithelial). (D) H&E staining and spatial heatmaps showing NSUN2 distribution in tumor and peritumoral regions. (E) Meta-analysis of univariate Cox regression of NSUN2 expression across multiple CRC bulk transcriptomic cohorts. (F) Kaplan–Meier survival curves for overall survival (OS) or disease-free survival (DFS) in CRC patients stratified by NSUN2 high and low expression across multiple CRC bulk transcriptomic cohorts. (G) Box plots of NSUN2 expression in different CRC sample groups. (H) Western blotting of NSUN2 protein in paired CRC tumor (T) and adjacent normal (N) tissues *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Membranes were blocked with 5% non-fat milk dissolved in TBST at room temperature for 1 h, and then incubated with primary antibodies against NSUN2 (20854-1-AP, Proteintech), GPX4 (ab125066, Abcam), DHODH (ab24689, Abcam), and β-actin (66009-1-Ig, Proteintech) at 4 °C for 12 h. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (EpiZyme) at room temperature for 1 h. Bands were visualized using an ECL detection system (Bio-Rad) and quantified by integrated density using ImageJ, with β-actin used as a loading control.

Techniques: Expressing, Gene Expression, Staining, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Targeting the NSUN2–DHODH axis reverses ferroptosis resistance and oxaliplatin resistance in colorectal cancer

doi: 10.3389/fphar.2026.1739981

Figure Lengend Snippet: Knockdown of NSUN2 inhibits CRC progression in vitro and in vivo . (A) NSUN2 knockdown efficiency in SW480 and HCT116 cells assessed by Western blotting. (B) Proliferation of NSUN2 knockdown and control cells by colony formation assay. (C) Migration ability of NSUN2 knockdown and control cells detected by wound healing assays. (D) Invasion ability of NSUN2 knockdown and control cells detected by transwell assays. (E) Tumor volume and weight of xenografts derived from NSUN2 knockdown and control cells. (F) Dot heatmap of pathway enrichment analysis based on DEGs between high and low NSUN2 expression groups (dot size, adjusted p-value; color, normalized enrichment score [NES]). (G) UMAP visualization of the CRC tissue atlas and dot heatmap of reference marker expression across cell types (dot size, fraction of expressing cells; color, average normalized expression levels). (H) GSVA showing activity of tumor-promoting pathways in cancer cell clusters stratified by NSUN2 expression and exposure to FOLFOX-Bevacizumab treatment *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Membranes were blocked with 5% non-fat milk dissolved in TBST at room temperature for 1 h, and then incubated with primary antibodies against NSUN2 (20854-1-AP, Proteintech), GPX4 (ab125066, Abcam), DHODH (ab24689, Abcam), and β-actin (66009-1-Ig, Proteintech) at 4 °C for 12 h. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (EpiZyme) at room temperature for 1 h. Bands were visualized using an ECL detection system (Bio-Rad) and quantified by integrated density using ImageJ, with β-actin used as a loading control.

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Control, Colony Assay, Migration, Derivative Assay, Expressing, Marker, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Targeting the NSUN2–DHODH axis reverses ferroptosis resistance and oxaliplatin resistance in colorectal cancer

doi: 10.3389/fphar.2026.1739981

Figure Lengend Snippet: NSUN2 promotes oxaliplatin resistance in CRC. (A) Box plots of NSUN2 expression in CRC patients with different therapeutic responses (CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease) in GSE72970 . (B) ROC analysis of NSUN2 expression predicting PD in GSE72970 . (C) Box plots of NSUN2 expression in responders (R) and non-responders (NR) in GSE72970 and GSE28702 . (D) SubMap analysis predicting chemotherapy response of high- and low-NSUN2 subgroups in GSE72970 and GSE28702 . (E) Correlation between NSUN2 expression and oxaliplatin IC50 across CRC cohorts (point size, sample size; color, correlation strength). (F) Scatter plots of GSE14333 showing positive correlations between NSUN2 expression and oxaliplatin IC50. (G) NSUN2 knockdown efficiency in SW480 and HCT116 cells assessed by Western blotting. (H,I) Flow cytometry analysis of apoptosis in SW480 and HCT116 cells after NSUN2 knockdown and oxaliplatin treatment. (J) Cell viability assays assessing the effect of NSUN2 on oxaliplatin sensitivity *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Membranes were blocked with 5% non-fat milk dissolved in TBST at room temperature for 1 h, and then incubated with primary antibodies against NSUN2 (20854-1-AP, Proteintech), GPX4 (ab125066, Abcam), DHODH (ab24689, Abcam), and β-actin (66009-1-Ig, Proteintech) at 4 °C for 12 h. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (EpiZyme) at room temperature for 1 h. Bands were visualized using an ECL detection system (Bio-Rad) and quantified by integrated density using ImageJ, with β-actin used as a loading control.

