Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 2. Regulation of Smad signaling response to TGF- by BMP-7. HK-2 cells were transfected with Smad3-responsive (A and B) or Smad2-responsive (C) plasmids and then were incubated with BMP-7 for various times or doses, before incubation with 1 ng/ml TGF- (black bar, control medium followed by TGF-; gray bars, BMP-7 followed by TGF-) or control medium (white bars) for 6 hours. A: Effect on Smad3 signaling of time course of 50 ng/ml BMP-7 incubation. B: Effect on Smad3 signaling of 24 hours preincubation with a BMP-7 dose range of 0 to 2000 ng/ml. C: Effect on Smad2 signaling of a 24-hour preincubation with BMP-7 at a dose range of 0 to 2000 ng/ml. *P 0.05 versus TGF-. RLU, relative light units.

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Transfection, Incubation, Control

Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 3. Time course of Smad phosphorylation and dephosphorylation/ degradation in response to TGF- and BMP-7. A: HK-2 cells were incubated with 50 ng/ml BMP-7 for 0 to 24 hours before incubation with 1 ng/ml TGF- for 1 hour. Whole-cell lysates were immunoblotted with antibodies against Phospho-Smads (PSmad) 1, 2, 3, and total Smads 1 and 3. Stripping and reprobing for glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used to confirm approximately equal loading. B–D: Time course of Smad1/3 dephosphorylation/degradation. HK-2 cells were incubated with control medium (B and C) or 50 ng/ml BMP-7 (D) for 24 hours before incubation with 1 ng/ml TGF- for 30 minutes. Cells were washed extensively and incubated in cytokine-free control medium (B) or medium containing the Alk5 kinase inhibitor SB431542 (C and D) for time points up to 5 hours. Residual Phospho-Smad3 activity was detected by immunoblot before strip- ping and reprobing for total Smad3.

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Incubation, Stripping Membranes, Control, Activity Assay, Western Blot

Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 4. Nuclear accumulation of Smad3. HK-2 cells were incubated with 50 ng/ml BMP-7 or control medium for 24 hours before incubation with 1 ng/ml TGF- or control medium for 1 hour. A: Immunoblotting of nuclear extracts for Phospho-Smad (PSmad) 1/3 and subsequent reprobing for c-Jun to confirm approximately equal loading. B: Immunofluorescent localization of Smad3. HK-2 cells were incubated with 50 ng/ml BMP-7 or control medium for 24 hours in eight-well chamber slides before incubation with 1 ng/ml TGF- or control medium for 1 hour and detection of Smad3 by immunofluorescence microscopy.

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Incubation, Control, Western Blot, Immunofluorescence, Microscopy

Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 5. BMP-7 inhibits Smad3 DNA binding. A and B: Electrophoretic mobility shift assay with a consensus Smad binding element probe. HK-2 cells were incubated with BMP-7 or control (Ctrl) medium for 24 hours before incubation with 1 ng/ml TGF- or control medium for 1 hour. Bold arrow, retarded probe; arrow, supershifted probe. A: Electrophoretic mo- bility shift assay performed with nuclear protein extract and consensus Smad binding element (SBE) probe. B: Supershift assay performed with antibodies to Smad3, 4, and 5. Sm, Smad. C and D: ChIP: Smad3 binding to the PAI-1 promoter. After chromatin immunoprecipitation with Smad3 antibody or pre-immune globulin, PAI-1 promoter Smad binding elements (SBE) were detected by qRT-PCR. Data are presented as Smad3-precipitated signal/pre- immune globulin-precipitated signal, normalized to control. C: HK-2 cells were incubated with TGF- for time points to 24 hours before ChIP. D: HK-2 cells were incubated with BMP-7 and TGF- as indicated for 6 hours before ChIP. RE, Relative Expression.

