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: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) UMAP projection of all endothelial cells from Tsukui et al.(22) (light blue-healthy red-IPF)(4,338 cells) (B) Volcano plot comparing genes differentially expressed across healthy and IPF endothelial cells. (C) UMAP projection of all endothelial cells from Tsukui et al. and proportion analysis (D) Heatmap of immune, hypoxic and cytoskeletal genes enriched in ACKR1pos VECs. (E) UMAP projection with gene expression for ACKR1 and COL15A1 split between healthy and IPF cells. (F) IF for ACKR1 and SELP in human IPF precision cut lung slices (large scale 50µm, small scale 20µm) (G) IF for ACKR1 and VCAM1 in human IPF precision cut lung slices (large scale 50µm, small scale 10µm) (H) IF for ACKR1 and HIF1A in healthy and IPF lungs (large scale 50µm, small scale 5µm). (I) Schematic for precision cut lung slices (F) IF for ACKR1, CD45 and COL1A1 in human IPF precision cut lung slices (large scale 50µm, small scale 20µm)(FF=Fibroblastic Foci). (K) IF for ACKR1 and aSMA in healthy and IPF lungs (large scale 50µm)

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) UMAP projection of all endothelial cell markers from Tsukui et al. (B) Differentially expressed genes between all endothelial subpopulations. (C) Violin plot with ACKR1 normalized expression in healthy and IPF endothelial cells. (D) Violin plot with ACKR1 normalized expression across all lung lineages. (E) GO enrichment of upregulated and downregulated genes.

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) Circle plot showing CellChat analysis of outgoing and incoming signaling in IPF lungs and (B) heatmap depicting relative interaction strength between senders and receivers. (line thickness indicates relative communication probability). (C) Enrichment and spatial mapping of the ACKR1 VECs gene signature in human lung spatial transcriptomics data (Franzén et al.(29)). (D) Heatmap displaying the mean spatial proximity scores between source endothelial populations CPE+ / CDH5+ double-positive spots in Healthy Controls (left) and ACKR1 signature-scoring spots in IPF(right) and various target cell types. (E) Enrichment of the ACKR1 VEC-CTHRC1 gene signature across healthy and IPF samples from the Franzén et al (29) dataset and proximity quantification. F) Enrichment of the ACKR1 VEC-CD14 gene signature across healthy and IPF samples from the Franzén et al (29) dataset and proximity quantification. (G) IF for ACKR1 in an IPF lung (large scale 500µm, small scale 50µm). (H) HE, ACKR1 VEC signature enrichment and fibrosis score enrichment on mild and severe IPF biopsy spatial RNA. Statistical significance: (C,E,F) non-parametric Wilcoxon rank-sum test; (D) two-sided Mann–Whitney U test.

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques: Spatial Transcriptomics, MANN-WHITNEY

Journal: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) Spatial RNA sequencing experimental set up. (B) Bar plot of average gene content of spatial RNA sequencing samples. (C) Bar plot of spot count of spatial RNA sequencing samples. ( D ) Spatial mapping and quantification of proximity between ACKR1 VECs and ligands in healthy and IPF lungs from the Franzén et al (29) dataset. Statistical significance was evaluated using a non-parametric Wilcoxon rank-sum test.

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) Circle plot depicting CellChat analysis of outgoing and incoming signaling in IPF lungs and heatmap showing relative interaction strength between senders and receivers (line thickness indicates relative communication probability). (B) Bubble plot with Ligand-Receptor pair interactions between ACKR1to macrophages (left), Macrophages to ACKR1 VECs and Monocytes to ACKR1 VECs. Color indicates normalized interaction strength. (C) IF for ACKR1 and CD68 in IPF precision cut lung slices (scale 50µm). (D) IF for ACKR1 and CCR5 in IPF precision cut lung slices (scale 50µm). (E) IF for ACKR1 and SPP1 in IPF precision cut lung slices (scale 50µm).

