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: 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: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 regulates NSCLC cell viability and proliferation (A–C) Western blot analysis of transfection efficiency for SPI1 overexpression and knockdown ( n = 3, n represents biological replicates). (D) CCK-8 assay assessing the effects of SPI1 overexpression and knockdown on NSCLC cell viability ( n = 6, n represents biological replicates). (E) Colony formation assay evaluating the impact of SPI1 overexpression and knockdown on NSCLC cell proliferation ( n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: Western Blot, Transfection, Over Expression, Knockdown, CCK-8 Assay, Colony Assay

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 regulates NSCLC cell migration, invasion, and EMT (A) Transwell assay assessing the effects of SPI1 on cell migration and invasion ( n = 3, n represents biological replicates), scale bars, 200 μm. (B) Western blot analysis of the impact of SPI1 on the expression levels of EMT-related proteins (N-cadherin, E-cadherin, and vimentin, n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Ⅰ, N-cadherin. Ⅱ, E-cadherin. Ⅲ, vimentin. Ⅳ, GAPDH.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: Migration, Transwell Assay, Western Blot, Expressing

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 promotes NSCLC cell invasion and migration by transcriptionally regulating miR-616-5p (A and B) RT-PCR analysis of miR-616-5p expression following SPI1 overexpression or knockdown ( n = 3, n represents biological replicates). (C–E) Transwell assay evaluating cell migration and invasion ( n = 3, n represents biological replicates), scale bars, 200 μm. Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Knockdown, Transwell Assay

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SF downregulates miR-616-5p activity by directly binding to and inhibiting SPI1 (A) Molecular docking analysis of SF with SPI1. (B) SPRi binding curve of SF to SPI1. (C and D) Western blot analysis of SPI1 expression in cells ( n = 3, n represents biological replicates). (E) RT-PCR analysis of miR-616-5p expression in cells ( n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: Activity Assay, Binding Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: In vivo targeting and anti-NSCLC efficacy of MSNs@SF-HA-FA (A) In vivo fluorescence imaging analysis ( n = 5, n represents the number of mice). (B) Ex vivo fluorescence imaging of major organs ( n = 5, n represents the number of mice). (C) Images of lung tumor tissues from mice ( n = 5, n represents the number of mice). (D) H&E-stained images and immunohistochemical analysis of SPI1 expression in xenograft tumors ( n = 5, n represents the number of mice), scale bars, 100 μm.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: In Vivo, Fluorescence, Imaging, Ex Vivo, Staining, Immunohistochemical staining, Expressing

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 regulates NSCLC cell viability and proliferation (A–C) Western blot analysis of transfection efficiency for SPI1 overexpression and knockdown ( n = 3, n represents biological replicates). (D) CCK-8 assay assessing the effects of SPI1 overexpression and knockdown on NSCLC cell viability ( n = 6, n represents biological replicates). (E) Colony formation assay evaluating the impact of SPI1 overexpression and knockdown on NSCLC cell proliferation ( n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: Western Blot, Transfection, Over Expression, Knockdown, CCK-8 Assay, Colony Assay

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 regulates NSCLC cell migration, invasion, and EMT (A) Transwell assay assessing the effects of SPI1 on cell migration and invasion ( n = 3, n represents biological replicates), scale bars, 200 μm. (B) Western blot analysis of the impact of SPI1 on the expression levels of EMT-related proteins (N-cadherin, E-cadherin, and vimentin, n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Ⅰ, N-cadherin. Ⅱ, E-cadherin. Ⅲ, vimentin. Ⅳ, GAPDH.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: Migration, Transwell Assay, Western Blot, Expressing

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 promotes NSCLC cell invasion and migration by transcriptionally regulating miR-616-5p (A and B) RT-PCR analysis of miR-616-5p expression following SPI1 overexpression or knockdown ( n = 3, n represents biological replicates). (C–E) Transwell assay evaluating cell migration and invasion ( n = 3, n represents biological replicates), scale bars, 200 μm. Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Knockdown, Transwell Assay

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SF downregulates miR-616-5p activity by directly binding to and inhibiting SPI1 (A) Molecular docking analysis of SF with SPI1. (B) SPRi binding curve of SF to SPI1. (C and D) Western blot analysis of SPI1 expression in cells ( n = 3, n represents biological replicates). (E) RT-PCR analysis of miR-616-5p expression in cells ( n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: Activity Assay, Binding Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: In vivo targeting and anti-NSCLC efficacy of MSNs@SF-HA-FA (A) In vivo fluorescence imaging analysis ( n = 5, n represents the number of mice). (B) Ex vivo fluorescence imaging of major organs ( n = 5, n represents the number of mice). (C) Images of lung tumor tissues from mice ( n = 5, n represents the number of mice). (D) H&E-stained images and immunohistochemical analysis of SPI1 expression in xenograft tumors ( n = 5, n represents the number of mice), scale bars, 100 μm.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: In Vivo, Fluorescence, Imaging, Ex Vivo, Staining, Immunohistochemical staining, Expressing