Journal: International endodontic journal

Article Title: Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-β1 secretion.

doi: 10.1111/iej.13943

Figure Lengend Snippet: FIGURE 2 The expression of smooth muscle cell-specific markers in dental pulp stem cells (DPSCs) monoculture and HUVEC+DPSC cocultures. (a) Western blot analysis of the expression of α-SMA, SM22α, Calponin, PDGFR-β and NG2 in DPSCs monoculture and HUVEC+DPSC cocultures at day 3 and day 6. (b–f) Quantitative analysis of bands intensities in (a) with Image J. (g) Representative fluorescent images of DPSCs monoculture and HUVEC+DPSC cocultures probed for α-SMA, SM22α and Calponin (red), CD31 (green) and nuclei (DAPI, blue), the scale bar represents 100 μm. Data are represented as mean ± standard error (n = 3), *p < .05, **p < .01, ***p < .001. HUVEC, human umbilical vascular endothelial cells.

Article Snippet: Then, cells were incubated with primary antibodies against α- SMA (19245S, Cell Signaling Technology), SM22α (ab14106, Abcam), Calponin (ab46794, Abcam) and CD31 (ab199012, Abcam) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: International endodontic journal

Article Title: Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-β1 secretion.

doi: 10.1111/iej.13943

Figure Lengend Snippet: FIGURE 3 The expression of smooth muscle cell-specific markers in dental pulp stem cells (DPSCs) and E-DPSCs. (a–d) Western blot analysis of the expression of α-SMA, SM22α and Calponin in DPSCs monoculture and DPSCs coculture with HUVECs separated by Transwell (TW) inserts (E-DPSC-TW) for 6 days. (e–h) Western blot analysis of the expression of α-SMA, SM22α and Calponin in DPSCs and E-DPSCs. (i) Representative fluorescent images of DPSCs and E-DPSCs probed for α-SMA and SM22α (red), and nuclei (DAPI, blue), the scale bar represents 100 μm. Data are represented as mean ± standard error (n = 3), ns = no significant difference, **p < .01, ****p < .0001. HUVEC, human umbilical vascular endothelial cells.

Article Snippet: Then, cells were incubated with primary antibodies against α- SMA (19245S, Cell Signaling Technology), SM22α (ab14106, Abcam), Calponin (ab46794, Abcam) and CD31 (ab199012, Abcam) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: International endodontic journal

Article Title: Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-β1 secretion.

doi: 10.1111/iej.13943

Figure Lengend Snippet: FIGURE 4 The expression of α-SMA, SM22α and Calponin in dental pulp stem cell (DPSC) after treatment with conditioned media (CM). (a–d) Western blot analysis of the expression of α-SMA, SM22α and Calponin in DPSC after treatment with CM of HUVECs monoculture (E-CM), CM of DPSCs monoculture (D-CM) and CM of HUVEC+DPSC cocultures (E+D-CM) for 6 days. (e) Representative fluorescent images of DPSCs probed for α-SMA and Calponin (red), and nuclei (DAPI, blue) after treatment with E-CM, D-CM or E+D- CM for 6 days, the scale bar represents 100 μm. Data are represented as mean ± standard error (n = 3), **p < .01. HUVEC, human umbilical vascular endothelial cells.

Article Snippet: Then, cells were incubated with primary antibodies against α- SMA (19245S, Cell Signaling Technology), SM22α (ab14106, Abcam), Calponin (ab46794, Abcam) and CD31 (ab199012, Abcam) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: International endodontic journal

Article Title: Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-β1 secretion.

