Journal: Journal of translational autoimmunity

Article Title: Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells.

doi: 10.1016/j.jtauto.2023.100202

Figure Lengend Snippet: Fig. 3. Immunofluorescent antibody staining in naïve and treated HUVECs. AK. The mixture of APS total IgG and β2GPI induces a pronounced increase of the protein levels of the proinflammatory cytokines IL-6, IL-8 as well the transcription factor NF-κB1 and cell adhesion molecules Tissue Factor, ICAM-1, VCAM-1, Eselectin, P- selectin and TGFR1. There was no significant difference for the TGFR1 molecule (3 J). Visual analysis revealed that all the inflammatory mediators and adhesion molecules presented statistically significant difference between the untreated and treated endothelial cells (3 K).

Article Snippet: Coverslips were incubated overnight at 4 ◦C with primary antibodies against IL-6 (5 μg/ ml, CSB-PA06757A0Rb, Cusabio), IL-8 (5 μg/ml, CSB-MA083271A0m, Cusabio), NF-κB1 (5 μg/ml, CSB-PA190132, Cusabio), TGF-β2 (5 μg/ ml, CSB- PA07319A0Rb, Cusabio), Tissue Factor (5 μg/ml, 4509, American Diagnostica), ICAM-1 (5 μg/ml, AF796, R&D Systems), VCAM-1 (4 μg/ml, sc-18854, Santa Cruz Biotechnology), E-selectin (4 μg/ml, sc-271267, Santa Cruz Biotechnology), P-selectin (4 μg/ml,sc137054, Santa Cruz Biotechnology) and TGFR1 (5 μg/ml,CSBPA023451LA01HU, Cusabio).

Techniques: Staining

Journal: Journal of translational autoimmunity

Article Title: Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells.

doi: 10.1016/j.jtauto.2023.100202

Figure Lengend Snippet: Fig. 5. Immunofluorescent antibody staining in placenta biopsies from APS patients and healthy women. A-K Placenta biopsies derived from APS patients as well as Healthy Donors show increased signal intensity for IL-6, IL-8, NF-κB1, ICAM1, VCAM-1, E-selectin, P-selectin, TGF-β2, and TGFR1 (5A-5D, 5F-5J). Slight difference in fluorescence intensity between HD and APS patient was observed for Tissue Factor (5E). Increased signal intensity was observed as well for the TNF-α molecule in the APS placenta biopsies (5 K).

Article Snippet: Coverslips were incubated overnight at 4 ◦C with primary antibodies against IL-6 (5 μg/ ml, CSB-PA06757A0Rb, Cusabio), IL-8 (5 μg/ml, CSB-MA083271A0m, Cusabio), NF-κB1 (5 μg/ml, CSB-PA190132, Cusabio), TGF-β2 (5 μg/ ml, CSB- PA07319A0Rb, Cusabio), Tissue Factor (5 μg/ml, 4509, American Diagnostica), ICAM-1 (5 μg/ml, AF796, R&D Systems), VCAM-1 (4 μg/ml, sc-18854, Santa Cruz Biotechnology), E-selectin (4 μg/ml, sc-271267, Santa Cruz Biotechnology), P-selectin (4 μg/ml,sc137054, Santa Cruz Biotechnology) and TGFR1 (5 μg/ml,CSBPA023451LA01HU, Cusabio).

Techniques: Staining, Derivative Assay, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Endoplasmic reticulum protein TXNDC5 promotes renal fibrosis by enforcing TGF- β signaling in kidney fibroblasts

doi: 10.1172/JCI143645

Figure Lengend Snippet: (A) Immunoblots showed that treatment of TGF-β1 (10 ng/mL) induced upregulation of TGFBR1 and phosphorylation of SMAD3, whereas TGFBR2 was not affected. Knockdown of TXNDC5 abolished TGFBR1 upregulation and SMAD3 phosphorylation was induced by TGF-β1 in HKFs (n = 5–11). (B) Overexpression of TXNDC5 was sufficient to upregulate TGFBR1 and phospho-SMAD3 in HKFs (n = 5–6). (C) Knockdown of TGFBR1 abolished the upregulation of fibroblast activation markers and ECM proteins induced by TXNDC5 overexpression (n = 6–12). Data are representative of 3 or more independent experimental replicates. For all panels, data are presented as mean ± SEM. The statistical significance of differences for 2 groups was determined by 2-sided t test and among 3 or more groups it was determined using 1-way ANOVA, followed by Sidak’s post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Briefly, 4% PFA–fixed HKFs were permeabilized and blocked with 0.1% Tween 20 in 2% BSA for 1 hour, followed by incubation with anti-TXNDC5 (1:1500, Proteintech, 19834-1-AP) and anti-TGFBR1 (1:100, Abnova, H00007046-A01) overnight at 4°C.

