Journal: The Journal of investigative dermatology

Article Title: Increased expression of Wnt2 and SFRP4 in Tsk mouse skin: role of Wnt signaling in altered dermal fibrillin deposition and systemic sclerosis.

doi: 10.1038/sj.jid.5701101

Figure Lengend Snippet: Figure 5. Wnt3a stimulates Fbn-1 matrix formation. (a) MEFs were cultured for 10 days in the absence or presence of 100 ng/ml Wnt3a. Fbn-1 and fibronectin matrix (red) were analyzed by immunofluorescence. Nuclei were stained with Hoechst (blue). Bar ¼ 10 mm. The graph shows quantification of fibrils of the matrix by densitometric analysis of the fluorescent staining. (b) MEFs cultured for 16 hours in the absence or presence of 100 ng ml1 Wnt3a. Fbn-1 mRNA was analyzed by northern blot (left panels), and Fbn-1 and fibronectin protein levels in media by western blot (right panels). (c) Human dermal fibroblasts from normal or SSc skin were left untreated (left panel) or were treated with Wnt3a (right panel) for 10 days on chamber slides and then stained for Fbn-1 or fibronectin by immunofluorescence. Results are representative of three independent experiments performed on normal cells and SSc cells. (d) MEF-TE cells overexpressing MAGP-2 and tropoelastin-enhanced green fluorescent protein were cultured as in (a). Fbn-1 (red), MAGP-2 (red), elastic fibers (green), and fibronectin (red) were analyzed in parallel by immunofluorescence and enhanced green fluorescent protein fluorescence. Bar ¼ 10 mm. (e) MEFs were serum-starved for 3 hours and then treated for 1 hour with TGF-b1 (5 ng/ml) or Wnt3a (100 ng/ml). Total lysates were analyzed by western blot for phospho-Smad3 as described in Materials and Methods.

Article Snippet: Total lysates and secreted proteins were resolved on an SDS-PAGE gel, transferred to polyvinylidine difluoride membranes (Immobilon-P; Millipore) using standard methods, and then stained with Ponceau S. Membranes were then blocked in 5% milk or 5% BSA 0.05% Tween- trisbuffered saline for 1 hour, incubated overnight at 41C with monoclonal mouse anti-SFRP4 antibody (Berndt et al., 2003), polyclonal rabbit anti-Fbn-1 (Ab9543, gift from L. Sakai), anti- bovine fibronectin (Calbiochem, San Diego, CA), anti-phospho Smad3 (Rockland, Gilbertsville, PA), or anti-Smad2/3 (Upstate, Billerica, MA) and then with a horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma, St. Louis, MO) or donkey anti- rabbit IgG (The Jackson Laboratory) for 1 hour.

Techniques: Cell Culture, Immunofluorescence, Staining, Northern Blot, Western Blot, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity

doi: 10.1016/j.jbc.2021.100545

Figure Lengend Snippet: Figure 1. EMT-associated cellular responses that are dependent on Smad3. A, SMAD3 knockout A549 cells (A549-S3KO) were prepared using CRISPR/ Cas9-mediated genome editing. A549-S3KO cells were infected with lentivirus carrying SMAD3 cDNA. Expression and TGF-β-induced phosphorylation of Smad3 were verified by immunoblotting with the indicated antibodies; α-tubulin was used as a loading control. B–E, A549 cells, A549-S3KO cells, or those expressing wild-type Smad3 were incubated in either the presence or absence of 1 ng/ml TGF-β1 for 24 h. B, light microscopic photographs. C, expression of E-cadherin was determined by immunoblotting with anti-E-cadherin; α-tubulin was used as a loading control. D, immunofluorescence detection of E-cadherin. E, formation of actin stress fibers in A549-S3KO cells expressing wild-type Smad3. F-actin was stained using Rhodamine-phalloidin. F, chamber migration assay or (G) wound healing assay of A549 cells, A549-S3KO cells, or those expressing wild-type Smad3 in either the presence or absence of 1 ng/ ml TGF-β1. Quantification is shown in the right. H, to evaluate growth rates of A549 cells, A549-S3KO cells, or those expressing wild-type Smad3 in either the presence or absence of 1 ng/ml TGF-β1, cell numbers were counted. TGF-β was added at day1. Scale bars: 10 μm (B and D–F) and 200 μm (G). Error bars represent SD (n = 5 for F, G). p values were determined by Student’s t-test. *p < 0.01. One representative result from two independent experiments is shown (F–H).

