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

Journal: International Dental Journal

Article Title: Chemokine-Like Receptor 1 Knockdown Suppresses Oral Squamous Cell Carcinoma Progression by Reducing Oxidative Phosphorylation

doi: 10.1016/j.identj.2026.109479

Figure Lengend Snippet: CMKLR1 is overexpressed in OSCC and is associated with a poor prognosis. Relative mRNA expression of CMKLR1 in (A) HNSCC tissues ( n = 520) versus adjacent normal tissues ( n = 44) and in (B) non–lymph node (N0) versus lymph node (N1) samples, according to TCGA database analysis. (C) Kaplan–Meier survival analysis of OS based on CMKLR1 expression in patients with HNSCC, with data from TCGA. (D) Representative IHC images depicting CMKLR1 expression in OSCC tissues and adjacent normal oral mucosal tissues ( n = 68). Scale bar = 20 μm. (E) Quantitative analysis of CMKLR1 IHC staining intensity in OSCC samples ( n = 68) versus normal tissue samples, with subgroup comparisons based on T stage, clinical stage, and lymph node metastasis. (F) Kaplan–Meier survival analysis of patients with OSCC with high versus low CMKLR1 expression according to IHC scores. (G) RT-qPCR analysis of CMKLR1 mRNA expression in 50 paired OSCC and adjacent normal tissues. (H) Western blot analysis of CMKLR1 protein levels in five representative paired OSCC (T) and adjacent nontumorous (N) tissues. N, adjacent nontumorous tissues; T, OSCC tumor tissues; TCGA, The Cancer Genome Atlas. * P < .05; ** P < .01; *** P < .001.

Article Snippet: Short hairpin RNA (shRNA) lentiviral vectors targeting human CMKLR1 were purchased from OBiO Technology (Shanghai, China).

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: International Dental Journal

Article Title: Chemokine-Like Receptor 1 Knockdown Suppresses Oral Squamous Cell Carcinoma Progression by Reducing Oxidative Phosphorylation

doi: 10.1016/j.identj.2026.109479

Figure Lengend Snippet: CMKLR1 promotes OSCC cell proliferation and tumor growth in vivo. (A) Western blot analysis confirming CMKLR1 knockdown in Cal-27 cells (Sh1 and Sh2) and CMKLR1 overexpression in SCC-9 cells (OE). (B) CCK-8 proliferation assays of the proliferation of CMKLR1-silenced Cal-27 cells and CMKLR1-overexpressing SCC-9 cells. (C, D) Colony formation assays and quantification in the indicated groups. (E) Representative images of xenograft tumors derived from OSCC cells with CMKLR1 knockdown (Sh), OSCC cells with CMKLR1 overexpression (OE), and control cells (NC). (F) Tumor growth curves demonstrating tumor volume changes over time. (G) Tumor weight measurements at the endpoint. * P < .05; ** P < .01; *** P < .001.

Article Snippet: Short hairpin RNA (shRNA) lentiviral vectors targeting human CMKLR1 were purchased from OBiO Technology (Shanghai, China).

Techniques: In Vivo, Western Blot, Knockdown, Over Expression, CCK-8 Assay, Derivative Assay, Control

Journal: International Dental Journal

Article Title: Chemokine-Like Receptor 1 Knockdown Suppresses Oral Squamous Cell Carcinoma Progression by Reducing Oxidative Phosphorylation

doi: 10.1016/j.identj.2026.109479

Figure Lengend Snippet: CMKLR1 promotes OSCC cell migration, invasion, and EMT. Representative images and quantification of wound healing assays in (A) Cal-27 cells with CMKLR1 knockdown and (B) SCC-9 cells with CMKLR1 overexpression. Transwell invasion assays and quantification in (C) Cal-27 and (D) SCC-9 cells, revealing a decreased and increased invasive capacity after CMKLR1 knockdown and overexpression, respectively. Scale bar = 100 μm. (E) Western blot analysis of EMT markers (E-cadherin, N-cadherin, vimentin, Snail, and Slug) in CMKLR1-modified OSCC cells. * P < .05; ** P < .01; *** P < .001.

Article Snippet: Short hairpin RNA (shRNA) lentiviral vectors targeting human CMKLR1 were purchased from OBiO Technology (Shanghai, China).