Techniques: Western Blot, Knockdown, Over Expression, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Endoplasmic reticulum protein TXNDC5 promotes renal fibrosis by enforcing TGF- β signaling in kidney fibroblasts

doi: 10.1172/JCI143645

Figure Lengend Snippet: (A) Cycloheximide chase assay showed that knockdown of TXNDC5 accelerated degradation of TGFBR1 in HKFs (n = 6). (B) Overexpression of TXNDC5 slowed down the degradation of TGFBR1 in HKFs. The 2 groups of samples were loaded and run on the same gel but not in neighboring lanes (n = 6). (C) Treatment of proteasome inhibitor MG132 restored the downregulation of TGFBR1 induced by TXNDC5 depletion in HKFs (n = 11). (D) Protein Co-IP experiments in HEK cells with ectopic expression of TXNDC5 and Myc-tagged TGFBR1 showed physical interaction between TXNDC5 and TGFBR1. (E) A dual fluorescence–labeled human TGFBR1 construct (CFP-TGFBR1-YFP) was used for FRET-based protein folding assay in HEK293 cells. Overexpression of TXNDC5 showed increased FRET efficiency compared with empty vector–transfected control cells, whereas AAA-mutant TXNDC5 transfection failed to increase the FRET signal (n = 10). Data are representative of 3 or more independent experimental replicates. For all panels, data are presented as mean ± SEM. The statistical significance of differences for 2 groups was determined by 2-sided t test and among 3 or more groups it was determined using 1-way ANOVA, followed by Sidak’s post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Briefly, 4% PFA–fixed HKFs were permeabilized and blocked with 0.1% Tween 20 in 2% BSA for 1 hour, followed by incubation with anti-TXNDC5 (1:1500, Proteintech, 19834-1-AP) and anti-TGFBR1 (1:100, Abnova, H00007046-A01) overnight at 4°C.

Techniques: Knockdown, Over Expression, Co-Immunoprecipitation Assay, Expressing, Fluorescence, Labeling, Construct, Plasmid Preparation, Transfection, Control, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Endoplasmic reticulum protein TXNDC5 promotes renal fibrosis by enforcing TGF- β signaling in kidney fibroblasts

doi: 10.1172/JCI143645

Figure Lengend Snippet: (A) Illustration of experimental design to induce Txndc5 deletion specifically in renal fibroblasts in mouse kidneys with established fibrosis. (B) Picrosirius red staining of kidney sections from Col1a2-Cre and Txndc5cKO mice. Ten days after UUO, Col1a2-Cre and Txndc5cKO mice showed a similar extent of renal fibrosis prior to tamoxifen injection. Eleven days after tamoxifen treatment, the fibrotic areas more than doubled (increased from 5.1% to 10.6%) in Col1a2-Cre, but barely changed in Txndc5cKO (changed from 5.3% to 6.9%) mouse kidneys (n = 5–6). Scale bar: 50 μm. (C) Protein expression levels of fibroblast activation marker (periostin), ECM (CCN2), and TGFBR1 in whole-kidney lysate from Col1a2-Cre and Txndc5cKO mice (n = 4–6). (D) Schematic summary of the proposed profibrotic mechanisms by which TXNDC5 contributes to the pathogenesis of renal fibrosis. TF: transcription factor. Data are representative of 3 or more independent experimental replicates. For all panels, data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-sided t test.

Article Snippet: Briefly, 4% PFA–fixed HKFs were permeabilized and blocked with 0.1% Tween 20 in 2% BSA for 1 hour, followed by incubation with anti-TXNDC5 (1:1500, Proteintech, 19834-1-AP) and anti-TGFBR1 (1:100, Abnova, H00007046-A01) overnight at 4°C.

Techniques: Staining, Injection, Expressing, Activation Assay, Marker

Journal: Signal Transduction and Targeted Therapy

Article Title: Engineered extracellular vesicles for targeted reprogramming of cancer-associated fibroblasts to potentiate therapy of pancreatic cancer

doi: 10.1038/s41392-024-01872-7

Figure Lengend Snippet: FERMT2 interacts with TGFBR1 and PYCR1 in cancer-associated fibroblasts (CAFs). a Correlation of FERMT2 expression with the TGF-β pathway in The Cancer Genome Atlas (TCGA) pancreatic cancer cohort. b Single-sample gene set enrichment analysis (ssGSEA) of FERMT2 in TCGA pancreatic cancer cohort. c Analysis of the interaction of FERMT2 with TGF-β signaling-related proteins using STRING database ( https://cn.string-db.org/ ). d Co-immunoprecipitation (Co-IP) analysis of FERMT2 and TGFBR1. e Western blotting analysis of the effect of FERMT2 knockdown and miR-138-5p inhibitor on the TGFBR1 and p-SMAD2/3 expression levels. f Correlation of FERMT2 with ACTA2, COL1A1, FAP, and FSP in TCGA pancreatic cancer dataset. g Western blotting analysis of the effect of FERMT2 knockdown and miR-138-5p inhibitor on the ACTA2, collagen1, FAP, and FSP expression levels. h Correlation of FERMT2 expression with the collagen production pathway in TCGA pancreatic cancer dataset. i Co-IP analysis of FERMT2 and TGFBR1. j Western blotting analysis of the impact of FERMT2 knockdown and miR-138-5p inhibitor on PYCR1 expression. k Effect of FERMT2 knockdown and PYCR1 overexpression on the proline content. Data are presented as mean (±SD); n = 3 per group. l Western blotting analysis of the effect of FERMT2 knockdown and PYCR1 overexpression on the PYCR1 and collagen1 expression levels

Article Snippet: Thereafter, the samples were subjected to western blotting using rabbit anti-FERMT2 (Proteintech), rabbit anti-PYCR1 (Proteintech), or rabbit anti-TGFBR1 (Servicebio) antibodies.

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Knockdown, Over Expression

Journal: Signal Transduction and Targeted Therapy

Article Title: Engineered extracellular vesicles for targeted reprogramming of cancer-associated fibroblasts to potentiate therapy of pancreatic cancer

doi: 10.1038/s41392-024-01872-7

Figure Lengend Snippet: Role of extracellular vesicles (EVs) loaded with miR-138-5p and pirfenidone (PFD) and subjected to surface modification with integrin α5-targeting peptides (IEVs-PFD/138) in cancer-associated fibroblast (CAF) phenotype reversal, cancer cell inhibition, and enhanced drug penetration. a Immunoprecipitation (IP) analysis of the FERMT2-TGFBR1 and FERMT2-PYCR1 complexes. b Western blotting analysis of the expression levels of FERMT2, PYCR1, TGFBR1, p-SMAD2/3, ACTA2, FAP, FSP, and collagen1. c Enzyme-linked immunosorbent assay (ELISA) for analyzing the secretion of IL6, TGFB1, and CXCL12 by CAFs. Data are presented as mean (± SD); n = 3 per group. d Proline levels in CAFs. Data represent the mean (± SD); n = 3 per group. e Scheme of the effect of conditioned medium (CM) derived from EV-treated CAFs on PANC-1 cells. f Western blotting analysis of PCNA, VIM, and N-cad in PANC-1 cells treated with CM derived from EV-treated CAFs. Data are presented as mean (±SD); n = 3 per group. g Evaluation of the proliferation, migration, and invasion of PANC-1 cells treated with CM derived from CAFs treated with EVs (Red: EdU-positive cells, blue: cell nuclei), (scale bar = 100 μm). Data are presented as mean (±SD); n = 3 per group. h – j Enhanced fluorescence intensity and penetration depth of Hoechst 33258 in stroma-rich three-dimensional (3D) multicellular tumor spheroids treated with IEVs-PFD/138 (scale bar = 100 μm). k – m Comparison of 3D multicellular tumor spheroid volume after treatment with the combination of IEVs-PFD/138 and gemcitabine (GEM) (scale bar = 100 μm). Data represent the mean (± SD); n = 3 per group

Article Snippet: Thereafter, the samples were subjected to western blotting using rabbit anti-FERMT2 (Proteintech), rabbit anti-PYCR1 (Proteintech), or rabbit anti-TGFBR1 (Servicebio) antibodies.

Techniques: Modification, Inhibition, Immunoprecipitation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Migration, Fluorescence, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Engineered extracellular vesicles for targeted reprogramming of cancer-associated fibroblasts to potentiate therapy of pancreatic cancer

doi: 10.1038/s41392-024-01872-7

Figure Lengend Snippet: Effect of treatment with extracellular vesicles (EVs) loaded with miR-138-5p and pirfenidone (PFD) and subjected to surface modification with integrin α5-targeting peptides (IEVs-PFD/138) in the subcutaneous desmoplastic pancreatic cancer model. a Scheme of IEVs-PFD/138 treatment. b Images of the mouse model after treatment on day 21. c , d Tumor growth curves of the mouse models. Data are presented as mean (± SD); n = 5 per group. e Tumor weight of the mouse models. Data are presented as mean (± SD); n = 5 per group. f Effect of treatment on the survival of the mouse model. g Ki67 immunofluorescence staining, Masson’s staining, ACTA2 immunofluorescence staining, FAP immunofluorescence staining, FSP immunofluorescence staining, PYCR1 immunofluorescence staining, and collagen1 immunohistochemical staining of tumors from mice under different treatments (scale bar = 100 μm). h Western blotting analysis of the FERMT2, PYCR1, TGFBR1, and p-SMAD2/3 expression levels in tumors from mice administered various treatments. i Hematoxylin and eosin (HE) staining of vital organs from mice administered various treatments (scale bar = 100 μm)

Article Snippet: Thereafter, the samples were subjected to western blotting using rabbit anti-FERMT2 (Proteintech), rabbit anti-PYCR1 (Proteintech), or rabbit anti-TGFBR1 (Servicebio) antibodies.

Techniques: Modification, Immunofluorescence, Staining, Immunohistochemical staining, Western Blot, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Engineered extracellular vesicles for targeted reprogramming of cancer-associated fibroblasts to potentiate therapy of pancreatic cancer

doi: 10.1038/s41392-024-01872-7

Figure Lengend Snippet: Effect of treatment with extracellular vesicles (EVs) loaded with miR-138-5p and pirfenidone (PFD) and subjected to surface modification with integrin α5-targeting peptides (IEVs-PFD/138) in the patient-derived xenograft (PDX) pancreatic cancer models. a Scheme of PDX model establishment and treatment. b Images of mouse models and tumors after treatment on day 24. c , d Tumor growth curves of the mouse models. Data are presented as mean (± SD); n = 5 per group. e Tumor weight of the mouse models. Data are presented as mean (±SD); n = 5 per group. f Relative gemcitabine (GEM) concentrations in tumors. Data are presented as mean (±SD); n = 3 per group. g Elastic modulus of tumors. Data are presented as mean (± SD); n = 3 per group. h Solid stress of tumors (three tumors with each tumor having three parts). i Effect of treatment on the survival of the mouse model. j Hematoxylin and eosin (HE) staining, Masson’s staining, collagen1 immunohistochemical staining, ACTA2 immunohistochemical staining, Ki67 immunohistochemical staining, and Tunel staining of tumors from mice administered various treatments (scale bar = 200 μm). k Western blotting analysis of the FERMT2, PYCR1, TGFBR1, P-SMAD2/3, HIF1A, and ENT1 expression levels in tumors from mice subjected to different treatments

Article Snippet: Thereafter, the samples were subjected to western blotting using rabbit anti-FERMT2 (Proteintech), rabbit anti-PYCR1 (Proteintech), or rabbit anti-TGFBR1 (Servicebio) antibodies.

