Journal: International Journal of Molecular Sciences

Article Title: MicroRNA-10 Family Promotes Renal Fibrosis through the VASH-1/Smad3 Pathway

doi: 10.3390/ijms25105232

Figure Lengend Snippet: Depletion of miR-10a and miR-10b alleviates RF and Smad3 phosphorylation induced by UUO. ( A ) The process of obtaining KO mice through hybridization. ( B ) Genotype identification: comparison of miR-10a and miR-10b gene-deficient mice with WT mice. ( C ) Representative images (superincumbent image scale bars, 50 µm) of kidney sections from the UUO model stained with H&E and Masson, showing day 7 and day 14; sham serves as the control. ( D ) Expression results of Fibronectin, Col-III, and p-Smad3 in Western blot experiments, with β-actin as the control. ( E ) Expression results of Fibronectin and Col-III in RT-qPCR experiments, with β-actin as the control. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Louis, MO, USA) via electrophoresis, followed by blocking with 5% BSA at room temperature for 2 h. Primary antibodies for each protein were diluted in 5% BSA as follows: Fibronectin (1:1000, 26836S, CST), Col-III (1:1000, 66887S, CST), VASH-1 (1:1000, sc-365541, Santa Cruz), p-Smad3 (1:1000, 9520S, CST), Smad3 (1:1000, 9513S, CST), β-actin (1:1000, #AF7018, Affinity), and EGFP (1:5000, SRP15324, Saier); the secondary antibodies used were anti-rabbit (1:40,000, S0001, Affinity) and anti-mouse (1:40,000, S0002, Affinity).

Techniques: Hybridization, Comparison, Staining, Control, Expressing, Western Blot, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA-10 Family Promotes Renal Fibrosis through the VASH-1/Smad3 Pathway

doi: 10.3390/ijms25105232

Figure Lengend Snippet: Overexpression of miR-10 promotes TGF-β1-induced fibrosis and Smad3 phosphorylation in HK-2 cells. ( A ) Morphological changes in HK-2 cells under 20 ng/mL TGF-β1 stimulation. ( B ) Western blot analysis of Fibronectin, Col-III, and p-Smad3 expression in HK-2 cells under TGF-β1 stimulation, with β-actin as a control. ( C ) RT-qPCR analysis of miR-10a, miR-10b, Fibronectin, and Col-III expression in HK-2 cells under TGF-β1 stimulation, with β-actin as a control. ( D ) Western blot examination of relevant target molecules in HK-2 cells transfected with the corresponding plasmids under TGF-β1 stimulation. * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Article Snippet: Louis, MO, USA) via electrophoresis, followed by blocking with 5% BSA at room temperature for 2 h. Primary antibodies for each protein were diluted in 5% BSA as follows: Fibronectin (1:1000, 26836S, CST), Col-III (1:1000, 66887S, CST), VASH-1 (1:1000, sc-365541, Santa Cruz), p-Smad3 (1:1000, 9520S, CST), Smad3 (1:1000, 9513S, CST), β-actin (1:1000, #AF7018, Affinity), and EGFP (1:5000, SRP15324, Saier); the secondary antibodies used were anti-rabbit (1:40,000, S0001, Affinity) and anti-mouse (1:40,000, S0002, Affinity).

Techniques: Over Expression, Western Blot, Expressing, Control, Quantitative RT-PCR, Transfection

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA-10 Family Promotes Renal Fibrosis through the VASH-1/Smad3 Pathway