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Control, Shift Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Expressing

Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 8. Proposed mechanism of regulation of Smad3 signaling by BMP7. A: In the absence of TGF-, Smads shuttle into and out of the nucleus. SnoN binds to Smad binding elements and prevents R-Smad binding. B: After ligand binding, active TGF- receptor complex leads to R-Smad phosphorylation. R-Smad-Arkadia-SnoN complexes lead to SnoN degradation. R-Smad-Smad4 complexes accumulate in the nucleus and bind to DNA. C: BMP7 prevents loss of SnoN expression. R-Smad-Smad4 com- plexes accumulate in the nucleus, but DNA binding to consensus Smad binding elements is prevented by SnoN. Arkadia-dependent SnoN degrada- tion appears necessary for Smad3 (or Smad2 -exon3)-dependent responses, but not Smad1/Smad4-dependent responses or responses driven by Smad2- Smad4-FoxH1 complexes (see Levy et al26).

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Binding Assay, Ligand Binding Assay, Phospho-proteomics, Expressing

Journal: Cancers

Article Title: Silibinin Overcomes EMT-Driven Lung Cancer Resistance to New-Generation ALK Inhibitors

doi: 10.3390/cancers14246101

Figure Lengend Snippet: Targeted effects of silibinin against the TGFβ/TGFβR/SMAD signaling pathway. ( A ) Expression levels of E-cadherin, SNAIL, vimentin, phospho-SMAD2/3, total SMAD2/3 were detected by immunoblotting in H2228/H2228CR and H3122/H3122CR cells using specific antibodies. The intensity of the bands was measured using the ImageJ software. Fold-change of each protein relative to parental cells was calculated using GAPDH as a loading control. The figure shows representative immunoblots of multiple ( n ≥ 5) independent experiments. E: Epithelial; M: Mesenchymal. ( B ) Top: Relative luciferase activity using SBE Reporter–HEK293 cells pre-incubated during 4–5 h with graded concentrations of SB525334 and silibinin before stimulation with TGFβ1. Bottom: Expression levels of phospho-SMAD2/3 and total SMAD2/3 were detected by immunoblotting in HEK293 cells stimulated with TGFβ1 (0, 6, and 24 h) in the absence/presence of either silibinin or SB431542 using specific antibodies. The intensity of the bands was measured using the ImageJ software. Fold-change of each protein relative to untreated samples was calculated using GAPDH as a loading control. The figure shows representative immunoblots of multiple ( n ≥ 3) independent experiments. ( C ) Volcano plots of the results from analyses of the Applied Biosystems TM TaqMan TM Array Human TGFβ Pathway in H2228/H2228CR cells cultured in the absence/presence of silibinin (100 μmol/L) for 48 h. Each dot represents a transcript with its corresponding mean Log2 fold-change (FC) ( x axis) and Benjamini–Hochberg corrected p -value (−log10, y axis). Colored dots illustrate differential lipid species, using a cutoff of p < 0.05 and log2FC > 1 or < 1. ( D ) The figure depicts the backbone of the overall crystal structure of TGFβR1 (5E8S) and TGFβR2 (5E8Y) with rainbow colors showing the best docked poses of silibinin A and silibinin B at the catalytic site. The uncropped western blot figures were presented in .

Article Snippet: The SBE Reporter–HEK293 cell line (Cat. #60653; BPS Bioscience, San Diego, CA, USA) was employed for monitoring the impact of silibinin on the activity of the TGFβ/SMAD signaling pathway.

