Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: RhoGEF17 is essential for cell-cell contacts and adherens junctions (AJ) protein regulation in EC. ( A , B , D ) Human umbilical vein endothelial cells (HUVEC) were transduced with adenoviruses encoding EGFP alone (EGFP) or in addition to a shRNA against RhoGEF17 (sh17-1). ( A ) After 48 h, RhoGEF17 was detected in cell lysates. Shown are representative immunoblots of RhoGEF17 and α-tubulin and the quantitative analysis. Values are normalized and given as means + SEM with the single data points, n = 7, * p < 0.05 analyzed by paired t-testing. ( B ). Depicted are bright field/EGFP overlay images of transduced HUVEC. Scale bar = 100 µm. ( C ) Rat fat pad endothelial cells (RFPEC) were transduced for 48 h and then used to generate spheroids. Bright field and fluorescence images are shown. Scale bar = 200 µm. ( D ) VE-cadherin, p120-catenin, and α-tubulin were detected by immunoblot in lysates of transduced HUVEC. Shown are representative immunoblots and the quantified data normalized by α-tubulin and relative to EGFP as means + SEM with the single data points, n = 4 − 7, * p < 0.05 analyzed by paired t-testing.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Transduction, shRNA, Western Blot, Fluorescence

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: Loss of RhoGEF17 results in the proteasomal degradation of AJ proteins in RFPEC. RFPEC were transduced with adenoviruses encoding EGFP (EGFP), or EGFP and a shRNA against RhoGEF17 (sh17-1). ( A ) Immunoblot analysis of RhoGEF17, pan-cadherin and α-tubulin was performed at the indicated time points. The values were normalized by α-tubulin and are given as means ± SEM relative to the time point 0 h, n = 3–21. ( B ) Immunofluorescence analysis was performed 48 h after transduction. Depicted are EGFP images, immunofluorescence staining of N-cadherin (left), p120-catenin (right) and the overlays with DAPI. Scale bar = 20 µm. ( C ) The transcript levels of RhoGEF17, N-cadherin, p120-catenin and the housekeeping gene PBGD were determined by qPCR after 48 h of transduction. The values are given as means + SEM with the single data points, n = 9, * p < 0.05 assessed by paired t-testing. ( D ) The proteasome was inhibited with 100 nM Bortezomib for 2 h. Non-transduced (nt) cells were used as additional control. RhoGEF17, N-cadherin and p120-catenin were detected by immunoblot in whole cell lysates. Shown are representative immunoblots of RhoGEF17, N-cadherin, p120-catenin and α-tubulin (left) and the quantitative analyses. Values were normalized by α-tubulin and are given relative to non-transduced cells treated with DMSO only. Shown are means + SEM and the single data points; n = 3–5, * p < 0.05 assessed by 2-way ANOVA with Tukey’s multiple comparison testing.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Transduction, shRNA, Western Blot, Immunofluorescence, Staining

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: Loss of RhoGEF17 results in the accumulation of phosphorylated β-catenin and increases β-catenin target gene expression in EC. ( A ) HUVEC or ( B ) RFPEC were transduced for 48 h. Representative immunoblots of β-catenin and α-tubulin are shown. The intensity of both detected β-catenin bands (β-cat) as well as the intensity of the upper β-catenin band (mod-β-cat) were quantified and normalized by α-tubulin. The values are given relative to the EGFP control as means + SEM with the single data points, n = 3 (HUVEC), n = 3–14 (RFPEC), * p < 0.05 vs. EGFP control (not shown) assessed by paired t-testing. ( C ) The proteasome was inhibited with 100 nM Bortezomib for 2 h in RFPEC. β-catenin and phosphorylated β-catenin were detected by immunoblot. Shown are representative immunoblots of β-catenin (Ab1 = Santa Cruz, Ab2 = Zymo Research), p -β-catenin (S33/37/T41) and α-tubulin (left) and the quantitative analyses. The values are normalized and given as means + SEM with the single data points, n = 8–11, * p < 0.05 assessed by 2-way ANOVA with Tukey’s multiple comparison test. ( D ) Axin1, survivin, cyclin D1 and α-tubulin were detected by immunoblot in RFPEC lysates. Shown are representative immunoblots (left) and the corresponding analyses. The values are given relative to the EGFP control as means + SEM with the single data points, n = 3, * p < 0.05 vs. EGFP control (not shown) assessed by paired t-testing. ( E ) Cell fractionation experiments were performed with transduced RFPEC. β-catenin, histone H1 and GAPDH were detected by immunoblot in different cell fractions.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Expressing, Western Blot, Cell Fractionation