Article Snippet: PANC-1 cells were cloned and used as a parental cell line for SMAD3 knockout using Double Nickase plasmid (catalog no. sc-4000069-NIC-2; Santa Cruz).

Techniques: Knock-Out, CRISPR, Infection, Expressing, Phospho-proteomics, Western Blot, Control, Incubation, Staining, Migration, Wound Healing Assay

Journal: Journal of Biological Chemistry

Article Title: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity

doi: 10.1016/j.jbc.2021.100545

Figure Lengend Snippet: Figure 2. Alignment of Smad1, Smad2, Smad3, and Smad5 protein sequences. Diverged residues are shown in bold. Arg-104 and Ala-105 in Smad3 are shown in red. Smad1-derived regions in Smad1/3 chimeras are shown in line.

Article Snippet: PANC-1 cells were cloned and used as a parental cell line for SMAD3 knockout using Double Nickase plasmid (catalog no. sc-4000069-NIC-2; Santa Cruz).

Techniques: Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity

doi: 10.1016/j.jbc.2021.100545

Figure Lengend Snippet: Figure 3. Signaling of TGF-β-induced cell motility is transmitted through a distinct region of Smad3 from other EMT-associated responses. A, schematic presentation of N1133 and N3311 chimeric proteins. N1133 (Smad3 with Met-1 to Val-65 from Smad1); N3311 (Smad3 with Gln-98 to Ser-144 from Smad1). B, A549-S3KO cells were infected with lentivirus carrying cDNA expressing either the N1133 or N3311 chimera. Expression and TGF-β-induced phosphorylation of N1133 and N3311 were verified by immunoblotting with indicated antibodies; α-tubulin was used as a loading control. C, CAGA-Luc assay of A549-S3KO cells expressing N1133 or N3311. Luciferase assay was carried out using (CAGA)12-MLP-Luc as a reporter plasmid. Error bars repre- sent SD (n = 3). D, light microscopic images, (E) expression of E-cadherin detected by immunoblotting, (F) formation of actin stress fibers visualized by Rhodamine-phalloidin staining, (G) immunofluorescence labeling of E-cadherin, and (H) chamber migration assay in A549-S3KO cells expressing wild-type Smad3, N1133, or N3311 in either the presence or absence of 1 ng/ml TGF-β1. Scale bars: 10 μm. Error bars represent SD (n = 5). p values were determined by Student’s t-test. *p < 0.01. I, DNA affinity precipitation assay (DNAP) was performed using biotinylated 3xCAGA as a probe. Total cell lysates (input) and proteins in cell lysates bound to the probe were analyzed by immunoblotting with indicated antibodies; α-tubulin was used as a loading control. One representative result from two independent experiments is shown (C, H, I).

Article Snippet: PANC-1 cells were cloned and used as a parental cell line for SMAD3 knockout using Double Nickase plasmid (catalog no. sc-4000069-NIC-2; Santa Cruz).

Techniques: Infection, Expressing, Phospho-proteomics, Western Blot, Control, Luciferase, Plasmid Preparation, Staining, Labeling, Migration

Journal: Journal of Biological Chemistry

Article Title: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity

doi: 10.1016/j.jbc.2021.100545

Figure Lengend Snippet: Figure 4. The β4 region of Smad3 is indispensable for TGF-β-enhanced cell motility. A, schematic presentation of Smad1/3 chimeric proteins. N3133 (Smad3 with Lys-45 to Val-65 replacement from Smad1); N3331a (Smad3 with Lys-104 to Ser-144 replacement from Smad1); N3331b (Smad3 with Cys-109 to Ser-144 replacement from Smad1). B, A549-S3KO cells were infected with lentivirus carrying cDNA encoding the N3133, N3331a, or N3331b chimera. Expression and TGF-β-induced phosphorylation of N3133, N3331a, and N3331b were verified by immunoblotting with indicated antibodies; α-tubulin was used as a loading control. C, CAGA-Luc assay, (D) light microscopic images, (E) expression of E-cadherin detected by immunoblotting, (F) formation of actin stress fibers visualized by Rhodamine-phalloidin staining, (G) immunofluorescence labeling of E-cadherin, and (H) chamber migration assay in A549-S3KO cells expressing wild-type Smad3, N3133, N3331a, or N3331b in either the presence or absence of 1 ng/ml TGF-β1. Scale bars: 10 μm. Error bars represent SD (n = 3 for C and n = 5 for H). p values were determined by Student’s t-test. *p < 0.01. One representative result from two independent experiments is shown (C, H).