Techniques: Migration, Knockdown, Over Expression, Western Blot, Modification

Journal: International Dental Journal

Article Title: Chemokine-Like Receptor 1 Knockdown Suppresses Oral Squamous Cell Carcinoma Progression by Reducing Oxidative Phosphorylation

doi: 10.1016/j.identj.2026.109479

Figure Lengend Snippet: CMKLR1 knockdown suppresses mitochondrial OXPHOS in OSCC cells. (A) Volcano plot of DEPs in CMKLR1-knockdown and control Cal-27 cells, depicting upregulated proteins (red), downregulated proteins (blue), and nonsignificant proteins (gray). (B) Heatmap of the top DEPs identified through proteomic analysis. (C) Subcellular localization pie chart of DEPs, revealing a large proportion localized to mitochondria. (D, E) GO and KEGG pathway enrichment analyses of DEPs, highlighting enrichment in OXPHOS-related processes. (F) Gene set enrichment analysis demonstrating the significant downregulation of OXPHOS in CMKLR1-silenced cells. (G) Seahorse XF analysis of mitochondrial respiration indicating a reduced OCR in CMKLR1-knockdown OSCC cells. (H) Quantification of basal respiration, ATP production, maximal respiration, and spare respiratory capacity in CMKLR1-knockdown cells. (I) Western blot analysis of representative OXPHOS complex subunits (CI-NDUFB8, CII-SDHB, CIII-UQCRC1, CIV-MTCO2, and CV-ATP5A1), with β-actin as loading control. (J) Representative MitoTracker images and quantification of fluorescence intensity (MitoTracker Red, scale bar = 20 μm). * P < .05; ** P < .01; *** P < .001.

Article Snippet: Short hairpin RNA (shRNA) lentiviral vectors targeting human CMKLR1 were purchased from OBiO Technology (Shanghai, China).

Techniques: Knockdown, Control, Western Blot, Fluorescence

Journal: International Dental Journal

Article Title: Chemokine-Like Receptor 1 Knockdown Suppresses Oral Squamous Cell Carcinoma Progression by Reducing Oxidative Phosphorylation

doi: 10.1016/j.identj.2026.109479

Figure Lengend Snippet: CMKLR1 promotes mitochondrial OXPHOS through the PI3K/AKT/PGC-1α axis. (A) Immunoblot analysis of p-PI3K, PI3K, p-AKT, AKT, and PGC-1α in OSCC cells with CMKLR1 knockdown. (B) Protein levels of p-PI3K, p-AKT, and PGC-1α were measured in OSCC cells overexpressing CMKLR1, with or without LY294002 treatment (10 μM). (C) Western blot analysis of representative OXPHOS complex subunits (CI-NDUFB8, CII-SDHB, CIII-UQCRC1, CIV-MTCO2, and CV-ATP5A1) in CMKLR1-overexpressing cells after transfection with siPGC-1α or siNC. (D) Seahorse XF analysis of OCR curves. (E) Quantification of basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. (F) Representative MitoTracker images and quantification of fluorescence intensity (MitoTracker Red, scale bar = 20 μm). * P < .05; ** P < .01; *** P < .001.

Article Snippet: Short hairpin RNA (shRNA) lentiviral vectors targeting human CMKLR1 were purchased from OBiO Technology (Shanghai, China).

Techniques: Western Blot, Knockdown, Transfection, Fluorescence

Journal: International Dental Journal

Article Title: Chemokine-Like Receptor 1 Knockdown Suppresses Oral Squamous Cell Carcinoma Progression by Reducing Oxidative Phosphorylation

doi: 10.1016/j.identj.2026.109479

Figure Lengend Snippet: Inhibition of OXPHOS reverses the impact of CMKLR1-mediated tumor-promoting effects. (A) CCK-8 assay of the proliferation of CMKLR1-overexpressing (OE) OSCC cells treated with IACS-010759. (B) Colony formation assay of NC, OE, and OE + IACS-010759 cells. The right panel presents the number of colonies. (C, D) Transwell assay revealing significantly impaired cell invasion and migration after OXPHOS inhibitor treatment in CMKLR1-overexpressing cells. Scale bar = 100 μm. (E) Representative images of xenograft tumors derived from NC, OE, and OE + IACS-010759 cells. (F) Tumor growth curves demonstrating tumor volume changes over time. (G) Tumor weight measurements at the endpoint. (H) Representative images of IHC staining of tumor sections with Ki-67. Scale bar = 50 μm. (I) Quantification of Ki67 expression in xenograft tumors. * P < .05; ** P < .01; *** P < .001.