doi: 10.1111/iej.13943

Figure Lengend Snippet: FIGURE 6 The expression of α-SMA, SM22α and Calponin in dental pulp stem cells (DPSCs) after treatment with activin A or TGF-β1. (a–d) Western blot analysis of the expression of α-SMA, SM22α and Calponin in DPSCs after treatment with activin A (25 ng/mL) for 3 and 6 days. (e–h) Western blot analysis of the expression of α-SMA, SM22α and Calponin in DPSCs after treatment with TGF-β1 (10 ng/mL) for 3 and 6 days. (i) Representative fluorescent images of DPSCs and T-DPSCs probed for α-SMA and SM22α (red), and nuclei (DAPI, blue), the scale bar represents 100 μm. (j) ELISA analysis of the TGF-β1 level in CM of HUVECs and DPSCs from monoculture and cocultures. (k) Contraction assay analysis of the contractility of DPSC, E-DPSC and T-DPSC in collagen gel. (l) The gel diameters were measured and analysed with Image J software after cells were seeded in the gels for 48 h. Data are represented as mean ± standard error (n = 3), ns = no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. HUVEC, human umbilical vascular endothelial cells.

Article Snippet: Then, cells were incubated with primary antibodies against α- SMA (19245S, Cell Signaling Technology), SM22α (ab14106, Abcam), Calponin (ab46794, Abcam) and CD31 (ab199012, Abcam) overnight at 4°C.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Contraction Assay, Software

Journal: International endodontic journal

Article Title: Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-β1 secretion.

doi: 10.1111/iej.13943

Figure Lengend Snippet: FIGURE 7 The expression of smooth muscle cell-specific markers in HUVEC+DPSC cocultures after blocking of TGF-β1/ALK5 signalling pathway. (a) Western blot analysis of the expression of p-Smad2, Smad2, α-SMA, SM22α and Calponin in HUVEC+DPSC cocultures with or without TGF-β RI kinase inhibitor (150 nM) for 6 days. (b–f) Quantitative analysis of bands intensities in (a) with Image J. (g) Representative fluorescent images of DPSC monoculture and HUVEC+DPSC cocultures probed for α-SMA and SM22α (red), CD31 (green) and nuclei (DAPI, blue) with or without TGF-β RI kinase inhibitor (150 nM) for 6 days, the scale bar represents 100 μm. Data are represented as mean ± standard error (n = 3), *p < .05, **p < .01, ***p < .001. DPSC, dental pulp stem cell; HUVEC, human umbilical vascular endothelial cells.

Article Snippet: Then, cells were incubated with primary antibodies against α- SMA (19245S, Cell Signaling Technology), SM22α (ab14106, Abcam), Calponin (ab46794, Abcam) and CD31 (ab199012, Abcam) overnight at 4°C.

Techniques: Expressing, Blocking Assay, Western Blot

Journal: International endodontic journal

Article Title: Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-β1 secretion.

doi: 10.1111/iej.13943

Figure Lengend Snippet: FIGURE 8 The expression of α-SMA, SM22α and Calponin in dental pulp stem cells (DPSCs) after blocking of TGF-β1/ALK5 signalling pathway. (a) Western blot analysis of the expression of α-SMA, SM22α and Calponin in DPSCs pretreated with TGF-β RI kinase inhibitor (150 nM) followed by treatment with E+D-CM or treated with E+D-CM supplemented with anti-TGF-β1 IgG for 6 days. (b–f) Quantitative analysis of bands intensities in (a) with Image J. (g) Representative fluorescent images of DPSC probed for α-SMA and SM22α (red), and nuclei (DAPI, blue) pretreated with TGF-β RI kinase inhibitor (150 nM) followed by treatment with E+D-CM or treated with E+D-CM supplemented with anti-TGF-β1 IgG for 6 days; the scale bar represents 100 μm. Data are represented as mean ± standard error (n = 3), ns = no significant difference, *p < .05, **p < .01, ***p < .001, ****p < .0001.

Article Snippet: Then, cells were incubated with primary antibodies against α- SMA (19245S, Cell Signaling Technology), SM22α (ab14106, Abcam), Calponin (ab46794, Abcam) and CD31 (ab199012, Abcam) overnight at 4°C.