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: The RhoGEF17 knockdown alters the adhesion and migration behavior of RFPEC. RFPEC were transduced for 48 h. ( A ) The cells were detached and reseeded. Adhesion was monitored by fluorescence microscopy over a time course of 24 h. Depicted are the percentages of transduced (EGFP + ), adherent cells given as means ± SEM, n = 6, * p < 0.05. ( B ) The surface area of the cells was determined at the end (24 h) of the adhesion assay. Given are the means + SEM with the single data points, n = 4, * p < 0.05 assessed by t-testing. ( C ) Shown are EGFP images, immunofluorescence staining of vinculin and the merges of EGFP (green), vinculin (red), and DAPI (blue). Scale bar = 20 µm. ( D ) The quantification of the number of central focal adhesions in transduced (EGFP + ) and non-transduced (EGFP - ) adjacent cells are given as means + SEM with the single data points, n = 4, p < 0.05, * vs. EGFP-transduced, # EGFP + vs. EGFP - in sh17-1 transduced cells. ( E ) Vinculin was detected by immunoblot. Shown are representative immunoblots of vinculin and α-tubulin. ( F ) Fluorescence imaging of the transduced cells was performed. Shown are EGFP and TRITC-phalloidin images together with the merges of EGFP (green), phalloidin (red), and DAPI (blue). Scale bar = 20 µm. ( G ) Confluent transduced cells were scratched and imaged at the indicated time points. Left: Representative bright field/fluorescent images are show. Middle: The migration distance of the sheet was measured as indicated in the left images by the arrow. Given are the quantified data as mean ± SEM, n = 3 with 10 replicates per experiment, * p < 0.05 vs. EGFP assessed by 2-way ANOVA with Sidak’s multiple comparison test. Single, EGFP + cells in the wound were counted at the end of the assay. The number of cells per mm 2 are given as means + SEM of all measured 30 wells, * p < 0.05 assessed an unpaired t-test.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Migration, Fluorescence, Microscopy, Cell Adhesion Assay, Immunofluorescence, Staining, Western Blot, Imaging

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: The reduction of RhoGEF17 prevents apoptosis and induces a cell cycle block in EC. HUVEC ( A , E , H ) or RFPEC ( B – D , F , G ) were transduced. ( A , B ) Apoptotic cells were detected by annexin-V in semi-efficiently transduced cells after 48 h. The number of apoptotic transduced (EGFP + ) and non-transduced (EGFP - ) adjacent cells are given as means + SEM with the single data points, n = 3–4, p < 0.05 * vs. EGFP-transduced, # EGFP + vs. EGFP - in sh17 transduced cells assessed by 1-way ANOVA with Tukey’s multiple comparison test. C ) Pro- and cleaved caspase 3 expression was analyzed by immunoblot in 100% transduced cells after 48 h. Representative immunoblots of both variants and β-actin ( B ) and the analyses ( C ) are shown. Quantified values are normalized by β-actin and are given relative to EGFP. Shown are the means + SEM with the single data points, n = 5, * p < 0.05 assessed by paired t-test. ( D ) Phosphorylation of Akt was detected in whole cell lysates by immunoblot 48h after transuction. Shown are representative immunoblots of pAkt (S473), total Akt and α-tubulin and the analysis. Quantified p-Akt was normalized by Akt and is given relative to EGFP. Shown are the means + SEM with the single data points, n = 7, * p < 0.05 assessed by paired t-test. ( E , F ) Cell cycle progression was analyzed by flow cytometry in cells transduced for 48 h. Calculation of G1, S, and G2/M phases are given as means + SEM with the single data points, n = 4–8, * p < 0.05 assessed by 2-way ANOVA with Sidak’s or Tukey’s multiple comparison test. ( G ) RFPEC were 100% transduced and replated. After the indicated time points cell proliferation was assessed by automated nuclei counting. The absolute cell number is given as means + SEM with single data points, n = 3, * p < 0.05 assessed by 2-way ANOVA with Sidak’s multiple comparison test. ( H ) HUVEC were seeded and transduced with different amounts of viruses in 24-well plates. One, two, and three days later images were taken and the non-transduced (EGFP - ) and transduced (EGFP + ) cells were counted manually in 160x magnification images. Left: The results were clustered according to the transduction efficiencies in low, medium, and high transduction experiments. Right: Given are the number of cells per field of view in the different conditions, n = 3–4, * p < 0.05 vs. the corresponding Day1 data as detected by 1-way ANOVA with a Dunnett’s multiple comparison test.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Blocking Assay, Expressing, Western Blot, Flow Cytometry, Transduction