Techniques: Expressing, Knockdown, Western Blot, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Targeting the NSUN2–DHODH axis reverses ferroptosis resistance and oxaliplatin resistance in colorectal cancer

doi: 10.3389/fphar.2026.1739981

Figure Lengend Snippet: NSUN2 knockdown induces ferroptosis in CRC. (A) KEGG enrichment analysis based on DEGs between high and low NSUN2 expression groups (dot size, adjusted p-value; color, normalized enrichment score [NES]). (B) UMAP visualization of meta-single-cell CRC datasets showing NSUN2 expression in naïve and treated samples; dot heatmap of NSUN2 expression across treated clusters; bar plot of NSUN2-positive cell proportions. (C) Heatmap of ferroptosis-related pathway activity between NSUN2-positive and NSUN2-negative cancer cell clusters. (D) Correlations between NSUN2 expression and Beyondcell score (BCS; higher BCS indicates greater predicted drug sensitivity) for oxaliplatin and ferroptosis inducers (erastin, ML162) across four CRC spatial transcriptomic samples in GSE225867 . (E) Spatial distribution of NSUN2 expression and ML162 sensitivity in GSE225867 -C3; scatter plots showing the correlation between NSUN2 expression and BCS. (F) Correlation between NSUN2 expression and IC50 values of ferroptosis inducers in bulk CRC samples. (G) Scatter plots showing correlations between NSUN2 expression and erastin/ML162 IC50 in GSE29621 and GSE29638 . (H) Dot blot of global RNA m5C modification in CRC cells with or without erastin treatment; methylene blue (MB) staining as loading control. (I,J) Cell viability assays showing the effect of Ferrostatin-1 (Fer-1) on erastin (I) and oxaliplatin (J) cytotoxicity in NSUN2 knockdown cells. (K) Cell viability assays showing the effect of NSUN2 on erastin sensitivity. (L) Flow cytometry of lipid ROS in CRC cells after NSUN2 silencing and erastin treatment. (M) Malondialdehyde (MDA) levels in control and NSUN2 knockdown CRC cells after 24 h erastin treatment. (N) Transmission electron microscopy (TEM) of mitochondrial morphology in NSUN2 knockdown and control cells. (O) Photograph of excised SW480 xenografts and tumor growth kinetics during the 14-day treatment period. (P) Tumor weights of excised SW480 xenografts at the end of the 14-day treatment period. (Q) Tumor lipid peroxidation assessed by malondialdehyde (MDA) quantification in xenografts collected at the end of treatment *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Membranes were blocked with 5% non-fat milk dissolved in TBST at room temperature for 1 h, and then incubated with primary antibodies against NSUN2 (20854-1-AP, Proteintech), GPX4 (ab125066, Abcam), DHODH (ab24689, Abcam), and β-actin (66009-1-Ig, Proteintech) at 4 °C for 12 h. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (EpiZyme) at room temperature for 1 h. Bands were visualized using an ECL detection system (Bio-Rad) and quantified by integrated density using ImageJ, with β-actin used as a loading control.