doi: 10.3390/ijms25105232

Figure Lengend Snippet: MiR-10 family promotes fibrosis by modulating the VASH-1/Smad3 pathway. ( A ) Western blot results of VASH-1 in mouse kidneys, with β-actin as a control. ( B ) After stimulation with 20 ng/mL TGF-β1, Western blot and RT-qPCR were performed to detect VASH-1 expression in HK-2 cells, with β-actin as a control. ( C ) After the transfection of HK-2 cells with overexpression plasmids, the results of Western blot and RT-qPCR experiments on the expression of VASH-1, with β-actin as a control, were determined. ( D ) After the transfection of HK-2 cells with knockdown plasmids, the results of Western blot and RT-qPCR experiments on the expression of VASH-1, with β-actin as a control, were determined. ( E ) Under stimulation with 20 ng/mL TGF-β1, after transfection with VASH-1-related plasmids, the protein expression results of Fibronectin, Col-III, and p-Smad3 in HK-2 cells, with β-actin as a control, were determined. ( F ) Under stimulation with 20 ng/mL TGF-β1, after transfection with relevant plasmids, the protein expression results of Fibronectin, Col-III, and p-Smad3 in HK-2 cells, with β-actin as a control, were determined. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Louis, MO, USA) via electrophoresis, followed by blocking with 5% BSA at room temperature for 2 h. Primary antibodies for each protein were diluted in 5% BSA as follows: Fibronectin (1:1000, 26836S, CST), Col-III (1:1000, 66887S, CST), VASH-1 (1:1000, sc-365541, Santa Cruz), p-Smad3 (1:1000, 9520S, CST), Smad3 (1:1000, 9513S, CST), β-actin (1:1000, #AF7018, Affinity), and EGFP (1:5000, SRP15324, Saier); the secondary antibodies used were anti-rabbit (1:40,000, S0001, Affinity) and anti-mouse (1:40,000, S0002, Affinity).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Transfection, Over Expression, Knockdown

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: DCM-Exos induced up-regulation of fibrotic genes in CFs (A) Representative fluorescent images of CTL-Exos or DCM-Exos (PKH26-labeled) treatment in CFs. Endocytosed Exos (orange) were observed in the cytoplasm of CFs (bar = 200 μm (left); 900 nm (right)). (B) Quantifications of Exo uptake in CFs. The uptake ratio was evaluated by the number of Exo uptaken cells (orange) divided by the total number of nuclei (blue) in the image (N = 22 pics/group), NS, nonsignificant. (C) qRT-PCR of fibrotic genes in CFs. N = 6; ∗ = p< 0.05. (D) Representative immunoblots. The CF cell lysis of different treatment groups was probed with specific antibodies (Col1a, Col3a, and CTGF). (E) Quantification of protein expression in immunoblots. Col1a, col3a, and CTGF expression were normalized to the loading control (GAPDH), N = 6. Data represented as mean ± SD; ∗ = p< 0.05; one-way ANOVA followed by Tukey post hoc test.

Article Snippet: Col3a , Cell Signaling Technology (H) , 66887.

Techniques: Labeling, Quantitative RT-PCR, Western Blot, Lysis, Expressing

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: Intracardial injection of DCM-Exo promoted fibrosis in mouse hearts (A) Representative echocardiographic images. Images were taken respectively from PBS, CTL-Exo, or DCM-Exo injection groups on day 0 and day 14. (B) LVEF was determined by echocardiography. The injection of DCM-Exo significantly decreased LVEF on day 14 (N = 8/group; ∗ = p< 0.05). Injection of PBS, or CTL-Exo resulted in nonsignificant change between day 14 and baseline. (C) Representative pictures of picrosirius red/fast green staining in series sections of the mouse hearts. The fibrotic area (Red) was quantified by ImageJ. N = 8/group, ∗∗ = p< 0.01. (D) Representative immunoblots of mouse hearts (PBS, CTL-Exo, or DCM-Exo treatments). Fibrotic markers, Col1a, Col3a, and CTGF were detected by specific antibodies. (E) Quantification of fibrotic markers expression. DCM-Exos injection resulted in significant upregulation of fibrotic markers (N= 8/group). GAPDH was used as a protein loading control. Data represented as mean ± SD; ∗ = p< 0.05; one-way ANOVA followed by Tukey post hoc test.

Article Snippet: Col3a , Cell Signaling Technology (H) , 66887.