Techniques: Expressing, Blocking Assay, Western Blot

Journal: Dose-response : a publication of International Hormesis Society

Article Title: Radiation Protection of Polydatin Against Radon Exposure Injury of Epithelial Cells and Mice.

doi: 10.1177/15593258231172271

Figure Lengend Snippet: Figure 4. PD can regulate the expression of EMT markers in radon-exposed cells. (A) The protein expression of E-cad, Vimentin, N-cad, FN1, α-SMA, and Snail in radon exposure model of 16HBE and BEAS-2B cells with directly adding PD. (C) The protein expression of E-cad, Vimentin, N-cad, FN1, α-SMA, and Snail in radon-exposed 16HBE and BEAS-2B cells with PD added before and after radon exposure. (B, D) The expression level of EMT markers in different groups was quantified by Image J software. *: Compared with Rn6, P < .05; * *: Compared with Rn6, P < .01.

Article Snippet: The membrane was sealed with 5% skim milk powder at room temperature and incubated overnight at 4°C with primary antibodies (E-cad (1: 3000, Abcam, USA), Vimentin (1: 5000, Abcam), FN1 (1: 5000, Abcam), Snail (1: 1000, CST, USA), α-SMA (1: 1000, CST), N-cad (1:1000, CST), p-PI3K (1: 500, CST), p-AKT (1: 1000, CST), p-mTOR (1: 800, CST), and GAPDH/ β-tubulin).

Techniques: Expressing, Software

Journal: iScience

Article Title: Profibrotic role of transcription factor SP1 in cross-talk between fibroblasts and M2 macrophages

doi: 10.1016/j.isci.2023.108484

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Smad7 , Proteintech , cat# 25840-1-AP; RRID: AB_2848137.

Techniques: Recombinant, cDNA Synthesis, CCK-8 Assay, Luciferase, Reporter Gene Assay, Methylation, Mass Spectrometry, Real-time Polymerase Chain Reaction, Knockdown, Plasmid Preparation, Software

Journal: Frontiers in Nutrition

Article Title: Anti-fibrotic effect of extracellular vesicles derived from tea leaves in hepatic stellate cells and liver fibrosis mice

doi: 10.3389/fnut.2022.1009139

Figure Lengend Snippet: The effects of TEVs on the proliferation of LX-2 cells. (A) The effect of TEVs on TGF-β1 non activated LX-2 cells proliferation. (B) The effect of TEVs on TGF-β1 activated LX-2 cells proliferation. (C) mRNA expression of α -SMA was analyzed by Real-time PCR. (D) mRNA expression of COL I was analyzed by Real-time PCR. (E) Protein expression of α-SMA was determined by western blotting. (F) Protein expression of COL I was determined by western blotting. Error bar represents SD ( n = 3; * P < 0.05, ** P < 0.01, indicating significantly different from control group).

Article Snippet: Primary antibodies used included: α-SMA rabbit polyclonal (1:100; proteintech, Wuhan, China), COL I rabbit polyclonal (1:150; Abcam, UK).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Frontiers in Nutrition

Article Title: Anti-fibrotic effect of extracellular vesicles derived from tea leaves in hepatic stellate cells and liver fibrosis mice

doi: 10.3389/fnut.2022.1009139

Figure Lengend Snippet: The effects of TEVs on the expression of fibrotic markers in mice. (A) Immunofluorescence detection α-SMA protein in the different experimental groups. Scale bar = 50 μm. (B) The relative fluorescence intensity in (A) was quantified using Image J software. (C) Immunofluorescence detection COL I protein in the different experimental groups. Scale bar = 50 μm. (D) The relative fluorescence intensity in (C) was quantified using Image J software. Error bar represents SD ( n = 3; * P < 0.05, ** P < 0.01, indicating significantly different from positive control group).

Article Snippet: Primary antibodies used included: α-SMA rabbit polyclonal (1:100; proteintech, Wuhan, China), COL I rabbit polyclonal (1:150; Abcam, UK).

