Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: NRDC is a direct target of miR-136-3p in human myotubes. Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24). * p < 0.05, ** p < 0.005. GSE = gene set enrichment; HEK293 = human embryonic kidney; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; nTPM = normalized transcripts per million; si NRDC = small interfering RNA of NRDC ; UTR = untranslated region.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative Proteomics, Negative Control, Small Interfering RNA

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: Cellular metabolism in human myotubes after miR-136-3p transfection or NRDC silencing. Mitochondrial respiration in miR-136-3p-transfected or NRDC- silenced human myotubes was monitored using the Mitochondrial Stress Test. (A) OCR and (B) ECAR were measured using the Seahorse XFe24 Extracellular Flux Analyzer. The trace shows representative data. (C) Quantification of the mitochondrial respiration data for basal respiration, maximal respiration, ATP production, and spare respiratory capacity obtained from 3 independent experiments. Human myotubes were transfected with miR-136-3p or siRNA against NRDC before determination of (D) uptake of radiolabeled glucose, (E) rates of radiolabeled glucose oxidation, (F) conversion of radiolabeled glucose into glycogen, (G) rate of radiolabeled palmitic acid oxidation, (H) protein synthesis as assessed by incorporation of puromycin, and (I) lactate release into the media. Results are expressed as mean ± standard error of the mean. * p < 0.05, ** p < 0.005 vs. control cells. ECAR = extracellular acidification rate; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; ns = no significance; OCR = oxygen consumption rate; OigoA = oligomycin A; Rot/AA = rotenone and antimycin A; si NRDC = small interfering RNA of NRDC; siRNA = small interfering RNA; scr = negative control for small interfering RNA.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Transfection, Control, Negative Control, Small Interfering RNA

Journal: Non-coding RNA Research

Article Title: The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor

doi: 10.1016/j.ncrna.2026.04.001

Figure Lengend Snippet: lncRNA TDRG1 acts as a piRNA precursor to generate piRNAs A. RT‒qPCR analysis of TDRG1 overexpression in GC-2 spd(ts) cells. Gapdh was used as the internal reference; empty vector transfection as the control (Ctrl). The relative expression was calculated relative to the expression level of Gapdh . Relative expression was normalized to Gapdh , and calculated versus shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗∗∗ P < 0.001). B. Expression levels of TDRG1 -related piRNAs after TDRG1 overexpression in GC-2 spd(ts), detected by RT‒qPCR. U6 snRNA was used as the internal reference; empty vector transfection as the control (Ctrl). Three piRNAs (piR-128687, piR-115293 and piR-103374) with no sequences overlap to 1700008K24Rik were used as negative controls. Relative expression was normalized to U6 , and calculated versus shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS: no significant difference).

Article Snippet: Reverse transcription of mRNAs or lncRNAs was performed using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01), while reverse transcription of piRNAs was carried out using a piRNA reverse transcription kit (miRNA First Strand cDNA Synthesis Kit (Tailing Reaction), Sangon Biotech, Cat# B532451). qPCR was performed via qPCR reagent (AceQ qPCR SYBR Green Master Mix, Vazyme, Cat# Q111-02) on a qPCR detection system (Bio-Rad) using the specific primers listed in in the “Multimedia component 3” file. mRNA or lncRNA expression was calculated, referring to that of the gene Gapdh , while piRNA expression was calculated, referring to that of the gene U6 .

Techniques: Over Expression, Plasmid Preparation, Transfection, Control, Expressing