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques:

Journal: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) Schematic for ACKR1 + VEC isolation (B) Brightfield image of ACKR1 + and ACKR1 - ECs. (scale 125 pixels) (C) Western blot for ACKR1 (D) Boxplots showing normalized mRNA expression for inflammatory and hypoxic markers (E) Schematic for conditional media experimental set up ( F) Boxplots showing normalized mRNA expression for CTHRC1, COL1A1 and ACTA2 (G) Schematic for immune cell adhesion experimental set up (H) Fluorescently labeled THP1 cells adhered to ACKR1 - and ACKR1 + ECs and quantification of adhered cells (scale 250 pixels). (I) Schematic for immune cell migration experimental set up (J) Quantification of migrated THP1 cells. Statistical analysis: (D,H,J) two-tailed Student’s t-test and (F) a one-way ANOVA.

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques: Isolation, Western Blot, Expressing, Labeling, Migration, Two Tailed Test

Journal: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) Boxplot showing normalized mRNA expression for ACKR1 (B) Fluorescent labeled THP1 cells adhered to Scramble or ACKR1-siRNA treated ACKR1pos ECs and quantification (scale 250 pixels) (C) Boxplots showing normalized mRNA expression for COL1A1, FN1, CTHRC1, ACTA2 and TNC (D) Schematic for collagen contraction assay experimental set up (E) Representative images of collagen contraction and quantification of collagen area (F) Heatmap for immune recruiting, profibrotic and cytoskeletal genes (G) GO enrichment of upregulated/downregulated genes. (H) IF staining for p65 and ACKR1 in healthy and IPF lungs (top scale 20µm, bottom scale 5µm). (I) Western blot for P-p65 and p65 Statistical significance: Statistical analysis: (B,C) two-tailed Student’s t-test and (A,E) a one-way ANOVA.

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques: Expressing, Labeling, Contraction Assay, Staining, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) IF for ACKR1 and Slc6a2 in bleomycin treated mouse lungs (scale 20 µm). (B) IF for ACKR1 and CD45 in bleomycin treated mouse lungs (large scale 20µm, small scale 10µm). (C) IF for ACKR1 and CD45 in mouse lungs twenty-one days after bleomycin installation (scale 50µm). (D) IF for ACKR1, CD68 and Col1a1 in mouse lungs twenty-one days after bleomycin installation (scale 200 µm). (E) IF for ACKR1, Slc6a2 and Col1a1 in mouse lungs twenty-one days after bleomycin installation (scale 100 µm). (F) scRNAseq set-up and UMAP projection of all venous endothelial cells (purple-sham, orange-seven days post bleomycin administration (cells) (G) UMAP projection of ACKR1 positive and ACKR1 negative cells and proportion plot (H) Heatmap with differentially expressed genes. (I) Bubble plot with inflammatory and immune recruiting genes (G) GO enrichment of upregulated genes. (K) Day seven ACKR1 signature on the Adams et al(11)., Habermann et al.(12) and Tsukui et al. (22) datasets. Statistical significance was evaluated using a two-sided Wilcoxon rank-sum test.

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques:

Journal: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) UMAP projection of all endothelial cells from sham and day seven post bleomycin and proportion plot. (B) UMAP projection of endothelial cells split between sham (purple) and day seven post bleomycin (orange). (C) Bubble plot with representative markers for each endothelial lineage. (D) Differential gene expression in each endothelial lineage. (E) UMAP plots with normalized gene expression across venous endothelial cells (F) Human and mouse ACKR1 VEC signature enrichment on all endothelial lineages from sham and bleomycin injured mice. (G) KEGG enrichment on mouse ACKR1pos VECs . (H) IF for ACKR1 and SPP1 in day day21 mouse bleomycin treated lungs (scale= µm). (I) IF for ACKR1 and EdU in day day7 mouse bleomycin treated lungs (scale= 10µm).