Techniques: Injection, Staining, Western Blot, Expressing

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: MiR-218-5p increased Smad2 phosphorylation in CFs (A) Immunoblots of cell lysates derived from CFs treated with TGF-β, miR-218-5p, and a TGF-β inhibitor (SB525334). Specific antibodies were used to detect Col1a, Col3a, CTGF, phospo-Smad2 ( p -SMAD2), and GAPDH. (B) Quantitation of protein expression in immunoblots. GAPDH was used as a protein loading control. TGF-β inhibitor (SB525334) significantly reduced TGF-β1-induced fibrotic effect (lane 3). MiR-218-5p increased the phosphorylation of Smad2. N= 3; one-way ANONVA followed by Tukey’s post hoc test, ∗ = p< 0.05; ∗∗ = p< 0.01; ∗∗∗ = p< 0.001. Data represented as mean ± SD.

Article Snippet: Col3a , Cell Signaling Technology (H) , 66887.

Techniques: Western Blot, Derivative Assay, Quantitation Assay, Expressing

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: MiR-218-5p targeted to TNFAIP3 (A) Targeted sequence prediction of miR-218-5p in RNA22. (B) Immunoblots (left) indicated TNFAIP3 expression was significantly repressed in CFs transfected with miR-218-5p. (C) Luciferase assay of miR-218-5p and TNFNAIP3 5′UTR region. Co-transfection of miR-218-5p + WT sequence significantly reduced luciferase activity compared with the scramble CTL + WT. One-way ANOVA followed by Tukey’s post hoc test, ∗∗ = p< 0.01; NS = nonsignificant. (D) Representative immunoblots of CFs treatment. (E) Quantitation of expression of TNFAIP3, Col1a, Col3a, and CTGF. One-way ANOVA followed by Tukey’s post hoc test; ∗∗ = p<0.01, ∗∗∗ = p< 0.001. Data represented as mean ± SD.

Article Snippet: Col3a , Cell Signaling Technology (H) , 66887.

Techniques: Sequencing, Western Blot, Expressing, Transfection, Luciferase, Cotransfection, Activity Assay, Quantitation Assay

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: Restoration of TNFAIP3 ameliorated the DCM-Exo-induced fibrotic effect (A) Representative immunoblots of CFs treated with either CTL-Exos or DCM-Exos. DCM-Exo treatment significantly decreased TNFAIP3 expression. Student’s t test; ∗∗ = p <0.01. (B) Representative immunoblots of TNFAIP3, Col1a, Col3a, CTGF, and GAPDH detection in CFs treated with CTL-Exos, DCM-Exos, or overexpression of TNFAIP3. (C–F) Quantitation of protein expression. TNFAIP3 overexpression significantly reduced the expression of fibrotic markers. One-ANOVA followed by Tukey’s post hoc test; N= 3; ∗∗ = p< 0.01. (G) Schematic picture showing the working model of our study. Data represented as mean ± SD.

Article Snippet: Col3a , Cell Signaling Technology (H) , 66887.

Techniques: Western Blot, Expressing, Over Expression, Quantitation Assay

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet:

Article Snippet: Col3a , Cell Signaling Technology (H) , 66887.

Techniques: Recombinant, Mutagenesis, Software

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: DCM-Exos induced up-regulation of fibrotic genes in CFs (A) Representative fluorescent images of CTL-Exos or DCM-Exos (PKH26-labeled) treatment in CFs. Endocytosed Exos (orange) were observed in the cytoplasm of CFs (bar = 200 μm (left); 900 nm (right)). (B) Quantifications of Exo uptake in CFs. The uptake ratio was evaluated by the number of Exo uptaken cells (orange) divided by the total number of nuclei (blue) in the image (N = 22 pics/group), NS, nonsignificant. (C) qRT-PCR of fibrotic genes in CFs. N = 6; ∗ = p< 0.05. (D) Representative immunoblots. The CF cell lysis of different treatment groups was probed with specific antibodies (Col1a, Col3a, and CTGF). (E) Quantification of protein expression in immunoblots. Col1a, col3a, and CTGF expression were normalized to the loading control (GAPDH), N = 6. Data represented as mean ± SD; ∗ = p< 0.05; one-way ANOVA followed by Tukey post hoc test.