Journal: Non-coding RNA Research

Article Title: The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor

doi: 10.1016/j.ncrna.2026.04.001

Figure Lengend Snippet: RNA-seq analysis of 1700008K24Rik -overexpressing GC-2 spd(ts) cells A . Volcano plot displaying differentially expressed genes (DEGs) in GC-2 spd(ts) cells overexpressing 1700008K24Rik versus control, based on RNA-seq data. The x-axis shows log 2 (fold change) the y-axis shows -log 10 ( P value). Red, green, and gray dots indicate up-regulated, down-regulated and non-DEGs, respectively. B-C . Gene Ontology (GO) enrichment analysis of up-regulated (B) and down-regulated (C) genes after 1700008K24Rik overexpression in GC-2 spd(ts) cells. Selected biological processes that were potentially involved are listed on the right. D . KEGG pathway enrichment analysis of down-regulated genes after 1700008K24Rik overexpression in GC-2 spd(ts) cells. Related pathways that were potentially involved are listed on the right. E-F . RT‒qPCR validation of RNA-seq results for up-regulated (E) and down-regulated (F) genes following 1700008K24Rik overexpression in GC-2 spd(ts) cells. Gapdh was used as the internal control; empty vector transfectionas the experimental control (Ctrl). Relative expression was calculated versus Ctrl. Data are presented as the mean ± SD; n = 3. Statistical significance was determined by unpaired t- test (∗ P < 0.05, ∗∗ P < 0.01, NS: not significant). G . Predicted targets of the 1700008K24Rik-related piRNAs among the differentially expressed genes, analyzed using miRanda algorithm. Shown are eight spermatogenesis-related genes harboring potential piRNA targeting sites.

Article Snippet: Reverse transcription of mRNAs or lncRNAs was performed using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01), while reverse transcription of piRNAs was carried out using a piRNA reverse transcription kit (miRNA First Strand cDNA Synthesis Kit (Tailing Reaction), Sangon Biotech, Cat# B532451). qPCR was performed via qPCR reagent (AceQ qPCR SYBR Green Master Mix, Vazyme, Cat# Q111-02) on a qPCR detection system (Bio-Rad) using the specific primers listed in in the “Multimedia component 3” file. mRNA or lncRNA expression was calculated, referring to that of the gene Gapdh , while piRNA expression was calculated, referring to that of the gene U6 .

Techniques: RNA Sequencing, Control, Over Expression, Biomarker Discovery, Plasmid Preparation, Expressing

Journal: Non-coding RNA Research

Article Title: The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor

doi: 10.1016/j.ncrna.2026.04.001

Figure Lengend Snippet: Knockdown of 1700008K24Rik impairs mouse spermatogenesis. A . Visualization of AAV9 virus delivery into mouse seminiferous tubules, with trypan blue indicating successful injection. B . RT‒qPCR analysis of 1700008K24Rik knockdown efficiency in mouse testes. Three-week-old mice were injected with AAV9-shRNA-GFP (shRNA) and analyzed after one month. The contralateral testis injected nontargeted shRNA (shCtrl) served as the control. Gapdh was used as the internal reference. Relative expression was calculated versus Ctrl. Data are presented as the mean ± SD; n = 3. Statistical significance was determined by unpaired t- test (∗∗ P < 0.01, NS: not significant). C . Representative images of testis size AAV9-shRNA-GFP (shRNA) and AAV9-shCtrl-GFP (shCtrl) injected mice. Viruses were delivered at 3 weeks of age; tissues were collected one month post-injection. n = 3. D . Testis weight quantification of shRNA and shCtrl groups. Relative testis weight was normalized to shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗∗∗ P < 0.001), n = 3. E . H&E staining of paraffin-embedded testis sections from shRNA and shCtrl groups. Scale bars: 100 μm (overview) and 20 μm (magnified). F . Seminiferous epithelium thickness measurement in shRNA and shCtrl groups. Thickness was assessed from five H&E-stained sections per testis. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗ P < 0.05, ∗∗ P < 0.01). G . Epididymal sperm counts in shRNA and shCtrl groups. Relative sperm count was calculated versus shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗∗ P < 0.01), n = 3. H . Expression levels of 1700008K24Rik -related piRNAs in shRNA or shCtrl testes, detected by RT‒qPCR. Three piRNAs (piR-128687, piR-115293 and piR-103374) with no sequence overlap to 1700008K24Rik were used as negative controls. Relative expression was calculated versus Ctrl. Data are presented as the mean ± SD; n = 3. Statistical significance was determined by unpaired t- test (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS: not significant).

