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