Journal: PLoS ONE

Article Title: Purinergic Signaling as a Regulator of Th17 Cell Plasticity

doi: 10.1371/journal.pone.0157889

Figure Lengend Snippet: ATP hydrolysis by CD39 on Th17 TGF-β1 cells promotes their conversion into IL-10-producing cells. (A) IL-10 production by IL-17-GFP+ Th17 TGF-β1 and Th17 IL-23 cells restimulated for 3 days with anti-CD3 and anti-CD28 antibodies in the presence and absence of Tr1 polarizing cytokines (TGF-β1, IL-21 and IL-27), 50 μM ATP and 250 μM ARL67156. IL-10 production was analyzed by CBA (n = 4). (B) Th17 IL-23 cells from wild-type and P2X7R knockout mice were restimulated for 3 days with anti-CD3 and anti-CD28 antibodies and IL-10 production was analyzed by CBA (n = 3). Data are presented as mean ± S.E.M. *p

Article Snippet: To generate Th17TGF-β1 cells, CD4+ T cells were differentiated in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience), 20 ng/ml recombinant mouse IL-6 (eBioscience), 10 ng/ml IL-1β (eBioscience) and 5 μg/ml of anti-IFN-γ (clone XMG1.2, Biolegend) and then reactivated for another 3 days in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience) and 20 ng/ml recombinant mouse IL-6 (eBioscience).

Techniques: Crocin Bleaching Assay, Knock-Out, Mouse Assay

Journal: PLoS ONE

Article Title: Purinergic Signaling as a Regulator of Th17 Cell Plasticity

doi: 10.1371/journal.pone.0157889

Figure Lengend Snippet: In vitro -generated Th17 TGF-β1 but not Th17 IL-23 cells express CD39 and CD73 ectonucleotidases. (A) IL-17-GFP expression in Th17 cells differentiated in the presence of TGF-β1 and IL-6 (Th17 TGF-β1 cells) or IL-23, TGF-β3 and IL-1β (Th17 IL-23 cells). (B) Percentage of IL-17-GFP+ cells among total CD4+ cells (n = 12). (C and D) FACS analysis and mean fluorescence intensity for RORγt, T-bet, GATA-3 and Foxp3 transcription factors in IL-17-GFP+ Th17 cells (n = 4). (E and F) Representative FACS analysis and mean fluorescence intensity for CD39 and CD73 ectonucleotidases (n = 7). (G) CD49b and Lag-3 (n = 4) expression in IL-17-GFP+ Th17 cells. (H) Percentage of CD49b+/Lag-3+ cells and percentage of CD49b-/Lag-3+ cells in IL-17-GFP+ Th17 cells (n = 4). (I and J) CCR6 expression in IL-17-GFP+ Th17 cells (n = 6). Data are presented as mean ± S.E.M. *p

Article Snippet: To generate Th17TGF-β1 cells, CD4+ T cells were differentiated in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience), 20 ng/ml recombinant mouse IL-6 (eBioscience), 10 ng/ml IL-1β (eBioscience) and 5 μg/ml of anti-IFN-γ (clone XMG1.2, Biolegend) and then reactivated for another 3 days in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience) and 20 ng/ml recombinant mouse IL-6 (eBioscience).

Techniques: In Vitro, Generated, Expressing, FACS, Fluorescence

Journal: PLoS ONE

Article Title: Purinergic Signaling as a Regulator of Th17 Cell Plasticity

doi: 10.1371/journal.pone.0157889

Figure Lengend Snippet: Th17 TGF-β1 cells present a regulatory phenotype. (A) IL-17-GFP+ Th17 TGF-β1 and Th17 IL-23 cells were sorted and then analyzed by real-time PCR for mRNA expression of several transcription factors and cytokines (n = 3). (B) IL-17-GFP+ Th17 TGF-β1 and Th17 IL-23 cells were sorted and then reactivated for 4 hrs with PMA plus ionomycin to assess cytokine production by CBA or (C) in the presence of PMA, ionomycin and brefeldin A to analyze GM-CSF production by FACS (n = 5). (D) Percentage of GM-CSF+ cells within IL-17-GFP+ Th17 TGF-β1 and Th17 IL-23 cells (n = 5). Data are presented as mean ± S.E.M. *p

Article Snippet: To generate Th17TGF-β1 cells, CD4+ T cells were differentiated in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience), 20 ng/ml recombinant mouse IL-6 (eBioscience), 10 ng/ml IL-1β (eBioscience) and 5 μg/ml of anti-IFN-γ (clone XMG1.2, Biolegend) and then reactivated for another 3 days in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience) and 20 ng/ml recombinant mouse IL-6 (eBioscience).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Crocin Bleaching Assay, FACS

Journal: PLoS ONE

Article Title: Purinergic Signaling as a Regulator of Th17 Cell Plasticity

doi: 10.1371/journal.pone.0157889

Figure Lengend Snippet: Th17 TGF-β1 but not Th17 IL-23 cells hydrolyze ATP to adenosine in a CD39-and CD73-dependent manner and survive in the presence of high doses of ATP. IL-17-GFP+ Th17 TGF-β1 and Th17 IL-23 cells were sorted and cultured for 1 hr with 10 μM ATP in the presence of 50 μM ARL67156 or 50 μM APCP. Supernatants were then analyzed by HPLC to assess (A and B) ATP and (C and D) AMP hydrolysis (n = 5). (E) Representative FACS analysis of Th17 cell survival (Annexin V-/PI-) in the presence of graded doses of ATP. (F) Percentage of Th17 cell survival in the presence of ATP (n = 3). (G) IL-17-GFP+ Th17 TGF-β1 and Th17 IL-23 cells were sorted and then analyzed by real-time PCR to assess mRNA encoding P2X7 receptor (n = 4). Data are presented as mean ± S.E.M. *p

Article Snippet: To generate Th17TGF-β1 cells, CD4+ T cells were differentiated in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience), 20 ng/ml recombinant mouse IL-6 (eBioscience), 10 ng/ml IL-1β (eBioscience) and 5 μg/ml of anti-IFN-γ (clone XMG1.2, Biolegend) and then reactivated for another 3 days in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience) and 20 ng/ml recombinant mouse IL-6 (eBioscience).

Techniques: Cell Culture, High Performance Liquid Chromatography, FACS, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Purinergic Signaling as a Regulator of Th17 Cell Plasticity

doi: 10.1371/journal.pone.0157889

Figure Lengend Snippet: Th17 TGF-β1 cells are less colitogenic than Th17 IL-23 cells and produce IL-10 and IFN-γ in vivo . 1.3x10 6 IL-17-GFP+ Th17 TGF-β1 and Th17 IL-23 cells were transferred to Rag1 -/- mice. (A) The weight of mice was measured over the course of 6 weeks after adoptive transfer of Th17 cells (n = 5–8 mice per group). (B) Colon length was measured 6 weeks following transfer of Th17 cells (n = 5). (C) Clinical score was calculated based on weight loss and colon length 6 weeks after adoptive transfer of Th17 cells (n = 5). (D) Colonic histopathology. H E and alcian blue staining, original magnification 20X. Scale bar 100 μm (E) To determine intestinal cytokine production, intestinal tissues of Th17 recipient mice were cultured for 24 hs at 37°C and 5% CO 2 and production of several cytokines was analyzed by CBA (n = 6). (F and G) Representative FACS analysis of IL-17, IL-10 and IFN-γ production by Th17 TGF-β1 and Th17 IL-23 cells 6 weeks after adoptive transfer to Rag1 -/- mice. Data are presented as mean ± S.E.M. *p

Article Snippet: To generate Th17TGF-β1 cells, CD4+ T cells were differentiated in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience), 20 ng/ml recombinant mouse IL-6 (eBioscience), 10 ng/ml IL-1β (eBioscience) and 5 μg/ml of anti-IFN-γ (clone XMG1.2, Biolegend) and then reactivated for another 3 days in the presence of 2 ng/ml recombinant human TGF-β1 (eBioscience) and 20 ng/ml recombinant mouse IL-6 (eBioscience).