Article Snippet: PANC-1 cells were cloned and used as a parental cell line for SMAD3 knockout using Double Nickase plasmid (catalog no. sc-4000069-NIC-2; Santa Cruz).

Techniques: Infection, Expressing, Phospho-proteomics, Western Blot, Control, Staining, Labeling, Migration

Journal: Journal of Biological Chemistry

Article Title: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity

doi: 10.1016/j.jbc.2021.100545

Figure Lengend Snippet: Figure 5. Smad3(RA/KP) fails to transmit signals for cell motility. A549-S3KO cells were infected with lentivirus carrying cDNA encoding either Smad3 with an Arg104Lys substitution (R/K) or the Smad3 Arg104Lys/Ala105Pro double mutant (RA/KP). A, expression and TGF-β-induced phosphorylation of Smad3(R/K) and Smad3(RA/KP) were verified by immunoblotting with indicated antibodies; α-tubulin was used as a loading control. B, CAGA-Luc assay, (C) chamber migration assay, and (D) wound healing assay in A549-S3KO cells expressing wild-type Smad3, Smad3(R/K), or Smad3(RA/KP) in either the presence or absence of 1 ng/ml TGF-β1. Scale bars: 10 μm (C), 200 μm (D). Error bars represent SD (n = 3 for B and n = 5 for C, D). p values were determined by Student’s t-test. *p < 0.01. **p < 0.05. E, molecular model of the β4 region in the 3D structure of Smad3’s MH1 domain bound to DNA (PDB ID 1OZJ). Arg- 104 and Ala-105 are shown in yellow and red, respectively. The DNA-binding β-hairpin containing strands β2 and β3 is shown in cyan. Image of 3D structure was generated with PyMOL (http://pymol.org). One representative result from two independent experiments is shown (B–D).

Article Snippet: PANC-1 cells were cloned and used as a parental cell line for SMAD3 knockout using Double Nickase plasmid (catalog no. sc-4000069-NIC-2; Santa Cruz).

Techniques: Infection, Mutagenesis, Expressing, Phospho-proteomics, Western Blot, Control, Migration, Wound Healing Assay, Binding Assay, Generated

Journal: Journal of Biological Chemistry

Article Title: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity

doi: 10.1016/j.jbc.2021.100545

Figure Lengend Snippet: Figure 6. Activation of Rac1 during TGF-β–induced motility. A, time course showing Rac1 activation after TGF-β stimulation. A549 cells were stimulated with 1 ng/ml TGF-β1 for 1–12 h. The amount of active, GTP-loaded Rac1 was determined by using a GST pull-down assay. Rac1 was detected by immunoblotting. B, Rac1 activation assay using A549, A549-S3KO cells, or those expressing Smad3 or Smad3(RA/KP). Cells were stimulated with 1 ng/ml TGF-β1 for 12 h before harvesting. Cell lysates were subsequently prepared and subjected to a Rac1 activation assay as in A. C, chamber migration assay using A549 cells pretreated with either a TβRI kinase inhibitor SB431542 (5 μM), a PI3K inhibitor LY294002 (10 μM), or 0.1% DMSO (vehicle-only control) for 1 h. Scale bars: 10 μm. Error bars represent SD (n = 5). p values were determined by Student’s t-test. *p < 0.01. D, Rac1 activation assay using A549 cells pretreated with LY294002, SB431542, or 0.1% DMSO, followed by stimulation with 1 ng/ml TGF-β1 for 12 h. Cell lysates were subsequently prepared and subjected to a Rac1 activation assay as in (A). E, TGF-β-induced phosphorylation of Akt (S473) in A549-S3KO cells expressing Smad3(RA/KP). Cells were pretreated with either 10 μM LY294002 or 0.1% DMSO for 1 h and stimulated with 1 ng/ml TGF-β1 for either 1 h or 4 h. Cell lysates were analyzed by immunoblotting using phospho-Akt or phospho-Smad2 antibodies; α-tubulin was used as a loading control. F, effects of a ROCK inhibitor Y27632 on TGF-β- induced Rac1 activation in A549-S3KO expressing either Smad3 or Smad3(RA/KP). Cells were treated with TGF-β (1 ng/ml) and/or Y27632 (25 μM) for 12 h on collagen-coated plate. Cell lysates were subsequently prepared and subjected to a Rac1 activation assay as in (A). One representative result from two independent experiments is shown (B–F).