Techniques: Expressing, Immunofluorescence, Fluorescence, Software, Positive Control

Journal: Frontiers in Nutrition

Article Title: Anti-fibrotic effect of extracellular vesicles derived from tea leaves in hepatic stellate cells and liver fibrosis mice

doi: 10.3389/fnut.2022.1009139

Figure Lengend Snippet: The miR-44 in TEVs was identified to inhibit liver fibrosis. (A) The miR-44 can inhibit liver fibrosis. 20 nM of miRNA mimic or miRNA (miR) negative control (NC) were transfected. The protein expression levels of α-SMA were detected. (B) The miR-44 dose-dependently decreased α-SMA protein expression. Different amounts of miRNA negative control were co-transfected with miR-44 to ensure the total transfected RNAs of 20 nM. (C) The miR-44 had an effect on Smads signal. 20 nM of miRNA mimic or miR-NC were transfected. The protein expression levels of Smad2 and Smad3 were detected. (D) The miR-44 dose-dependently decreased Smad2 and Smad3 protein expression. Different amounts of miRNA negative control were co-transfected with miR-44 to ensure the total transfected RNAs of 20 nM.

Article Snippet: Primary antibodies used included: α-SMA rabbit polyclonal (1:100; proteintech, Wuhan, China), COL I rabbit polyclonal (1:150; Abcam, UK).

Techniques: Negative Control, Transfection, Expressing

Journal: Cell Reports Methods

Article Title: Modeling alcohol-associated liver disease in humans using adipose stromal or stem cell-derived organoids

doi: 10.1016/j.crmeth.2024.100778

Figure Lengend Snippet:

Article Snippet: Smooth Muscle Actin Polyclonal antibody , Proteintech , Cat# 14395-1-AP; RRID: AB_2223009.

Techniques: Control, Virus, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Diagnostic Assay, Detection Assay, Quantitative RT-PCR, Software

Journal: Burns & Trauma

Article Title: Overexpression of miR-101 suppresses collagen synthesis by targeting EZH2 in hypertrophic scar fibroblasts

doi: 10.1093/burnst/tkab038

Figure Lengend Snippet: Overexpression of miR-101 decreased fibrosis-related protein expression and proliferation. miR-101 RNA levels were analyzed using qRT-PCR normalized to U6 in HS ( a ) and HSF ( b ), compared with NS and NSF. Each data point was normalized against its corresponding U6 level. ( c ) FISH staining for miR-101 using serial sections of clinical HS and paired NS. Scale bar = 50 μm. ( d ) qRT-PCR analysis of the effects of mimics control, miR-101 mimics, inhibitor control and miR-101 inhibitor on the level of miR-101. qRT-PCR analysis ( e , f ) and representative immunoblots ( g ) showing the mRNA level and protein level changes of Col1, Col3 and α-SMA in HSF transfected with mimics control, miR-101 mimics, inhibitor control and miR-101 inhibitor. ( h , i ) Col1 and Col3 supernatant protein levels were determined using ELISA in the same treatment. Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, * * p < 0.01. qRT-PCR quantitative reverse-transcriptase polymerase chain reaction, U6 U6 small nuclear 1, HS hypertrophic scar, HSF hypertrophic scar fibroblast, NS normal skin, NSF normal skin fibroblast, FISH fluorescence in situ hybridization, ELISA enzyme-linked immunosorbent assay.

Article Snippet: Antibodies were as follows: anti-Col1 (rabbit, 1:1000; Abcam), anti-Col3 (rabbit, 1:3000; Abcam), anti-α-SMA (rabbit, 1:1000; Abcam), anti-EZH2 (rabbit, 1:1000; Abcam) and anti-GAPDH (Rabbit, 1:3000; Abcam).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Staining, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Fluorescence, In Situ Hybridization