Article Snippet: Short hairpin RNA (shRNA) lentiviral vectors targeting human CMKLR1 were purchased from OBiO Technology (Shanghai, China).

Techniques: Inhibition, CCK-8 Assay, Colony Assay, Transwell Assay, Migration, Derivative Assay, Immunohistochemistry, Expressing

Journal: International Dental Journal

Article Title: Chemokine-Like Receptor 1 Knockdown Suppresses Oral Squamous Cell Carcinoma Progression by Reducing Oxidative Phosphorylation

doi: 10.1016/j.identj.2026.109479

Figure Lengend Snippet: Schematic diagram of the mechanism by which CMKLR1 facilitates OSCC progression.

Article Snippet: Short hairpin RNA (shRNA) lentiviral vectors targeting human CMKLR1 were purchased from OBiO Technology (Shanghai, China).

Techniques:

Journal: NPJ Precision Oncology

Article Title: NAALADL1 modulates cellular resistance to Tumor Treating Fields in colorectal cancer

doi: 10.1038/s41698-026-01492-0

Figure Lengend Snippet: A Boxplot analysis of gene expression levels across all samples confirmed successful normalization. B PCA revealed a clear separation between the high-efficacy and low-efficacy groups. C Clustering analysis did not detect extreme outliers. D A soft thresholding power of β = 7 with a scale-free topology fit index (R² > 0.85) was selected to construct a scale-free co-expression network. E A hierarchical clustering dendrogram based on the TOM identified 28 co-expression modules. F The red module (r = 0.56, P = 0.002) was positively associated with the low-efficacy group, while the orange module (r = –0.54, P = 0.003) was negatively associated. G Significant correlation of MM and GS was detected in the red module (cor = 0.36, P < 0.001). H as well as in the orange module (cor = 0.23, P = 0.008). I Differential gene expression analysis found that NAALADL1 was significantly upregulated in the low-efficacy group. J Venn diagram analysis identified NAALADL1 as the only gene overlapping between the upregulated genes in the low-efficacy group and the red module.

Article Snippet: Commercially available lentiviral particles targeting NAALADL1 (Catalog No. TL311278V, OriGene Technologies GmbH, Herford, Germany; A-D mix in equal volumes) and corresponding control vectors (Catalog No. TR30021V, OriGene Technologies GmbH), which contain random sequences of the same length as the shRNA but without human targets, were used to infect the HROC217 and HROC300 cell lines (HROC217shRNANC, HROC217shNAALADL1, HROC300shRNANC, and HROC300shNAALADL1).

Techniques: Gene Expression, Construct, Expressing

Journal: NPJ Precision Oncology

Article Title: NAALADL1 modulates cellular resistance to Tumor Treating Fields in colorectal cancer

doi: 10.1038/s41698-026-01492-0

Figure Lengend Snippet: A WB analysis of NAALADL1 expression in 14 HROC cell lines. NAALADL1 expression in the high-efficacy group was statistically lower than in the low-efficacy group ( P < 0.001). B WB analysis showed successful knockdown of NAALADL1 in HROC217 and HROC300 cells using shRNA lentiviral transduction. C Viability assays demonstrated that NAALADL1 knockdown enhanced sensitivity to TTFields treatment, as indicated by decreased cell viability in both HROC217 ( P < 0.001) and HROC300 ( P < 0.001).

Article Snippet: Commercially available lentiviral particles targeting NAALADL1 (Catalog No. TL311278V, OriGene Technologies GmbH, Herford, Germany; A-D mix in equal volumes) and corresponding control vectors (Catalog No. TR30021V, OriGene Technologies GmbH), which contain random sequences of the same length as the shRNA but without human targets, were used to infect the HROC217 and HROC300 cell lines (HROC217shRNANC, HROC217shNAALADL1, HROC300shRNANC, and HROC300shNAALADL1).

Techniques: Expressing, Knockdown, shRNA, Transduction

Journal: NPJ Precision Oncology

Article Title: NAALADL1 modulates cellular resistance to Tumor Treating Fields in colorectal cancer

doi: 10.1038/s41698-026-01492-0

Figure Lengend Snippet: A Structural analysis of NAALADL1. Left: Ribbon diagram of NAALADL1 dimer. Middle: Identification of a major binding pocket (highlighted in blue) based on geometric parameters. Right: Tunnel detection within the protein structure using CaverWeb; a key tunnel connecting the protein surface to the binding pocket is shown with a highlighted bottleneck. B Molecular docking results for top-ranked FDA-approved compounds—Lumacaftor, Doxepin N-Oxide Glucuronide, and Estradiol Benzoate. Each panel shows the chemical structure of the compound and the corresponding docking affinity plot, indicating potential binding strength and binding trajectory characteristics within the NAALADL1 tunnel. C Cell viability assays of HROC217 and HROC300 treated with DMSO (control), TTFields, NAALADL1 inhibitors alone (50 µM), or in combination with TTFields.

Article Snippet: Commercially available lentiviral particles targeting NAALADL1 (Catalog No. TL311278V, OriGene Technologies GmbH, Herford, Germany; A-D mix in equal volumes) and corresponding control vectors (Catalog No. TR30021V, OriGene Technologies GmbH), which contain random sequences of the same length as the shRNA but without human targets, were used to infect the HROC217 and HROC300 cell lines (HROC217shRNANC, HROC217shNAALADL1, HROC300shRNANC, and HROC300shNAALADL1).

Techniques: Binding Assay, Control

Journal: NPJ Precision Oncology

Article Title: NAALADL1 modulates cellular resistance to Tumor Treating Fields in colorectal cancer

doi: 10.1038/s41698-026-01492-0

Figure Lengend Snippet: A NAALADL1 was significantly downregulated in tumor tissues compared with normal tissues based on dataset analysis from public databases ( P < 0.001). B Representative immunohistochemical staining demonstrated reduced NAALADL1 protein expression in CRC tissues compared to normal tissues. C GSEA revealed NAALADL1 was significantly enriched in the Reactome pathways Mitotic Prophase, Cell Cycle Checkpoints, G2/M Checkpoints, and Rho GTPases Activate PKNs. D Crystal violet assays showed that NAALADL1 knockdown significantly suppressed the proliferation of HROC217 and HROC300. E FC analysis revealed a significant increase in the proportion of G2/M phase cells upon NAALADL1 knockdown in both HROC217 ( P < 0.01) and HROC300 ( P < 0.01). F WB analysis of cleaved caspase-3 expression in NC and NAALADL1 knockdown cell lines revealed no significant difference. G WB analysis demonstrated that NAALADL1 knockdown upregulated Ace-tubulin levels in HROC217 ( P < 0.001) and HROC300 ( P < 0.05). H Immunofluorescence staining showed significantly increased microtubule polymerization in HROC217 cells following NAALADL1 knockdown ( P < 0.05). Scale bar = 5 µm.

Article Snippet: Commercially available lentiviral particles targeting NAALADL1 (Catalog No. TL311278V, OriGene Technologies GmbH, Herford, Germany; A-D mix in equal volumes) and corresponding control vectors (Catalog No. TR30021V, OriGene Technologies GmbH), which contain random sequences of the same length as the shRNA but without human targets, were used to infect the HROC217 and HROC300 cell lines (HROC217shRNANC, HROC217shNAALADL1, HROC300shRNANC, and HROC300shNAALADL1).

Techniques: Immunohistochemical staining, Staining, Expressing, Knockdown, Immunofluorescence

Journal: NPJ Precision Oncology

Article Title: NAALADL1 modulates cellular resistance to Tumor Treating Fields in colorectal cancer

doi: 10.1038/s41698-026-01492-0

Figure Lengend Snippet: NAALADL1 inhibition potentiates TTFields by stabilizing microtubules, inducing spindle defects and G2/M arrest - thereby increasing the vulnerable cell population susceptible to TTFields’ mitotic interference.

Article Snippet: Commercially available lentiviral particles targeting NAALADL1 (Catalog No. TL311278V, OriGene Technologies GmbH, Herford, Germany; A-D mix in equal volumes) and corresponding control vectors (Catalog No. TR30021V, OriGene Technologies GmbH), which contain random sequences of the same length as the shRNA but without human targets, were used to infect the HROC217 and HROC300 cell lines (HROC217shRNANC, HROC217shNAALADL1, HROC300shRNANC, and HROC300shNAALADL1).

Techniques: Inhibition