Journal: Renal Failure

Article Title: miR-29a-3p/ Vegfa axis modulates high phosphate-induced vascular smooth muscle cell calcification

doi: 10.1080/0886022x.2025.2489712

Figure Lengend Snippet: Figure 1. Identification of Vegfa as a direct target of miR-29a-3p in VSMCs using bioinformatics and dual-luciferase assay. (A) Venn diagram depicting the overlap of miR-29a-3p target genes predicted by four miRNA target prediction tools: miRTarBase, miRWalk, TargetScan, and miRDB. The box highlights the seven genes identified as common targets across all tools. (B) Sequence alignment of miR-29a-3p with the 3′-UTR region of wild-type and mutated Vegfa mRNA. The predicted binding site and seed region are marked in red. (C) Dual-luciferase reporter assay results for VSMCs co-transfected with a control vector, a vector containing the wild type (WT) Vegfa 3′-UTR sequence, or the MUT Vegfa sequence, along with miR-29a-3p mimics or a negative control miRNA (NT). Relative luciferase activity was measured, and data are presented as mean ± SD from three independent experiments.

Article Snippet: VSMCs recovered by centrifugation were homogenized and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (China National Pharmaceutical Group Corporation Chemical reagent Co., Ltd., China; Cat. #30166428). the proteins were then transferred to polyvinylidene fluoride membranes (Millipore Co., uSa; Cat. #ISEQ00010). the blots were stained with rabbit polyclonal antibodies against VEGFa (1:10,000; Proteintech, uSa; Cat. #19003-1-aP), and mouse monoclonal antibodies against β-actin (1:10,000, Servicebio, China; Cat. #Gb15001), followed by anti-rabbit IgG (1:10,000, SIMubIotECH, China; Cat. #S2001), and anti-mouse IgG horseradish peroxidase conjugates (1:10,000, abclonal, China; Cat. #aS003).

Techniques: Luciferase, Sequencing, Binding Assay, Reporter Assay, Transfection, Control, Plasmid Preparation, Negative Control, Activity Assay

Journal: Renal Failure

Article Title: miR-29a-3p/ Vegfa axis modulates high phosphate-induced vascular smooth muscle cell calcification

doi: 10.1080/0886022x.2025.2489712

Figure Lengend Snippet: Figure 2. miR-29a-3p overexpression suppresses Vegfa expression in VSMCs. (A) Representative Western blot analysis of VEGFA protein levels in VSMCs under standard culture conditions (control), high phosphate conditions (Pi), and high phosphate conditions following miR-29a-3p overexpression (Pi + miR-29a-3p). β-actin served as a loading control. Quantitative analysis of VEGFA protein levels is shown as relative fold change normalized to β-actin. (B) Vegfa mRNA levels quantified using RT-qPCR in the same experimental groups. GAPDH was used as an internal control for normalization. Data represent the mean ± SD of five independent biological replicates.

Article Snippet: VSMCs recovered by centrifugation were homogenized and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (China National Pharmaceutical Group Corporation Chemical reagent Co., Ltd., China; Cat. #30166428). the proteins were then transferred to polyvinylidene fluoride membranes (Millipore Co., uSa; Cat. #ISEQ00010). the blots were stained with rabbit polyclonal antibodies against VEGFa (1:10,000; Proteintech, uSa; Cat. #19003-1-aP), and mouse monoclonal antibodies against β-actin (1:10,000, Servicebio, China; Cat. #Gb15001), followed by anti-rabbit IgG (1:10,000, SIMubIotECH, China; Cat. #S2001), and anti-mouse IgG horseradish peroxidase conjugates (1:10,000, abclonal, China; Cat. #aS003).

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

Journal: Renal Failure

Article Title: miR-29a-3p/ Vegfa axis modulates high phosphate-induced vascular smooth muscle cell calcification

doi: 10.1080/0886022x.2025.2489712

Figure Lengend Snippet: Figure 3. Vegfa knockdown and miR-29a-3p overexpression mitigate high phosphate-induced VSMCs calcification. (A–C) Analysis of Vegfa knockdown effects in VSMCs cultured in normal media (control) or high phosphate conditions (Pi). (A) Vegfa mRNA levels were quantified via RT-qPCR. (B) ARS with corresponding spectrophotometric quantification to assess cellular mineralization. (C) Quantification of intracellular Ca2+ content. Cells were treated with small interfering RNA targeting Vegfa (siVegfa) or a NT control. (D–F) Investigation of the role of miR-29a-3p in VSMCs cultured under high phosphate conditions. (D) Vegfa mRNA expression measured using RT-qPCR, (E) mineralization assessed using ARS and spectrophotometric quantification, and (F) intracellular Ca2+ levels were quantified. Cells were transfected with either an empty vector (VE) or a Vegfa-overexpressing construct (OE), combined with a NT miRNA mimic or a miR-29a-3p mimic. All experiments were performed in five replicates, and error bars represent the SD. GAPDH was used as an internal control in RT-qPCR experiments. Scale bar = 100 µm.