Techniques: In Vivo, Mouse Assay, Adoptive Transfer Assay, Histopathology, Staining, Cell Culture, Crocin Bleaching Assay, FACS

Journal: Scientific Reports

Article Title: Interferon-γ enhances the therapeutic effect of mesenchymal stem cells on experimental renal fibrosis

doi: 10.1038/s41598-020-79664-6

Figure Lengend Snippet: Anti-fibrotic effects of IFN-γ-treated mesenchymal stem cells (MSCs) in the kidney of IRI rats. IFN-γ-treated or untreated rat MSCs were injected immediately after IRI induction. Twenty-one days later, protein levels of fibrosis markers were evaluated by western blot ( a ) and immunohistochemical ( b , c ) analyses. ( a ) Western blot analysis of α-SMA and TGF-β1 in the rat kidney cortex. Protein levels were normalized to GAPDH levels (n = 5 in each group). The blots are the cropped images from different parts of the same gel. Full-length gel images are provided in the supplementary file. The samples derive from the same experiment and that gels were processed in parallel. ( b ) Representative immunohistochemical staining of α-SMA, Collagen type I (Col-I), and Collagen type III (Col-III) in kidney sections. (scale bar, 100 µm). ( c ) Quantification of α-SMA-, Col-I-, and Col-III-positive areas (n = 5 in each group). Data are presented as the mean ± SD. ** p

Article Snippet: Cell treatment with TGF-β1After incubation in DMEM supplemented with 0.1% FBS, CM from control hMSCs, or CM from hMSCs treated with IFN-γ for 24 h, human HK-2 cells were treated with 10 ng/ml recombinant human TGF-β1 (R & D Systems, Minneapolis, MN).

Techniques: Injection, Western Blot, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Interferon-γ enhances the therapeutic effect of mesenchymal stem cells on experimental renal fibrosis

doi: 10.1038/s41598-020-79664-6

Figure Lengend Snippet: Inhibitory effects of PTGES siRNA on the anti-fibrotic effects of IFN-γ-treated rMSCs in IRI rats. After rMSCs transfected with NC siRNA or PTGES siRNA were treated with IFN-γ, the cells were injected into rats immediately after IRI induction. Twenty-one days later, protein levels of fibrosis markers in the kidney were evaluated by western blot and immunohistochemical analyses. ( a ) Secreted PGE2 levels in CM from IFN-γ-treated rMSCs transfected with NC siRNA or PTGES siRNA were evaluated by an ELISA (n = 5 in each group). ( b , c ) Western blot analysis of α-SMA and TGF-β1 in the rat kidney cortex of IRI rats. Protein levels were normalized to GAPDH levels (n = 4 in each group). The blots are the cropped images from different parts of the same gel. Full-length gel images are provided in the supplementary file. The samples derive from the same experiment and that gels were processed in parallel. Sham, non-IRI procedure; PBS, PBS injection; NC siRNA, injection of IFN-γ rMSCs transfected with NC siRNA; PTGES siRNA, injection of IFN-γ rMSCs transfected with PTGES siRNA. ( d ) Representative immunohistochemical staining of α-SMA and Col-III in kidney sections (scale bar, 100 µm). ( e ) Quantification of α-SMA- and Col-III-positive areas (n = 4 in each group). Data are presented as the mean ± SD. * p

Article Snippet: Cell treatment with TGF-β1After incubation in DMEM supplemented with 0.1% FBS, CM from control hMSCs, or CM from hMSCs treated with IFN-γ for 24 h, human HK-2 cells were treated with 10 ng/ml recombinant human TGF-β1 (R & D Systems, Minneapolis, MN).

Techniques: Transfection, Injection, Western Blot, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Staining

Journal: Scientific Reports

Article Title: Interferon-γ enhances the therapeutic effect of mesenchymal stem cells on experimental renal fibrosis

doi: 10.1038/s41598-020-79664-6

Figure Lengend Snippet: DAMPs and the effects of MSCs treated with IFN-γ on renal fibrosis. ( a ) HMGB1 and IL-18 are members of DAMPs—HMGB1 was reported to promote the migration of MSCs, whereas IL-18 contributed to the secretion of IFN-γ. IFN-γ secreted from natural killer cells in injured tissues activates MSCs that exert anti-inflammatory effects. However, such activation require a long period of time, which delays the effect of MSCs administered for therapeutic purposes. ( b ) IFN-γ stimulation promotes the secretion of prostaglandin E2 from MSCs, and increased prostaglandin E2 induces polarization of immunosuppressive CD163-positive macrophages, suppressing the persistence of inflammation. MSCs treated with IFN-γ also directly inhibit profibrogenic TGF-β1/Smad signaling, leading to the prevention of fibrosis.

Article Snippet: Cell treatment with TGF-β1After incubation in DMEM supplemented with 0.1% FBS, CM from control hMSCs, or CM from hMSCs treated with IFN-γ for 24 h, human HK-2 cells were treated with 10 ng/ml recombinant human TGF-β1 (R & D Systems, Minneapolis, MN).

Techniques: Migration, Activation Assay

Journal: Scientific Reports

Article Title: Interferon-γ enhances the therapeutic effect of mesenchymal stem cells on experimental renal fibrosis

doi: 10.1038/s41598-020-79664-6

Figure Lengend Snippet: Paracrine effects of IFN-γ-treated human MSCs (hMSCs) on the TGF-β/Smad signaling pathway and phenotypic changes of macrophages. ( a , b ) After HK-2 cells, a human proximal tubular cell line , were incubated with conditioned medium (CM) obtained from IFN-γ-treated or untreated hMSCs for 24 h, the cells were treated with TGF-β1 for 30 min ( a ) or 24 h ( b ). ( a ) Western blot analysis of phosphorylated Smad2 (p-Smad2) and Smad2 in HK-2 cells. p-Smad2 protein levels were normalized to Smad2 levels (n = 5 in each group). ( b ) Western blot analysis of α-SMA in HK-2 cells. α-SMA protein levels were normalized to GAPDH levels (n = 5 in each group). The blots are the cropped images from different parts of the same gel. Full-length gel images are provided in the supplementary file. The samples derive from the same experiment and that gels were processed in parallel. Control hMSCs, CM from hMSCs without IFN-γ treatment; IFN-γ hMSCs, CM from hMSCs treated with IFN-γ. ( c ) Concentration of PGE2 in CM from hMSCs was evaluated by an ELISA at 24 or 48 h after IFN-γ stimulation. HK-2 cells were used as a negative control (n = 5 in each group). HK-2, CM from HK-2 cells. ( d ) Phenotypic changes of macrophages were evaluated by CD163 and CD68 protein levels. To induce differentiation of monocytic THP-1 cells into macrophages, the cells were treated with PMA for 48 h. The induced THP-1 cells were incubated with CM from IFN-γ-treated or untreated hMSCs. After 48 h, the cells were harvested and subjected to western blot analysis of CD163, CD206 and CD68. CD163 and CD206 protein levels were normalized to the GAPDH and CD68 levels (n = 5 in each group). The blots are the cropped images from different parts of the same gel. Full-length gel images are provided in the supplementary file. The samples derive from the same experiment and that gels were processed in parallel. Data are presented as the mean ± SD. * p

Article Snippet: Cell treatment with TGF-β1After incubation in DMEM supplemented with 0.1% FBS, CM from control hMSCs, or CM from hMSCs treated with IFN-γ for 24 h, human HK-2 cells were treated with 10 ng/ml recombinant human TGF-β1 (R & D Systems, Minneapolis, MN).

Techniques: Incubation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Scientific Reports

Article Title: Up-regulated miR-133a orchestrates epithelial-mesenchymal transition of airway epithelial cells

doi: 10.1038/s41598-018-33913-x

Figure Lengend Snippet: TGFβ1 and miR-133a target ESRP1 pathways in airway epithelial cells. ( a ) Beas-2b cells were treated with 5 ng/ml TGFβ1 for the indicated time. ( b ) Beas-2b and NHBE cells were mock transfected, or transfected with control or miR-133a mimics. Cells were harvested 3 days later for western blot analysis of indicated proteins. Experiments were conducted at least three times, and representative results are shown. The grouped blots in ( a ) were cropped from different parts of the same gel. The anti-p120ctn and anti-GAPDH blots in ( b .

Article Snippet: To examine TGFβ1-induced EMT, Beas-2b cells were seeded a day prior in 6 well or 12 well plates, and then stimulated at ~20% confluence without or with 5 ng/ml of recombinant human TGFβ1 (R & D Systems, Minneapolis, MN) in complete BEGM medium.

Techniques: Transfection, Western Blot

Journal: Nature medicine

Article Title: Epithelial to Mesenchymal Transition induces cell cycle arrest and parenchymal damage in renal fibrosis

doi: 10.1038/nm.3902

Figure Lengend Snippet: EMT program G2 cell cycle arrest. ( a ) Percent E–cadherin + pH3 + cells in kidneys from the indicated experimental groups. WT contralat., n = 4; Twist cKO contralat., n = 5; WT UUO, n = 4; Twist cKO UUO, n = 5. WT contralat., n = 3; Snail cKO contralat., n = 3; WT UUO, n = 4; Snail cKO UUO, n = 3. ( b ) Representative images (5 visual fields for each tissue analyzed) of immunolabeling for YFP, AQP1 and pH3 and percentage of αSMA + cells out of the YFP + pH3 + TECs (contralat., n = 6; UUO n = 3). Scale bar, 100 ~m. ( c ) Relative Twist1 expression in indicated cells. ( d ) Representative images of brightfield imaging in indicated cells. ( e ) Representative images (3 visual fields for each tissue analyzed) of immunolabeling for β–catenin and percent nuclear accumulation. Scale bar, 50 ~m. ( f,g ) Representative images (3 visual fields for each tissue analyzed) of immunolabeling for Ki67 and pH3 ( f ) and percent cells in G2 ( g ). Scale bar, 50 ~m. ( h ) Representative images of brightfield imaging in indicated cells. ( i ) Representative images (3 visual fields for each tissue analyzed) of immunolabeling for β–catenin and percent nuclear accumulation. Scale bar, 50 ~m. ( j ) Representative images (3 visual fields for each tissue analyzed) of immunolabeling for Ki67 and pH3 ( f) and percent cells in G2 ( g ). Scale bar, 50 ~m. Vehicle (vh) and TGF-β1 treatment were conducted for 24 hours, followed by vehicle or TGF-β1 withdrawal for 24 hours, n = 3. Data is represented as mean ± SEM. Hoechst: nucleus. One–way ANOVA with Tukey post–hoc analysis. b , unpaired one–tailed t–test. * P

Article Snippet: For the induction of EMT, MCT cells were treated with 5 ng/ml human recombinant TGF-β1 (R & D Systems) for 24 hours and HK2 cells were treated with 10 ng/ml TGF-β1 for 72 hours in serum free medium.

Techniques: Immunolabeling, Expressing, Imaging, One-tailed Test

Journal: Nature medicine

Article Title: Epithelial to Mesenchymal Transition induces cell cycle arrest and parenchymal damage in renal fibrosis

doi: 10.1038/nm.3902

Figure Lengend Snippet: EMT program is associated with deregulated expression and functionality of TEC transporter in subjects with kidney disease. ( a ) Relative expression of the indicated genes in renal biopsies from healthy individuals and subjects with renal fibrosis. ( b–c ) Representative images of H E ( b ) and MTS ( c ) staining of kidneys from human biopsies. ( d–e ) Representative images (3 visual fields for each tissue analyzed) of co–immunolabeling for Collagen IV and AQP1 ( d ), and Collagen IV and Na + /K + ATPase ( e ) and associated quantification. Scale bar, 50 ~m. ( f ) Relative expression of the indicated genes in TECs isolated from renal biopsies from healthy individuals and subjects with renal fibrosis. ( g ) Na + /K + ATPase activity assay, Pi: inorganic phosphate. ( h ) Relative expression of the indicated genes in HK2 cells with and without TGF-β1 treatment, n = 3. ( i ) Na + /K + ATPase activity assay of HK2 cells treated with vehicle or TGF-β1. ( j ) Transporter uptake assays of vehicle or TGF-β1–treated HK2 cells cultured with radio–labeled substrates in the presence or absence of unlabeled inhibitors. Data is presented as mean ± SEM. Vehicle (vh) and TGF-β1 treatment were conducted for 72 hours, n = 3. a and f , error bars denote variation in technical replicates, all other error bars depict variation in biological replicates. d,e and h,i , unpaired two–tailed t–test was used. * P

Article Snippet: For the induction of EMT, MCT cells were treated with 5 ng/ml human recombinant TGF-β1 (R & D Systems) for 24 hours and HK2 cells were treated with 10 ng/ml TGF-β1 for 72 hours in serum free medium.

Techniques: Expressing, Staining, Immunolabeling, Isolation, Activity Assay, Cell Culture, Labeling, Two Tailed Test

Journal: Nature medicine

Article Title: Epithelial to Mesenchymal Transition induces cell cycle arrest and parenchymal damage in renal fibrosis

doi: 10.1038/nm.3902

Figure Lengend Snippet: p21 controls EMT program G2 cell cycle arrest. ( a ) Relative expression of Twist1 Snai1 and Cdkn1a (p21) in MCT cells transfected with empty vector, Twist or Snail overexpression vectors, 24 hours post transfection, n = 3. ( b ) Representative images (3 visual fields for each tissue analyzed) of vehicle or MCT cells transfected with empty vector, Twist or Snail overexpression vectors, 24 hours post transfection, immunolabeled for Ki67 and pH3 and respective quantification of the percentage of cells in G2 phase, n = 3. Scale bar: 50 ~m. ( c ) Phase contrast light microscopy and immunolabeling for Ki67 and pH3 of MCT shScrbl and MCT shTwist cells transfected with empty or Twist overexpression (OE Twist) plasmids and treated with vehicle or TGF-β1. ( d ) Quantification of the percentage of empty or Twist OE transfected MCT shScrbl and MCT shTwist cells in G2 phase of the cell cycle comparing cells treated with vehicle or TGF-β1, n = 3. ( e ) Relative expression of Cdkn1a (p21). ( f ) Representative images (3 visual fields for each tissue analyzed) of immunolabeling for Ki67 and pH3 of control or p21 siRNA–transfected MCT cells and percent of cells in G2 phase. Scale bar, 50 ~m. Data is represented as mean ± SEM. Hoechst: nucleus. One–way ANOVA with Tukey post–hoc analysis. b and e , unpaired one–tailed t–test. * P

Article Snippet: For the induction of EMT, MCT cells were treated with 5 ng/ml human recombinant TGF-β1 (R & D Systems) for 24 hours and HK2 cells were treated with 10 ng/ml TGF-β1 for 72 hours in serum free medium.

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Immunolabeling, Light Microscopy, One-tailed Test

Journal: Hepatology Communications

Article Title: Role of monocyte chemoattractant protein‐1 in liver fibrosis with transient myeloproliferative disorder in down syndrome

doi: 10.1002/hep4.1150

Figure Lengend Snippet: Monocyte chemoattractant protein‐1 mediates tumor growth factor β1‐primed fibrogenic activation of hepatic stellate cells. (A) Experimental approach to analyze the fibrogenic activation of hSCs. LX‐2 cells were treated with TGF‐ß1 for 1 hour and then supplied with fresh medium. MCP‐1 concentration in the culture supernatant was assessed at the indicated times. During cell cultivation, the culture medium was changed daily with or without the presence of MCP‐1 blocking antibody. At the end of the culturing phase, the cell count and type IV collagen protein levels were analyzed. (B) Assessment of extracellular secretion of MCP‐1 in the culture supernatant. Data represent mean ± SD. (C) Cell growth at 120 hours after transient TGF‐β1 stimulation (0, 10 ng/mL and 100 ng/mL) in the presence of either IgG control antibody or MCP‐1 blocking antibody ( # P

Article Snippet: Cells were stimulated with either recombinant human MCP‐1 (Z028029, GenScript, Piscataway, NJ) or TGF‐β1 (240‐B‐002, R & D Systems, Minneapolis, MN) and then incubated with either 1 μg/mL MCP‐1 blocking antibody (M2420; Sigma, St. Louis, MO) or mouse IgG (278‐810; Ancell, Bayport, MN).

Techniques: Activation Assay, Concentration Assay, Blocking Assay, Cell Counting

Journal: Scientific Reports

Article Title: Proline-rich tyrosine kinase 2 mediates transforming growth factor-beta-induced hepatic stellate cell activation and liver fibrosis

doi: 10.1038/s41598-020-78056-0

Figure Lengend Snippet: Inhibition of PYK2, but not FAK, suppresses upregulation of pro-fibrotic CTGF expression on stimuli of TGF-β1. ( A , B ) qPCR of fibrogenic gene expression in in vitro activated primary mouse hepatic stellate cell (mHSC) ( A ) and in LX2 ( B ), treated as indicated and shown as fold change (n = 3). ( C ) Western blot of α-SMA in mHSC and LX2 after treatments. ( D ) Western blot of activated forms of both FAK and PYK2, and CTGF in LX2 treated as indicated (n = 3). ( E ) qPCR of CTGF expression in LX2 that were treated with either of two FAK/PYK2 inhibitors (n = 3). ( F , G ) After transfection with siRNAs of negative control, FAK or PYK2, qPCR of the indicated gene expression ( F ) and western blot ( G ) were performed. Two FAK/PYK2 dual inhibitors were used: PF271, PF-573271; PF228, PF573228. Student's t-test; * p

Article Snippet: To activate TGF-β1 signaling in vitro, serum-starved cells were stimulated by 5 ng/ml recombinant human TGF-β1 protein (R & D Systems) for 1.5 h in most experiments, with the exception that 10 h or overnight treatment was required to induce expression of α-SMA and type I collagen.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, In Vitro, Western Blot, Transfection, Negative Control

Journal: Scientific Reports

Article Title: Proline-rich tyrosine kinase 2 mediates transforming growth factor-beta-induced hepatic stellate cell activation and liver fibrosis

doi: 10.1038/s41598-020-78056-0

Figure Lengend Snippet: Profibrogenic TGF-β1 activates YAP in PYK2-dependent manner in vivo and in vitro. ( A ) qPCR of YAP target genes expression in activated mHSC that were pretreated with DMSO or Verteporfin before TGF-β1 stimuli, and shown as fold change (n = 3). ( B ) qPCR of YAP target genes and SMAD7 mRNA expression (shown as fold change, n = 3) and western blot of YAP and TAZ (n = 2) in LX2 transfected with siRNA of control, YAP, or TAZ, followed by TGF-β1 stimuli. ( C ) YAP reporter assay in LX2 transfected with either IFN-luc or YAP-luc (8xGTIIC-luc) followed by TGF-β1 stimuli as indicated (n = 3). ( D ) qPCR of CTGF and CYR61 mRNA expression in LX2 that were transfected with either vector alone or YAP(wt) and then followed by the treatments as indicated (n = 3). ( E ) YAP reporter assay in LX2 transfected with siRNA of either control or PYK2, followed by 2nd transfection with vector alone or YAP(wt) (n = 2). ( F ) IHC co-staining of α-SMA and YAP in paraffin liver sections of CCl 4 -treated mouse (n = 4). Arrows indicate the co-localized regions. ( G ) Western of YAP expression in primary mHSCs at the indicated times on in vitro culture. ( H ) IHC of YAP in CCl 4 -treated mouse livers (n = 3) and western blot of YAP expression in livers from O + V (n = 3), O + CCl 4 (n = 3), CCl 4 + PF (n = 3). ( I ) IHC of α-SMA, p-PYK2(402), and YAP expression in serial liver sections from patients with cirrhosis (n = 4). The enlarged insets were shown below. The luciferase activities were normalized with co-transfected renilla luciferase activity. Scale bar: 200 μm in ( F , H ), 500 μm in ( I ), and 200 μm in insets of ( I ), Student's t-test; * p

Article Snippet: To activate TGF-β1 signaling in vitro, serum-starved cells were stimulated by 5 ng/ml recombinant human TGF-β1 protein (R & D Systems) for 1.5 h in most experiments, with the exception that 10 h or overnight treatment was required to induce expression of α-SMA and type I collagen.

Techniques: In Vivo, In Vitro, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Reporter Assay, Plasmid Preparation, Immunohistochemistry, Staining, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: Proline-rich tyrosine kinase 2 mediates transforming growth factor-beta-induced hepatic stellate cell activation and liver fibrosis

doi: 10.1038/s41598-020-78056-0

Figure Lengend Snippet: Diagram of PYK2-Src-RhoA triad functioning between TGF-β1 and YAP activation. TGF-β1 activates PYK2, and autophosphorylated PYK2 recruits and activates Src, which results in activation of RhoA. Upon RhoA activation, PYK2 and Src is further activated in a feedforward manner leading to signal amplification.

Article Snippet: To activate TGF-β1 signaling in vitro, serum-starved cells were stimulated by 5 ng/ml recombinant human TGF-β1 protein (R & D Systems) for 1.5 h in most experiments, with the exception that 10 h or overnight treatment was required to induce expression of α-SMA and type I collagen.

Techniques: Activation Assay, Amplification

Journal: Scientific Reports

Article Title: Proline-rich tyrosine kinase 2 mediates transforming growth factor-beta-induced hepatic stellate cell activation and liver fibrosis

doi: 10.1038/s41598-020-78056-0

Figure Lengend Snippet: PYK2 mediated CTGF induction by TGF-β1 is independent of Smad activation. ( A ) Western blot of activated forms of Smad2 and Smad3 upon TGF-β1 stimuli in LX2 transfected with siRNAs of negative control, FAK, or PYK2 (n = 3). ( B ) IF of Smad2 in LX2 grown on glass and treated as indicated (n = 4). Scale bar: 50 μm. ( C ) Western blot of Smad2 and Smad3 (n = 2) and ( D ) qPCR of indicated genes (n = 3) in LX2 transfected with siRNAs of control, Smad2, Smad3, or Pyk2. Student's t-test; * p

Article Snippet: To activate TGF-β1 signaling in vitro, serum-starved cells were stimulated by 5 ng/ml recombinant human TGF-β1 protein (R & D Systems) for 1.5 h in most experiments, with the exception that 10 h or overnight treatment was required to induce expression of α-SMA and type I collagen.

Techniques: Activation Assay, Western Blot, Transfection, Negative Control, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Proline-rich tyrosine kinase 2 mediates transforming growth factor-beta-induced hepatic stellate cell activation and liver fibrosis

doi: 10.1038/s41598-020-78056-0

Figure Lengend Snippet: PYK2-Src activation is required in TGF-β1 mediated CTGF upregulation. ( A ) Western blot of different phosphorylated forms of PYK2 as well as CTGF in LX2 pretreated with two SRC inhibitors (PP2 and Saracatinib) (n = 3). ( B ) qPCR of CTGF mRNA expression (shown as fold change, n = 3) and ( C ) Western blot of α-SMA in LX2 treated as indicated (n = 2). ( D ) Western blot of CTGF and different phosphorylated forms of PYK2 (n = 2) and ( E ) qPCR of CTGF, TGFB1, and SMAD7 mRNA expression (shown as fold change, n = 3) in LX2 transfected with siRNA of control or Src. Student's t-test; * p

Article Snippet: To activate TGF-β1 signaling in vitro, serum-starved cells were stimulated by 5 ng/ml recombinant human TGF-β1 protein (R & D Systems) for 1.5 h in most experiments, with the exception that 10 h or overnight treatment was required to induce expression of α-SMA and type I collagen.

Techniques: Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection

Journal: Scientific Reports

Article Title: Proline-rich tyrosine kinase 2 mediates transforming growth factor-beta-induced hepatic stellate cell activation and liver fibrosis

doi: 10.1038/s41598-020-78056-0

Figure Lengend Snippet: Activation of PYK2-Src-RhoA triad is essential for TGF-β1 mediated upregulation of CTGF. ( A ) G-LISA activation assay of Rho Small GTPase family in LX2 at different time points after TGF-β1 stimuli and shown as fold change (n = 4). ( B ) qPCR of CTGF mRNA expression (shown as fold change, n = 3) and ( C ) western of CTGF (n = 2) in LX2 transfected to overexpress the dominant-negative forms of Rho GTPase family members and followed by TGF-β1 stimuli. ( D ) qPCR of CTGF mRNA expression (shown as fold change, n = 3) and ( E ) western of activated forms of both PYK2 and Src, and CTGF (n = 2) in both activated mouse hepatic stellate cells and LX2 pretreated with Rho family inhibitors. ( F ) G-LISA assay of RhoA activation by TGF-β1 stimuli for 30 min in LX2 transfected with siRNA of control, PYK2, or Src (n = 3). ( G ) Western of α-SMA in both LX2 and activated primary mouse hepatic stellate cells that were pretreated with ROCK inhibitor Y27632 before TGF-β1 stimuli (n = 2). Student's t-test; * p

Article Snippet: To activate TGF-β1 signaling in vitro, serum-starved cells were stimulated by 5 ng/ml recombinant human TGF-β1 protein (R & D Systems) for 1.5 h in most experiments, with the exception that 10 h or overnight treatment was required to induce expression of α-SMA and type I collagen.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Dominant Negative Mutation

Journal: International Journal of Molecular Sciences

Article Title: Non-Canonical Regulation of Type I Collagen through Promoter Binding of SOX2 and Its Contribution to Ameliorating Pulmonary Fibrosis by Butylidenephthalide

doi: 10.3390/ijms19103024

Figure Lengend Snippet: BP reduced collagen I production in lung fibroblast pre-stimulated with recombinant TGF-β1. TGF-β (5 ng/mL) stimulate for 12 h and ( A ) mRNA and ( B ) protein expression levels of BP treatment in several dosages (0, 10, 20, and 30 µg/mL) for 24 h on type I collagen, and SOX2 expressions. ( C ) mRNA and ( D ) protein expression levels of BP treatment for several time points (0, 1, 3, 6, 12, 24, and 48 h) in the dosage of 30 µg/mL on type I collagen and SOX2 expressions. The same amount of DMSO was added in 0 µg/mL groups as a vehicle control. Data showed 3 independent qPCR experiments and presented are mean ± SD. * denotes a significant decrease with the 0 µg/ml group of p

Article Snippet: In vitro studies of lung fibrosis were performed in normal Human lung fibroblast (NHLF), which was stimulated into fibrogenesis by recombinant human TGF-β1 (PeproTech, Catalog Number: 100-21, 5 ng/mL) for 12 h.

Techniques: Recombinant, Expressing, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Transforming Growth Factor-β Promotes Morphomechanical Effects Involved in Epithelial to Mesenchymal Transition in Living Hepatocellular Carcinoma

doi: 10.3390/ijms20010108

Figure Lengend Snippet: Representative confocal images of PLC/PRF/5 CTR ( A ) and TGF-β treated for 48 h, PLC/PRF/5 TGF-β1 ( B ); the scale bar in the figures correspond to 20 µm. In the panel the value of F-actin coherency ( C ), nuclear area ( D ), circularity ( E ) and roundness ( F ) were reported. The analysis of F-actin and nuclear morphology of control and TGF-β1 treated PLC/PRF/5 cells, by using ImageJ software. Results were statistically significant for p

Article Snippet: After 24 h, some petri dishes of HepG2 and PLC/PRF/5 cells were stimulated for a whole 48 h with 5 ng/mL of human recombinant TGF-β1 (PEPROTEC, Rocky Hill, NJ, USA), specified as HepG2TGF-β1 , PLC/PRF/5TGF-β1 .

Techniques: Planar Chromatography, Software

Journal: International Journal of Molecular Sciences

Article Title: Transforming Growth Factor-β Promotes Morphomechanical Effects Involved in Epithelial to Mesenchymal Transition in Living Hepatocellular Carcinoma

doi: 10.3390/ijms20010108

Figure Lengend Snippet: The Young’s modulus values with standard deviation, calculated from the nuclear region and the cytoskeletal area respectively, were reported for untreated PLC/PRF/5 CTR and TGF-β1 treated PLC/PRF/5 TGF-β1 cells. Results were statistically significant for p

Article Snippet: After 24 h, some petri dishes of HepG2 and PLC/PRF/5 cells were stimulated for a whole 48 h with 5 ng/mL of human recombinant TGF-β1 (PEPROTEC, Rocky Hill, NJ, USA), specified as HepG2TGF-β1 , PLC/PRF/5TGF-β1 .

Techniques: Standard Deviation, Planar Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Transforming Growth Factor-β Promotes Morphomechanical Effects Involved in Epithelial to Mesenchymal Transition in Living Hepatocellular Carcinoma

doi: 10.3390/ijms20010108

Figure Lengend Snippet: The Young’s modulus values with standard deviation, calculated from the nuclear region and the cytoskeletal area respectively, were reported for Galunisertib and TGF-β1 concurrently treated HepG2 cells. Results were statistically significant for p

Article Snippet: After 24 h, some petri dishes of HepG2 and PLC/PRF/5 cells were stimulated for a whole 48 h with 5 ng/mL of human recombinant TGF-β1 (PEPROTEC, Rocky Hill, NJ, USA), specified as HepG2TGF-β1 , PLC/PRF/5TGF-β1 .

Techniques: Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Transforming Growth Factor-β Promotes Morphomechanical Effects Involved in Epithelial to Mesenchymal Transition in Living Hepatocellular Carcinoma

doi: 10.3390/ijms20010108

Figure Lengend Snippet: The Young’s modulus values with standard deviation, calculated from the nuclear region and the cytoskeletal area respectively, were reported for untreated HepG2 CTR and TGF-β1 treated HepG2 TGF-β1 cells. Results were statistically significant for p

Article Snippet: After 24 h, some petri dishes of HepG2 and PLC/PRF/5 cells were stimulated for a whole 48 h with 5 ng/mL of human recombinant TGF-β1 (PEPROTEC, Rocky Hill, NJ, USA), specified as HepG2TGF-β1 , PLC/PRF/5TGF-β1 .

Techniques: Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Transforming Growth Factor-β Promotes Morphomechanical Effects Involved in Epithelial to Mesenchymal Transition in Living Hepatocellular Carcinoma

doi: 10.3390/ijms20010108

Figure Lengend Snippet: Representative confocal images of HepG2 CTR ( A ) and transforming growth factor (TGF)-β treated for 48 h HepG2 TGF-β1 ( B ), the scale bar in the figures correspond to 20 µm. In the panel, the value of F-actin coherency ( C ), nuclear area ( D ), circularity ( E ) and roundness ( F ) were reported. The analysis of F-actin and nuclear morphology of control and TGF-β1 treated HepG2 cells, were performed by using ImageJ software. Results were statistically significant for p

Article Snippet: After 24 h, some petri dishes of HepG2 and PLC/PRF/5 cells were stimulated for a whole 48 h with 5 ng/mL of human recombinant TGF-β1 (PEPROTEC, Rocky Hill, NJ, USA), specified as HepG2TGF-β1 , PLC/PRF/5TGF-β1 .

Techniques: Software

Journal: PLoS Biology

Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

doi: 10.1371/journal.pbio.2004408

Figure Lengend Snippet: Mitochondrial p27 restores the impaired αSMA up-regulation in p27-deficient cardiac fibroblasts. Fibroblasts isolated from the hearts of p27-deficient mice were lentivirally transduced with an expression vector for mitochondrially targeted p27 (“mito p27”) or a corresponding empty vector (“EV”). (A) Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the mitochondrially targeted p27 by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. (B, C) Myofibroblast differentiation was induced by treatment with 2 ng/ml TGFβ1 for 48 hours, and αSMA was detected by immunoblot. (B) Representative immunoblots, Vimentin served as loading control. (C) Semiquantitative analysis of αSMA normalized to Vimentin. Data are mean ± SEM, n = 5, * p . αSMA; α smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; n.s., not significant; TGFβ1, transforming growth factor β1; TIM23, translocase of inner mitochondrial membrane 23.

Article Snippet: For the induction of myofibroblast differentiation, the cells were grown for 24 hours in DMEM GlutaMAX/1% fetal bovine serum/1% penicillin/streptomycin before recombinant human TGFβ1 (2 ng/ml; Peprotech) was added for another 48 hours.

Techniques: Isolation, Mouse Assay, Transduction, Expressing, Plasmid Preparation, Staining, Fluorescence, Western Blot

Journal: PLoS Biology

Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

doi: 10.1371/journal.pbio.2004408

Figure Lengend Snippet: p27 is required for myofibroblast differentiation of cardiac fibroblasts. Cardiac fibroblasts were isolated from hearts of wild-type (“wt”) mice and p27-deficient (“p27 −/−”) littermates. Myofibroblast differentiation was induced by treatment with 2 ng/ml TGFβ1 for 48 hours in the presence or absence of 50 μM caffeine. Induction of αSMA was detected by immunoblot and immunostaining. (A) Representative immunoblots, Vimentin served as loading control. (B) Semiquantitative analysis of αSMA normalized to Vimentin. Data are mean ± SEM, n = 8: wt untreated, wt +TGFβ1, p27 −/− untreated, p27 −/− +TGFβ1; n = 5: all others, * p

Article Snippet: For the induction of myofibroblast differentiation, the cells were grown for 24 hours in DMEM GlutaMAX/1% fetal bovine serum/1% penicillin/streptomycin before recombinant human TGFβ1 (2 ng/ml; Peprotech) was added for another 48 hours.

Techniques: Isolation, Mouse Assay, Immunostaining, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Distinct Anti-Fibrotic Effects of Exosomes Derived from Endothelial Colony-Forming Cells Cultured Under Normoxia and Hypoxia

doi: 10.12659/MSM.911306

Figure Lengend Snippet: In vitro effects of nor-exo and hyp-exo on Col-1α and α-SMA expression in cardiac fibroblasts. Cardiac fibroblasts were incubated with nor-exo (75 μg/ml) or GW4869-treated nor-exo (75 mg/ml) and then stimulated with TGF-β 1 (15 ng/ml) for 48 h ( A ). In part B , the anti-fibrotic effects of hyp-exo (75 mg/ml) and nor-exo (75 μg/ml) in combination with ATA (25 μM) were also detected. Col-1α was observed in cardiac fibroblasts treated with different exosomes using a fluorescence microscope ( C ). Mean ±SD, n=3, ANOVA followed by Tukey’s post-test. * P

Article Snippet: After the initiation of TGF-β (catalog no. 100-21, PeproTech, USA) stimulation (15 ng/ml), cardiac fibroblasts were cultured in basal medium with 0.1% fetal bovine serum for 48 h. Next, 75 μg/ml nor-exo or hyp-exo were added, and the effects of the exosomes were tested in vitro (see for dose testing).

Techniques: In Vitro, Expressing, Incubation, Fluorescence, Microscopy

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Distinct Anti-Fibrotic Effects of Exosomes Derived from Endothelial Colony-Forming Cells Cultured Under Normoxia and Hypoxia

doi: 10.12659/MSM.911306

Figure Lengend Snippet: Targeting of HDAC4 and Smurf1 mRNAs by miR-10b-5p. Predicted mRNA targets of miR-10b-5p that overlapped with the TGF-β signaling pathway ( A, B ). Validation of mRNA targets of miR-10b-5p using a dual-luciferase reporter gene assay ( C ). Effects of nor-exo and miR-10b-5p on HDAC4 and Smurf1 expression ( D–F ). Mean ±SD, n=3, ANOVA followed by Tukey’s post-test. * P

Article Snippet: After the initiation of TGF-β (catalog no. 100-21, PeproTech, USA) stimulation (15 ng/ml), cardiac fibroblasts were cultured in basal medium with 0.1% fetal bovine serum for 48 h. Next, 75 μg/ml nor-exo or hyp-exo were added, and the effects of the exosomes were tested in vitro (see for dose testing).

Techniques: Luciferase, Reporter Gene Assay, Expressing

Journal: Respiratory Research

Article Title: Human alveolar epithelial cells type II are capable of TGFβ-dependent epithelial-mesenchymal-transition and collagen-synthesis

doi: 10.1186/s12931-018-0841-9

Figure Lengend Snippet: Transcriptional analysis of hAECII upon TGFβ stimulation. hAECII different donors were stimulated each with 5 ng/ml TGβ1, 10 μM SB431542, 5 ng/ml TGFβ1 and 10 μM SB431542 or left untreated for 48 h ( N = 4). Significantly regulated genes were computed by Repeated-Measures ANOVA (RM-ANOVA) with a Benjamini-Hochberg multiple testing correction cut-off of ≤0.05. Averaged gene expression data of results from RM-ANOVA is shown as a Heatmap with Hierarchical Clustering of samples and entities according to Pearson Centered algorithm with Ward’s linkage rule. A Fold-Change Filter was applied to further analyze only probes that were at least ≥2 fold up-regulated compared to medium control ( a ). Out of those genes, a list of targets was extracted that was exclusively regulated by TGFβ and designated TGFβ fingerprint ( b ). The TGFβ fingerprint genes were further investigated for mutual interactions by querying the String protein-protein interaction database with those genes that exhibited an annotated GeneSymbol. A global map of these genes was constructed based on interaction scores within Cytoscape ( c ). The presence of intrinsic modules within the interactions of the TGFβ fingerprint w as computed by a spectral partition based cluster algorithm from the Reactome FIViz app and respective modules encoded by different colours. Further details of interactions within each module are shown in ( d ). Global enrichment of GSEA Hallmark gene sets ( e ) and Reactome pathways ( f ) from whole list of TGFβ fingerprint genes are displayed by their FDR q-value

Article Snippet: The cells were stimulated with 5 ng/ml of human recombinant TGFβ1 (Peprotech, Hamburg, Germany) as well as 10 μM of TGFβ receptor I kinase inhibitor SB431542 (Sigma Aldrich, Germany) for 48 h in an incubator with 37 °C and 5% CO2 ; each compound was reconstituted according to manufacturer’s instructions.

Techniques: Expressing, Construct

Journal: PLoS ONE

Article Title: The oligosaccharides 6’-sialyllactose, 2’-fucosyllactose or galactooligosaccharides do not directly modulate human dendritic cell differentiation or maturation

doi: 10.1371/journal.pone.0200356

Figure Lengend Snippet: TGFβ and VitD3 induce phenotypic distinct DCs. CD14+ monocytes were cultured in the presence of IL-4 and GM-CSF for six days in the presence or absence of breast milk components. Surface marker expression was measured by flow cytometry. A) A multi-colour overlay of CD14 expression versus CD1a expression on moDC (black), TGFβDC (blue) or VitD3 (red) of one representative donor is shown. The percentage of B) CD14+ and C) CD1a+ DC and relative surface marker expression of D) HLA-DR, E) CD80, F) CD86 or G) PD-L1 on immature DC differentiated in the presence of TGFβ, VitD3, 6’SL, 2’FL or GOS was shown. Relative fold change was calculated by dividing the MFI (median fluorescence intensity) of DC differentiated in the presence of a breast milk component/MFI of moDC of each respective donor.

Article Snippet: Immature moDCs were generated by differentiating monocytes for 6 days in the presence of 20 ng/ml IL-4 (Peptrotech; 200–04) and GM-CSF (Peprotech; 300–03) in the presence or absence of TGFβ (3 ng/ml TGFβ1 + 10ng/ml TGFβ2, Peprotech; 100-21/100-35), 10nM VitD3 (Sigma-Aldrich, D1530), 0.5 mg/ml 2’FL (Inalco S.p.A.) or GOS (FrieslandCampina), or 0.375 mg/ml 6’SL (Carbosynth, OS04398).

Techniques: Cell Culture, Marker, Expressing, Flow Cytometry, Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: The oligosaccharides 6’-sialyllactose, 2’-fucosyllactose or galactooligosaccharides do not directly modulate human dendritic cell differentiation or maturation

doi: 10.1371/journal.pone.0200356

Figure Lengend Snippet: TGFβ and VitD3 differentially modulate the production of IL-6 and IL-8 during differentiation. 6’SL was treated with an optimized Triton X-114 method to remove LPS traces. Immature DC were cultured for six days in the presence or absence of VitD3, TGFβ, 6’SL, LPS-free 6’SL, 2’FL or GOS. A,C) IL-6 and B,C) IL-8 were measured in the supernatant by CBA.

Article Snippet: Immature moDCs were generated by differentiating monocytes for 6 days in the presence of 20 ng/ml IL-4 (Peptrotech; 200–04) and GM-CSF (Peprotech; 300–03) in the presence or absence of TGFβ (3 ng/ml TGFβ1 + 10ng/ml TGFβ2, Peprotech; 100-21/100-35), 10nM VitD3 (Sigma-Aldrich, D1530), 0.5 mg/ml 2’FL (Inalco S.p.A.) or GOS (FrieslandCampina), or 0.375 mg/ml 6’SL (Carbosynth, OS04398).

Techniques: Cell Culture, Crocin Bleaching Assay

Journal: BMC Complementary and Alternative Medicine

Article Title: Oleanolic acid attenuates TGF-β1-induced epithelial-mesenchymal transition in NRK-52E cells

doi: 10.1186/s12906-018-2265-y

Figure Lengend Snippet: Effects of OA on the expression of pSmad2/3 in NRK-52E cells. The cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of OA (0, 2, 4, 8 μM). a The expression level of Smad2/3 and pSmad2/3 was determined by Western blotting. b The pSmad2/3 was quantitatively analyzed with Image J software. The data showing mean ± SD. # P

Article Snippet: Recombinant TGF-β1 was purchased from Peprotech (Cat. No. 100-21C, USA).

Techniques: Expressing, Incubation, Western Blot, Software

Journal: BMC Complementary and Alternative Medicine

Article Title: Oleanolic acid attenuates TGF-β1-induced epithelial-mesenchymal transition in NRK-52E cells

doi: 10.1186/s12906-018-2265-y

Figure Lengend Snippet: Effects of OA on the ILK and Snail expression in NRK-52E cells. The cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of OA (0, 2, 4, 8 μM). a The expression level of ILK and Snail was determined by Western blotting. b , c The expression level was quantitatively analyzed with Image J software. The data showing mean ± SD. # P

Article Snippet: Recombinant TGF-β1 was purchased from Peprotech (Cat. No. 100-21C, USA).

Techniques: Expressing, Incubation, Western Blot, Software

Journal: BMC Complementary and Alternative Medicine

Article Title: Oleanolic acid attenuates TGF-β1-induced epithelial-mesenchymal transition in NRK-52E cells

doi: 10.1186/s12906-018-2265-y

Figure Lengend Snippet: Effects of OA on TGF-β1-induced Morphological changes in NRK-52E cells. The cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of OA (0, 2, 4, 8 μM). a Untreated NRK-52E cells showed a pebble-like shape is clearly observed. b TGF-β1-treated cells showed a more elongated morphological shape. c , d , e showed OA reversed TGF-β1-induced morphological changes (Magnification of 200×)

Article Snippet: Recombinant TGF-β1 was purchased from Peprotech (Cat. No. 100-21C, USA).

Techniques: Incubation

Journal: BMC Complementary and Alternative Medicine

Article Title: Oleanolic acid attenuates TGF-β1-induced epithelial-mesenchymal transition in NRK-52E cells

doi: 10.1186/s12906-018-2265-y

Figure Lengend Snippet: Effects of OA on the Nrf2 and klotho expression in NRK-52E cells. The cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of OA (0, 2, 4, 8 μM). a The expression level of Nrf2 and klotho was determined by Western blotting. b , c The expression level was quantitatively analyzed with Image J software. The data showing mean ± SD. # P

Article Snippet: Recombinant TGF-β1 was purchased from Peprotech (Cat. No. 100-21C, USA).

Techniques: Expressing, Incubation, Western Blot, Software

Journal: BMC Complementary and Alternative Medicine

Article Title: Oleanolic acid attenuates TGF-β1-induced epithelial-mesenchymal transition in NRK-52E cells

doi: 10.1186/s12906-018-2265-y

Figure Lengend Snippet: Effects of OA on TGF-β1-induced EMT in NRK-52E cells. The cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of OA (0, 2, 4, 8 μM). a The expression of Fibronectin, α-SMA and E-cadherin was determined by Western blotting. b , c , d The expression level was quantitatively analyzed with Image J software. The data showing mean ± SD. # P

Article Snippet: Recombinant TGF-β1 was purchased from Peprotech (Cat. No. 100-21C, USA).

Techniques: Incubation, Expressing, Western Blot, Software

Journal: Stem Cell Research & Therapy

Article Title: Sialylation regulates myofibroblast differentiation of human skin fibroblasts

doi: 10.1186/s13287-017-0534-1

Figure Lengend Snippet: Defect in raft localization of CD44 during the process to senescence was restored by treatment with a sialidase inhibitor. a Cell surface glycans in early passage ( EP ), late passage ( LP ), and zanamivir (2 mM)-treated LP fibroblasts were analyzed by FACS using lectins. Mean fluorescent intensities ( MFIs ) relative to those of control cells are shown. Results are presented as means ± SD from three independent experiments. b Total cell lysates from EP, LP, and zanamivir-treated LP fibroblasts were pulled down with ECA. Immunoblotting of CD44 was performed on ECA-binding proteins ( left ). In addition, immunoprecipitation ( IP ) of CD44, followed by immunoblotting of ECA, MAL-II, and CD44, was performed ( right ). Representative images are shown. c Immunocytochemical staining was performed in EP, LP, and zanamivir-treated LP fibroblasts after transforming growth factor ( TGF )-β1 treatment. Representative images are shown (GM1, red ; CD44, green ; DAPI, blue ; colocalization, yellow ). d The histogram shows the mean ± SD percentage of GM1-CD44 colocalized cells colored yellow , as shown in ( c ), from two independent experiments (total of six fields); *** P

Article Snippet: For myofibroblast differentiation, cells were cultured in serum-free medium containing 10 ng/ml human transforming growth factor (TGF)-β1 (Peprotech, Rocky Hill, NJ, USA).