Journal: Burns & Trauma

Article Title: Overexpression of miR-101 suppresses collagen synthesis by targeting EZH2 in hypertrophic scar fibroblasts

doi: 10.1093/burnst/tkab038

Figure Lengend Snippet: The role of EZH2 in regulating collagen and α-SMA expression in HSF. EZH2 mRNA ( a ) and protein expression ( b ) were measured using qRT-PCR and western blot analyses, respectively, in siRNA-EZH2 and siRNA-control transfected HSF. ( c ) RNA sequencing profiling of HSF in which EZH2 was knocked down. Shades of red represent increased gene expression; shades of blue represent decreased gene expression. ( d ) Pathway enrichment was done using the ClueGO and CluePedia plugins of the Cytoscape software. This analysis identified seven enriched pathways based on statistical analysis, with no duplication between clusters. Cluster one mainly includes positive regulation of collagen production ( e ) and fibroblasts responsible for TGF-β activation ( f ). Immunoblotting ( g ) or qRT-PCR ( h ) assessing the effect of EZH2-siRNA on the expression of Col1, Col3 and α-SMA in HSF. ( i , j ) ELISA of Col1 and Col3 supernatant protein levels in EZH2 knocked down HSF. EZH2 enhancer of zeste homolog 2, qRT-PCR quantitative reverse-transcriptase polymerase chain reaction, HSF hypertrophic scar fibroblast, ELISA enzyme-linked immunosorbent assay

Article Snippet: Antibodies were as follows: anti-Col1 (rabbit, 1:1000; Abcam), anti-Col3 (rabbit, 1:3000; Abcam), anti-α-SMA (rabbit, 1:1000; Abcam), anti-EZH2 (rabbit, 1:1000; Abcam) and anti-GAPDH (Rabbit, 1:3000; Abcam).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, RNA Sequencing Assay, Software, Activation Assay, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction

Journal: Burns & Trauma

Article Title: Overexpression of miR-101 suppresses collagen synthesis by targeting EZH2 in hypertrophic scar fibroblasts

doi: 10.1093/burnst/tkab038

Figure Lengend Snippet: miR-101 targets EZH2 in HSF. Expression levels of miR-101 are negatively correlated with EZH2 expression in HS ( a ) and HSF ( b ). Pearson’s correlation analysis of the relative expression levels of miR-101 (normalized to U6) and EZH2 (normalized to GAPDH) was determined using qRT-PCR in nine paired HS and HSF samples. ( c ) Binding of miR-101 to the wild type EZH2 3′ UTR (WT-EZH2-UTR) and the designed mutant (Mut-EZH2-UTR). ( d ) Dual-luciferase reporter assay showing that miR-101 mimics reduced luciferase activity in HEK293A cells after transfection with WT- EZH2-UTR, but not with the Mut- EZH2-UTR vector. Analysis of expression levels of EZH2 in HSF infection with miR-101 and miR-101 inhibitor using qRT-PCR ( e ) and western blot ( f ) analyses. Western blot analysis of Col1, Col3 and α-SMA protein expression in HSF treated with miR-101 mimics or infected with lentivirus overexpressing EZH2. ( h ) Western blot analysis of Col1, Col3 and α-SMA protein expression in HSF treated with miR-101 inhibitor or EZH2 siRNA. ELISA analysis of Col1 ( i ) and Col3 ( j ) supernatant protein level treated with miR-101 mimics or infected with lentivirus overexpressing EZH2. ELISA analysis of Col1 ( k ) and Col3 ( l ) supernatant protein level treated with miR-101 inhibitor or EZH2 siRNA. EZH2 enhancer of zeste homolog 2, HS hypertrophic scar, HSF hypertrophic scar fibroblast, U6 U6 small nuclear 1, UTR untraslated region, WT wild type, Mut mutant, HEK293A human embryonic kidney 293A cell, ELISA enzyme-linked immunosorbent assay

Article Snippet: Antibodies were as follows: anti-Col1 (rabbit, 1:1000; Abcam), anti-Col3 (rabbit, 1:3000; Abcam), anti-α-SMA (rabbit, 1:1000; Abcam), anti-EZH2 (rabbit, 1:1000; Abcam) and anti-GAPDH (Rabbit, 1:3000; Abcam).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Microparticles derived from human erythropoietin mRNA-transfected mesenchymal stem cells inhibit epithelial-to-mesenchymal transition and ameliorate renal interstitial fibrosis

doi: 10.1186/s13287-020-01932-z

Figure Lengend Snippet: qRT-PCR primers

Article Snippet: After blocking with 5% skim milk, the membranes were incubated with primary antibodies against E-cadherin (1:500; BD Biosciences, Bedford, UK), α-SMA (1:10000; R&D, Minneapolis, IL, USA), fibronectin (1:1000; Santa Cruz, CA, USA), phospho-p38 (1:1000; Santa Cruz), p38 (1:1000; Abcam, Cambridge, UK), phospho-Smad2 (1:500; Millipore), Smad2 (1:1000; Santa Cruz), phospho-Smad3 (1:500; Abcam), Smad3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Microparticles derived from human erythropoietin mRNA-transfected mesenchymal stem cells inhibit epithelial-to-mesenchymal transition and ameliorate renal interstitial fibrosis

doi: 10.1186/s13287-020-01932-z

Figure Lengend Snippet: MPs and rhEPO attenuate TGF-β1-induced EMT in MDCK cells. A Morphological changes in TGF-β1-treated MDCK cells observed by phase contrast microscopy. Compared to control MDCK cells (a), TGF-β1 treatment changed MDCK cell morphology from cuboidal to an elongated spindle-like shape (b). These morphological changes were reversed by co-treatment with MOCK-MPs (c), hEPO-MPs (d), and rhEPO (e). Fluorescence microscopy of red fluorescent CellTracker™-labeled MP incorporation into MDCK cells (m, n, r, s). Blue staining represents nuclear counterstaining with DAPI (f–j, p–t). B RT-PCR of hEPO mRNA expression in MDCK cells co-treated with hEPO-MPs, demonstrating that MPs mediated the horizontal transfer of hEPO mRNA into target MDCK cells. C Immunofluorescence microscopy of E-cadherin, vimentin, α-SMA, and fibronectin in MDCK cells after co-treatment with TGF-β1 and MPs or rhEPO, indicating reduced E-cadherin immunostaining intensity in response to TGF-β1-treatment (b) and its reversal by co-treatment with MOCK-MPs (c), hEPO-MPs (d), and rhEPO (e). TGF-β1 treatment increased vimentin (g), α-SMA (l), and fibronectin (q) immunostaining intensity which was reversed by co-treatment with MOCK-MPs (h, m, r), hEPO-MPs (i, n, s), and rhEPO (j, o, t). Magnification, × 400. Scale bars, 100 μm (white)

Article Snippet: After blocking with 5% skim milk, the membranes were incubated with primary antibodies against E-cadherin (1:500; BD Biosciences, Bedford, UK), α-SMA (1:10000; R&D, Minneapolis, IL, USA), fibronectin (1:1000; Santa Cruz, CA, USA), phospho-p38 (1:1000; Santa Cruz), p38 (1:1000; Abcam, Cambridge, UK), phospho-Smad2 (1:500; Millipore), Smad2 (1:1000; Santa Cruz), phospho-Smad3 (1:500; Abcam), Smad3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Microscopy, Fluorescence, Labeling, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Immunostaining

Journal: Stem Cell Research & Therapy

Article Title: Microparticles derived from human erythropoietin mRNA-transfected mesenchymal stem cells inhibit epithelial-to-mesenchymal transition and ameliorate renal interstitial fibrosis