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: Scheme of RhoGEF17 function in EC. RhoGEF17 stabilizes AJ in EC. Its loss leads to AJ protein degradation via the proteasome and an accumulation of β-catenin in its destruction complex (phosphorylated and ubiquitinated) form. A part of the β-catenin pool, which might be phosphorylated by Akt, is translocated to the nucleus and induces β-catenin/TCF dependent gene transcription of, for example, survivin and cyclin D1. Besides impaired cell adhesion and migration due to the disruption of the AJ, RhoGEF17-depleted EC can escape anoikis and end in cell cycle arrest. In contrast neighboring cells, without RhoGEF17 knockdown, enter apoptosis due to the loss of cell–cell contacts. EC = Endothelial cell, RhoGEF17 = Rho-specific guanine nucleotide exchange factor 17, p120 = p120-catenin, α = α-catenin, TCF = Transcription factor, Ub = ubiquitinated, P = phosphorylated.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Migration

Journal: Cells

Article Title: Heat-Treated Lacticaseibacillus rhamnosus Strains Modulate Inflammatory and Metabolic Processes in In Vitro Systems Relevant to Canine Osteoarthritis

doi: 10.3390/cells15040336

Figure Lengend Snippet: Screening of the anti-inflammatory effect of different heat-treated strains in canine chondrocytes. IL-6 secretion by primary canine chondrocytes stimulated with IL-1β was measured in the presence of a collection of candidates. Data are represented as the percentage of IL-6 secretion relative to IL-1β stimulated condition without inactivated cells. Control condition refers to chondrocytes stimulated with IL-1β, whereas negative control (NC) corresponds to chondrocytes without stimulation. Data are presented as the mean ± SD of three independent experiments. Statistical test: One-way ANOVA with Dunnett’s multiple comparisons test (Postbiotic vs. Control). Significance levels were indicated as follows: p -value > 0.05 (ns: non-significant), p -value < 0.01(**), p -value < 0.0001 (****).

Article Snippet: Alginate beads were washed 5 times with NaCl solution (0.9%) and finally, one more time in chondrocyte differentiation medium (Cell Applications Inc., San Diego, CA, USA).

Techniques: Control, Negative Control

Journal: Cells

Article Title: Heat-Treated Lacticaseibacillus rhamnosus Strains Modulate Inflammatory and Metabolic Processes in In Vitro Systems Relevant to Canine Osteoarthritis

doi: 10.3390/cells15040336

Figure Lengend Snippet: Insoluble type II collagen production by primary canine chondrocytes cultured in spheroids structure. Data are presented as the mean ± SD of three independent experiments and expressed as the percentage of insoluble type II collagen production relative to the control condition. In the control condition, canine chondrocytes were cultured without supplementation of heat-treated strains. Statistical test: Kruskal-Wallis test, followed by Dunn’s multiple comparison test (Control vs. Postbiotic). Significance levels were indicated as follows: p -value < 0.05 (*).

Article Snippet: Alginate beads were washed 5 times with NaCl solution (0.9%) and finally, one more time in chondrocyte differentiation medium (Cell Applications Inc., San Diego, CA, USA).

Techniques: Cell Culture, Control, Comparison

Journal: Nature Communications

Article Title: Capsaicin diet drives gut inflammation and exosomal miR-17-3p elevation in idiopathic short stature

doi: 10.1038/s41467-025-67883-2

Figure Lengend Snippet: a Morphological analysis of the extracted material was conducted using transmission electron microscopy ( n = 3 biological replicates). b Nano-Sight analysis confirmed that the particles had an average size of around 100 nm. c Western blotting demonstrated the presence of exosome markers CD63, CD9, and CD81 in the samples. ( n = 3 biological replicates per group) e Successful uptake of FAM-labeled exosomes (indicated by green fluorescence) by chondrocytes (blue fluorescence) was verified. d , f, g CCK8 ( d ) and EdU ( f , g ) staining results indicated significant suppression of chondrocyte proliferation following co-culture with ISS plasma exosomes ( n = 3 biological replicates per group). h , i Flow cytometry analysis showed a cell cycle arrest at the G0/G1 phase ( n = 3 biological replicates per group). j –l Western blot and RT-qPCR results revealed a notable downregulation of genes associated with chondrocyte differentiation (COL10A1, RUNX2) and osteogenic marker gene (OPN, OCN) after co-culture with ISS plasma exosomes ( n = 3 biological replicates per group). m The activity of ALP was reduced, Alizarin Red and Von Kossa staining revealed a decrease in mineralization after the co-culture of human chondrocytes with ISS plasma exosomes. The arrow indicates the positive particles ( n = 3 biological replicates and 4 technical replicates per group). Data are shown as mean ± SD. Statistical comparisons were made using a two-tailed Welch’s t-test, with ** P < 0.01, *** P < 0.001 and **** P < 0.0001 versus control.

Article Snippet: Fig. 2 miR-17-3p as a Key Mediator in ISS Exosome-Induced Inhibition of Chondrocyte Proliferation and Bone Formation. a Experimental Grouping: Group A (Gray) consisted of chondrocytes co-cultured with NC-Exosomes; Group B (Pine) included chondrocytes co-cultured with ISS-Exosomes; Group C (Blue) involved chondrocytes co-cultured with ISS-Exosomes and siRNA to silence miR-17-3p expression. b The expression of miR-17-3p was significantly elevated in Group B compared to Group A, while siRNA effectively silenced miR-17-3p expression following co-culture with ISS-Exosomes( n = 3 biological replicates per group). c , f , g CCK8 ( c ) and EdU ( f , g ) assays demonstrated a marked improvement in chondrocyte proliferation upon silencing of miR-17-3p ( n = 3 biological replicates per group). d , e Flow cytometric analysis indicated a restoration of the cell cycle to the G2/M phase following downregulation of miR-17-3p ( n = 3 biological replicates per group). h –j RT-qPCR and Western blot analyses revealed significant upregulation of marker genes associated with chondrocyte hypertrophic differentiation (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) after the downregulation of miR-17-3p ( n = 3 biological replicates per group). k Alkaline phosphatase (ALP) activity and Alizarin Red and Von Kossa staining showed enhanced mineralization following the reduction of miR-17-3p levels( n = 3 biological replicates and 4 technical replicates per group).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Labeling, Fluorescence, Staining, Co-Culture Assay, Clinical Proteomics, Flow Cytometry, Quantitative RT-PCR, Marker, Activity Assay, Two Tailed Test, Control

Journal: Nature Communications

Article Title: Capsaicin diet drives gut inflammation and exosomal miR-17-3p elevation in idiopathic short stature

doi: 10.1038/s41467-025-67883-2

Figure Lengend Snippet: a Experimental Grouping: Group A (Gray) consisted of chondrocytes co-cultured with NC-Exosomes; Group B (Pine) included chondrocytes co-cultured with ISS-Exosomes; Group C (Blue) involved chondrocytes co-cultured with ISS-Exosomes and siRNA to silence miR-17-3p expression. b The expression of miR-17-3p was significantly elevated in Group B compared to Group A, while siRNA effectively silenced miR-17-3p expression following co-culture with ISS-Exosomes( n = 3 biological replicates per group). c , f , g CCK8 ( c ) and EdU ( f , g ) assays demonstrated a marked improvement in chondrocyte proliferation upon silencing of miR-17-3p ( n = 3 biological replicates per group). d , e Flow cytometric analysis indicated a restoration of the cell cycle to the G2/M phase following downregulation of miR-17-3p ( n = 3 biological replicates per group). h –j RT-qPCR and Western blot analyses revealed significant upregulation of marker genes associated with chondrocyte hypertrophic differentiation (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) after the downregulation of miR-17-3p ( n = 3 biological replicates per group). k Alkaline phosphatase (ALP) activity and Alizarin Red and Von Kossa staining showed enhanced mineralization following the reduction of miR-17-3p levels( n = 3 biological replicates and 4 technical replicates per group). The arrow indicates the positive particles. Data are presented as mean ± SD. Statistical significance was assessed using a one-way ANOVA and a two-tailed Welch’s t-test. Results were considered not significant (ns) unless * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001 compared to the control group.

Article Snippet: Fig. 2 miR-17-3p as a Key Mediator in ISS Exosome-Induced Inhibition of Chondrocyte Proliferation and Bone Formation. a Experimental Grouping: Group A (Gray) consisted of chondrocytes co-cultured with NC-Exosomes; Group B (Pine) included chondrocytes co-cultured with ISS-Exosomes; Group C (Blue) involved chondrocytes co-cultured with ISS-Exosomes and siRNA to silence miR-17-3p expression. b The expression of miR-17-3p was significantly elevated in Group B compared to Group A, while siRNA effectively silenced miR-17-3p expression following co-culture with ISS-Exosomes( n = 3 biological replicates per group). c , f , g CCK8 ( c ) and EdU ( f , g ) assays demonstrated a marked improvement in chondrocyte proliferation upon silencing of miR-17-3p ( n = 3 biological replicates per group). d , e Flow cytometric analysis indicated a restoration of the cell cycle to the G2/M phase following downregulation of miR-17-3p ( n = 3 biological replicates per group). h –j RT-qPCR and Western blot analyses revealed significant upregulation of marker genes associated with chondrocyte hypertrophic differentiation (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) after the downregulation of miR-17-3p ( n = 3 biological replicates per group). k Alkaline phosphatase (ALP) activity and Alizarin Red and Von Kossa staining showed enhanced mineralization following the reduction of miR-17-3p levels( n = 3 biological replicates and 4 technical replicates per group).

Techniques: Cell Culture, Expressing, Co-Culture Assay, Quantitative RT-PCR, Western Blot, Marker, Activity Assay, Staining, Two Tailed Test, Control

Journal: Nature Communications

Article Title: Capsaicin diet drives gut inflammation and exosomal miR-17-3p elevation in idiopathic short stature

doi: 10.1038/s41467-025-67883-2

Figure Lengend Snippet: a Experimental Grouping: Group A (Gray) consisted of normal control rats (WT); Group B (Pine) comprised rats with exosomal overexpression of miR-17-3p ; Group C (Blue) included rats with exosomal overexpression of miR-17-3p alongside overexpression of Znf148; Group D (Green) included rats with exosomal overexpression of miR-17-3p alongside overexpression of Sos1. b –e Although body height and femur and tibia lengths were significantly shorter in Group B compared to Group A, no significant differences were observed in body height or bone lengths between Group A and Group C or D, indicating that overexpression of Znf148 or Sos1 counteracted the inhibitory effects of miR-17-3p on growth plate proliferation and bone formation( n = 5 biological replicates per group). f, h Calcein staining demonstrated that exosomal miR-17-3p overexpression inhibited new bone formation (area between the two red lines), as observed in Group B. However, overexpression of Znf148 or Sos1 significantly restored bone formation, as shown in Groups C and D( n = 3 biological replicates per group). g , i Safranin O/Fast Green staining revealed that growth plate height was reduced in Group B ( miR-17-3p overexpression) compared to Group A, while overexpression of Znf148 or Sos1 restored growth plate height to normal levels, as observed in Groups C and D( n = 3 biological replicates per group). j –p Immunofluorescence staining and Western blot analyses showed that exosomal miR-17-3p overexpression significantly suppressed the expression of chondrocyte hypertrophic differentiation markers (Col10a1, Runx2) and osteogenic markers genes (Ocn, Opn). However, overexpression of Znf148 or Sos1 reversed this suppression, restoring the expression of these genes, as observed in Groups C and D. These findings indicate that exosomal miR-17-3p inhibits growth plate chondrocyte proliferation and bone formation by modulating the Znf148/Sos1 axis( n = 3 biological replicates per group). Data are presented as mean ± SD. Statistical significance was assessed using a one-way ANOVA and a two-tailed Welch’s t-test. Results were considered not significant (ns) unless * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001 compared to the control group.

Article Snippet: Fig. 2 miR-17-3p as a Key Mediator in ISS Exosome-Induced Inhibition of Chondrocyte Proliferation and Bone Formation. a Experimental Grouping: Group A (Gray) consisted of chondrocytes co-cultured with NC-Exosomes; Group B (Pine) included chondrocytes co-cultured with ISS-Exosomes; Group C (Blue) involved chondrocytes co-cultured with ISS-Exosomes and siRNA to silence miR-17-3p expression. b The expression of miR-17-3p was significantly elevated in Group B compared to Group A, while siRNA effectively silenced miR-17-3p expression following co-culture with ISS-Exosomes( n = 3 biological replicates per group). c , f , g CCK8 ( c ) and EdU ( f , g ) assays demonstrated a marked improvement in chondrocyte proliferation upon silencing of miR-17-3p ( n = 3 biological replicates per group). d , e Flow cytometric analysis indicated a restoration of the cell cycle to the G2/M phase following downregulation of miR-17-3p ( n = 3 biological replicates per group). h –j RT-qPCR and Western blot analyses revealed significant upregulation of marker genes associated with chondrocyte hypertrophic differentiation (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) after the downregulation of miR-17-3p ( n = 3 biological replicates per group). k Alkaline phosphatase (ALP) activity and Alizarin Red and Von Kossa staining showed enhanced mineralization following the reduction of miR-17-3p levels( n = 3 biological replicates and 4 technical replicates per group).

Techniques: Control, Over Expression, Staining, Immunofluorescence, Western Blot, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Capsaicin diet drives gut inflammation and exosomal miR-17-3p elevation in idiopathic short stature

doi: 10.1038/s41467-025-67883-2

Figure Lengend Snippet: a , c , d , e Capsaicin diet led to the shortened femur, tibia, and overall stunted growth in rats, compared to the wild-type (WT) control group ( n = 6 biological replicates per group). b , f Safranin O/Fast Green staining showed reduced growth plate size in capsaicin-fed rats compared to the WT group ( n = 3 biological replicates per group). g , h Calcein staining indicated inhibited new bone formation in the growth plates of capsaicin-fed rats ( n = 3 biological replicates per group). i –m Immunofluorescence staining, RT-qPCR, and Western blot analyses demonstrated suppressed expression of hypertrophic chondrocyte differentiation markers (Col10a1, Runx2) and osteogenic markers (Ocn, Opn) in the capsaicin group ( n = 3 biological replicates per group). As a result, capsaicin-fed rats exhibited a short-stature phenotype. Data are presented as mean ± SD. Statistical significance was assessed using a one-way ANOVA and a two-tailed Welch’s t-test. Results were considered not significant (ns) unless * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001 compared to the control group.