Article Snippet: Primary antibodies used were: mouse anti-human CD63 (1:1,000; ab59479; Abcam); anti-CD9 (1:1000; #13174 cell signaling); anti-HSP70 (1:1000, #4872 cell signaling); anti-Col1a (1:1,000; 91144; Cell Signaling Technology), anti-Col3a (PA5-27828; cell signaling #66887); anti-CTGF (1:1000, Abcam6992), anti-TNFAIP3 (1:1000, #5630, #4625 cell signaling); anti-pSMAD2 (1:1000, #3108 cell signaling); anti-GAPDH (MA1-16757) .

Techniques: Labeling, Quantitative RT-PCR, Western Blot, Lysis, Expressing

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: Intracardial injection of DCM-Exo promoted fibrosis in mouse hearts (A) Representative echocardiographic images. Images were taken respectively from PBS, CTL-Exo, or DCM-Exo injection groups on day 0 and day 14. (B) LVEF was determined by echocardiography. The injection of DCM-Exo significantly decreased LVEF on day 14 (N = 8/group; ∗ = p< 0.05). Injection of PBS, or CTL-Exo resulted in nonsignificant change between day 14 and baseline. (C) Representative pictures of picrosirius red/fast green staining in series sections of the mouse hearts. The fibrotic area (Red) was quantified by ImageJ. N = 8/group, ∗∗ = p< 0.01. (D) Representative immunoblots of mouse hearts (PBS, CTL-Exo, or DCM-Exo treatments). Fibrotic markers, Col1a, Col3a, and CTGF were detected by specific antibodies. (E) Quantification of fibrotic markers expression. DCM-Exos injection resulted in significant upregulation of fibrotic markers (N= 8/group). GAPDH was used as a protein loading control. Data represented as mean ± SD; ∗ = p< 0.05; one-way ANOVA followed by Tukey post hoc test.

Article Snippet: Primary antibodies used were: mouse anti-human CD63 (1:1,000; ab59479; Abcam); anti-CD9 (1:1000; #13174 cell signaling); anti-HSP70 (1:1000, #4872 cell signaling); anti-Col1a (1:1,000; 91144; Cell Signaling Technology), anti-Col3a (PA5-27828; cell signaling #66887); anti-CTGF (1:1000, Abcam6992), anti-TNFAIP3 (1:1000, #5630, #4625 cell signaling); anti-pSMAD2 (1:1000, #3108 cell signaling); anti-GAPDH (MA1-16757) .

Techniques: Injection, Staining, Western Blot, Expressing

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: MiR-218-5p increased Smad2 phosphorylation in CFs (A) Immunoblots of cell lysates derived from CFs treated with TGF-β, miR-218-5p, and a TGF-β inhibitor (SB525334). Specific antibodies were used to detect Col1a, Col3a, CTGF, phospo-Smad2 ( p -SMAD2), and GAPDH. (B) Quantitation of protein expression in immunoblots. GAPDH was used as a protein loading control. TGF-β inhibitor (SB525334) significantly reduced TGF-β1-induced fibrotic effect (lane 3). MiR-218-5p increased the phosphorylation of Smad2. N= 3; one-way ANONVA followed by Tukey’s post hoc test, ∗ = p< 0.05; ∗∗ = p< 0.01; ∗∗∗ = p< 0.001. Data represented as mean ± SD.

Article Snippet: Primary antibodies used were: mouse anti-human CD63 (1:1,000; ab59479; Abcam); anti-CD9 (1:1000; #13174 cell signaling); anti-HSP70 (1:1000, #4872 cell signaling); anti-Col1a (1:1,000; 91144; Cell Signaling Technology), anti-Col3a (PA5-27828; cell signaling #66887); anti-CTGF (1:1000, Abcam6992), anti-TNFAIP3 (1:1000, #5630, #4625 cell signaling); anti-pSMAD2 (1:1000, #3108 cell signaling); anti-GAPDH (MA1-16757) .