Techniques: Knockdown, Expressing, Single Cell, Activity Assay, Dot Blot, Modification, Staining, Control, Flow Cytometry, Transmission Assay, Electron Microscopy

Journal: Frontiers in Pharmacology

Article Title: Targeting the NSUN2–DHODH axis reverses ferroptosis resistance and oxaliplatin resistance in colorectal cancer

doi: 10.3389/fphar.2026.1739981

Figure Lengend Snippet: NSUN2 regulates ferroptosis resistance in CRC through stabilization of DHODH mRNA. (A) Correlations between NSUN2 and major ferroptosis-related genes across CRC cohorts. (B) Violin plot showing preferential DHODH expression in NSUN2-positive malignant epithelial populations at the single-cell level. (C) Co-expression heatmap showing spatial overlap between NSUN2 and DHODH. (D,E) Cell viability assays of NSUN2 knockdown and control cells treated with GPX4 inhibitor RSL3 (D) or DHODH inhibitor Brequinar (BQR) (E). (F) qRT-PCR analysis of GPX4 and DHODH mRNA in NSUN2 knockdown and control cells. (G) Western blotting of GPX4 and DHODH protein in NSUN2 knockdown and control cells. (H) Western blotting of DHODH protein in NSUN2-silenced cells re-expressing wild-type or enzymatically inactive NSUN2 mutants (C271A, C321A). (I) Dot blot of total RNA m5C levels in control cells, shNSUN2 cells, and shNSUN2 cells re-expressing wild-type NSUN2. (J) DHODH mRNA decay measured after actinomycin D treatment *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Membranes were blocked with 5% non-fat milk dissolved in TBST at room temperature for 1 h, and then incubated with primary antibodies against NSUN2 (20854-1-AP, Proteintech), GPX4 (ab125066, Abcam), DHODH (ab24689, Abcam), and β-actin (66009-1-Ig, Proteintech) at 4 °C for 12 h. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (EpiZyme) at room temperature for 1 h. Bands were visualized using an ECL detection system (Bio-Rad) and quantified by integrated density using ImageJ, with β-actin used as a loading control.

Techniques: Expressing, Single Cell, Knockdown, Control, Quantitative RT-PCR, Western Blot, Dot Blot

Journal: Frontiers in Pharmacology

Article Title: Targeting the NSUN2–DHODH axis reverses ferroptosis resistance and oxaliplatin resistance in colorectal cancer

doi: 10.3389/fphar.2026.1739981

Figure Lengend Snippet: NSUN2 promotes oxaliplatin resistance via DHODH-mediated ferroptosis suppression. (A) Western blotting of NSUN2 and DHODH in NSUN2 knockdown CRC cells. (B,C) Colony formation (B) and proliferation (C) assays of NSUN2 knockdown cells treated with erastin with or without DHODH overexpression. (D) Flow cytometry of apoptosis in NSUN2 knockdown cells with or without DHODH overexpression after oxaliplatin treatment. (E,F) Cell viability assays of NSUN2 knockdown cells with or without DHODH overexpression treated with erastin (E) or oxaliplatin (F) . (G,H) Quantification of MDA levels (G) and flow cytometric analysis (H) of lipid ROS in NSUN2 knockdown cells with or without DHODH overexpression *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Membranes were blocked with 5% non-fat milk dissolved in TBST at room temperature for 1 h, and then incubated with primary antibodies against NSUN2 (20854-1-AP, Proteintech), GPX4 (ab125066, Abcam), DHODH (ab24689, Abcam), and β-actin (66009-1-Ig, Proteintech) at 4 °C for 12 h. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (EpiZyme) at room temperature for 1 h. Bands were visualized using an ECL detection system (Bio-Rad) and quantified by integrated density using ImageJ, with β-actin used as a loading control.

Techniques: Western Blot, Knockdown, Over Expression, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Targeting the NSUN2–DHODH axis reverses ferroptosis resistance and oxaliplatin resistance in colorectal cancer

doi: 10.3389/fphar.2026.1739981

Figure Lengend Snippet: Schematic illustration of NSUN2 function in CRC. Proposed model of NSUN2-mediated m5C modification of DHODH mRNA promoting ferroptosis resistance and oxaliplatin resistance in CRC. Created with BioRender.com .

Article Snippet: Membranes were blocked with 5% non-fat milk dissolved in TBST at room temperature for 1 h, and then incubated with primary antibodies against NSUN2 (20854-1-AP, Proteintech), GPX4 (ab125066, Abcam), DHODH (ab24689, Abcam), and β-actin (66009-1-Ig, Proteintech) at 4 °C for 12 h. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (EpiZyme) at room temperature for 1 h. Bands were visualized using an ECL detection system (Bio-Rad) and quantified by integrated density using ImageJ, with β-actin used as a loading control.

Techniques: Modification