Techniques: Expressing, Western Blot, Software, Luciferase, Activity Assay, Incubation, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: PLK1 inhibition dampens NLRP3 inflammasome–elicited response in inflammatory disease models

doi: 10.1172/JCI162129

Figure Lengend Snippet: ( A – C ) After 7 days of differentiation, murine BMDMs were primed (100 ng/mL LPS, 5 hours) and activated (5 μM nigericin, for up to 2 hours). Phosphohistone H3 (PHH3) was used as a mitotic marker ( A and B ), and cells with high ASC fluorescence (ASC hi ) were identified as an activated speck-forming subpopulation ( A and C ). n = 3/group. PE, phycoerythrin. ( D and E ) Geometric (geom) MFI of PLK1 was measured in mitotic cells (dark green) and in nonmitotic cells under the untreated condition (green) and in nonmitotic cells under the primed condition (orange). An isotype antibody control (Isotype Ab. Ctrl) and a secondary antibody control (Sec. Ab. Ctrl) were used. n = 3/group. ( F – H ) Primed (100 ng/mL LPS, 5 hours) BMDMs were activated (5 mM ATP, 30 minutes), with or without selective PLK1 inhibitors (3 μM cyclapolin 9 [C9]; 10 nM SBE13; 50 nM Ro3280; 0.8 nM BI6727) at the activation stage. The supernatants were collected to measure the IL-1β concentration ( F ), cell death ( G ), and the TNF-α concentration ( H ). The caspase 1 inhibitor Ac-YVAD-FMK or no inhibitor (n.i.) treatment was used as a control. Results are representative of 4 ( A – E ) or 3 ( F – H ) independent experiments. n = 9, 7, 5, 5, 5, and 5 (in order from the left to the right bars in F and H ). n = 4/group ( G ). One-way ANOVA with Tukey’s post hoc test was used for statistical analysis. All data are the mean ± SEM.

Article Snippet: BMDMs were treated with the following PLK1 inhibitors: 3 μM cyclapolin 9 (Tocris, 3316), 10nM SBE13 (Selleckchem, S7720), 50nM Ro3280 (Stratech, S7248-SEL), and 0.4 or 0.8 nM BI6727 (Selleckchem, S2235).

Techniques: Marker, Fluorescence, Control, Activation Assay, Concentration Assay

Journal:

Article Title: Novel tumor growth inhibition mechanism by cell cycle regulator cdk2ap1 involves anti-angiogenesis modulation

doi: 10.1016/j.mvr.2010.06.001

Figure Lengend Snippet: (A) Activin A and TGFβ1 production by SCC1 cells as detected using either a human/rat/mouse Activin A or human TGFβ1 quantikine ELISA kits at either 24h or 48h following dox treatment. (B) Blocking of TGFβ signaling by using a neutralizing antibody resulted in reversal of several cdk2ap1 antiangiogenic phenotypes, as assessed by HUVEC tube formation. HUVEC2 cells were incubated either with conditioned media (CCM)-plus anti-TGFβ1 antibody or CCM containing isotype control antibody. (C) Representative images of HUVEC tube formation assay showing the effect of incubating HUVEC with anti-TGFβ1 antibody (+) or isotype control (−). The TGFβ antibody reversed the antiangiogenesis phenotype of cdk2ap1. (D) HUVEC CCK8 cell viability assay. In a CCK8 HUVEC proliferation assay, blocking TGFβ reverted cdk2ap1 antigrowth effects on HUVEC cells on day 4 post-treatment (p<0.03). (E) A CaspGlo luminescence assay for assessing Casp3/7 activity as a measure of HUVEC apoptosis was performed on day 4 post-treatment in a 96-well format. No effect on caspase activity was observed with TGFβ blockage.

Article Snippet: The signaling pathway-responsive reporter (luc) constructs used were obtained from Addgene (Cambridge, MA): pG13-luc (p53 sites, #16442), FBE/SBE-Luc (Smad4 sites, TGFβ1 pathway, #16525), k3(long)-luc (TNFα promoter, #11110), 10-4 cycE-luc (E2F1 sites, #8458), 16xTCF/LEF-luc (TCF/LEF sites, #17165), and NFkB-luc and AP1-luc were obtained from Clontech (Mountain View, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation, HUVEC Tube Formation Assay, Viability Assay, Proliferation Assay, Luminescence Assay, Activity Assay