Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: ADAMTS1 levels were increased in the serum of patients with myocardial fibrosis, in the hearts of mice after CFPMI and in cardiac fibroblasts stimulated with fibrotic factors. A . ADAMTS1 levels in clinical serum samples were evaluated via ELISA. N = 30. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. B . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. C . Survival rate of mice in each group. D . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. E and F . ADAMTS1 expression in mouse heart tissue was determined through Western blot and IHC. Human cardiac fibroblasts were treated with 10 ng/mL TGF-β1 or 0.1 μM Ang II for 12, 24, or 48 h to construct in vitro models. The cells were divided into four groups: the Control, TGF-β1/Ang II (12 h), TGF-β1/Ang II (24 h), and TGF-β1/Ang II (48 h) groups. G and H . Western blot detection of the expression of ADAMTS1, Collagen I and FN. I. The expression levels of ADAMTS1, Collagen I and FN were measured via qRT‒PCR. After the optimal time point (48 h) was determined, the mice were further divided into the control, TGF-β1 and Ang II groups. J . ADAMTS1 expression in cells was assessed by IF. Human cardiac fibroblasts were treated with 10 ng/mL TGF-β1 or 0.1 μM Ang II for 15, 30, 60, 90, and 120 min. K. qRT-PCR detection of the mRNA expression of ADAMTS1, Collagen I and FN. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Construct, Staining, Expressing, Western Blot, In Vitro, Control, Quantitative RT-PCR

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: SMAD2 regulated ADAMTS1 expression in human and mouse cardiac fibroblasts induced by TGF-β1. We used 10 ng/mL TGF-β1 to induce human and mouse cardiac fibroblasts for 15, 30, 60, 90, and 120 min. A and B . Western blot analysis of the expression of SMAD2, p-SMAD2 and ADAMTS1. Human and mouse cardiac fibroblasts were treated with 10 ng/mL TGF-β1 for 48 h, and the cells were divided into Control and TGF-β1 groups. C . Human and mouse cardiac fibroblasts were induced with 10 ng/mL TGF-β1 for 12 h, 24 h, 48 h, and 72 h. qRT‒PCR was performed to detect the mRNA levels of ADAMTS1, Collagen I, and FN. D . IF staining was conducted to determine the expression of p-SMAD2 and ADAMTS1 at 48 h. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. E . p-SMAD2 expression in mouse heart tissue was measured by IHC. F . ChIP verification of the interaction between SMAD2 and ADAMTS1. Then, we interfered with SMAD2 expression. The groups were the si-NC, si-SMAD2-1, and si-SMAD2-2 groups. G . SMAD2 expression was assessed via qRT‒PCR. After the best si-SMAD2 was selected, the cells were further divided into the si-NC, TGF-β1 (48 h), si-SMAD2, and TGF-β1 + si-SMAD2 groups. H . Western blot detection of ADAMTS1, Collagen I, and FN expression in cells. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Expressing, Western Blot, Control, Staining, Construct

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: Overexpression of ADAMTS1 enhanced the production of collagen fiber proteins in human and mouse cardiac fibroblasts induced by TGF-β1. We overexpressed ADAMTS1 and divided the cells into the oe-NC, TGF-β1 (48 h), oe-ADAMTS1, and TGF-β1 + oe-ADAMTS1 groups. A . Western blot detection of ADAMTS1, Collagen I, FN, and α-SMA expression. B-D . IF staining of Collagen I and FN expression. Additionally, we interfered with ADAMTS1; the cells were grouped as follows: si-NC, si-ADAMTS1-1, and si-ADAMTS1-2. E . ADAMTS1 expression was determined through qRT‒PCR. After the best si-ADAMTS1 was selected, the cells were further divided into the si-NC, TGF-β1 (48 h), si-ADAMTS1, and TGF-β1 + si-ADAMTS1 groups. F and G . Western blot detection of ADAMTS1, Collagen I, FN, and α-SMA expression. H and I . IF staining of Collagen I and FN expression. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Over Expression, Western Blot, Expressing, Staining

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: Knockdown of ADAMTS1 alleviated TGF-β1-induced fibrosis by downregulating HDAC6 protein expression. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. A . HDAC6 expression in mouse heart tissue was determined through IHC. After the best si-ADAMTS1 was selected, the cells were further divided into the si-NC and si-ADAMTS1 groups. B . ADAMTS1 and HDAC6 expression was assessed via qRT‒PCR. C . Western blot analysis of ADAMTS1 and HDAC6 expression. Furthermore, we overexpressed ADAMTS1 and divided the cells into the oe-NC, TGF-β1, oe-ADAMTS1, and TGF-β1 + oe-ADAMTS1 groups. D . Western blot detection of HDAC6 expression. After the si-ADAMTS1 group was selected, the cells were further divided into the si-NC, TGF-β1, si-ADAMTS1, and TGF-β1 + si-ADAMTS1 groups. E. Western blot detection of HDAC6 expression. Moreover, cells were treated with the HDAC6 inhibitor ACY1215 (5 μM) for 48 h and then divided into the si-NC, si-ADAMTS1, si-NC + ACY1215, and si-ADAMTS1 + ACY1215 groups. F. Western blot detection of TGF-β1, Collagen I, FN, and ADAMTS1 expression. Subsequently, experiments were performed in human and mouse cardiac fibroblasts with ADAMTS1 overexpression followed by treatment with the HDAC6 inhibitor ACY1215. G. Western blot analysis was conducted to detect the expression levels of ADAMTS1 and HDAC6. H. Western blot analysis was performed to determine the expression levels of TGF-β1, Collagen I, and FN. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Knockdown, Expressing, Construct, Western Blot, Over Expression