Article Snippet: Reverse transcription of mRNAs or lncRNAs was performed using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01), while reverse transcription of piRNAs was carried out using a piRNA reverse transcription kit (miRNA First Strand cDNA Synthesis Kit (Tailing Reaction), Sangon Biotech, Cat# B532451). qPCR was performed via qPCR reagent (AceQ qPCR SYBR Green Master Mix, Vazyme, Cat# Q111-02) on a qPCR detection system (Bio-Rad) using the specific primers listed in in the “Multimedia component 3” file. mRNA or lncRNA expression was calculated, referring to that of the gene Gapdh , while piRNA expression was calculated, referring to that of the gene U6 .

Techniques: Knockdown, Virus, Injection, shRNA, Control, Expressing, Staining, Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: Metastasis-related 12-gene signature, and identification of UPK1B and CGB5 as independent prognostic markers associated with advanced metastatic GC. (A) Venn diagram showing the intersection of genes upregulated in N1-3 stage and M1 stage GC tissues. (B) LASSO Cox regression analysis based on TCGA-STAD data identified a metastasis-associated prognostic risk signature. LASSO coefficient profiles of the signature genes in the merged dataset are shown (top) and the coefficient profile plot is generated against the log(λ) sequence (bottom). (C) The high-risk group of patients with GC exhibited a poor prognosis (overall survival). High expression of (D) CGB5 and (E) UPK1B was associated with poor overall survival in patients with GC based on data from the TCGA-STAD cohort. (F) Elevated UPK1B expression predicted poor prognosis (overall survival) of patients with GC in the Kaplan-Meier plotter database. UPK1B expression was increased in (G) M1 compared with M0 and in (H) N2 metastatic stage GC tissues compared with N0 stage. UPK1B, uroplakin 1B; CGB5, chorionic gonadotropin subunit β-5; GC, gastric cancer; LASSO, Least Absolute Shrinkage and Selection Operator; TCGA-STAD, The Cancer Genome Atlas Stomach Adenocarcinoma; HR, hazard ratio; TPM, transcripts per million.

Article Snippet: For gene knockdown experiments, cells were transfected with a GV493 short hairpin RNA (sh) plasmid targeting UPK1B ( ) or a negative control scrambled plasmid (sh-NC), both obtained from GeneChem, Inc.

Techniques: Generated, Sequencing, Expressing, Selection

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B drives GC cell invasion and migration in a PI3K/AKT-dependent manner. (A) Gene set enrichment analysis indicated that genes upregulated in the UPK1B-high group were enriched in the PI3K/AKT pathway. (B) Protein levels of UPK1B in GC cell lines. (C) Knockdown of UPK1B reduced PI3K/AKT activation in MKN45 cells. Silencing UPK1B suppressed the (D) migration/invasion capacity and (E) wound closure rate of MKN45 cells. (F) Overexpression of UPK1B enhanced PI3K/AKT pathway activation in AGS cells, which was attenuated by the PI3K inhibitor LY294002. Inhibition of PI3K/AKT signaling reversed UPK1B-induced (G) migration/invasion capacity and (H) wound closure rate of AGS cells. UPK1B, uroplakin 1B; GC, gastric cancer; p-, phosphorylated; sh, short hairpin RNA; NC, negative control; OE, overexpression.

Article Snippet: For gene knockdown experiments, cells were transfected with a GV493 short hairpin RNA (sh) plasmid targeting UPK1B ( ) or a negative control scrambled plasmid (sh-NC), both obtained from GeneChem, Inc.

Techniques: Migration, Knockdown, Activation Assay, Over Expression, Inhibition, shRNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: CDX2 acts as a transcriptional repressor of UPK1B and its high expression is associated with favorable prognosis of patients with GC. (A) Venn diagram showing overlapping predicted transcriptional regulators of UPK1B from ChEA and ChEA3 databases. (B) Knockdown of CDX2 in AGS cells resulted in increased UPK1B (C) mRNA and (D) protein expression. (E) Overexpression of CDX2 in MKN45 cells reduced UPK1B protein levels. Data from (F) The Cancer Genome Atlas Stomach Adenocarcinoma cohort and (G) the Kaplan-Meier plotter database indicated that high CDX2 expression was associated with improved prognosis of patients with GC. UPK1B, uroplakin 1B; GC, gastric cancer; si, small interfering RNA; NC, negative control; OE, overexpression; HR, hazard ratio; CDX2, caudal-related homeobox transcription factor 2; ChEA, ChIP-X Enrichment Analysis.