Techniques: FACS, Binding Assay, Immunoprecipitation, Staining

Journal: Stem Cell Research & Therapy

Article Title: Sialylation regulates myofibroblast differentiation of human skin fibroblasts

doi: 10.1186/s13287-017-0534-1

Figure Lengend Snippet: Myofibroblast differentiation was inhibited by sialidase. a , b Cell surface glycans in control ( Ctr ; non-treated EP fibroblasts) and 100 U/ml sialidase-treated EP fibroblasts were analyzed by FACS using lectins. Three independent experiments were performed and representative results are shown ( a ). Controls are presented in gray . Mean fluorescent intensities ( MFIs ) relative to those of control cells are shown ( b ). Results are presented as means ± SD from three independent experiments. c , d Western blot analysis of α-smooth muscle actin (α -SMA ) was performed 3 days after myofibroblast differentiation in control and sialidase-treated EP fibroblasts. The histogram ( d ) shows the mean densitometric analysis ± SD of α-SMA normalized to the loading control (β-actin). The values were obtained from three independent experiments. e Immunocytochemical staining was performed in control and sialidase-treated EP fibroblasts after transforming growth factor ( TGF )-β1 treatment. Representative images are shown (GM1, red ; CD44, green ; DAPI, blue ; colocalization, yellow ). f The histogram shows the mean ± SD percentage of GM1-CD44 colocalized cells colored yellow , as shown in ( e ), from two independent experiments (total of six fields); *** P

Article Snippet: For myofibroblast differentiation, cells were cultured in serum-free medium containing 10 ng/ml human transforming growth factor (TGF)-β1 (Peprotech, Rocky Hill, NJ, USA).

Techniques: FACS, Western Blot, Staining

Journal: Stem Cell Research & Therapy

Article Title: Sialylation regulates myofibroblast differentiation of human skin fibroblasts

doi: 10.1186/s13287-017-0534-1

Figure Lengend Snippet: Raft localization of CD44 after TGF-β1 stimulation was inhibited by reduced sialylation with BGN. a , b Western blot analysis of phosphorylated extracellular signal-related kinase ( pERK ) was performed in control ( Ctr ; vehicle-treated EP fibroblasts in DMSO) and GalNAc-α- O -benzyl ( BGN ; 2 mM)-treated EP fibroblasts. The histogram ( b ) shows the mean densitometric analysis ± SD for the phosphorylated proteins normalized to the loading control. Values were obtained from three independent experiments. **P

Article Snippet: For myofibroblast differentiation, cells were cultured in serum-free medium containing 10 ng/ml human transforming growth factor (TGF)-β1 (Peprotech, Rocky Hill, NJ, USA).

Techniques: Western Blot

Journal: Cancers

Article Title: TGFβ Imprinting During Activation Promotes Natural Killer Cell Cytokine Hypersecretion

doi: 10.3390/cancers10110423

Figure Lengend Snippet: TGFβi generates NK cells with increased degranulation but transiently impairs cytotoxicity. ( A ) Control and TGFβi NK cells from K562mbIL-21 expansions were rested overnight in IL-2 or IL-2 + TGFβ and subsequently stimulated with tumor targets for 3 h (MG63, n = 6; DAOY, n = 4; HOS, n = 5) and assessed for degranulation by CD107a. %CD107a + NK cells are corrected for no target controls. ( B ) The control and TGFβi NK cell cytotoxicity was measured using a 4-h calcein-release cytotoxicity assay, following overnight treatment in IL-2 alone or IL-2 plus TGFβ. (MG63, n = 9; DAOY, n = 3; HOS, n = 5; CHLA-255, n = 3). ( C ) TGFβi NK cells were removed from TGFβ for 7 days (±1 day) and cytotoxicity against CHLA-255, MG63, and DAOY following overnight treatment with IL-2 or IL-2 plus TGFβ was measured using a calcein-release assay ( n = 3). Individual data points depicted for all. Lines and bars represent Mean ± SD. Statistical differences were determined by two-way repeated measures ANOVA with Holm-Sidak’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Control expanded NK cells were supplemented with 50 IU/mL recombinant human IL-2, and TGFβi expanded NK cells received 50 IU/mL IL-2 (Prometheus, 22mmu NDC 65483-0116-07, San Diego, CA, USA) plus 10 ng/mL TGFβ (Biolegend, 580706, San Diego, CA, USA) every 2–3 days.

Techniques: Cytotoxicity Assay, Release Assay

Journal: Cancers

Article Title: TGFβ Imprinting During Activation Promotes Natural Killer Cell Cytokine Hypersecretion

doi: 10.3390/cancers10110423

Figure Lengend Snippet: NK cell activation is required for TGFβ induced cytokine hypersecretion. NK cells were cultured with IL-2 alone or IL-2 plus 10 ng/mL TGFb and ( A ) K562mbIL-15 (IFNγ: n = 4, TNFα: n = 3) or ( B ) no feeder cell ( n = 6), or ( C ) parental K562 ( n = 4) for 7–14 days. Following culture, anti-tumor cytokine secretion or production by CBA or intracellular flow cytometry was assessed against an MG63 tumor target. ( D ) NK cells were stimulated overnight with 10 ng/mL IL-12, 50 ng/mL IL-15, and 50 ng/mL IL-18 plus or minus IL-2 and TGFβ. Following overnight stimulation, the NK cells were cultured with 1 ng/mL IL-15 plus or minus IL-2 and TGFβ. After 7–14 days of culture, anti-tumor IFNγ and TNFα production in response to MG63 was measured by intracellular flow cytometry ( n = 4). Percent anti-tumor IFNγ+ and TNFα+ NK cells normalized to no target depicted for (B) and (D). Individual data points depicted for all. Lines and bars represent Mean ± SD. Statistical differences were determined by paired t-test for 2B and two-way repeated measures ANOVA with Holm–Sidak’s multiple comparisons test for all other graphs. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p .

Article Snippet: Control expanded NK cells were supplemented with 50 IU/mL recombinant human IL-2, and TGFβi expanded NK cells received 50 IU/mL IL-2 (Prometheus, 22mmu NDC 65483-0116-07, San Diego, CA, USA) plus 10 ng/mL TGFβ (Biolegend, 580706, San Diego, CA, USA) every 2–3 days.

Techniques: Activation Assay, Cell Culture, Crocin Bleaching Assay, Flow Cytometry, Cytometry

Journal: Biomaterials

Article Title: Heterogeneous engineered cartilage growth results from gradients of media-supplemented active TGF-β and is ameliorated by the alternative supplementation of latent TGF-β

doi: 10.1016/j.biomaterials.2015.10.018

Figure Lengend Snippet: Distribution of media-supplemented exogenous active TGF-β1 as a function of distance from the media-exposed construct surface in (A) acellular agarose after 18 and 72 h, (B) freshly-cast chondrocyte-seeded constructs after 72 h, (C) mature (28-day cultured) chondrocyte-seeded constructs after 72 h, and (D) freshly-cast MSC-seeded constructs after 72 h. Control levels represent endogenous active TGF-β1 levels in constructs. Dashed line represents active TGF-β1 media bath concentration. *p

Article Snippet: For all experiments, exogenously supplemented active TGF-β1, active TGF-β3, and latent TGF-β1 (100 kDa small latent complex form) were human recombinant (R & D Systems).

Techniques: Construct, Cell Culture, Concentration Assay

Journal: Biomaterials

Article Title: Heterogeneous engineered cartilage growth results from gradients of media-supplemented active TGF-β and is ameliorated by the alternative supplementation of latent TGF-β

doi: 10.1016/j.biomaterials.2015.10.018

Figure Lengend Snippet: ]. All concentrations are normalized to day 0 construct volume. For all groups, active TGF-β1 was not detected in conditioned media or tissue constructs. *p

Article Snippet: For all experiments, exogenously supplemented active TGF-β1, active TGF-β3, and latent TGF-β1 (100 kDa small latent complex form) were human recombinant (R & D Systems).

Techniques: Construct