doi: 10.1186/s13287-020-01932-z

Figure Lengend Snippet: Effect of MPs or rhEPO on EMT markers in MDCK cells. A – C qRT-PCR of E-cadherin, vimentin, and α-SMA mRNA in MDCK cells co-treated with TGF-β1 and MPs or rhEPO. Compared to vehicle-treated MDCK cells, TGF-β1 treatment significantly decreased E-cadherin and increased vimentin and α-SMA mRNA expression. MOCK-MPs, hEPO-MPs, and rhEPO reversed these changes in E-cadherin, vimentin, and α-SMA mRNA expression. D Western blotting for E-cadherin, α-SMA, and fibronectin protein levels in MDCK cells co-treated with TGF-β1 and MPs or rhEPO. Compared to vehicle-treated MDCK cells, TGF-β1 significantly decreased E-cadherin expression ( E ) and increased α-SMA and fibronectin expression ( F , G ). Co-treatment with MOCK-MPs, hEPO-MPs, and rhEPO significantly ameliorated these changes induced by TGF-β1. Western blotting data were normalized to GAPDH. Molecular weights are shown in kDa. Data are presented as mean ± SD; n = 4~5 for each experimental group. a P < 0.05 vs. vehicle, b P < 0.05 vs. TGF-β1, c P < 0.05 vs. TGF-β1+MOCK-MPs, d P < 0.05 vs. TGF-β1+hEPO-MPs, e P < 0.05 vs. TGF-β1+rhEPO

Article Snippet: After blocking with 5% skim milk, the membranes were incubated with primary antibodies against E-cadherin (1:500; BD Biosciences, Bedford, UK), α-SMA (1:10000; R&D, Minneapolis, IL, USA), fibronectin (1:1000; Santa Cruz, CA, USA), phospho-p38 (1:1000; Santa Cruz), p38 (1:1000; Abcam, Cambridge, UK), phospho-Smad2 (1:500; Millipore), Smad2 (1:1000; Santa Cruz), phospho-Smad3 (1:500; Abcam), Smad3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Microparticles derived from human erythropoietin mRNA-transfected mesenchymal stem cells inhibit epithelial-to-mesenchymal transition and ameliorate renal interstitial fibrosis

doi: 10.1186/s13287-020-01932-z

Figure Lengend Snippet: Anti-fibrotic effects of MPs or rhEPO in UUO kidneys. A Microscopic analysis of Masson’s trichrome and Sirius red-stained UUO kidney sections. Sham-operated control (a, f), obstructed kidney treated with vehicle control (b, g), and UUO kidneys treated with MOCK-MPs (c, h), hEPO-MPs (d, i), and rhEPO (e, j). MPs and rhEPO significantly attenuated renal interstitial fibrosis as quantified using Image J and MetaMorph software ( B , C ). Immunohistochemical analysis of α-SMA expression in sham-operated control (k) and UUO kidneys treated with the vehicle control (l), MOCK-MPs (m), hEPO-MPs (n), and rhEPO (o). MPs and rhEPO significantly attenuated α-SMA expression in obstructed kidneys ( D ). Immunohistochemical analysis of F4/80-positive cells in sham-operated control (p) and UUO kidneys treated with vehicle control (q), MOCK-MPs (r), hEPO-MPs (s), and rhEPO (t). MPs and rhEPO significantly attenuated the infiltration of F4/80-positive macrophage/inflammatory cells in obstructed kidneys. Quantitative assessment of renal F4/80-positive macrophage/inflammatory cells ( E ). Magnification, × 400 and × 1000 (for inserts). Scale bars, 50 μm (black). Data are presented as the mean ± SD; n = 5 for each experimental group. a P < 0.05 vs. sham control, b P < 0.05 vs. UUO+vehicle, c P < 0.05 vs. UUO+MOCK-MPs, d P < 0.05 vs. UUO+hEPO-MPs

Article Snippet: After blocking with 5% skim milk, the membranes were incubated with primary antibodies against E-cadherin (1:500; BD Biosciences, Bedford, UK), α-SMA (1:10000; R&D, Minneapolis, IL, USA), fibronectin (1:1000; Santa Cruz, CA, USA), phospho-p38 (1:1000; Santa Cruz), p38 (1:1000; Abcam, Cambridge, UK), phospho-Smad2 (1:500; Millipore), Smad2 (1:1000; Santa Cruz), phospho-Smad3 (1:500; Abcam), Smad3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Staining, Software, Immunohistochemical staining, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Microparticles derived from human erythropoietin mRNA-transfected mesenchymal stem cells inhibit epithelial-to-mesenchymal transition and ameliorate renal interstitial fibrosis