Article Snippet: For gene knockdown experiments, cells were transfected with a GV493 short hairpin RNA (sh) plasmid targeting UPK1B ( ) or a negative control scrambled plasmid (sh-NC), both obtained from GeneChem, Inc.

Techniques: Expressing, Knockdown, Over Expression, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B activates PI3K/AKT signaling by antagonizing the inhibitory regulator PIK3IP1 in gastric cancer cells. (A) Venn diagram showing that PIK3IP1 was identified as a putative UPK1B-interacting partner based on BioGRID and HIPPIE protein-protein interaction databases. (B) UPK1B and PIK3IP1 co-localized in the cytoplasm and plasma membrane of MKN45 cells. (C) Interaction between UPK1B and PIK3IP1 in MKN45 cells. (D) Knockdown of PIK3IP1 in MKN45 cells. (E) Silencing PIK3IP1 in UPK1B-knockdown MKN45 cells restored PI3K/AKT pathway activation. Knockdown of PIK3IP1 reversed the decrease in (F) migration/invasion and (G) wound-healing capacity in UPK1B-silenced MKN45 cells. UPK1B, uroplakin 1B; p-, phosphorylated; si, small interfering RNA; sh, short hairpin RNA; NC, negative control; PIK3IP1, PI3K inhibitor interacting protein 1; HIPPIE, Human Integrated Protein-Protein Interaction Reference; IP, immunoprecipitation.

Article Snippet: For gene knockdown experiments, cells were transfected with a GV493 short hairpin RNA (sh) plasmid targeting UPK1B ( ) or a negative control scrambled plasmid (sh-NC), both obtained from GeneChem, Inc.

Techniques: Clinical Proteomics, Membrane, Knockdown, Activation Assay, Migration, Small Interfering RNA, shRNA, Negative Control, Immunoprecipitation

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B activates PI3K/AKT signaling by antagonizing the inhibitory regulator PIK3IP1 in gastric cancer cells. (A) Venn diagram showing that PIK3IP1 was identified as a putative UPK1B-interacting partner based on BioGRID and HIPPIE protein-protein interaction databases. (B) UPK1B and PIK3IP1 co-localized in the cytoplasm and plasma membrane of MKN45 cells. (C) Interaction between UPK1B and PIK3IP1 in MKN45 cells. (D) Knockdown of PIK3IP1 in MKN45 cells. (E) Silencing PIK3IP1 in UPK1B-knockdown MKN45 cells restored PI3K/AKT pathway activation. Knockdown of PIK3IP1 reversed the decrease in (F) migration/invasion and (G) wound-healing capacity in UPK1B-silenced MKN45 cells. UPK1B, uroplakin 1B; p-, phosphorylated; si, small interfering RNA; sh, short hairpin RNA; NC, negative control; PIK3IP1, PI3K inhibitor interacting protein 1; HIPPIE, Human Integrated Protein-Protein Interaction Reference; IP, immunoprecipitation.

Article Snippet: Cells were also transfected with small interfering RNAs (siRNAs) targeting CDX2 or PIK3IP1 , with a universal non-targeting scrambled siRNA (si-NC) as the negative control , obtained from GeneChem, Inc. For UPK1B and CDX2 overexpression, the p-TSB-CMV-UPK1B and p-TSB-CMV-CDX2 expression vectors [Shanghai Genomeditech Co., Ltd.] and the corresponding empty p-TSB-CMV vector (negative control) were used.