Article Snippet: Fig. 2 miR-17-3p as a Key Mediator in ISS Exosome-Induced Inhibition of Chondrocyte Proliferation and Bone Formation. a Experimental Grouping: Group A (Gray) consisted of chondrocytes co-cultured with NC-Exosomes; Group B (Pine) included chondrocytes co-cultured with ISS-Exosomes; Group C (Blue) involved chondrocytes co-cultured with ISS-Exosomes and siRNA to silence miR-17-3p expression. b The expression of miR-17-3p was significantly elevated in Group B compared to Group A, while siRNA effectively silenced miR-17-3p expression following co-culture with ISS-Exosomes( n = 3 biological replicates per group). c , f , g CCK8 ( c ) and EdU ( f , g ) assays demonstrated a marked improvement in chondrocyte proliferation upon silencing of miR-17-3p ( n = 3 biological replicates per group). d , e Flow cytometric analysis indicated a restoration of the cell cycle to the G2/M phase following downregulation of miR-17-3p ( n = 3 biological replicates per group). h –j RT-qPCR and Western blot analyses revealed significant upregulation of marker genes associated with chondrocyte hypertrophic differentiation (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) after the downregulation of miR-17-3p ( n = 3 biological replicates per group). k Alkaline phosphatase (ALP) activity and Alizarin Red and Von Kossa staining showed enhanced mineralization following the reduction of miR-17-3p levels( n = 3 biological replicates and 4 technical replicates per group).

Techniques: Control, Staining, Immunofluorescence, Quantitative RT-PCR, Western Blot, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Capsaicin diet drives gut inflammation and exosomal miR-17-3p elevation in idiopathic short stature

doi: 10.1038/s41467-025-67883-2

Figure Lengend Snippet: a –d Transmission electron microscopy, Nano-Sight analysis, zeta potential, and Western blot analyses confirmed the successful extraction of exosomes derived from chondrocytes ( n = 3 biological replicates per group). e –j RT-qPCR, Western blot, confocal microscopy, and immunofluorescence analyses demonstrated the expression of exosomal membrane fusion proteins. Additionally, siRNA and growth hormone were successfully enriched within the exosomes ( n = 3 biological replicates per group). l The targeted exosomes (ET-Exo) efficiently entered chondrocytes and rats’ growth plate cartilage. k Compared to control exosomes, ET-Exo exhibited bone-targeting solid capability. Data are presented as mean ± SD. Statistical significance was assessed using a one-way ANOVA and a two-tailed Welch’s t-test. Results were considered not significant (ns) unless * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001 compared to the control group.

Article Snippet: Fig. 2 miR-17-3p as a Key Mediator in ISS Exosome-Induced Inhibition of Chondrocyte Proliferation and Bone Formation. a Experimental Grouping: Group A (Gray) consisted of chondrocytes co-cultured with NC-Exosomes; Group B (Pine) included chondrocytes co-cultured with ISS-Exosomes; Group C (Blue) involved chondrocytes co-cultured with ISS-Exosomes and siRNA to silence miR-17-3p expression. b The expression of miR-17-3p was significantly elevated in Group B compared to Group A, while siRNA effectively silenced miR-17-3p expression following co-culture with ISS-Exosomes( n = 3 biological replicates per group). c , f , g CCK8 ( c ) and EdU ( f , g ) assays demonstrated a marked improvement in chondrocyte proliferation upon silencing of miR-17-3p ( n = 3 biological replicates per group). d , e Flow cytometric analysis indicated a restoration of the cell cycle to the G2/M phase following downregulation of miR-17-3p ( n = 3 biological replicates per group). h –j RT-qPCR and Western blot analyses revealed significant upregulation of marker genes associated with chondrocyte hypertrophic differentiation (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) after the downregulation of miR-17-3p ( n = 3 biological replicates per group). k Alkaline phosphatase (ALP) activity and Alizarin Red and Von Kossa staining showed enhanced mineralization following the reduction of miR-17-3p levels( n = 3 biological replicates and 4 technical replicates per group).