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: ADAMTS1 interacted with HDAC6 during fibrosis. A . Co-IP verification of the interaction of ADAMTS1 with HDAC6, with ADAMTS1 as the bait protein. In the experiment, we first captured the ADAMTS1 protein using a specific antibody and then used Co-IP technology to detect its interaction with HDAC6. B . Co-IP verification of the interaction of HDAC6 with ADAMTS1, with HDAC6 as the bait protein. In the experiment, we captured the HDAC6 protein using a specific antibody and then used Co-IP technology to detect its interaction with ADAMTS1. We used 10 ng/mL TGF-β1 to induce human and mouse cardiac fibroblasts for 48 h, and the cells were further divided into control and TGF-β1 groups. C . IF staining was performed to evaluate the colocalization of ADAMTS1 and HDAC6 in TGF-β1-treated human and mouse cardiac fibroblasts. D . Changes in the level of ubiquitinated HDAC6 protein in human and mouse cardiac fibroblasts transfected with oe-ADAMTS1/si-ADAMTS1 or oe-NC/si-NC in the presence of 10 μM MG132. E. In si-ADAMTS1-treated human and mouse cardiac fibroblasts, MG132 (10 μM) was added to detect the ubiquitination levels of ADAMTS1 protein. F. Human and mouse cardiac fibroblasts were treated with TGF-β1 and subjected to SMAD2 knockdown to determine the ubiquitination levels of ADAMTS1 protein. N = 3. *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Co-Immunoprecipitation Assay, Control, Staining, Transfection, Ubiquitin Proteomics, Knockdown

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: AAV-shRNA-ADAMTS1 treatment alleviated myocardial fibrosis and improved cardiac function after CFPMI. The mice were randomly divided into the Sham, CFPMI, and CFPMI + sh-ADAMTS1 groups. A . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. B . Survival rate of mice in each group. C . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. D . HDAC6 expression was determined through qRT‒PCR. E. Western blot analysis of the expression of TGF-β1, SMAD2, and p-SMAD2 in mouse heart tissue. F. Western blot detection of Collagen I, and FN expression. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: shRNA, Staining, Expressing, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: AAV-shRNA-HDAC6 transfection combined with ADAMTS1 inhibitor treatment alleviated myocardial fibrosis and improved cardiac function after CFPMI. The mice were randomly divided into the Sham, CFPMI, CFPMI + anti-ADAMTS1, CFPMI + sh-HDAC6, and CFPMI + anti-ADAMTS1 + sh-HDAC6 groups. A . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. B . Survival rate of mice in each group. C and D . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. E. HDAC6 expression was determined through qRT‒PCR. F. Western blot analysis of the expression of TGF-β1, SMAD2, and p-SMAD2 in mouse heart tissue. G. Western blot detection of Collagen I and FN expression. N = 3. ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: shRNA, Transfection, Staining, Expressing, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: TGF-β1/SMAD2 regulated ADAMTS1 by mediating CFPMI through HDAC6 ubiquitination

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Effect of RBE-Exos on the adhesion of NK-92 cells. ( A ) Growth status of NK-92 cells. NK cells treated with RBE-Exos lost the normal cluster growing. ( B ) CD11a protein levels. ( C ) CD18 protein levels. ( D ) CD54 protein levels. Western blot showed that the protein levels of CD11a, CD18 and, CD54 were obviously decreased in NK-92 cells treated with RBE-Exos. ( E ) Relative transcriptional levels of CD11a , CD18 , CD54 and, CD2 in NK-92 cells. qRT-PCR displayed that RBE-Exos downregulated the transcriptional levels of CD11a , CD18 , CD54 , but not CD2 . Data were presented as mean ± SEM for three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The primary antibodies were as follows: rabbit anti-human CD63, BCL-2, BCL-XL, CD11a, β-actin (Abcam, USA), mouse anti-human HSP70 (Santa-Cruz, USA), rabbit anti-human CD18 (Proteintech, China), mouse anti-human CD54 (Proteintech, China).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Cytotoxicity and expression of adhesion molecules in primary NK cells. ( A ) Cytotoxicity of primary NK cells from CCA patients and healthy people against target cells. LDH detection displayed the cytotoxicity of NK cells from CCA patients was significantly lower than that of NK cells healthy people. Data are presented as the mean ± SEM from n = 3 independent experiments. ( B ) Protein levels of CD18 in primary NK cells from CCA patients and healthy people. Data are presented as the mean ± SEM from n = 3 independent experiments. ( C ) Protein levels of CD54 in primary NK cells from CCA patients and healthy people. Data are presented as the mean ± SEM from n = 3 independent experiments. ** P < 0.01.