Techniques: Clinical Proteomics, Membrane, Knockdown, Activation Assay, Migration, Small Interfering RNA, shRNA, Negative Control, Immunoprecipitation

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with siRNA targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Expressing, Methylation, MSP Assay, Over Expression, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Immunoprecipitation, Western Blot, Control, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: Inhibition of DNMT1 abrogates LINC00184-induced PI3K/AKT pathway activation and functional phenotypes. (A) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells following overexpression of LINC00184 and treatment with 5-AZA. (B) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (A). (C) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (A). (D) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells following overexpression of LINC00184 and treatment with 5-AZA. (E) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (D). (F) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (D). (G) Western blotting analysis of p-AKT and total AKT levels in esophageal squamous cell carcinoma cells under the following conditions: Control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein expression level corresponding to the immunoblots presented in (G). Data are presented as mean ± SEM (n=3). Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Inhibition, Activation Assay, Functional Assay, Western Blot, Expressing, Over Expression, Derivative Assay, Control, Negative Control, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: DNMT1 inhibition reverses LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell viability of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (B) Representative images of migrated KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (C) Representative flow cytometry dot plots showing the apoptosis rate of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (D) Statistical counting of migrated KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative analysis of the apoptosis rate of KYSE-150 cells corresponding to (C) (n=3). (F) Cell viability of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (G) Representative images of migrated TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (H) Representative flow cytometry dot plots showing the apoptosis rate of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (I) Statistical counting of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative analysis of the apoptosis rate of TE-1 cells corresponding to (H) (n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in ESCC cells under the conditions of control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3), Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous cell carcinoma.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Inhibition, Over Expression, Flow Cytometry, Cell Counting, Control, Negative Control, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with siRNA targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.

Article Snippet: In total, three siRNA duplexes targeting distinct regions of LINC00184 (sequences listed in ) were chemically synthesized by Sangon Biotech Co., Ltd. KYSE-150 and TE-1 cells were transfected in 6-well plates using Lipofectamine ® RNAiMAX (Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Methylation, MSP Assay, Over Expression, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Immunoprecipitation, Western Blot, Control, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: Inhibition of DNMT1 abrogates LINC00184-induced PI3K/AKT pathway activation and functional phenotypes. (A) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells following overexpression of LINC00184 and treatment with 5-AZA. (B) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (A). (C) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (A). (D) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells following overexpression of LINC00184 and treatment with 5-AZA. (E) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (D). (F) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (D). (G) Western blotting analysis of p-AKT and total AKT levels in esophageal squamous cell carcinoma cells under the following conditions: Control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein expression level corresponding to the immunoblots presented in (G). Data are presented as mean ± SEM (n=3). Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.

Article Snippet: In total, three siRNA duplexes targeting distinct regions of LINC00184 (sequences listed in ) were chemically synthesized by Sangon Biotech Co., Ltd. KYSE-150 and TE-1 cells were transfected in 6-well plates using Lipofectamine ® RNAiMAX (Thermo Fisher Scientific, Inc.).

Techniques: Inhibition, Activation Assay, Functional Assay, Western Blot, Expressing, Over Expression, Derivative Assay, Control, Negative Control, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: DNMT1 inhibition reverses LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell viability of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (B) Representative images of migrated KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (C) Representative flow cytometry dot plots showing the apoptosis rate of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (D) Statistical counting of migrated KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative analysis of the apoptosis rate of KYSE-150 cells corresponding to (C) (n=3). (F) Cell viability of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (G) Representative images of migrated TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (H) Representative flow cytometry dot plots showing the apoptosis rate of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (I) Statistical counting of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative analysis of the apoptosis rate of TE-1 cells corresponding to (H) (n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in ESCC cells under the conditions of control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3), Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous cell carcinoma.

Article Snippet: In total, three siRNA duplexes targeting distinct regions of LINC00184 (sequences listed in ) were chemically synthesized by Sangon Biotech Co., Ltd. KYSE-150 and TE-1 cells were transfected in 6-well plates using Lipofectamine ® RNAiMAX (Thermo Fisher Scientific, Inc.).

Techniques: Inhibition, Over Expression, Flow Cytometry, Cell Counting, Control, Negative Control, Small Interfering RNA

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