Techniques: Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Western Blot, Extraction, Derivative Assay, Quantitative RT-PCR, Confocal Microscopy, Immunofluorescence, Expressing, Membrane, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Capsaicin diet drives gut inflammation and exosomal miR-17-3p elevation in idiopathic short stature

doi: 10.1038/s41467-025-67883-2

Figure Lengend Snippet: a Group A (Gray): Chondrocytes overexpressing the mock vector (Mock); Group B (Pine): Overexpression of miR-17-3p followed by treatment with cartilage-targeted exosomes loaded with siRNA and growth hormone (GH), labeled as OE- miR-17-3p + ET-Exo (si- miR-17-3p + GH); Group C (Blue): Overexpression of miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with siRNA and GH, labeled as OE- miR-17-3p + Exo (si- miR-17-3p + GH); Group D (Green): Overexpression of miR-17-3p only; Group E (Yellow): Overexpression of miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with siRNA, labeled as OE- miR-17-3p + Exo (si- miR-17-3p ); Group F (Purple): Overexpression of miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with GH, labeled as OE- miR-17-3p + Exo (GH). b , c , e CCK8 ( b ) and EdU assays ( c , e ) showed that while miR-17-3p overexpression significantly inhibited chondrocyte proliferation, this inhibitory effect was restored upon treatment with ET-Exo (si- miR-17-3p + GH), as demonstrated in Group B. Furthermore, the cartilage-targeted exosomes (ET-Exo) displayed superior efficacy to non-cartilage-targeted exosomes (OE- miR-17-3p + Exo), as seen in Groups B and C. Additionally, growth hormone alone could not reverse the proliferation inhibition caused by miR-17-3p overexpression, suggesting that miR-17-3p overexpression induces growth hormone resistance( n = 3 biological replicates per group). d , f Flow cytometry revealed that cell cycle progression was restored following treatment with ET-Exo (si- miR-17-3p + GH), with better results than Exo (si- miR-17-3p + GH), as shown in Groups B and C( n = 3 biological replicates per group). g –j, l–p RT-qPCR and Western blot analyses demonstrated that miR-17-3p overexpression (Group D) significantly suppressed the expression of hypertrophic chondrocyte differentiation markers (COL10A1, RUNX2) and osteogenic markers gene (OCN, OPN). However, these suppressions were reversed following treatment with ET-Exo (si- miR-17-3p + GH), with better outcomes observed in Group B compared to Group C ( n = 3 biological replicates per group). k Alkaline phosphatase (ALP) activity, Alizarin Red, and Von Kossa staining indicated that while miR-17-3p overexpression reduced mineralization, these effects were restored upon treatment with ET-Exo (si- miR-17-3p + GH), again showing that ET-Exo performed better than Exo, as shown in Groups B and C. The arrow indicates the positive particles ( n = 3 biological replicates and 4 technical replicates per group). Data are presented as mean ± SD. Statistical significance was assessed using a one-way ANOVA and a two-tailed Welch’s t-test. Results were considered not significant (ns) unless * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001 compared to the control group.

Article Snippet: Fig. 2 miR-17-3p as a Key Mediator in ISS Exosome-Induced Inhibition of Chondrocyte Proliferation and Bone Formation. a Experimental Grouping: Group A (Gray) consisted of chondrocytes co-cultured with NC-Exosomes; Group B (Pine) included chondrocytes co-cultured with ISS-Exosomes; Group C (Blue) involved chondrocytes co-cultured with ISS-Exosomes and siRNA to silence miR-17-3p expression. b The expression of miR-17-3p was significantly elevated in Group B compared to Group A, while siRNA effectively silenced miR-17-3p expression following co-culture with ISS-Exosomes( n = 3 biological replicates per group). c , f , g CCK8 ( c ) and EdU ( f , g ) assays demonstrated a marked improvement in chondrocyte proliferation upon silencing of miR-17-3p ( n = 3 biological replicates per group). d , e Flow cytometric analysis indicated a restoration of the cell cycle to the G2/M phase following downregulation of miR-17-3p ( n = 3 biological replicates per group). h –j RT-qPCR and Western blot analyses revealed significant upregulation of marker genes associated with chondrocyte hypertrophic differentiation (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) after the downregulation of miR-17-3p ( n = 3 biological replicates per group). k Alkaline phosphatase (ALP) activity and Alizarin Red and Von Kossa staining showed enhanced mineralization following the reduction of miR-17-3p levels( n = 3 biological replicates and 4 technical replicates per group).