Article Snippet: PANC-1 cells were cloned and used as a parental cell line for SMAD3 knockout using Double Nickase plasmid (catalog no. sc-4000069-NIC-2; Santa Cruz).

Techniques: Activation Assay, Pull Down Assay, Western Blot, Expressing, Migration, Control, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity

doi: 10.1016/j.jbc.2021.100545

Figure Lengend Snippet: Figure 7. Multiple ARHGAPs are downregulated during sustained Rac1 activation. A, TGF-β-induced downregulation of ARHGAPs. A549 cells, A549- S3KO cells, or A549-S3KO cells expressing either wild-type Smad3 or Smad3(RA/KP) were stimulated with 1 ng/ml TGF-β1 for 2–24 h. Total RNAs were extracted and analyzed by quantitative real-time PCR to determine endogenous ARHGAP22, ARHGAP24, ARHGAP27, and SERPINE1 levels; GAPDH was used for normalization. B, effect of a protein synthesis inhibitor on downregulation of ARHGAPs by TGF-β. A549 cells pretreated with either 5 μM cycloheximide (CHX) or 0.1% DMSO for 1 h were stimulated with 1 ng/ml TGF-β1 for 6 h. ARHGAP22, ARHGAP24, ARHGAP27, or SMAD7 mRNA expression was measured by quantitative real-time PCR. SMAD7 is a direct target gene of TGF-β that is not affected by CHX. C, knockdown of ARHGAP24 in A549 cells. Cells were treated with control siRNA (siControl) or ARHGAP24 siRNA for 24 h. The expression level of mRNA was measured by quantitative real-time PCR. D, effect of ARHGAP24 knockdown on TGF-β-induced cell motility in A549-S3KO cells expressing Smad3(RA/KP). Scale bars: 10 μm. Error bars represent SD (n = 3 for A–C or n = 5 for D). p values were determined by Student’s t-test. *p < 0.01. One representative result from three (A) or two (B–D) independent experiments is shown.

Article Snippet: PANC-1 cells were cloned and used as a parental cell line for SMAD3 knockout using Double Nickase plasmid (catalog no. sc-4000069-NIC-2; Santa Cruz).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Knockdown, Control

Journal: Molecular Medicine Reports

Article Title: Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats

doi: 10.3892/mmr.2026.13815

Figure Lengend Snippet: (A) Masson's trichrome staining and immunohistochemistry results. Masson's trichrome staining detected changes in collagen fibers in urethral tissues, with collagen fibers appearing blue and muscle fibers, fibrin and red blood cells appearing red. Immunohistochemistry staining showed the cell nuclei in blue and positive protein staining for the fibrosis markers α-SMA, TGF-β1, collagen I, collagen III and Smad3 in brown. Images are presented at a magnification of ×200. (B) Statistical analysis of Masson's trichrome staining and immunohistochemistry results. Staining results were analyzed with Image-Pro Plus software(version 6.0; Media Cybernetics, Inc.), followed by bar chart construction using GraphPad Prism. *P<0.05, **P<0.01 and ***P<0.001. (C) Western blot analysis results. Western blot analysis was used to detect the protein expression of fibrosis markers in urethral tissue. (D) Statistical analysis of western blotting results. Band densities were analyzed using Image-Pro Plus software(version 6.0; Media Cybernetics, Inc.), and bar charts were drawn with GraphPad Prism. *P<0.05, **P<0.01 and ***P<0.001. α-SMA, α-smooth muscle actin; TDN, tetrahedral DNA nanostructure.

Article Snippet: The membranes were blocked with 5% BSA for 1 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: α-SMA (1:1,000; cat. no. AF1032; Affinity Biosciences), TGF-β1 (1:5,000; cat. no. 21898-1-AP; Proteintech Group, Inc.), collagen I (1:1,000; cat. no. AF7001; Affinity Biosciences), collagen III (1:1,000; cat. no. AF5457; Affinity Biosciences) and Smad3 (1:10,000; cat. no. 66516-1-Ig; Proteintech Group, Inc.), and β-actin (1:25,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.), which was used as a loading control.