Article Snippet: VSMCs recovered by centrifugation were homogenized and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (China National Pharmaceutical Group Corporation Chemical reagent Co., Ltd., China; Cat. #30166428). the proteins were then transferred to polyvinylidene fluoride membranes (Millipore Co., uSa; Cat. #ISEQ00010). the blots were stained with rabbit polyclonal antibodies against VEGFa (1:10,000; Proteintech, uSa; Cat. #19003-1-aP), and mouse monoclonal antibodies against β-actin (1:10,000, Servicebio, China; Cat. #Gb15001), followed by anti-rabbit IgG (1:10,000, SIMubIotECH, China; Cat. #S2001), and anti-mouse IgG horseradish peroxidase conjugates (1:10,000, abclonal, China; Cat. #aS003).

Techniques: Knockdown, Over Expression, Cell Culture, Control, Quantitative RT-PCR, Small Interfering RNA, Expressing, Transfection, Plasmid Preparation, Construct

Journal: Stem Cell Research & Therapy

Article Title: MSC-exosomes pretreated by Danshensu extracts pretreating to target the hsa-miR-27a-5p and STAT3-SHANK2 to enhanced antifibrotic therapy

doi: 10.1186/s13287-025-04181-0

Figure Lengend Snippet: Primer sequences

Article Snippet: The following primary antibodies were used: Rabbit polyclonal VEGFA (19,003-1-AP; Proteintech), α-SMA (14,395-1-AP; Proteintech), HIF-1α (20,960-1-AP; Proteintech), E-cadherin (20,874-1-AP; Proteintech), STAT3 (10,253-2-AP; Proteintech), and p-STAT3 (AP0705; ABclonal).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: MSC-exosomes pretreated by Danshensu extracts pretreating to target the hsa-miR-27a-5p and STAT3-SHANK2 to enhanced antifibrotic therapy

doi: 10.1186/s13287-025-04181-0

Figure Lengend Snippet: Function and morphology of peritoneal. A Masson staining for collagen (blue); B Collagen area percentage (Masson); C HE staining for general tissue morphology; D Nuclear counting (HE); E Sirius Red staining for collagen (red); F Collagen area percentage (Sirius Red); G Peritoneal thickness; H Ultrafiltration volume; I BUN clearance rate; J Glucose clearance rate; K Fibronectin concentration; L – Q Immunohistochemical analysis; L , M α-SMA expression; N , O E-Cadherin expression; P , Q VEGFA expression. Ns: P > 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001; #: P < 0.0001

Article Snippet: The following primary antibodies were used: Rabbit polyclonal VEGFA (19,003-1-AP; Proteintech), α-SMA (14,395-1-AP; Proteintech), HIF-1α (20,960-1-AP; Proteintech), E-cadherin (20,874-1-AP; Proteintech), STAT3 (10,253-2-AP; Proteintech), and p-STAT3 (AP0705; ABclonal).

Techniques: Staining, Concentration Assay, Immunohistochemical staining, Expressing

Journal: Stem Cell Research & Therapy

Article Title: MSC-exosomes pretreated by Danshensu extracts pretreating to target the hsa-miR-27a-5p and STAT3-SHANK2 to enhanced antifibrotic therapy

doi: 10.1186/s13287-025-04181-0

Figure Lengend Snippet: The expression of STAT3, HIF-1α and VEGFA in vivo. A – F Immunohistochemical analysis of tissue sections; A STAT3 expression, C p-STAT3 expression, E HIF-1α expression; B , D , F Quantitative analysis of mean density (IOD value) for STAT3, STAT3 (S727), and HIF-1α, respectively; G – I Immunofluorescence, G DAPI (nuclei, blue), STAT3 (green), VEGFA (red), and overlay images; H DAPI (nuclei, blue), HIF-1α (green), STAT3 (red), and overlay images; I DAPI (nuclei, blue), HIF-1α (green), VEGFA (red), and overlay images. Ns: P > 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001; #: P < 0.0001

Article Snippet: The following primary antibodies were used: Rabbit polyclonal VEGFA (19,003-1-AP; Proteintech), α-SMA (14,395-1-AP; Proteintech), HIF-1α (20,960-1-AP; Proteintech), E-cadherin (20,874-1-AP; Proteintech), STAT3 (10,253-2-AP; Proteintech), and p-STAT3 (AP0705; ABclonal).

Techniques: Expressing, In Vivo, Immunohistochemical staining, Immunofluorescence

Journal: Stem Cell Research & Therapy

Article Title: MSC-exosomes pretreated by Danshensu extracts pretreating to target the hsa-miR-27a-5p and STAT3-SHANK2 to enhanced antifibrotic therapy