Techniques: Plasmid Preparation, Over Expression, Labeling, Inhibition, Flow Cytometry, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay, Staining, Two Tailed Test, Control

Journal: Nature Communications

Article Title: Capsaicin diet drives gut inflammation and exosomal miR-17-3p elevation in idiopathic short stature

doi: 10.1038/s41467-025-67883-2

Figure Lengend Snippet: a Group A (Gray): Wild-type control rats (WT); Group B (Pine): Rats overexpressing miR-17-3p followed by treatment with cartilage-targeted exosomes loaded with siRNA and growth hormone (Gh), labeled as OE- miR-17-3p + ET-Exo (si- miR-17-3p + Gh); Group C (Blue): Rats overexpressing miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with siRNA and Gh, labeled as OE- miR-17-3p + Exo (si- miR-17-3p + Gh); Group D (Green): Rats overexpressing miR-17-3p only; Group E (Yellow): Rats overexpressing miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with siRNA, labeled as OE- miR-17-3p + Exo (si- miR-17-3p ); Group F (Purple): Rats overexpressing miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with GH, labeled as OE- miR-17-3p + Exo (GH). b –e Overexpression of miR-17-3p significantly inhibited rat growth, leading to shorter femurs, tibias, and reduced height (Group D). However, as demonstrated in Group B, this inhibitory effect was reversed following treatment with ET-Exo (si- miR-17-3p + GH). Additionally, the cartilage-targeted exosomes (ET-Exo) outperformed the non-cartilage-targeted exosomes (OE- miR-17-3p + Exo), as shown in Groups B and C. Notably, growth hormone alone was unable to counteract the proliferation inhibition caused by miR-17-3p overexpression, suggesting that miR-17-3p overexpression is a critical factor in growth hormone resistance. This experiment reveals for the first time the potential cause of growth hormone insensitivity in ISS children treated with growth hormone therapy ( n = 6 biological replicates per group). f , g Calcein staining demonstrated that miR-17-3p overexpression inhibited new bone formation in the growth plate (area between the two red lines), as observed in Group D. However, the rate of new bone formation was restored following treatment with ET-Exo (si- miR-17-3p + GH), with better results compared to Exo (si- miR-17-3p + GH), as seen in Groups B and C. Importantly, growth hormone alone could not restore bone formation impaired by miR-17-3p overexpression, indicating that growth hormone’s ability to promote growth requires first alleviating miR-17-3p -induced suppression in the growth plate( n = 3 biological replicates per group). h , i Safranin O/Fast Green staining revealed that although growth plate height was reduced in Group D ( miR-17-3p overexpression) compared to Group A, treatment with ET-Exo (si- miR-17-3p + GH) restored growth plate height to normal levels, with better outcomes than Exo (si- miR-17-3p + GH), as seen in Groups B and C ( n = 3 biological replicates per group). j , k Immunofluorescence staining indicated that miR-17-3p overexpression (Group D) significantly suppressed the expression of chondrocyte hypertrophic differentiation markers (Col10a1, Runx2) and osteogenic markers (Ocn, Opn). These suppressions were reversed following treatment with ET-Exo (si- miR-17-3p + Gh), with superior results in Group B compared to Group C ( n = 3 biological replicates per group). Data are presented as mean ± SD. Statistical significance was assessed using a one-way ANOVA and a two-tailed Welch’s t-test. Results were considered not significant (ns) unless * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001 compared to the control group.

Article Snippet: Fig. 2 miR-17-3p as a Key Mediator in ISS Exosome-Induced Inhibition of Chondrocyte Proliferation and Bone Formation. a Experimental Grouping: Group A (Gray) consisted of chondrocytes co-cultured with NC-Exosomes; Group B (Pine) included chondrocytes co-cultured with ISS-Exosomes; Group C (Blue) involved chondrocytes co-cultured with ISS-Exosomes and siRNA to silence miR-17-3p expression. b The expression of miR-17-3p was significantly elevated in Group B compared to Group A, while siRNA effectively silenced miR-17-3p expression following co-culture with ISS-Exosomes( n = 3 biological replicates per group). c , f , g CCK8 ( c ) and EdU ( f , g ) assays demonstrated a marked improvement in chondrocyte proliferation upon silencing of miR-17-3p ( n = 3 biological replicates per group). d , e Flow cytometric analysis indicated a restoration of the cell cycle to the G2/M phase following downregulation of miR-17-3p ( n = 3 biological replicates per group). h –j RT-qPCR and Western blot analyses revealed significant upregulation of marker genes associated with chondrocyte hypertrophic differentiation (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) after the downregulation of miR-17-3p ( n = 3 biological replicates per group). k Alkaline phosphatase (ALP) activity and Alizarin Red and Von Kossa staining showed enhanced mineralization following the reduction of miR-17-3p levels( n = 3 biological replicates and 4 technical replicates per group).

Techniques: Control, Labeling, Over Expression, Inhibition, Staining, Immunofluorescence, Expressing, Two Tailed Test