Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

Journal: Cell Reports Medicine

Article Title: Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis

doi: 10.1016/j.xcrm.2020.100056

Figure Lengend Snippet: Transcriptome Analysis Identifies a Common Metabolic Signature in Human Primary Cells Treated with TGF-β (A) Schematic overview of RNA-seq analyses of four human primary cell types with TGF-β treatment. 3 independent replicates were used for each time point collection. (B) RT-PCR analysis of αSMA gene expression in human primary cells upon TGF-β treatment (5 ng/mL for 24 h), which was almost completely reversed by the presence of ALK5 inhibitor, SB-525334 (10 μM). n = 3, Mean ± SD. (C) Common effects of TGF-β across four cell types. Shown are the 1,603 genes that met the ±1.2-fold change and FDR_BH p < 0.1 threshold at 24 h in all four cell types. The color gradient represents fold change compared to vehicle-treated cells (−3.0 to 3.0-fold). (D) GO term enrichment analysis of the 1,603 genes using the PANTHER enrichment test ( http://pantherdb.org ). (E) Gene expression ratios of fatty acid oxidation enzymes in human primary cells and fibrotic tissues. From left: cardiac fibroblasts, hepatic stellate cells (HSCs), NHLF, RPTEC, UUO kidney, CCl4 liver, and BDL liver. Heatmap was generated by using Morpheus software ( https://software.broadinstitute.org/morpheus/ ). The color gradient represents fold change compared treatment (TGF-β 24 h, surgery, or CCl4 treatment) to control (−8.5 to 8.5-fold). Gray, below detection limit. ∗Peroxisomal enzymes.

Article Snippet: The next day, 5ng/ml of recombinant human TGFβ1 (BioLegend, #580702) was added to the culture medium and cells were incubated at 37°C for the indicated time.

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Generated, Software, Control

Journal: Cell Reports Medicine

Article Title: Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis

doi: 10.1016/j.xcrm.2020.100056

Figure Lengend Snippet:

Article Snippet: The next day, 5ng/ml of recombinant human TGFβ1 (BioLegend, #580702) was added to the culture medium and cells were incubated at 37°C for the indicated time.

Techniques: Transduction, Virus, Ligation, Recombinant, Saline, Injection, Protease Inhibitor, Mass Spectrometry, Staining, cDNA Synthesis, Extraction, RNA Library Preparation, Enzyme-linked Immunosorbent Assay, Software, Simple Western

Journal: Cell death & disease

Article Title: TGFβ signaling sensitizes MEKi-resistant human melanoma to targeted therapy-induced apoptosis.

doi: 10.1038/s41419-024-07305-1

Figure Lengend Snippet: Fig. 2 Apoptosis is the mode of cell death induced by concomitant TGFβ1 and MEKi treatment in melanoma cells. A–C Representative FACS plots of the three melanoma cell lines M130830 (A), M170117 (B) and M010817 (C) treated with combinations of TGFβ1 (10 ng/mL), MEKi (10 nM) and ZVAD (40 µM) for 72 h as indicated and stained with Annexin V and Propidium Iodide (PI) solution. D–F Live cell quantification of the FACS staining from (A–C). Experiments were performed in 3 independent replicates. P-values were calculated by one-way ANOVA and multiple comparisons for selected pairs with *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. G–I Western blots for PARP, cCASP3, and β-ACTIN (ACTB) from whole cell lysates of one sensitive, M130830 (G) and two resistant melanoma cell lines, M170117 (H) and M010817 (I), treated with combinations of TGFβ1 and MEKi for 48 h as indicated. cCASP3 and cPARP levels were quantified relative to the loading control with the MEKi only condition set to 1 and shown below the blots.

Article Snippet: Stimulations were performed with TGFβ1 (RnD Systems 7754-BH-005, Stock: 10 μg/mL in 4 mM HCl+ 0.1% BSA, 1:5000 dilution of stock), and/or MEKi (trametinib, Stock: 50 μM Active Biochemicals—A-1258, 1:1000 dilution of stock), and/or DMSO 20 hours prior to harvest for CUT&RUN.

Techniques: Staining, Western Blot, Control

Journal: Cell death & disease

Article Title: TGFβ signaling sensitizes MEKi-resistant human melanoma to targeted therapy-induced apoptosis.

doi: 10.1038/s41419-024-07305-1

Figure Lengend Snippet: Fig. 3 Double treatment induces a distinctive gene expression signature as compared to the individual treatments. A, B Heatmaps of bulk RNAseq data of a “sensitive” (A) and a “resistant” (B) human melanoma cell line, representing differentially expressed genes (p adjusted < 0.05) that are part of previously defined [36, 39] melanoma programs: AXL, MITF and the resistance program. The expression values are z-score normalized per row and genes are clustered using hierarchical clustering with euclidean distance. C, D Gene set enrichment analyses (GSEAs) of each program (p adjusted value < 0.05) under each condition compared to the vehicle control (DMSO) in a sensitive (C) and a resistant cell line (D). Running Enrichment Score is shown on the Y-axis, gene number on the x-axis. E, F Scores of TGFβ1, MEKi or TGFβ1 + MEKi treatment signatures represented in cell subpopulations previously identified by single cell RNAseq analysis in a xenograft melanoma model during BRAFi/MEKi treatment [37]. Violin plots show cell score distribution within each cell population in a sensitive (E) and a resistant (F) cell line. “SMC” = starved-like melanoma cell state. “NCSC” = neural crest stem cell state.

Article Snippet: Stimulations were performed with TGFβ1 (RnD Systems 7754-BH-005, Stock: 10 μg/mL in 4 mM HCl+ 0.1% BSA, 1:5000 dilution of stock), and/or MEKi (trametinib, Stock: 50 μM Active Biochemicals—A-1258, 1:1000 dilution of stock), and/or DMSO 20 hours prior to harvest for CUT&RUN.

Techniques: Gene Expression, Expressing, Control

Journal: Cell death & disease

Article Title: TGFβ signaling sensitizes MEKi-resistant human melanoma to targeted therapy-induced apoptosis.

doi: 10.1038/s41419-024-07305-1

Figure Lengend Snippet: Fig. 6 Apoptosis signature genes cannot be used as predictive markers for favorable treatment with targeted therapy. A Schematic setup of patient-derived melanoma cell line processing. Melanoma cell biopsies were cultured in vitro before bulk RNAseq and the half maximal inhibitory concentration (IC50) for MEKi was determined by MTT assays. B Representation of MEKi IC50 values for the top 20 and bottom 20 patient-derived melanoma cell lines, grouped accordingly. P-values were calculated by Mann-Whitney test with *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. C Bar plots showing correlation of MEKi sensitive and MEKi resistant cell lines with average gene expression of all genes of the TGFβ1 + MEKi-associated apoptosis signature. P-values were calculated by unpaired t-test with *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: Stimulations were performed with TGFβ1 (RnD Systems 7754-BH-005, Stock: 10 μg/mL in 4 mM HCl+ 0.1% BSA, 1:5000 dilution of stock), and/or MEKi (trametinib, Stock: 50 μM Active Biochemicals—A-1258, 1:1000 dilution of stock), and/or DMSO 20 hours prior to harvest for CUT&RUN.

Techniques: Biomarker Discovery, Derivative Assay, Cell Culture, In Vitro, Concentration Assay, MANN-WHITNEY, Gene Expression

Journal: The Korean Journal of Internal Medicine

Article Title: Concomitant inhibition of renin angiotensin system and Toll-like receptor 2 attenuates renal injury in unilateral ureteral obstructed mice

doi: 10.3904/kjim.2015.004

Figure Lengend Snippet: The effects of TLR2-KO on UUO-induced injury. Renal mRNA expression of (A) MCP-1, (B) OPN, or (C) TGF-β were similar in TLR2-KO and WT UUO mice. There were no significant differences in renal expression of MCP-1 (A, p = 0.299), OPN (B, p = 0.549), and TGF-β (C, p = 0.654) between WT and TLR2-KO UUO kidneys. (D) There was no significant difference in tubulointerstitial injury sore, MT stained area, and TGF-β positive area between WT and TLR2-KO UUO kidneys (×200). UUO: UUO WT mice (n = 8), TLR2-KO UUO mice (n = 8). TLR2, Toll-like receptor 2; KO, knockout; UUO, unilateral ureteral obstruction; MCP-1, monocyte chemotactic protein 1; OPN, osteopontin; TGF-β, transforming growth factor β; WT, wild type; MT, Masson’s trichrome.

Article Snippet: The slides were processed for immunodetection of transforming growth factor β (TGF-β) and angiotensin II type 1 receptor (AT1R) with antibodies specific for TGF-β (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-AT1R (Santa Cruz Biotechnology) respectively.

Techniques: Expressing, Staining, Knock-Out

Journal: The Korean Journal of Internal Medicine

Article Title: Concomitant inhibition of renin angiotensin system and Toll-like receptor 2 attenuates renal injury in unilateral ureteral obstructed mice

doi: 10.3904/kjim.2015.004

Figure Lengend Snippet: The effects of inhibition of renin angiotensin system and TLR on renal fibrosis at 7 days after UUO. (A) Masson’s trichrome (MT) staining and (B) TGF-β staining of (a) sham-operated, (b) UUO, (c) aliskiren-treated WT UUO, and (d) aliskiren-treated TLR2-KO UUO kidneys (×200). Ae and Be show quantification of the data in each panel. (Ab) MT-stained and (Bb) TGF-β stained areas were larger in UUO kidneys compared with sham-operated kidneys. Aliskiren treatment reduced the MT-stained area and TGF-β stained area in UUO kidneys (Ac, Bc). Aliskiren-treated TLR2-KO UUO kidneys showed a significant decrease in MT-stained and TGF-β stained areas compared with aliskiren-treated WT UUO kidneys (Ad, Bd). Aliskiren treatment reduced the renal mRNA expressions of TGF-β in UUO kidneys. Aliskiren-treated TLR2-KO UUO kidneys showed significant decreases in renal mRNA expression of TGF-β compared with aliskiren-treated WT UUO kidneys by RT-PCR (Bf). (C) Renin expression. The expression of renin was increased in UUO, A-UUO, and A-TRL2-KO UUO kidney ( p < 0.05). (D) AT1R expression. (a) Sham-operated, (b) WT UUO, (c) aliskiren-treated WT UUO, (d) aliskiren-treated TLR2-KO UUO kidneys, (e) area of AT1R stain, (f) representative AT1R immunoblots: sham-operated (n = 3), WT-UUO (n = 3), A-UUO (n = 4), A-TLR2-KO-UUO (n = 4) (×200). The expression of AT1R was increased in UUO kidney. The area of AT1R stain in tubulointerstitium (yellow arrows). However, aliskiren treatment significantly decreased the AT1R expression in both WT UUO and TLR2-KO UUO kidneys compared with aliskiren-untreated WT UUO kidneys (Da-Dd and Df, p < 0.05). Although AT1R expression showed the increase tendency in aliskiren-treated TLR2-KO kidney compared with aliskiren-treated WT UUO kidney, there were no significant difference between two groups ( p = 0.075). TLR, Toll-like receptor; UUO, unilateral ureteral obstruction; MT, Masson’s trichrome; TGF-β, transforming growth factor β; WT, wild type; KO, knockout; RT-PCR, real-time polymerase chain reaction; A-UUO, aliskiren-treated UUO WT mice (n = 8); A-TLR2-KO UUO, aliskiren-treated TLR2-KO UUO mice (n = 8); AT1R, angiotensin II type 1 receptor; S, sham-operated mice (n = 7); UUO, UUO operated mice (n = 8). a,b,c p < 0.05.

Article Snippet: The slides were processed for immunodetection of transforming growth factor β (TGF-β) and angiotensin II type 1 receptor (AT1R) with antibodies specific for TGF-β (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-AT1R (Santa Cruz Biotechnology) respectively.

Techniques: Inhibition, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knock-Out, Real-time Polymerase Chain Reaction

Journal: Naunyn-Schmiedeberg's archives of pharmacology

Article Title: Alleviation of pulmonary fibrosis by the dual PPAR agonist saroglitazar and breast milk mesenchymal stem cells via modulating TGFß/SMAD pathway.

doi: 10.1007/s00210-024-03004-y

Figure Lengend Snippet: Fig. 3 Effect of BrMSCs and/or saroglitazar on lipid peroxidation parameters A MDA and B SOD, the pro-inflammatory cytokines C IL-1β, D IL-10, E TGF-ß, and F serum Beclin-1 in rats with BLM-induced lung fibrosis

Article Snippet: To localize alpha-smooth muscle actin (α-SMA) protein as a marker for myofibroblasts, anti-alpha-smooth muscle actin antibody (rabbit polyclonal antibody; No. ab5694; dilution 1/50; Abcam, Cambridge, UK) (Egger et al. 2013) and with a monoclonal rabbit anti-TGFβ1 antibody (Clone TB21, MCA797, Serotec, Oxford, UK) at 1:200 dilution for TGF-β1 detection (Attia et al. 2017).

Techniques:

Journal: Naunyn-Schmiedeberg's archives of pharmacology

Article Title: Alleviation of pulmonary fibrosis by the dual PPAR agonist saroglitazar and breast milk mesenchymal stem cells via modulating TGFß/SMAD pathway.

doi: 10.1007/s00210-024-03004-y

Figure Lengend Snippet: Fig. 4 Effect of BrMSCs and/or saroglitazar on the mRNA expression genes of A TGF-β, B SMAD-3, C SMAD-7, D PPAR-γ, E PPAR-α in rats’ lungs with BLM-induced pulmonary fibrosis

Article Snippet: To localize alpha-smooth muscle actin (α-SMA) protein as a marker for myofibroblasts, anti-alpha-smooth muscle actin antibody (rabbit polyclonal antibody; No. ab5694; dilution 1/50; Abcam, Cambridge, UK) (Egger et al. 2013) and with a monoclonal rabbit anti-TGFβ1 antibody (Clone TB21, MCA797, Serotec, Oxford, UK) at 1:200 dilution for TGF-β1 detection (Attia et al. 2017).

Techniques: Expressing

Journal: Naunyn-Schmiedeberg's archives of pharmacology

Article Title: Alleviation of pulmonary fibrosis by the dual PPAR agonist saroglitazar and breast milk mesenchymal stem cells via modulating TGFß/SMAD pathway.

doi: 10.1007/s00210-024-03004-y

Figure Lengend Snippet: Fig. 10 A photomicrograph of immune-stained sections of the study groups. A CN-G showing faint expression of TGF-β1 by some cells in the alveolar septa between the alveoli and alveolar sacs. B, C BLM-G sections showing [B] Numerous cytoplasmic reactions for TGF- β1 positive immune expressed cells (arrows) within the inter- alveolar septum. C Increased TGF-β1 positive immune expressed cells (arrows) within the inter-alveolar septum. D BrMSCs-G showing some positive immune expressed cells (arrows) for TGF-β1 within the inter-alveolar septum. E Saro-G showing few positive immune expressed cells (arrows) for TGF-β1 within the inter-alveo- lar septum. F BrMSCs + Saro-G showing faint weak positive TGF-β1 immune expressed cells (arrows) within the inter- alveolar septum. (TGF-β1 X 400) (scale bar = 40 μm). G Mean values of % area of TGF-ß1 among different studied groups, one way ANOVA was used for statistical analysis. “a” means value is statistical significance when comparing CN-G with other groups, “b” means the value is statistical significance when comparing BLM-G with other groups, “c” means the value is statistical significance when comparing BrMSCs + Saro-G with other groups. The same letters mean no statistical significance dif- ference

Article Snippet: To localize alpha-smooth muscle actin (α-SMA) protein as a marker for myofibroblasts, anti-alpha-smooth muscle actin antibody (rabbit polyclonal antibody; No. ab5694; dilution 1/50; Abcam, Cambridge, UK) (Egger et al. 2013) and with a monoclonal rabbit anti-TGFβ1 antibody (Clone TB21, MCA797, Serotec, Oxford, UK) at 1:200 dilution for TGF-β1 detection (Attia et al. 2017).

Techniques: Staining, Expressing

Journal: Diseases

Article Title: Impact of miR-181a on SIRT1 Expression and Senescence in Hutchinson–Gilford Progeria Syndrome

doi: 10.3390/diseases13080245

Figure Lengend Snippet: Interconnection between autophagy and inflammatory pathways. Under high-nutrient conditions, the mechanistic target of rapamycin (mTOR) is activated, leading to the inhibition of the autophagic pathway, which involves the initiation, elongation, maturation, and fusion of the autophagosome [ , , ]. Inflammation signaling typically results in the activation of NF-κB, which can occur through canonical or non-canonical mechanisms. The canonical activation of NF-κB is initiated by the binding of growth factors to tumor necrosis factor (TNF) or toll-like receptors (TLRs), which activate TNF receptor-associated factor (TRAF) proteins. This leads to the activation of the IKK complex, degradation of IKB, and translocation of the NF-ĸB1-RelA complex into the nucleus to activate proinflammatory genes. In contrast, the non-canonical pathway is triggered by signals from TNF receptors that activate the NF-ĸB-inducing kinase, leading to IKK activation [ , , , ]. TLR4 signaling also induces the JAK/STAT pathway, another critical component of inflammatory signaling [ , , ]. Autophagy and inflammation are connected through the DNA damage response. DNA damage activates the cytosolic DNA sensors cGAS and STING, which, in conjunction with ATM, can stimulate both inflammatory responses and autophagic vesicle formation, as well as the central cell-cycle regulator p53 [ , , , ]. Proteins analyzed by miRNA interactions are illustrated in green.

Article Snippet: Cultures were treated in the starving medium with 10 ng/ml transforming growth factor β1 (TGFβ1, HZ-1011, Proteintech, Rosemont, IL, USA) for 96 h and collected for RNA isolation or fixed for immunofluorescence staining. miR-181a-5p mimic (Thermo Fisher, miRVanaTM miR-181a-5p mimic, 4464066) and miR-181a-5p inhibitor (Thermo Fisher, miRVanaTM miR-181a-5p inhibitor, 4464084) were transfected in a concentration of 25 μM for 6 and 9 days and 10 nM for 6 and 9 days, respectively, at a cell density of 30–50%.

Techniques: Inhibition, Activation Assay, Binding Assay, Translocation Assay

Journal: Diseases

Article Title: Impact of miR-181a on SIRT1 Expression and Senescence in Hutchinson–Gilford Progeria Syndrome

doi: 10.3390/diseases13080245

Figure Lengend Snippet: Identification and profiling of miRNAs regulating autophagy and inflammation pathways in control and HGPS. ( A ) Literature search procedure. Proteins from both autophagy and inflammation pathways were examined for miRNA regulation, and overlapping miRNAs were analyzed using RT-qPCR. ( B ) RT-qPCR analysis. miRNAs identified through text mining were analyzed in normal and HGPS fibroblasts during replicative senescence. Control fibroblasts (GMO1651c, GMO1652c, GMO3349c, GMO1582B) and HGPS fibroblasts (HGADFN127, HGADFN188, HGADFN003) were compared at passages with a replicative senescence under 5% (young) and over 20% (old) intragroup, as well as same-aged groups. Relative expression levels were normalized to U6 small nuclear 1 (RNU-6). Graphs show mean values ± standard deviation. Statistical significance was calculated using ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01, (control: n = 4, HGPS: n = 3)). Non-significant changes are not indicated.

Article Snippet: Cultures were treated in the starving medium with 10 ng/ml transforming growth factor β1 (TGFβ1, HZ-1011, Proteintech, Rosemont, IL, USA) for 96 h and collected for RNA isolation or fixed for immunofluorescence staining. miR-181a-5p mimic (Thermo Fisher, miRVanaTM miR-181a-5p mimic, 4464066) and miR-181a-5p inhibitor (Thermo Fisher, miRVanaTM miR-181a-5p inhibitor, 4464084) were transfected in a concentration of 25 μM for 6 and 9 days and 10 nM for 6 and 9 days, respectively, at a cell density of 30–50%.

Techniques: Control, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: Diseases

Article Title: Impact of miR-181a on SIRT1 Expression and Senescence in Hutchinson–Gilford Progeria Syndrome

doi: 10.3390/diseases13080245

Figure Lengend Snippet: Elevated TGFβ1 levels in HGPS promote upregulation of miR-181a-5p expression. ( A ) TGFβ1 mRNA expression level in three control (GMO5565, GMO5757c, HGFDFN369) and HGPS (HGADFN127, HGADFN003, HGADFN164) fibroblast cell strains. Comparisons were made between the young and old stages intragroup as well as same-aged stages. Expression was normalized to GAPDH. ( B ) Representative immunofluorescence staining of the control cell line 5757c P18 with α-SMA following TGFβ1 treatment. Scale bar = 50 µm ( C ) Quantification of immunofluorescence signal in control (GMO5757c P18) and HGPS (HGADFN003 P12) cell strains. Comparisons were made between treated and non-treated samples. ( D ) Western blot of α-SMA and GAPDH as a loading control in control (GMO5757c P18) and HGPS (HGADN271 P11) cell strains. ( E ) Relative miR-181a-5p levels following TGFß1 treatment in three control (HGFDFN369, GMO5757c, GMO3349c) and HGPS (HGADFN271, HGADFN003, HGADFN164) cell strains with a senescence index <5%. Expression was normalized to U6 small nuclear 1 (RNU-6). Comparisons were made between treated and non-treated samples. ( A , C , E ) Graphs show mean values ± standard deviation. Statistical significance was calculated using ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01, **** p < 0.0001 ( n = 3)).

Article Snippet: Cultures were treated in the starving medium with 10 ng/ml transforming growth factor β1 (TGFβ1, HZ-1011, Proteintech, Rosemont, IL, USA) for 96 h and collected for RNA isolation or fixed for immunofluorescence staining. miR-181a-5p mimic (Thermo Fisher, miRVanaTM miR-181a-5p mimic, 4464066) and miR-181a-5p inhibitor (Thermo Fisher, miRVanaTM miR-181a-5p inhibitor, 4464084) were transfected in a concentration of 25 μM for 6 and 9 days and 10 nM for 6 and 9 days, respectively, at a cell density of 30–50%.

Techniques: Expressing, Control, Immunofluorescence, Staining, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Long-Term Gene Therapy with Thrombospondin 2 Inhibits TGF-β Activation, Inflammation and Angiogenesis in Chronic Allograft Nephropathy

doi: 10.1371/journal.pone.0083846

Figure Lengend Snippet: In gene therapy, treated rats with renal transplants TGF-β were evaluated by immunohistochemistry. Active TGF-β was either detected directly by using an antibody recognizing active TGF-β (A) or indirectly by evaluation of phosphorylation of the TGF-β signaling molecule smad2/3 (B) or expression of the TGF-β downstream target PAI-1 within the glomeruli (C) or the cortex (D). Representative microphotographs of PAI-1 staining in renal grafts treated with control (E) and TSP-2 overexpressing plasmid (F) are shown. Total TGF-β1 (G) and TGF-β2 (H) was similar in both groups. Control (n = 8) vs. TSP-2 treated (n = 8); *p<0,05.

Article Snippet: The TGF-β system was studied using antibodies to TGF-β1 (rabbit anti-human TGF-β1, Santa Cruz Biotechnology Inc. ); TGF-β2 (rabbit anti-human TGF-β2, Santa Cruz ); active TGF-β1 (chicken anti-human active TGF-β1, (R&D systems, Germany , , Phospho-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435, Santa Cruz ) or PAI-1, a rabbit polyclonal to human plasminogen activator inhibitor-1 (Santa Cruz).

Techniques: Immunohistochemistry, Phospho-proteomics, Expressing, Staining, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Long-Term Gene Therapy with Thrombospondin 2 Inhibits TGF-β Activation, Inflammation and Angiogenesis in Chronic Allograft Nephropathy

doi: 10.1371/journal.pone.0083846

Figure Lengend Snippet: Representative microphotographs from immunohistological staining of kidney grafts for active TGF-β (A, control plasmid; B, TSP-2 therapy, brown cytosolic staining), P-smad 2/3 (C, control plasmid; D, TSP-2 therapy, brown nuclear staining), PAI-1 (E, control plasmid; F, TSP-2 therapy, brown staining), fibronectin (G, control plasmid; H, TSP-2 therapy, brown staining) and alpha-smooth muscle actin (I, control plasmid; J, TSP-2 therapy, brown staining) are shown.

Article Snippet: The TGF-β system was studied using antibodies to TGF-β1 (rabbit anti-human TGF-β1, Santa Cruz Biotechnology Inc. ); TGF-β2 (rabbit anti-human TGF-β2, Santa Cruz ); active TGF-β1 (chicken anti-human active TGF-β1, (R&D systems, Germany , , Phospho-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435, Santa Cruz ) or PAI-1, a rabbit polyclonal to human plasminogen activator inhibitor-1 (Santa Cruz).

Techniques: Staining, Control, Plasmid Preparation

Journal: Journal of cell science

Article Title: Integrin alpha8beta1 mediates adhesion to LAP-TGFbeta1.

doi: 10.1242/jcs.00145

Figure Lengend Snippet: Fig. 2. (i) α8β1 mediates spreading on LAP-TGFβ1. α8-transfected or mock-transfected AtT20 cells were plated on 5 µg/ml of recombinant LAP-TGFβ1 in serum-free media for 24 or 48 hours. At 24 hours, mock transfected AtT20 cells remain rounded and unattached on LAP-TGFβ1 (A), whereas α8-transfected cells attach and spread on LAP-TGFβ1 (B). Further spreading is seen at 48 hours (C). (ii) Adhesion of α8β1 to LAP-TGFβ1 results in FAK phosphorylation. α8-transfected or mock-transfected AtT20 cells were plated on 0.01% poly-L-lysine (PLL), 5 µg/ml fibronectin (FN), 5 µg/ml LAP-TGFβ1 (LAP1) or 5 µg/ml RGE-LAP-TGFβ1 (RGE) for 30 minutes in serum-free media. Cells were lysed in buffer containing phosphatase inhibitors, immunoprecipitated with anti- FAK antibody followed by western blotting with either anti- phosphotyrosine antibody PY20 (top panel) or anti-FAK antibody (bottom panel).

Article Snippet: Wells were washed with PBS0.05% Tween and then incubated with 50 μl of anti-LAP-TGFβ1 IgG antibody (0.5 μg/ml) (R&D Systems) at RT for 2 hours.

Techniques: Transfection, Recombinant, Phospho-proteomics, Immunoprecipitation, Western Blot

Journal: Journal of cell science

Article Title: Integrin alpha8beta1 mediates adhesion to LAP-TGFbeta1.

doi: 10.1242/jcs.00145

Figure Lengend Snippet: Fig. 3. (A) Proliferation of cells adherent to LAP-TGFβ1. Equal numbers of α8-transfected or mock-transfected AtT20 cells were plated on 5 µg/ml LAP-TGFβ1 or 5 µg/ml fibronectin (FN) in serum-free media. After 3 days, proliferation was assayed using the Roche Cell Proliferation kit (MTT). Results for four independent clones of AtΤ20α8 cells are shown. Data is reported as the mean absorbance of triplicate wells±s.d. (B) ERK phosphorylation on LAP-TGFβ1. α8-transfected or mock-transfected AtT20 cells were plated on 0.01% poly-L-lysine (PLL) 5 µg/ml fibronectin (FN), 5 µg/ml LAP-TGFβ1 (LAP1), or 5 µg/ml RGE-LAP-TGFβ1 (RGE) for 30 minutes in serum-free media and then lysed in buffer containing phosphatase inhibitors. Equal amounts of protein were loaded and probed with an antibody to phospho-ERK (top) or ERK (bottom).

Article Snippet: Wells were washed with PBS0.05% Tween and then incubated with 50 μl of anti-LAP-TGFβ1 IgG antibody (0.5 μg/ml) (R&D Systems) at RT for 2 hours.

Techniques: Transfection, Clone Assay, Phospho-proteomics

Journal: Journal of cell science

Article Title: Integrin alpha8beta1 mediates adhesion to LAP-TGFbeta1.

doi: 10.1242/jcs.00145

Figure Lengend Snippet: Fig. 5. LAP-TGFβ1 localization in adult lung tissue. Paraffin- embedded, formalin fixed tissues were stained with an antibody specific for LAP-TGFβ1 and counterstained with hematoxylin. Immunoreactivity for LAP-TGFβ1 is seen on a macrophage (arrowhead) and interstitial cells (arrows). Magnification: A, 4×; B,C, 20×; D, 40×.

Article Snippet: Wells were washed with PBS0.05% Tween and then incubated with 50 μl of anti-LAP-TGFβ1 IgG antibody (0.5 μg/ml) (R&D Systems) at RT for 2 hours.

Techniques: Staining