Techniques: Staining, Immunohistochemistry, Software, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Validation of the differential expression of miR-130a-3p in the livers of mice and patients. (a ) miR-130a-3p expression was decreased, and the expression of TGFBR1, TGFBR2, Col-1, and Col-4 was increased in the liver tissues of the MCD-fed mice. TGFBR1, TGFBR2, Col-1, and Col-4 expression was detected by IHC ( × 200 magnification), and miR-130a-3p expression was detected using an ISH ( × 400 magnification) assay. Positive staining is indicated by a brown color. (b) Effects of the MCD diet on serum ALT and AST levels. Values represent the mean±S.D., *** P <0.001 compared with control. (c) Hepatic mRNA and (d) protein expression of TGFBR1, TGFBR2, Smad2, Smad3, Col-1, and Col-4 were upregulated in the MCD-fed mice compared with the control mice. β -actin was used as a loading control. Values represent the mean±S.D., * P <0.05, ** P <0.01, *** P <0.001 compared with control. (e) Histopathological changes were evaluated with hematoxylin and eosin staining, and Masson’s trichrome staining. miR-130a-3p expression was decreased, and TGFBR1, TGFBR2, Col-1, and Col-4 levels were increased in the liver tissues of patients with NASH-related liver fibrosis. The expression of miR-130a-3p was detected by ISH ( × 400 magnification), and TGFBR1, TGFBR2, Col-1, and Col-4 were detected using an IHC ( × 200 magnification) assay. Positive staining is indicated by a brown color

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Biomarker Discovery, Quantitative Proteomics, Expressing, Immunohistochemistry, In Situ Hybridization, Staining, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: miR-130a-3p expression was downregulated and regulated the downstream expression of genes of the TGF-β/SMAD signaling pathway in activated HSCs. ( a ) miR-130a-3p expression was examined by real-time qRT-PCR during HSC activation. Values represent the mean±S.D., (* P <0.05, ** P <0.01). ( b ) The hepatic mRNA and ( c ) protein expression of TGF- β 1 , α -SMA, Smad2, and Smad3 were increased during the process of HSC activation. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). ( d ) HSC-T6 cells were transfected with miR-130a-3p mimics or mimics control for 48 h. The mRNA and protein ( e ) levels of TGF- β 1 , Smad2, and Smad3 were analyzed by qRT-PCR and western blotting, respectively. Mimics control is the negative control of mimics, and control is the blank control in this group. The result showed that there was no significant difference between control and mimics control. β -actin was used as a loading control. Values represent the mean±S.D., ** P <0.01, *** P <0.001 compared with control. ( f ) The mRNA and protein ( g ) levels of MMP-2, MMP-9, Col-1, and Col-4 were analyzed by qRT-PCR and western blotting, respectively. β -actin was used as a loading control. Values represent the mean±S.D., * P <0.05, ** P <0.01, *** P <0.001 compared with control

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Control, Transfection, Western Blot, Negative Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Knockdown of either TGFBR1 or TGFBR2 inhibited the TGF- β / SMAD signaling pathway and reduced HSC activation. ( a ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein expression levels ( b ) of TGFBR1 and TGFBR2. β-actin was used as a loading control. Values represent the mean ±SD, *** P < 0.001 compared with control. ( c ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein expression levels ( d ) of α -SMA, Smad2, and Smad3. β-actin was used as a loading control. Values represent the mean ±SD, * P <0.05, ** P <0.01, *** P <0.001 compared with control. ( e ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein levels ( f ) of MMP-2, MMP-9, Col-1, and Col-4. β -actin was used as a loading control. Values represent the mean±S.D., *** P <0.001 compared with control

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Knockdown, Activation Assay, Expressing, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Overexpression of TGFBR1 and TGFBR2 rescued miR-130a-3p-impaired proliferation and cell growth in HSCs. The HSC-T6 cells were transduced with the miR-130a-3p mimics and the TGFBR1-expressing and TGFBR2-expressing vectors. The rescue efficiency of TGFBR1 or TGFBR2 in the HSC-T6 cells was confirmed by ( a ) RT-PCR and ( b ) western blotting. β -actin was used as a loading control. Values represent the mean±S.D. (*** P <0.001). Overexpression of TGFBR1 and TGFBR2 rescued the (c) mRNA and ( d ) protein expression of Smad2 and Smad3. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). Overexpression of TGFBR1 and TGFBR2 rescued the ( e ) mRNA and ( f ) protein expression of Col-1 and Col-4. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). ( g ) A schematic diagram highlighting the regulation of HSCs by miR-130a-3p during the genesis and resolution of liver fibrosis

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Over Expression, Transduction, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Primers for quantitative real-time PCR analysis

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Real-time Polymerase Chain Reaction