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: ACKR1-expressing venous endothelial cells establish a pro-fibrotic niche in pulmonary fibrosis

doi: 10.64898/2026.07.31.742106

Figure Lengend Snippet: (A) Experimental set up. (B) Diagram with representative body weight. (C) Bar plot depicting left lobe wet weight. (D) Bar plot depicting left lobe hydroxyproline measurement (E) Masson’s trichrome staining of sham, carrier and amikacin treated lungs (scale 100 µm). (F) IF for ACKR1, CD45 and CD68 in sham, carrier and amikacin treated lungs (scale 20 µm). (G) IF for ACKR1, aSMA and Col1a1 in sham, carrier and amikacin treated lungs (scale 20 µm). Statistical significance: (C,D) one-way ANOVA.

Article Snippet: IPF-derived ACKR1+ VEC cells were cultured and treated with either scramble siRNA (Horizon Discovery/Dharmacon, D-001810-01-05) or siRNA targeting ACKR1 (MedChemExpress, 5500343490) for two days in OptiMEM media (Fisher, 31985070) for 48 hours, after which the cells were lysed and RNA extracted.Total RNA integrity was verified using RNA 6000 Pico Assay run on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).

Techniques: Staining

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: International Journal of Surgery (London, England)

Article Title: Therapeutic effects of Icariside II on radiation cystitis: revealing the mechanistic role of the FN1/Itgαvβ6-PI3K/AKT signaling pathway using single-cell RNA sequencing

doi: 10.1097/JS9.0000000000005212

Figure Lengend Snippet: ICA II attenuates radiation-induced cellular damage by disrupting FN1–Itgαvβ6 binding and regulating the PI3K-AKT signaling pathway in bladder cells. (A) Representative surface plasmon resonance (SPR) sensorgrams and kinetic fitting curves showing the binding of ICA II to Itgαvβ6. (B) SPR sensorgrams and kinetic fitting curves for the interaction between FN1 and Itgαvβ6. (C) SPR sensorgrams and kinetic fitting curves of FN1 binding to Itgαvβ6 in the presence of 10 μM ICA II. (D) SPR sensorgrams and kinetic fitting curves of FN1 binding to Itgαvβ6 in the presence of 100 μM ICA II. (E) Effects of different radiation doses on SV-HUC-1 cell proliferation. (F) Effects of different ICA II concentrations on SV-HUC-1 cell proliferation. (G) Effects of different radiation doses on human bladder fibroblast (HBF) cell proliferation. (H) Effects of different ICA II concentrations on HBF cell proliferation. (I) Effect of ICA II on the repair of radiation-induced damage in SV-HUC-1 cells, as shown by proliferation and morphological changes after treatment. (J) Inhibitory effect of ICA II on the proliferation of HBF cells caused by radiation damage, indicating reduced fibroblast proliferation after ICA II treatment. (K and L) Western blot analysis of the effect of ICA II on the protein expression of Itgαvβ6 in SV-HUC-1 cells; the results revealed a dose-dependent decrease in expression following ICA II treatment. (M and N) Western blot analysis of the effect of ICA II on the protein expression of FN1 in HBF cells, which revealed a significant reduction in FN1 expression after ICA II treatment. (O) Enzyme-Linked Immunosorbent Assay detection of FN1 expression levels in the culture supernatant of HBF cells, confirming the suppression of FN1 secretion in response to ICA II treatment. (P) RT‒qPCR analysis was used to determine the silencing efficiency of FN1-targeted siRNA (siFN1), which successfully knocked down FN1 expression at both the mRNA and protein levels. (Q) Co-immunoprecipitation showing the interaction between FN1 and Itgαv/Itgβ6 in SV-HUC-1 cells treated with fibroblast-conditioned media. (R and S) ICA II treatment reduced EMT and fibrosis-related protein expression and inhibited PI3K-AKT pathway activation in radiation-damaged cells, suggesting that ICA II plays a protective role through regulating FN1 and Itgαvβ6 interactions. Statistical significance is indicated using standard notation.

Article Snippet: HBF cells were transiently transfected with small interfering RNA (siRNA) targeting FN1 (MCE, HY-RS05013) via the siRNA Transfection Reagent (MCE, HY-K2017) according to the manufacturer’s instructions.

Techniques: Binding Assay, SPR Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Activation Assay

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