Techniques: Western Blot, Derivative Assay, Quantitation Assay, Expressing

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: MiR-218-5p targeted to TNFAIP3 (A) Targeted sequence prediction of miR-218-5p in RNA22. (B) Immunoblots (left) indicated TNFAIP3 expression was significantly repressed in CFs transfected with miR-218-5p. (C) Luciferase assay of miR-218-5p and TNFNAIP3 5′UTR region. Co-transfection of miR-218-5p + WT sequence significantly reduced luciferase activity compared with the scramble CTL + WT. One-way ANOVA followed by Tukey’s post hoc test, ∗∗ = p< 0.01; NS = nonsignificant. (D) Representative immunoblots of CFs treatment. (E) Quantitation of expression of TNFAIP3, Col1a, Col3a, and CTGF. One-way ANOVA followed by Tukey’s post hoc test; ∗∗ = p<0.01, ∗∗∗ = p< 0.001. Data represented as mean ± SD.

Article Snippet: Primary antibodies used were: mouse anti-human CD63 (1:1,000; ab59479; Abcam); anti-CD9 (1:1000; #13174 cell signaling); anti-HSP70 (1:1000, #4872 cell signaling); anti-Col1a (1:1,000; 91144; Cell Signaling Technology), anti-Col3a (PA5-27828; cell signaling #66887); anti-CTGF (1:1000, Abcam6992), anti-TNFAIP3 (1:1000, #5630, #4625 cell signaling); anti-pSMAD2 (1:1000, #3108 cell signaling); anti-GAPDH (MA1-16757) .

Techniques: Sequencing, Western Blot, Expressing, Transfection, Luciferase, Cotransfection, Activity Assay, Quantitation Assay

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet: Restoration of TNFAIP3 ameliorated the DCM-Exo-induced fibrotic effect (A) Representative immunoblots of CFs treated with either CTL-Exos or DCM-Exos. DCM-Exo treatment significantly decreased TNFAIP3 expression. Student’s t test; ∗∗ = p <0.01. (B) Representative immunoblots of TNFAIP3, Col1a, Col3a, CTGF, and GAPDH detection in CFs treated with CTL-Exos, DCM-Exos, or overexpression of TNFAIP3. (C–F) Quantitation of protein expression. TNFAIP3 overexpression significantly reduced the expression of fibrotic markers. One-ANOVA followed by Tukey’s post hoc test; N= 3; ∗∗ = p< 0.01. (G) Schematic picture showing the working model of our study. Data represented as mean ± SD.

Article Snippet: Primary antibodies used were: mouse anti-human CD63 (1:1,000; ab59479; Abcam); anti-CD9 (1:1000; #13174 cell signaling); anti-HSP70 (1:1000, #4872 cell signaling); anti-Col1a (1:1,000; 91144; Cell Signaling Technology), anti-Col3a (PA5-27828; cell signaling #66887); anti-CTGF (1:1000, Abcam6992), anti-TNFAIP3 (1:1000, #5630, #4625 cell signaling); anti-pSMAD2 (1:1000, #3108 cell signaling); anti-GAPDH (MA1-16757) .

Techniques: Western Blot, Expressing, Over Expression, Quantitation Assay

Journal: iScience

Article Title: Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway

doi: 10.1016/j.isci.2023.105963

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were: mouse anti-human CD63 (1:1,000; ab59479; Abcam); anti-CD9 (1:1000; #13174 cell signaling); anti-HSP70 (1:1000, #4872 cell signaling); anti-Col1a (1:1,000; 91144; Cell Signaling Technology), anti-Col3a (PA5-27828; cell signaling #66887); anti-CTGF (1:1000, Abcam6992), anti-TNFAIP3 (1:1000, #5630, #4625 cell signaling); anti-pSMAD2 (1:1000, #3108 cell signaling); anti-GAPDH (MA1-16757) .

Techniques: Recombinant, Mutagenesis, Software