Techniques: Modification, Derivative Assay, Staining, Immunohistochemical staining, TUNEL Assay, Western Blot, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Engineered extracellular vesicles for targeted reprogramming of cancer-associated fibroblasts to potentiate therapy of pancreatic cancer

doi: 10.1038/s41392-024-01872-7

Figure Lengend Snippet: Effects of treatment with extracellular vesicles (EVs) loaded with miR-138-5p and pirfenidone (PFD) and subjected to surface modification with integrin α5-targeting peptides (IEVs-PFD/138) in the orthotopic desmoplastic pancreatic cancer C57/B6 models. a Scheme of IEVs-PFD/138 and gemcitabine (GEM) treatment. b Sample in vivo bioluminescence images of mice. c Bioluminescence intensity quantification of mice with orthotopic pancreatic tumors. Data represent the mean (± SD); n = 3 per group. d Growth-inhibitory effects on tumor based on bioluminescence intensity. Data represent the mean (±SD); n = 3 per group. e Images of excised tumors with spleen and pancreas in different treatment groups on day 12. f Tumor volume. Data are presented as mean (±SD); n = 5 per group. g Western blotting analysis of the FERMT2, PYCR1, TGFBR1, P-SMAD2/3, and ENT1 expression levels in tumors from mice subjected to different treatments. h Masson’s staining (scale bar = 100 μm), Tunel (scale bar = 200 μm), KI67 immunofluorescence (scale bar = 100 μm), ACTA2 immunofluorescence (scale bar = 100 μm), and collagen1 immunofluorescence (scale bar = 100 μm) staining of tumors

Article Snippet: Thereafter, the samples were subjected to western blotting using rabbit anti-FERMT2 (Proteintech), rabbit anti-PYCR1 (Proteintech), or rabbit anti-TGFBR1 (Servicebio) antibodies.

Techniques: Modification, In Vivo, Western Blot, Expressing, Staining, TUNEL Assay, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors

doi: 10.3390/ijms23052780

Figure Lengend Snippet: ACVR1 ( a , a’ ), BMPR1A ( b , b’ ), BMPR1B ( c , c’ ), and TGFBR1 ( d , d’ ) mRNA and protein abundance in preantral (primordial, primary, and secondary follicles) and small antral follicles obtained from the control (CTR) and methoxychlor (MXC-) treated gilts. The mRNA expression (quantitative real-time PCR) is presented relative to GAPDH as mean ± SEM ( a – d ). Relative protein abundance was measured by the densitometric method and expressed as the ratio relative to β-actin abundance (mean ± SEM, a’ – d’ ). The fragment of membranes with bands corresponding to predicted molecular weights is shown above graphs. Asterisks denote significant differences between the CTR and treated animals (for preantral follicles pool n = 5, for small antral follicle n = 15, * p < 0.05, ** p < 0.01, *** p < 0.001, Mann–Whitney U test).

Article Snippet: Anti-TGFBR1 , 1:1000 , 1:50 , rabbit polyclonal , Abgent San Diego, CA, USA (AP7822c, Lot no. SA110808BQ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors

doi: 10.3390/ijms23052780

Figure Lengend Snippet: Immunolocalization of ACVR1, BMPR1A, BMPR1B, and TGFBR1 in preantral (primordial, primary, and secondary follicle) ( a ) and small antral ( b ) follicles of the control (CTR) and methoxychlor (MXC-) treated gilts. ACVR1, BMPR1A, BMPR1B, and TGFBR1 positive staining was observed in oocytes (asterisks) and granulosa cells (arrows) of preantral follicles, as well as granulosa (arrows) and theca cells (arrowheads) of small antral follicles, in both examined groups. All sections were counterstained with hematoxylin QS. There was no positive staining observed in the negative control sections ( a , b , insets). Prim—primordial follicles; SF—secondary follicles; AF—antral follicles. Bars = 50 µm.

Article Snippet: Anti-TGFBR1 , 1:1000 , 1:50 , rabbit polyclonal , Abgent San Diego, CA, USA (AP7822c, Lot no. SA110808BQ).

Techniques: Staining, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors

doi: 10.3390/ijms23052780

Figure Lengend Snippet: List of primary antibodies used for Western blot and immunohistochemistry.

Article Snippet: Anti-TGFBR1 , 1:1000 , 1:50 , rabbit polyclonal , Abgent San Diego, CA, USA (AP7822c, Lot no. SA110808BQ).