doi: 10.1186/s13287-020-01932-z

Figure Lengend Snippet: Western blotting of α-SMA, collagen I, fibronectin, and TGF-β1 in UUO kidneys. Compared to the sham-operated control, the obstructed kidneys of vehicle-treated mice showed significantly lower E-cadherin ( B ) and higher α-SMA ( C ), collagen I ( D ), fibronectin ( E ), and TGF-β1 ( F ) expression. Treatment with MOCK-MPs, hEPO-MPs, and rhEPO significantly reversed these changes. Protein levels were calculated using NIH Image J software. Western blot data were normalized to GAPDH. Molecular weights are shown in kDa. Data are presented as the mean ± SD; n = 5 for each experimental /group. a P < 0.05 vs. sham control, b P < 0.05 vs. UUO+vehicle, c P < 0.05 vs. UUO+MOCK-MPs, d P < 0.05 vs. UUO+hEPO-MPs, e P < 0.05 vs. UUO+rhEPO

Article Snippet: After blocking with 5% skim milk, the membranes were incubated with primary antibodies against E-cadherin (1:500; BD Biosciences, Bedford, UK), α-SMA (1:10000; R&D, Minneapolis, IL, USA), fibronectin (1:1000; Santa Cruz, CA, USA), phospho-p38 (1:1000; Santa Cruz), p38 (1:1000; Abcam, Cambridge, UK), phospho-Smad2 (1:500; Millipore), Smad2 (1:1000; Santa Cruz), phospho-Smad3 (1:500; Abcam), Smad3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Expressing, Software

Journal: Journal of the American Society of Nephrology

Article Title: Thrombospondin 2 Functions as an Endogenous Regulator of Angiogenesis and Inflammation in Experimental Glomerulonephritis in Mice

doi: 10.1681/asn.2006080873

Figure Lengend Snippet: Figure 2. During anti-GBM nephritis, the lack of TSP2 neither leads to compensatory upregulation of TSP1 nor influences TGF- activation. During anti-GBM nephritis, TSP1 was de- tected predominantly in cells of Bowman’s capsule, in the periglomerular tubulointerstitium (A), and in some cases within the glomerulus (B), as assessed by immunohistochem- istry (brown staining). Semiquantitative evaluation of cortical TSP1 revealed peak expression at approximately days 14 to 21 in both groups (C). Phosphorylated Smad 2/3, as an indirect indicator of activated TGF-, was evaluated by immunohisto- chemistry followed by computer-assisted analysis using Meta- Vue software (D). Magnifications: 400 in A and B.

Article Snippet: To perform immunostaining, we incubated tissue sections with the following primary and secondary antibodies: 19A2, a murine IgG mAb against proliferating cell nuclear antigen (PCNA; Chemicon, Temecula, CA), an indicator of actively proliferating cells; a rat monoclonal IgG2a to mouse CD4 antigen (Caltag Laboratory, Burlingame, CA); a rat monoclonal IgG2a to mouse CD8a antigen (Caltag Laboratory [18]); F4/80, a murine IgG1 mAb to a surface receptor that is present on monocytes, macrophages, and dendritic cells (Caltag Laboratory); MECA-32, a murine IgG1 mAb that is specific for detecting endothelial cells (a gift from R. Hallmann, University of Münster, Münster, Germany); a murine IgG2 mAb to -smooth muscle actin ( -SMA; Dako, Hamburg, Germany) (21); a rabbit polyclonal antibody to collagen I (Biogenesis, New Fields, UK); a biotinylated polyclonal antibody to human collagen IV (Southern Biotechnology Associates, Birmingham, UK); a murine IgG1 mAb against TSP1 (Dunn, Labortechnik, Asbach, Germany [21]); a rabbit polyclonal antibody to TSP2 (23); and a rabbit polyclonal antibody to phosphorylated Smad 2/3 (Santa Crutz Biotechnology, Santa Cruz, CA [10]).

Techniques: Activation Assay, Staining, Expressing, Immunohistochemistry, Software