doi: 10.1186/s13287-025-04181-0

Figure Lengend Snippet: The expression of STAT3/HIF-1α/VEGFA in vitro. A Western blot images for STAT3 (T721 and S727), HIF-1α, VEGFA expression with different treatments: (a) Control, (b) PD, (c) PD + DSS-Exo, (d) PD + DSS-Exo + Colivelin, (e) PD + Colivelin, (f) PD + DSS-Exo + Fenbendazole-d3, (g) PD + Fenbendazole-d3, (h) PD + DSS-Exo + Colivelin + Fenbendazole-d3, (i) PD + Colivelin + Fenbendazole-d3. B – E Quantitative analysis of the relative expression levels of proteins: B STAT3 (T721), C STAT3 (S727), D HIF-1α, E VEGFA. F Western blot images for STAT3 (T721 and S727), HIF-1α, and VEGFA expression with different treatments: (a) Control, (b) PD, (c) PD + DSS-Exo, (d) PD + DSS-Exo + Stattic + LW6, (e) PD + Stattic + LW6, (f) PD + DSS-Exo + Stattic, (g) PD + Stattic, (h) PD + DSS-Exo + LW6, (i) PD + LW6. G – J Quantitative analysis of the relative expression levels of proteins: G STAT3 (T721), H STAT3 (S727), I HIF-1α, J VEGFA. ns: P > 0.05; *: P < 0.05; **: P < 0.01;***: P < 0.001; #: P < 0.0001

Article Snippet: The following primary antibodies were used: Rabbit polyclonal VEGFA (19,003-1-AP; Proteintech), α-SMA (14,395-1-AP; Proteintech), HIF-1α (20,960-1-AP; Proteintech), E-cadherin (20,874-1-AP; Proteintech), STAT3 (10,253-2-AP; Proteintech), and p-STAT3 (AP0705; ABclonal).

Techniques: Expressing, In Vitro, Western Blot, Control

Journal: Stem Cell Research & Therapy

Article Title: MSC-exosomes pretreated by Danshensu extracts pretreating to target the hsa-miR-27a-5p and STAT3-SHANK2 to enhanced antifibrotic therapy

doi: 10.1186/s13287-025-04181-0

Figure Lengend Snippet: miRNA-sequencing analysis and network analysis. A Annotation of Rfam; B Gene Venn diagram of the differential miRNA; C Cluster analysis of the differential miRNA; D Volcano plot of RNA expression differences; E Correlation analysis of RNA expression; F KEGG pathway enrichment analysis; G hsa-miR-27a-5p_R-1-SHANK2 network; H hsa-miR-27a-5p_R-1-target gene interaction network; I hsa-miR-27a-5p_R-1-STAT3 network; J miRNA-gene-protein network; K GO enrichment analysis; L Western blot results of targets and EMT-related proteins, STAT3, p-STAT3, SHANK2, FN, α-SMA, VEGFA, E-cadherin; M Quantification of protein expression from panel L. P < 0.05, P < 0.01, P < 0.001, P < 0.0001

Article Snippet: The following primary antibodies were used: Rabbit polyclonal VEGFA (19,003-1-AP; Proteintech), α-SMA (14,395-1-AP; Proteintech), HIF-1α (20,960-1-AP; Proteintech), E-cadherin (20,874-1-AP; Proteintech), STAT3 (10,253-2-AP; Proteintech), and p-STAT3 (AP0705; ABclonal).

Techniques: Sequencing, RNA Expression, Western Blot, Expressing

Journal: Stem Cell Research & Therapy

Article Title: MSC-exosomes pretreated by Danshensu extracts pretreating to target the hsa-miR-27a-5p and STAT3-SHANK2 to enhanced antifibrotic therapy

doi: 10.1186/s13287-025-04181-0

Figure Lengend Snippet: Mechanism diagram. Annotation: Specific miRNAs upregulated or downregulated by DSS-Exo include hsa-miR-126-3p_R-1, hsa-miR-223-3p, hsa-miR-122-5p_R-1, among others, which modulate genes related to fibrosis such as α-SMA, CTGF, E-cadherin, Fibronectin, VEGFA, and HIF-1α. DSS-Exo modulate the STAT3 transcription factor, which binds to the SHANK2 promoter, influencing gene transcription

Article Snippet: The following primary antibodies were used: Rabbit polyclonal VEGFA (19,003-1-AP; Proteintech), α-SMA (14,395-1-AP; Proteintech), HIF-1α (20,960-1-AP; Proteintech), E-cadherin (20,874-1-AP; Proteintech), STAT3 (10,253-2-AP; Proteintech), and p-STAT3 (AP0705; ABclonal).

Techniques:

Journal: Animals : an Open Access Journal from MDPI

Article Title: Melatonin Regulates the Expression of VEGF and HOXA10 in Bovine Endometrial Epithelial Cells through the SIRT1/PI3K/AKT Pathway

doi: 10.3390/ani14192771

Figure Lengend Snippet: The antibody used in the present study.

Article Snippet: VEGFA rabbit Polyclonal antibody , 1:1000 , Proteintech, Wuhan, China , 19003-1-AP.

Techniques:

Journal: Animals : an Open Access Journal from MDPI

Article Title: Melatonin Regulates the Expression of VEGF and HOXA10 in Bovine Endometrial Epithelial Cells through the SIRT1/PI3K/AKT Pathway

doi: 10.3390/ani14192771

Figure Lengend Snippet: The antibody used in immunofluorescence assay.

Article Snippet: VEGFA rabbit Polyclonal antibody , 1:1000 , Proteintech, Wuhan, China , 19003-1-AP.

Techniques: Immunofluorescence