Techniques: Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Transforming growth factor (TGF)-β1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis

doi: 10.1038/s41419-019-1873-x

Figure Lengend Snippet: a KEGG analysis of Targetscan predicted miR-133a targets. HFL cells were transfected with miR-133a mimic or control mimic (CTL) for 24 h, and then stimulated without or with 1 ng/mL TGF-β1 for 48 h. Cells were harvested and subjected to western blot ( b – d ) or quantitative RT-PCR analysis ( e , f ). Transfection of miR-133a down-regulated TGFBR1 protein expression ( b ), blocked TGF-β1-stimulated Smad2/3 phosphorylation ( c ), CTGF protein expression ( d ), Col1a1 ( e ), and Col4a1 ( f ) mRNA expression. Data are mean ± SEM ( n = 3–4) with * P < 0.05; *** P < 0.001. g The 3′UTR of TGFBR1, CTGF or Col1a1 contains a putative miR-133a binding site that is conserved among different species (in red). miR-133a binding site mutations were created by deletion or nucleotide changes (in blue). h The 3′UTR fragments containing the putative miR-133a binding site (3’UTR-133a) or their mutants (3′UTR-133aM) of human TGFBR1 (inserted fragment: 1989–2403; seeding region: 2161–2167), CTGF (inserted fragment: 886–1065; seeding region: 1027–1033), or Col1a1 (inserted fragment 78–301; seeding region: 194–200) were subcloned into the pmirGLO reporter vector. i These luciferase reporter constructs were transfected into HEK293 cells, along with control or miR-133a mimic. Cells were harvested for firefly luciferase (fLuc) assays with Renilla luciferase (rLuc) as an internal control. Data are mean ± SEM ( n = 3–4) with ** P < 0.01

Article Snippet: The rabbit anti-TGFBR1 antibody was obtained from Merck Millipore (Billerica, MA, USA).

Techniques: Transfection, Control, Western Blot, Quantitative RT-PCR, Expressing, Phospho-proteomics, Binding Assay, Plasmid Preparation, Luciferase, Construct

Journal: Cell Death & Disease

Article Title: Transforming growth factor (TGF)-β1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis

doi: 10.1038/s41419-019-1873-x

Figure Lengend Snippet: a The cis-element of miR-133a expression vector driven by the FTS1 promoter. b Confocal laser scanning microscopy images of GFP expressed in NIH3T3 fibroblasts or epithelial 16HBE cells using pAAV-CMV-GFP or pAAV-FTS1-GFP vector. Scale bar: 50 μm. c pAAV-CMV-GFP-133a or pAAV-FTS1-GFP-133a vector was transfected into NIH3T3 fibroblasts or epithelial 16HBE cells. 24 h later, the expression levels of miR-133a were determined by quantitative RT-PCR. d Experimental scheme of the mouse model of bleomycin-induced pulmonary fibrosis. Mice were intratracheally injected with saline or bleomycin (50 mg/kg) at day 0. On day 5, mice were administrated pAAV-FTS1-GFP-miR-133a expression vector or its control vector by tail vein injection. This treatment was repeated every 4 days and mice were killed on day 15. e Photomicrographs showing H&E staining and Masson staining of lung tissues of mice treated with saline, bleomycin without or with miR-133a expression vector or its control vector. Scale bar: 200 μm. f Standardized quantification of pulmonary fibrosis in Masson staining samples by the modified Ashcroft method (score range 0–8). Data are mean ± SEM, n = 4, *** P < 0.001. g Graphical summary of results. TGF-β1-induced miR-133a functions as a feed-back negative regulator of TGF-β1 profibrogenic pathways. TGF-β1 induces miR-133a expression via Smad3 and p38MAPK signaling pathways. Upregulated miR-133a in turn negatively regulates myofibroblast differentiation via targeting multiple components of the TGF-β1 signal pathway such as TGFBR1, CTGF, and Col1a1

Article Snippet: The rabbit anti-TGFBR1 antibody was obtained from Merck Millipore (Billerica, MA, USA).

Techniques: Expressing, Plasmid Preparation, Confocal Laser Scanning Microscopy, Transfection, Quantitative RT-PCR, Injection, Saline, Control, Staining, Modification, Protein-Protein interactions