Article Snippet: The primary antibodies were as follows: rabbit anti-human CD63, BCL-2, BCL-XL, CD11a, β-actin (Abcam, USA), mouse anti-human HSP70 (Santa-Cruz, USA), rabbit anti-human CD18 (Proteintech, China), mouse anti-human CD54 (Proteintech, China).

Techniques: Expressing

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) HUVECs were transfected with either control mimic (con) or miR-30b mimic (30b) and levels of TGFβ1 and TGFβ2 mRNA were assessed by qRT-PCR. Expression levels relative to control mimic transfected cells and normalized to β-actin expression are presented as the mean ± SEM (n = 2). Overexpression of miR-30b significantly increases TGFβ2 expression. * P < 0.05, ** P < 0.01 as determined by unpaired Student’s t -test. (B) Cells were transfected with 20 nM of either control mimic (control) or miR-30b mimic (miR-30b) and protein lysates were collected after 48 hours for assessment of TGFβ2 protein levels by western blot. β-actin was used as endogenous control. (C) ELISAs for TGFβ1 and TGFβ2 were performed with 24 hour conditioned supernates from HUVECs transfected with 20 nM of either control or miR-30b mimic. Data represents the mean ± SEM (n = 2). Overexpression of miR-30b significantly increases TGFβ2 secretion into cell culture supernate. * P = 0.044 as determined by unpaired Student’s t -test. (D) HUVECs were transfected with 20 nM of either control mimic (control) or miR-30b mimic (miR-30b) and protein lysates were collected after 48 hours for assessment of Smad2 phosphorylation by western blot.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Control, Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Cell Culture, Phospho-proteomics

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) JDP2 mRNA expression was assessed in HUVECs transfected with miR-30b mimic (20 nM) as compared to control by qRT-PCR. Data represents the mean ± SEM (n = 3) normalized to β-actin as endogenous control. * P = 0.016 as determined by unpaired Student’s t -test. (B) HUVEC were transfected with 50 nM of either control siRNA or ATF2 siRNA 1 or 2 and RNA was isolated at 48 hours post transfection. Levels of ATF2 and TGFβ2 mRNA were assessed by qRT-PCR with β-actin as endogenous control. Data presented is mean ± SEM (n = 2). Statistically significant decreases in ATF2 and TGFβ2 expression were seen in ATF2 siRNA treated cells as compared to control siRNA treated cells. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by unpaired Student’s t -tests for each ATF2 siRNA compared to control siRNA. (C) Cells transfected with 5 nM of either control siRNA or ATF2 siRNA 1 were seeded onto growth factor reduced BME and the formation of capillary-like cord structures and number of loops was assessed after 24 hours. (D) A statistically significant increase in cord formation was observed in cells depleted of ATF2 through siRNA. Data represents the mean ± SEM (n = 2). * P = 0.041 as determined by unpaired Student’s t -test. (E) HUVECs were co-transfected with miRNA mimic (20 nM) and ATF2 siRNA 1 or 2 (50 nM) in the combinations displayed and cell lysates were collected at 48 hours post transfection and assessed for TGFβ2 mRNA expression. Data presented is mean ± SEM (n = 2). * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by ANOVA with post hoc analysis. (F) Cells were transfected as in (E) using miRNA mimic (20 nM) and ATF2 siRNA 1 (5 nM) and serum starved overnight in MCDB 131 with 0.5% FBS prior to protein expression analysis by western blot. Data is representative of expression levels observed in two independently performed experiments.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

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

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) HUVECs were serum starved overnight in MCDB 131 with 0.5% FBS and stimulated with VEGF (50 ng/ml) in the presence or absence of Avastin (1 μg/ml) for 24 hours. Data represents the mean ± SEM (n = 2) for expression of TGFβ1 and TGFβ2 assessed by qRT-PCR relative to β-actin endogenous control. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by ANOVA. (B) HUVECs were treated with 5 ng/ml of TGFβ2 for 3 days prior to seeding onto growth factor reduced BME for assessment of capillary-like cord formation after 24 hours. (C) A significant decrease in cord formation is observed in the TGFβ2 treated group. Data represents the mean ± SEM (n = 2). ** P = 0.0072 as determined by unpaired Student’s t -test. (D) HUVECs transfected with 1 nM control or miR-30b mimic were treated 4 hours post transfection with 0.8 μg/ml anti-TGFβ2 neutralizing antibody or rabbit IgG. Media was refreshed after 24 hours, again with rabbit IgG or anti-TGFβ2 antibody and cells were seeded onto growth factor reduced BME 24 hours later (ie. 48 hours post transfection) in media containing rabbit IgG or anti-TGFβ2 antibody. (E) Data represents the mean ± SEM (n = 3) of the number of capillary-like cord structures or number of loops formed after 24 hours on BME. * P < 0.05, ns denotes not significant as determined by ANOVA with post hoc analysis.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection