anti-smad2 Search Results


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  • 99
    Cell Signaling Technology Inc smad2 3
    BMP4 treatment attenuates TGF-β1-induced <t>Smad2/3</t> phosphorylation by increasing activating Smad1/5/9. GAPDH was used as the protein loading control. ( A ) Phosphorylated Smad 1/5/9 and ( B ) Phosphorylated Smad 2/3 was measured by Western blots and semi-quantified with relative total Smad protein. The data are presented as the mean values ± SEM. n = 3/group. *P
    Smad2 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho smad2
    GDF1 is not an active ligand but enhances Nodal activity. ( A – C ) The activity of the Nodal-responsive reporter (n2) 7 luc in the Xenopus animal cap assay was determined after injection of mRNAs for Nodal (10 pg), GDF1 (1000 pg), or the Nodal coreceptor Cripto (20 pg) ( A ); of mRNAs for Nodal (2 pg) or GDF1 (40 pg) ( B ); or of mRNAs for Nodal (2 pg), GDF1 (40 pg), Lefty1 (50 pg), or Lefty2 (50 pg) ( C ). All embryos in B and C were also injected with 100 pg of the mRNA for the Nodal coreceptor Cryptic. ( D ) Xenopus embryos were injected with mRNAs for Nodal (++, 50 pg; +, 10 pg), GDF1 (40 pg), or Cryptic (100 pg), as indicated, after which animal caps were subjected to immunoblot analysis with antibodies to <t>phospho-Smad2</t> (p-Smad2) or to α-tubulin (loading control). ( E , F ) The animal cap assay was also performed with mRNAs for zDVR1, Squint (Sqt), Cyclops (Cyc), or Flag-tagged OEP (OEP), as indicated. Injected mRNA amounts are shown in picograms (in parentheses).
    Phospho Smad2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1941 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti gapdh
    ADPL profiling of differential enzyme activity correlated with distinct phenotypes in native contexts. a Representative ADPL images measuring endogenous <t>NCEH1</t> activity in paired aggressive (SKOV3 and PC3) and non-aggressive (OVCAR3 and LNCaP) cancer cell lines from ovarian and prostate cancers, respectively. b Relative quantification of NCEH1 activity in each cell line from a . Statistical evaluations shown are comparing mean ADPL signal between non-aggressive and aggressive cells within each tissue of origin. Quantification in b : LNCaP ( n = 47), PC3 ( n = 43), OVCAR3 ( n = 60), SKOV3 ( n = 35). c α-Biotin western blot “gel-based” profiling of serine hydrolase activity in the four cell lines is shown. The two bands at ~42 and 45 kDa are glycoforms of NCEH1; the overlapped intermediate band is another enzyme family member. α-NCEH1 immunoblotting indicates protein abundance. <t>α-GAPDH</t> immunoblotting from the same experiment is shown as a loading control. Scale bars = 10 μm in all images. Blue channel: DAPI nuclear; red channel: ADPL signal; gray channel: DIC. *** P
    Anti Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 23621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc rabbit anti phospho smad2
    Ampkα1 sensitive up-regulation of Tgf-β-activated protein kinase 1 (Tak1) following unilateral ureteral obstruction. A. Representative original Western blots and arithmetic means ± SEM (n = 9) of normalized Tgf-β precursor/Gapdh protein ratio in renal tissue from non-obstructed control kidney (CTR) and obstructed kidney (UUO) of Ampkα1 knockout mice (black bars, Ampkα1 -/- ) and respective wild-type mice (white bars, Ampkα1 +/+ ) following 7 days of unilateral ureteral obstruction. B. Representative original Western blots and arithmetic means ± SEM (n = 9) of normalized phospho-Tak1 (Ser 412 )/ Gapdh protein ratio and total Tak1/ Gapdh protein ratio in renal tissue from non-obstructed control kidney (CTR) and obstructed kidney (UUO) of Ampkα1 knockout mice (black bars, Ampkα1 -/- ) and respective wild-type mice (white bars, Ampkα1 +/+ ) following 7 days of unilateral ureteral obstruction. C. Representative original Western blots and arithmetic means ± SEM (n = 9) of normalized <t>phospho-Smad2</t> (Ser 465/467 )/ Gapdh protein ratio and total Smad2/ Gapdh protein ratio in renal tissue from non-obstructed control kidney (CTR) and obstructed kidney (UUO) of Ampkα1 knockout mice (black bars, Ampkα1 -/- ) and respective wild-type mice (white bars, Ampkα1 +/+ ) following 7 days of unilateral ureteral obstruction. *(p
    Rabbit Anti Phospho Smad2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 267 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit anti smad2 3
    Transient activation of TGFβ signaling induced Snail up-regulation, which was required for anastasis, and caused increased migration in the late stage. (A) Cleavage of PARP1 and Snail protein in HeLa cells stably expressing scrambled shRNA or Snail shRNA treated with or without EtOH for 3 h. The white dotted line divides the lanes that were cropped from the same blot. (B) Recovery rate of HeLa-scrambled shRNA and HeLa-Snail shRNA cells after mock treatment or EtOH treatment ( n = 3). (C) Recovery rate of HeLa-scrambled shRNA and HeLa-Snail shRNA cells after control treatment or STS treatment ( n = 3). In all bar graphs, error bars represent the standard error of the mean. (D) Western blots of full-length PARP1 (FL PARP1) and cleaved PARP1 in HeLa-scrambled shRNA and HeLa-Snail shRNA cells treated with STS or control. The white dotted line divides the lanes that were cropped from the same blot. (E) Western blots of <t>phosphor-Smad2/3</t> (pSmad2/3), total Smad2/3, Snail in cells after mock treatment, EtOH treatment, or starvation (T) and in cells recovering from these treatments (recovery). (F) The level of pSmad2/3 and Smad2/3 in HeLa cells treated with STS or vehicle control (T) and in cells recovering from STS or control treatment (recovery). (G) The level of pSmad2/3, Smad2/3, and Snail in apoptotic cells (treatment); cells after 1 h of recovery (recovery 1 h); and cells after 4 h of recovery (recovery 4 h). The addition of LY364497 and EtOH is indicated. The numbers under the blots are the intensity of the bands or the indicated ratio relative to the mock-treated sample. (H) The mRNA expression of Snail (SNAI1) after 1 h of recovery. The addition of LY364947 and EtOH is indicated. (I) Mean migration speed of the indicated group of cells during wound-healing assay ( n = 8). Before wound-healing assay, cells were treated with or without EtOH together with 0.1% DMSO or 5 µM LY364947 for 3 h, followed by 4 h of recovery with 0.1% DMSO or 5 µM LY364947 and an additional 16 h of recovery without any inhibitor. Error bars represent 95% confidence interval. *, P
    Rabbit Anti Smad2 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Cell Signaling Technology Inc phospho smad2 ser465 467
    Transient activation of TGFβ signaling induced Snail up-regulation, which was required for anastasis, and caused increased migration in the late stage. (A) Cleavage of PARP1 and Snail protein in HeLa cells stably expressing scrambled shRNA or Snail shRNA treated with or without EtOH for 3 h. The white dotted line divides the lanes that were cropped from the same blot. (B) Recovery rate of HeLa-scrambled shRNA and HeLa-Snail shRNA cells after mock treatment or EtOH treatment ( n = 3). (C) Recovery rate of HeLa-scrambled shRNA and HeLa-Snail shRNA cells after control treatment or STS treatment ( n = 3). In all bar graphs, error bars represent the standard error of the mean. (D) Western blots of full-length PARP1 (FL PARP1) and cleaved PARP1 in HeLa-scrambled shRNA and HeLa-Snail shRNA cells treated with STS or control. The white dotted line divides the lanes that were cropped from the same blot. (E) Western blots of <t>phosphor-Smad2/3</t> (pSmad2/3), total Smad2/3, Snail in cells after mock treatment, EtOH treatment, or starvation (T) and in cells recovering from these treatments (recovery). (F) The level of pSmad2/3 and Smad2/3 in HeLa cells treated with STS or vehicle control (T) and in cells recovering from STS or control treatment (recovery). (G) The level of pSmad2/3, Smad2/3, and Snail in apoptotic cells (treatment); cells after 1 h of recovery (recovery 1 h); and cells after 4 h of recovery (recovery 4 h). The addition of LY364497 and EtOH is indicated. The numbers under the blots are the intensity of the bands or the indicated ratio relative to the mock-treated sample. (H) The mRNA expression of Snail (SNAI1) after 1 h of recovery. The addition of LY364947 and EtOH is indicated. (I) Mean migration speed of the indicated group of cells during wound-healing assay ( n = 8). Before wound-healing assay, cells were treated with or without EtOH together with 0.1% DMSO or 5 µM LY364947 for 3 h, followed by 4 h of recovery with 0.1% DMSO or 5 µM LY364947 and an additional 16 h of recovery without any inhibitor. Error bars represent 95% confidence interval. *, P
    Phospho Smad2 Ser465 467, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 334 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti smad2 3
    Inhibition of <t>Smad2/3</t> enhances reprogramming, but reduces Oct4 expression
    Anti Smad2 3, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti smad2
    NIT1 recruits <t>Smad2/3</t> and then activates the TGFβ-Smad2/3 pathway by interacting with SARA and SMAD2/3 in CRC. a Protein interactions among NIT1, SARA and SMAD2/3 using coimmunoprecipitation (co-IP) assays. b Western blot analysis of Smad3, p-Smad3, Smad2 and p-Smad2 after increasing or decreasing the expression of NIT1 in CRC cells as indicated. GAPDH served as the loading control. c Negative control or si-SARA was transfected into over-expressed NIT1 CRC cells to detect the expression levels of SARA, Smad3, p-Smad3, Smad2 and p-Smad2 using western blot assays. GAPDH served as the loading control
    Anti Smad2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 216 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    BMP4 treatment attenuates TGF-β1-induced Smad2/3 phosphorylation by increasing activating Smad1/5/9. GAPDH was used as the protein loading control. ( A ) Phosphorylated Smad 1/5/9 and ( B ) Phosphorylated Smad 2/3 was measured by Western blots and semi-quantified with relative total Smad protein. The data are presented as the mean values ± SEM. n = 3/group. *P

    Journal: Scientific Reports

    Article Title: Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition

    doi: 10.1038/srep32182

    Figure Lengend Snippet: BMP4 treatment attenuates TGF-β1-induced Smad2/3 phosphorylation by increasing activating Smad1/5/9. GAPDH was used as the protein loading control. ( A ) Phosphorylated Smad 1/5/9 and ( B ) Phosphorylated Smad 2/3 was measured by Western blots and semi-quantified with relative total Smad protein. The data are presented as the mean values ± SEM. n = 3/group. *P

    Article Snippet: The following antibodies were used for Western blotting and immunofluorescence: BMP4 (Thermo Scientific, Carlsbad, CA), α-Smooth muscle and fibronectin (Sigma-Aldrich, MO, USA), E-cadherin (BD biosciences, San Jose, CA), BMPR1A, BMPR1B, activin receptor type IA, Vimentin, smad1/5/9 and β-actin (Abcam Ltd., Cambridge, USA), ZO-1 (Invitrogen, Carlsbad, CA), phospho-Smad1/5/9, phospho-Smad2/3 and Smad2/3 (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Western Blot

    CD109 inhibits extracellular matrix protein production and Smad2/3 phosphorylation in systemic sclerosis and normal fibroblasts . (A, C) Systemic sclerosis (SSc) skin fibroblasts and (B, D) normal skin fibroblasts were transiently transfected with CD109-specific siRNA or control siRNA. Cell lysates were analyzed by western blot using (A, B) anti-CD109 (top panel), anti-fibronectin (second panel), anti-type I collagen (third panel) or anti-CCN2 (fourth panel) antibodies or (C, D) anti-phosphoSmad2, anti-phosphoSmad3, anti-Smad2 or anti-Smad3 antibodies. Membranes were reprobed with an anti-β-actin (bottom panel) antibody to confirm that equal amounts of protein were loaded in each lane. Results presented are representative of three independent experiments. The representative SSc fibroblast data shown correspond to (A) SSc Patient S2 and (C) SSc Patient S16.

    Journal: Arthritis Research & Therapy

    Article Title: CD109, a TGF-? co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

    doi: 10.1186/ar3877

    Figure Lengend Snippet: CD109 inhibits extracellular matrix protein production and Smad2/3 phosphorylation in systemic sclerosis and normal fibroblasts . (A, C) Systemic sclerosis (SSc) skin fibroblasts and (B, D) normal skin fibroblasts were transiently transfected with CD109-specific siRNA or control siRNA. Cell lysates were analyzed by western blot using (A, B) anti-CD109 (top panel), anti-fibronectin (second panel), anti-type I collagen (third panel) or anti-CCN2 (fourth panel) antibodies or (C, D) anti-phosphoSmad2, anti-phosphoSmad3, anti-Smad2 or anti-Smad3 antibodies. Membranes were reprobed with an anti-β-actin (bottom panel) antibody to confirm that equal amounts of protein were loaded in each lane. Results presented are representative of three independent experiments. The representative SSc fibroblast data shown correspond to (A) SSc Patient S2 and (C) SSc Patient S16.

    Article Snippet: Following blocking with 5% nonfat dry milk in Tris-buffered saline-Tween at room temperature for 1 hour, membranes were incubated overnight with antibodies against CD109 (R & D Systems Inc.), pSmad2, pSmad3 and Smad2 (all from Cell Signaling Technology, Danvers, MA, USA), collagen type I (Abcam, Cambridge, MA, USA), fibronectin (BD Biosciences, Mississauga, ON, Canada) and CCN2, Smad3 and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by incubation with a horseradish peroxidase-conjugated secondary antibody.

    Techniques: Transfection, Western Blot

    The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes breast cancer invasion and metastasis via inducing TGF-β1/Smad-mediated EMT. a-b Wound healing, transwell migration and invasion assays for circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μm. Representative images are shown at 0 and 24 h after gap creation. Scale bar = 20 μm. c Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with ESRP1, USF1 or TGF-β1 vector, or miR-148a/152-3p inhibitors. d Immunoblot analysis of p-Smad2, p-Smad3, Smad2/3, E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μM. GAPDH was used as a loading control. e The illustration summarizes our findings. CircANKS1B, as miR-148a-3p and miR-152-3p sponge, increases USF1 expression by eliminating miR-148a/152-3p-mediated repression of USF1, and then, USF1 can respectively transcriptionally up-regulate ESRP1 and TGF-β1 expression via directly binding to the E-box motifs in their promoter regions. Subsequently, ESRP1 promotes circANKS1B generation, and TGF-β1 activates its downstream Smad signaling to induce EMT, thereby enhancing breast cancer invasion and metastasis. Data were represented as means ± S.D. of at least three independent experiments. ** p

    Journal: Molecular Cancer

    Article Title: The pro-metastasis effect of circANKS1B in breast cancer

    doi: 10.1186/s12943-018-0914-x

    Figure Lengend Snippet: The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes breast cancer invasion and metastasis via inducing TGF-β1/Smad-mediated EMT. a-b Wound healing, transwell migration and invasion assays for circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μm. Representative images are shown at 0 and 24 h after gap creation. Scale bar = 20 μm. c Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with ESRP1, USF1 or TGF-β1 vector, or miR-148a/152-3p inhibitors. d Immunoblot analysis of p-Smad2, p-Smad3, Smad2/3, E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μM. GAPDH was used as a loading control. e The illustration summarizes our findings. CircANKS1B, as miR-148a-3p and miR-152-3p sponge, increases USF1 expression by eliminating miR-148a/152-3p-mediated repression of USF1, and then, USF1 can respectively transcriptionally up-regulate ESRP1 and TGF-β1 expression via directly binding to the E-box motifs in their promoter regions. Subsequently, ESRP1 promotes circANKS1B generation, and TGF-β1 activates its downstream Smad signaling to induce EMT, thereby enhancing breast cancer invasion and metastasis. Data were represented as means ± S.D. of at least three independent experiments. ** p

    Article Snippet: The antibodies we used are as follows: anti-E-cadherin (Abcam # ab40772), anti-Vimentin (Abcam # ab92547), anti-Fibronectin (Proteintech # 15613–1-AP), anti-AGO2 (Abcam # ab57113), anti-USF1 (Santa Cruz # sc-390,027), anti-RNA polymerase II (Santa Cruz # sc-47,701), anti-TGF-β1 (Abcam # ab92486), anti-ESRP1 (Abcam # ab107278), anti-p-Smad2 (Cell Signaling Technology # 8828), anti-p-Smad3 (Cell Signaling Technology # 9520), anti-Smad2/3 (Cell Signaling Technology # 8685), anti-GAPDH (Proteintech # 10494–1-AP) and anti-β-actin (Cell Signaling Technology # 4970).

    Techniques: Migration, Transfection, Concentration Assay, Multiple Displacement Amplification, Plasmid Preparation, Expressing, Binding Assay

    Cytoplasmic PML induces SMAD2/3 phosphorylation, EMT and cell invasion in vitro . ( a ) Immunoblotting of whole-cell extracts from DU145 and PC3 cells expressing PML mutant constructs using antibodies against phosphorylated forms of SMAD2/3 and β-actin (loading control). ( b ) Immunoblotting analysis of whole-cell extracts from DU145 and PC3 cells expressing the PML ΔNLS construct, treated with DMSO (control) or TGFβ-RI inhibitor (3 μ M for 48 h) using antibodies against HA, phosphorylated forms of SMAD2/3, E-Cadherin and β-actin (loading control). ( c ) Immunoblotting of whole-cell extracts from DU145 and PC3 cells expressing PML mutant constructs using antibodies against total SMAD2, SMAD3 and β-actin (loading control). ( d ) Immunofluorescence analysis of DU145 and PC3 cells expressing the PML ΔNLS construct, treated with DMSO (control) or TGFβ-RI inhibitor (3 μM for 48 h) using antibodies against phosphorylated forms of SMAD2/3, E-Cadherin and N-Cadherin. Scale bar=20 μm. ( e , f ) Wound healing assay (Scratch assay) using DU145 and PC3 cells expressing the PML ΔNLS construct, treated with DMSO (control) or TGFβ-RI inhibitor (3 μM for 48h) and the data relating to the percentages of gap closure at time points 0 and 24 h following scratching. Two-tailed paired test: * P =0.0451 and ** P =0.0088.

    Journal: Oncogene

    Article Title: Cytoplasmic PML promotes TGF-β-associated epithelial–mesenchymal transition and invasion in prostate cancer

    doi: 10.1038/onc.2015.409

    Figure Lengend Snippet: Cytoplasmic PML induces SMAD2/3 phosphorylation, EMT and cell invasion in vitro . ( a ) Immunoblotting of whole-cell extracts from DU145 and PC3 cells expressing PML mutant constructs using antibodies against phosphorylated forms of SMAD2/3 and β-actin (loading control). ( b ) Immunoblotting analysis of whole-cell extracts from DU145 and PC3 cells expressing the PML ΔNLS construct, treated with DMSO (control) or TGFβ-RI inhibitor (3 μ M for 48 h) using antibodies against HA, phosphorylated forms of SMAD2/3, E-Cadherin and β-actin (loading control). ( c ) Immunoblotting of whole-cell extracts from DU145 and PC3 cells expressing PML mutant constructs using antibodies against total SMAD2, SMAD3 and β-actin (loading control). ( d ) Immunofluorescence analysis of DU145 and PC3 cells expressing the PML ΔNLS construct, treated with DMSO (control) or TGFβ-RI inhibitor (3 μM for 48 h) using antibodies against phosphorylated forms of SMAD2/3, E-Cadherin and N-Cadherin. Scale bar=20 μm. ( e , f ) Wound healing assay (Scratch assay) using DU145 and PC3 cells expressing the PML ΔNLS construct, treated with DMSO (control) or TGFβ-RI inhibitor (3 μM for 48h) and the data relating to the percentages of gap closure at time points 0 and 24 h following scratching. Two-tailed paired test: * P =0.0451 and ** P =0.0088.

    Article Snippet: Antibodies For this study, we used antibodies to PML (1:250 for IB, 1:100 for IP, sc-966, Santa Cruz Biotechnology, Delaware Ave., Santa Cruz, CA, USA), PML (1:500 for immunohistochemistry (IHC), sc5621, Santa Cruz Biotechnology, Delaware Ave.), HA (1:1000 for IB, 1:200 for IF, A190-108 A, Bethyl Laboratories Inc., Montgomery, TX, USA), β-actin (1:5000 for IB, A5441, Sigma-Aldrich, St Louis, MO, USA), Exportin-1 (CRM1; 1:5000 for IB, 1:2000 for IHC, A300-469A, Bethyl Laboratories Inc.), E-Cadherin (1:500 for IF, 610181, BD Transduction Laboratories, San Jose, CA, USA), EMT antibody Sampler Kit (9782, Cell Signaling Technology, Danvers, MA, USA), which contains rabbit antibodies each used at 1:300 (IB), N-Cadherin and Vimentin antibodies were used at 1:500 for IF, Phospho-Smad2/Smad3 (1:300 for IB, 8828, Cell Signaling Technology), Phospho-Smad2/3 (1:100 for IHC, sc-11769, Santa Cruz Biotechnology), Anti-rabbit IgG, HRP-linked Antibody (1:1000 for IB, 7074, Cell Signaling Technology), Anti-TGFβ Receptor I (1:1000 for IB, 3712, Cell Signaling Technology), Anti-SMAD2 (1:1000 for IB, 3122, Cell Signaling Technology), Anti-SMAD3 (1:1000 for IB, 9513, Cell Signaling Technology), Anti-phospho-SMAD2 (1:500, SAB4504207, Sigma-Aldrich), Anti-phospho-SMAD3 (1:500, SAB4300253, Sigma-Aldrich), Anti-mouse IgG, HRP-linked antibody (1:1000 IB, 7076, Cell Signaling Technology).

    Techniques: In Vitro, Expressing, Mutagenesis, Construct, Immunofluorescence, Wound Healing Assay, Two Tailed Test

    Schematic representation of the role played by cytoplasmic PML in promoting TGF-β signalling in tumour microenvironment. PML is exported from the nucleus to the cytoplasm in a CRM1-dependent manner. Cytoplasmic PML interacts and promotes the association of SARA with SMAD2 and SMAD3, which activates their phosphorylation. Phosphorylated SMAD2 and SMAD3 interact with SMAD4 and translocate to the nucleus where they regulate gene expression of factors involved in promoting EMT and invasion. During EMT, epithelial cells characterized by the expression of epithelial markers and a baso-apical polarity start to lose their cell–cell contacts and to express mesenchymal markers characteristic of mesenchymal cells. The later have a high migratory and invasive properties and have a key role in cancer progression.

    Journal: Oncogene

    Article Title: Cytoplasmic PML promotes TGF-β-associated epithelial–mesenchymal transition and invasion in prostate cancer

    doi: 10.1038/onc.2015.409

    Figure Lengend Snippet: Schematic representation of the role played by cytoplasmic PML in promoting TGF-β signalling in tumour microenvironment. PML is exported from the nucleus to the cytoplasm in a CRM1-dependent manner. Cytoplasmic PML interacts and promotes the association of SARA with SMAD2 and SMAD3, which activates their phosphorylation. Phosphorylated SMAD2 and SMAD3 interact with SMAD4 and translocate to the nucleus where they regulate gene expression of factors involved in promoting EMT and invasion. During EMT, epithelial cells characterized by the expression of epithelial markers and a baso-apical polarity start to lose their cell–cell contacts and to express mesenchymal markers characteristic of mesenchymal cells. The later have a high migratory and invasive properties and have a key role in cancer progression.

    Article Snippet: Antibodies For this study, we used antibodies to PML (1:250 for IB, 1:100 for IP, sc-966, Santa Cruz Biotechnology, Delaware Ave., Santa Cruz, CA, USA), PML (1:500 for immunohistochemistry (IHC), sc5621, Santa Cruz Biotechnology, Delaware Ave.), HA (1:1000 for IB, 1:200 for IF, A190-108 A, Bethyl Laboratories Inc., Montgomery, TX, USA), β-actin (1:5000 for IB, A5441, Sigma-Aldrich, St Louis, MO, USA), Exportin-1 (CRM1; 1:5000 for IB, 1:2000 for IHC, A300-469A, Bethyl Laboratories Inc.), E-Cadherin (1:500 for IF, 610181, BD Transduction Laboratories, San Jose, CA, USA), EMT antibody Sampler Kit (9782, Cell Signaling Technology, Danvers, MA, USA), which contains rabbit antibodies each used at 1:300 (IB), N-Cadherin and Vimentin antibodies were used at 1:500 for IF, Phospho-Smad2/Smad3 (1:300 for IB, 8828, Cell Signaling Technology), Phospho-Smad2/3 (1:100 for IHC, sc-11769, Santa Cruz Biotechnology), Anti-rabbit IgG, HRP-linked Antibody (1:1000 for IB, 7074, Cell Signaling Technology), Anti-TGFβ Receptor I (1:1000 for IB, 3712, Cell Signaling Technology), Anti-SMAD2 (1:1000 for IB, 3122, Cell Signaling Technology), Anti-SMAD3 (1:1000 for IB, 9513, Cell Signaling Technology), Anti-phospho-SMAD2 (1:500, SAB4504207, Sigma-Aldrich), Anti-phospho-SMAD3 (1:500, SAB4300253, Sigma-Aldrich), Anti-mouse IgG, HRP-linked antibody (1:1000 IB, 7076, Cell Signaling Technology).

    Techniques: Expressing

    Expression of PML, CRM1, p-SMAD2/3, E-Cadherin and N-Cadherin in normal prostate and invasive adenocarcinoma tissues. ( a , b ) Immunohistochemistry/immunofluorescence using antibodies against PML, CRM1, p-SMAD2/3, E-Cadherin and N-Cadherin in normal prostate tissue ( a ) and invasive prostate adenocarcinoma tissue ( b ). Arrows indicates nuclear p-SMAD2/3. Scale bar=100 μ m and 50 μ m . DAPI, 4,6-diamidino-2-phenylindole.

    Journal: Oncogene

    Article Title: Cytoplasmic PML promotes TGF-β-associated epithelial–mesenchymal transition and invasion in prostate cancer

    doi: 10.1038/onc.2015.409

    Figure Lengend Snippet: Expression of PML, CRM1, p-SMAD2/3, E-Cadherin and N-Cadherin in normal prostate and invasive adenocarcinoma tissues. ( a , b ) Immunohistochemistry/immunofluorescence using antibodies against PML, CRM1, p-SMAD2/3, E-Cadherin and N-Cadherin in normal prostate tissue ( a ) and invasive prostate adenocarcinoma tissue ( b ). Arrows indicates nuclear p-SMAD2/3. Scale bar=100 μ m and 50 μ m . DAPI, 4,6-diamidino-2-phenylindole.

    Article Snippet: Antibodies For this study, we used antibodies to PML (1:250 for IB, 1:100 for IP, sc-966, Santa Cruz Biotechnology, Delaware Ave., Santa Cruz, CA, USA), PML (1:500 for immunohistochemistry (IHC), sc5621, Santa Cruz Biotechnology, Delaware Ave.), HA (1:1000 for IB, 1:200 for IF, A190-108 A, Bethyl Laboratories Inc., Montgomery, TX, USA), β-actin (1:5000 for IB, A5441, Sigma-Aldrich, St Louis, MO, USA), Exportin-1 (CRM1; 1:5000 for IB, 1:2000 for IHC, A300-469A, Bethyl Laboratories Inc.), E-Cadherin (1:500 for IF, 610181, BD Transduction Laboratories, San Jose, CA, USA), EMT antibody Sampler Kit (9782, Cell Signaling Technology, Danvers, MA, USA), which contains rabbit antibodies each used at 1:300 (IB), N-Cadherin and Vimentin antibodies were used at 1:500 for IF, Phospho-Smad2/Smad3 (1:300 for IB, 8828, Cell Signaling Technology), Phospho-Smad2/3 (1:100 for IHC, sc-11769, Santa Cruz Biotechnology), Anti-rabbit IgG, HRP-linked Antibody (1:1000 for IB, 7074, Cell Signaling Technology), Anti-TGFβ Receptor I (1:1000 for IB, 3712, Cell Signaling Technology), Anti-SMAD2 (1:1000 for IB, 3122, Cell Signaling Technology), Anti-SMAD3 (1:1000 for IB, 9513, Cell Signaling Technology), Anti-phospho-SMAD2 (1:500, SAB4504207, Sigma-Aldrich), Anti-phospho-SMAD3 (1:500, SAB4300253, Sigma-Aldrich), Anti-mouse IgG, HRP-linked antibody (1:1000 IB, 7076, Cell Signaling Technology).

    Techniques: Expressing, Immunohistochemistry, Immunofluorescence

    PAR-1-induced TGF-β activation on fibroblasts is mediated by FX A. Thrombin (FII), FX, Granzyme K (GZMK), MMP1, MMP13, KLK1, KLK4 and KLK6 mRNA levels in RAW264.7 cells as assessed by real-time reverse transcriptase PCR. Data are expressed relative to two housekeeping genes, GAPDH and TBP. Shown is the mean ± SEM, of an experiment performed three times. B. Western blot analysis of SMAD2 phosphorylation in NIH3T3 cells stimulated for 0, 15, 30 and 60 minutes with RAW264.7 CM in the absence or presence of the FXa inhibitor antistasin (40 μM). GAPDH served as loading control. C. Proposed mechanism by which macrophages promote lung fibrosis in a PAR-1 dependent manner. During lung injury, epithelial cells release mediators that potentiate PAR-1 dependent macrophage migration towards the injured site (1). The recruited macrophages subsequently secrete TGF-β and FX. The PAR-1 agonist (FX) than activates PAR-1 on fibroblasts (2) leading to TGF-β production and activation. Finally, TGF-β induces TGFBR signaling (3) on fibroblast thereby inducing their migration, differentiation and ECM deposition.

    Journal: Oncotarget

    Article Title: Protease activated receptor-1 regulates macrophage-mediated cellular senescence: a risk for idiopathic pulmonary fibrosis

    doi:

    Figure Lengend Snippet: PAR-1-induced TGF-β activation on fibroblasts is mediated by FX A. Thrombin (FII), FX, Granzyme K (GZMK), MMP1, MMP13, KLK1, KLK4 and KLK6 mRNA levels in RAW264.7 cells as assessed by real-time reverse transcriptase PCR. Data are expressed relative to two housekeeping genes, GAPDH and TBP. Shown is the mean ± SEM, of an experiment performed three times. B. Western blot analysis of SMAD2 phosphorylation in NIH3T3 cells stimulated for 0, 15, 30 and 60 minutes with RAW264.7 CM in the absence or presence of the FXa inhibitor antistasin (40 μM). GAPDH served as loading control. C. Proposed mechanism by which macrophages promote lung fibrosis in a PAR-1 dependent manner. During lung injury, epithelial cells release mediators that potentiate PAR-1 dependent macrophage migration towards the injured site (1). The recruited macrophages subsequently secrete TGF-β and FX. The PAR-1 agonist (FX) than activates PAR-1 on fibroblasts (2) leading to TGF-β production and activation. Finally, TGF-β induces TGFBR signaling (3) on fibroblast thereby inducing their migration, differentiation and ECM deposition.

    Article Snippet: Membranes were blocked for 1 hour in 4% milk in TBST and incubated overnight with monoclonal antibodies against a-SMA (1:1000, Santa Cruz, CA), GAPDH (1:1000, Santa Cruz, CA), collagen (1:800, SouthernBiotech, AL) or p-SMAD2 (1:1000, Cell Signaling Technology, Boston, MA) at 4°C.

    Techniques: Activation Assay, Polymerase Chain Reaction, Western Blot, Migration

    PAR-1 mediates TGF-β activation and production A. Representative Western blot analysis of SMAD2 phosphorylation in NIH3T3 cells stimulated for 0, 15, 30 and 60 minutes with RAW264.7 CM in the absence or presence of TGFBR inhibitors (10 μM SB-431542 and 10 μM LY-2157299) or P1pal-12 (10 μM). GAPDH served as loading control. B. Cell viability of NIH3T3s lentivirally transduced with a control shRNA construct (WT fibroblasts, down-pointing triangle) or a PAR-1 shRNA construct (PAR-1−/− fibroblasts, up-pointing triangle) as evaluated by MTT assays after 12 or 24 hours of incubation (Mean+/−SEM of an experiment performed two times in octoplo). C. Intracellular Ca 2+ fluxes in WT fibroblasts (circle) and PAR-1−/− fibroblasts (square) after stimulation with thrombin (1 U/ml). Ca 2+ fluxes are expressed as arbitrary units of fluorescent intensity after background correction. Shown is a representative experiment of three independent experiments. D. Western blot analysis of SMAD2 phosphorylation in WT fibroblasts or PAR-1−/− fibroblasts stimulated for 0, 15, 30 and 60 minutes with RAW264.7 CM or recombinant TGF-β (1 ng/ml). GAPDH served as loading control. E. Total TGF-β production of RAW264.7 cells and WT or PAR-1−/− fibroblasts stimulated without or with RAW264.7 conditioned medium (CM) after 24 hours (mean ± SEM, n = 6; *** P

    Journal: Oncotarget

    Article Title: Protease activated receptor-1 regulates macrophage-mediated cellular senescence: a risk for idiopathic pulmonary fibrosis

    doi:

    Figure Lengend Snippet: PAR-1 mediates TGF-β activation and production A. Representative Western blot analysis of SMAD2 phosphorylation in NIH3T3 cells stimulated for 0, 15, 30 and 60 minutes with RAW264.7 CM in the absence or presence of TGFBR inhibitors (10 μM SB-431542 and 10 μM LY-2157299) or P1pal-12 (10 μM). GAPDH served as loading control. B. Cell viability of NIH3T3s lentivirally transduced with a control shRNA construct (WT fibroblasts, down-pointing triangle) or a PAR-1 shRNA construct (PAR-1−/− fibroblasts, up-pointing triangle) as evaluated by MTT assays after 12 or 24 hours of incubation (Mean+/−SEM of an experiment performed two times in octoplo). C. Intracellular Ca 2+ fluxes in WT fibroblasts (circle) and PAR-1−/− fibroblasts (square) after stimulation with thrombin (1 U/ml). Ca 2+ fluxes are expressed as arbitrary units of fluorescent intensity after background correction. Shown is a representative experiment of three independent experiments. D. Western blot analysis of SMAD2 phosphorylation in WT fibroblasts or PAR-1−/− fibroblasts stimulated for 0, 15, 30 and 60 minutes with RAW264.7 CM or recombinant TGF-β (1 ng/ml). GAPDH served as loading control. E. Total TGF-β production of RAW264.7 cells and WT or PAR-1−/− fibroblasts stimulated without or with RAW264.7 conditioned medium (CM) after 24 hours (mean ± SEM, n = 6; *** P

    Article Snippet: Membranes were blocked for 1 hour in 4% milk in TBST and incubated overnight with monoclonal antibodies against a-SMA (1:1000, Santa Cruz, CA), GAPDH (1:1000, Santa Cruz, CA), collagen (1:800, SouthernBiotech, AL) or p-SMAD2 (1:1000, Cell Signaling Technology, Boston, MA) at 4°C.

    Techniques: Activation Assay, Western Blot, Transduction, shRNA, Construct, MTT Assay, Incubation, Recombinant

    GATA4 interacts with JMJD3 via a short linker between Zinc Finger domains including residue V266 A and B) Co-IPs (A) using nuclear lysates harvested HEK293Ts transfected with full length HA-JMJD3 and MYC-GATA4 truncation constructs (Schematic, B). Following Co-IP with anti-MYC, proteins were resolved by SDS-PAGE and immunoblotted for BRG1, HA, SMAD2/3, and MYC. NZF, N Terminal zinc finger. CZF, C Terminal zinc finger. +++, ++, +, +/-, and – represent strong, moderate, mild, minimal, and no binding. C and D) Co-IP (C) using nuclear lysates harvested HEK293Ts transfected with full length MYC-GATA4 and HA-JMJD3 truncation constructs (Schematic, D). Following Co-IP with anti-MYC, proteins were resolved by SDS-PAGE and immunoblotted for HA and MYC. JmjC, Jumonji C domain. +++ and + represent strong and mild binding. E) Co-IPs using nuclear lysates harvested HEK293Ts transfected with HA-JMJD3 and MYC-GATA4 wildtype (WT) or MYC-GATA4 V266M mutant. Following Co-IP with anti-MYC, proteins were resolved by SDS-PAGE and immunoblotted for HA and MYC. F) Electrophoretic mobility shift assay (EMSA) performed using nuclear extracts from HEK293Ts transfected with GFP, WT GATA4, GATA4Δ252-269, or GATA4 V266M. Arrows point to free probes, probes bound to GATA4, or probes bound to GATA4/MYC antibody (supershift) complex. G) Luciferase reporter assay performed in HeLa cells co-transfected with Nppa or Myh6 promoter vectors, Renilla vector, and WT GATA4, GATA4Δ252-269, or GATA4 V266M. Reporter activation was determined as a ratio of firefly luciferase to Renilla luciferase and normalized to the reporter alone group. N=3 per group. Data shown as mean ± SEM. * p

    Journal: bioRxiv

    Article Title: Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis

    doi: 10.1101/2020.02.12.945790

    Figure Lengend Snippet: GATA4 interacts with JMJD3 via a short linker between Zinc Finger domains including residue V266 A and B) Co-IPs (A) using nuclear lysates harvested HEK293Ts transfected with full length HA-JMJD3 and MYC-GATA4 truncation constructs (Schematic, B). Following Co-IP with anti-MYC, proteins were resolved by SDS-PAGE and immunoblotted for BRG1, HA, SMAD2/3, and MYC. NZF, N Terminal zinc finger. CZF, C Terminal zinc finger. +++, ++, +, +/-, and – represent strong, moderate, mild, minimal, and no binding. C and D) Co-IP (C) using nuclear lysates harvested HEK293Ts transfected with full length MYC-GATA4 and HA-JMJD3 truncation constructs (Schematic, D). Following Co-IP with anti-MYC, proteins were resolved by SDS-PAGE and immunoblotted for HA and MYC. JmjC, Jumonji C domain. +++ and + represent strong and mild binding. E) Co-IPs using nuclear lysates harvested HEK293Ts transfected with HA-JMJD3 and MYC-GATA4 wildtype (WT) or MYC-GATA4 V266M mutant. Following Co-IP with anti-MYC, proteins were resolved by SDS-PAGE and immunoblotted for HA and MYC. F) Electrophoretic mobility shift assay (EMSA) performed using nuclear extracts from HEK293Ts transfected with GFP, WT GATA4, GATA4Δ252-269, or GATA4 V266M. Arrows point to free probes, probes bound to GATA4, or probes bound to GATA4/MYC antibody (supershift) complex. G) Luciferase reporter assay performed in HeLa cells co-transfected with Nppa or Myh6 promoter vectors, Renilla vector, and WT GATA4, GATA4Δ252-269, or GATA4 V266M. Reporter activation was determined as a ratio of firefly luciferase to Renilla luciferase and normalized to the reporter alone group. N=3 per group. Data shown as mean ± SEM. * p

    Article Snippet: The following primary antibodies were used: anti-p-Smad2 (Cell Signaling 3108S, 1:1000), anti-total Smad2/3 (Cell Signaling 8685S, 1:1000), anti-HA (Rockland 600-401-384, 1:5000), anti-Myc (Santa Cruz sc-789, 1:500), anti-Brg1 (Santa Cruz sc-17796, 1:500), and anti-GAPDH (Life Technologies AM4300, 1:5000).

    Techniques: Transfection, Construct, Co-Immunoprecipitation Assay, SDS Page, Binding Assay, Mutagenesis, Electrophoretic Mobility Shift Assay, Luciferase, Reporter Assay, Plasmid Preparation, Activation Assay

    Recruitment mechanisms of epigenetic modifiers to cardiogenic genes by GATA4. Schematic summary of the regulation of interactions between GATA4 and epigenetic modifiers. Canonical TGF-β pathway activation or the congenital heart disease-associated mutation in GATA4 (V267M) impede the recruitment of epigenetic modifiers JMJD3 and BRG1 to cardiogenic genes by GATA4, resulting in impaired H3K27me3 demethylation and gene expression in cardiomyogenesis. TGF-β blockade prevents nuclear translocation of the SMAD2/3/4 complex, promoting the recruitment of epigenetic modifiers to cardiogenic genes by GATA4 resulting in efficient removal of H3K27me3 and gene expression.

    Journal: bioRxiv

    Article Title: Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis

    doi: 10.1101/2020.02.12.945790

    Figure Lengend Snippet: Recruitment mechanisms of epigenetic modifiers to cardiogenic genes by GATA4. Schematic summary of the regulation of interactions between GATA4 and epigenetic modifiers. Canonical TGF-β pathway activation or the congenital heart disease-associated mutation in GATA4 (V267M) impede the recruitment of epigenetic modifiers JMJD3 and BRG1 to cardiogenic genes by GATA4, resulting in impaired H3K27me3 demethylation and gene expression in cardiomyogenesis. TGF-β blockade prevents nuclear translocation of the SMAD2/3/4 complex, promoting the recruitment of epigenetic modifiers to cardiogenic genes by GATA4 resulting in efficient removal of H3K27me3 and gene expression.

    Article Snippet: The following primary antibodies were used: anti-p-Smad2 (Cell Signaling 3108S, 1:1000), anti-total Smad2/3 (Cell Signaling 8685S, 1:1000), anti-HA (Rockland 600-401-384, 1:5000), anti-Myc (Santa Cruz sc-789, 1:500), anti-Brg1 (Santa Cruz sc-17796, 1:500), and anti-GAPDH (Life Technologies AM4300, 1:5000).

    Techniques: Activation Assay, Mutagenesis, Expressing, Translocation Assay

    Activation of Canonical TGF-β signaling impairs cardiac reprogramming A and B) Immunoblot (A) and quantification (B) of phosphorylated SMAD2 (p-SMAD2) normalized to Total SMAD2 from whole cell extracts of Day 12 GHMT2m-reprogrammed cells co-infected with GFP, SMAD2, or SMAD7, or treated with 5 ng/mL TGF-β1 or 0.5 µM A-83-01. N = 3 per group. C) Beating cell counts per field (0.89 mm 2 ) from Day 10 and Day 13 GHMT2m-reprogrammed cells co-infected with GFP, SMAD2, or SMAD7, or treated with 5 ng/mL TGF-β1 or 0.5 µM A-83-01. N = 3 per group. D) Messenger RNA expression of cardiac genes Myh6 , Actc1 , and Pln harvested from Day 13 GHMT2m-reprogrammed MEFs co-infected with GFP, SMAD2, or SMAD7, or treated with 5 ng/mL TGF-β1 or 0.5 µM A-83-01. All data were normalized to the GFP group. N = 3 per group. Representative fluorescent images of indicated levels of sarcomere organization in Day 9 GHMT2m-reprogrammed MEFs. Red: α-actinin; Blue: Hoechst. Scale bar = 25 µM. E) Quantification of sarcomere organization in Day 9 GHMT2m-reprogrammed MEFs co-infected with GFP, SMAD2, or SMAD7. 10 fields of view per dish were collected across 3 individual experiments per group to analyze sarcomere organization. All data shown as mean ± SEM. * p

    Journal: bioRxiv

    Article Title: Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis

    doi: 10.1101/2020.02.12.945790

    Figure Lengend Snippet: Activation of Canonical TGF-β signaling impairs cardiac reprogramming A and B) Immunoblot (A) and quantification (B) of phosphorylated SMAD2 (p-SMAD2) normalized to Total SMAD2 from whole cell extracts of Day 12 GHMT2m-reprogrammed cells co-infected with GFP, SMAD2, or SMAD7, or treated with 5 ng/mL TGF-β1 or 0.5 µM A-83-01. N = 3 per group. C) Beating cell counts per field (0.89 mm 2 ) from Day 10 and Day 13 GHMT2m-reprogrammed cells co-infected with GFP, SMAD2, or SMAD7, or treated with 5 ng/mL TGF-β1 or 0.5 µM A-83-01. N = 3 per group. D) Messenger RNA expression of cardiac genes Myh6 , Actc1 , and Pln harvested from Day 13 GHMT2m-reprogrammed MEFs co-infected with GFP, SMAD2, or SMAD7, or treated with 5 ng/mL TGF-β1 or 0.5 µM A-83-01. All data were normalized to the GFP group. N = 3 per group. Representative fluorescent images of indicated levels of sarcomere organization in Day 9 GHMT2m-reprogrammed MEFs. Red: α-actinin; Blue: Hoechst. Scale bar = 25 µM. E) Quantification of sarcomere organization in Day 9 GHMT2m-reprogrammed MEFs co-infected with GFP, SMAD2, or SMAD7. 10 fields of view per dish were collected across 3 individual experiments per group to analyze sarcomere organization. All data shown as mean ± SEM. * p

    Article Snippet: The following primary antibodies were used: anti-p-Smad2 (Cell Signaling 3108S, 1:1000), anti-total Smad2/3 (Cell Signaling 8685S, 1:1000), anti-HA (Rockland 600-401-384, 1:5000), anti-Myc (Santa Cruz sc-789, 1:500), anti-Brg1 (Santa Cruz sc-17796, 1:500), and anti-GAPDH (Life Technologies AM4300, 1:5000).

    Techniques: Activation Assay, Infection, RNA Expression

    GATA4, JMJD3, and BRG1 form a large epigenetic complex which is disrupted by canonical TGF-β signaling A and B) Co-IPs using nuclear lysates harvested from HEK293T cells transfected with MYC-GHMT and HA-JMJD3. Following Co-IP with anti-MYC (A) or anti-HA (B), proteins were resolved by SDS-PAGE and immunoblotted for BRG1, HA, MYC and SMAD2/3. C) Co-IPs using nuclear lysates harvested from HEK293T cells co-transfected with MYC-GATA4 and either β-GAL (control) or HA-JMJD3. Following Co-IP with anti-MYC, proteins were resolved by SDS-PAGE and immunoblotted for BRG1, HA, and MYC. D) Co-IPs using nuclear lysates harvested from HEK293Ts co-transfected with HA-JMJD3, MYC-GATA4, and GFP, SMAD2, or SMAD7. Following Co-IP with anti-MYC, lysates were resolved by SDS-PAGE and immunoblotted for BRG1, HA, SMAD2/3, and MYC. E) Quantification of interactions between GATA4 and JMJD3 or BRG1 shown in panel D. N=3 per group. F) Luciferase reporter assay performed in HEK293Ts co-transfected with Myh6 promoter vector, Renilla vector, GHMT, pCMV-HA (empty vector control) or HA-JMJD3, and shGFP or sh Smarca4 (BRG1). Reporter activation was determined as a ratio of firefly luciferase to Renilla luciferase and normalized to HEK293Ts transfected with Myh6 promoter vector, Renilla vector, and pCMV-HA alone. N=3 per group. G) Luciferase reporter assay performed in HEK293Ts co-transfected with Myh6 promoter vector, Renilla vector, GHMT, HA-JMJD3, and GFP, SMAD2, or SMAD7. Reporter activation was determined as a ratio of firefly luciferase to Renilla luciferase and normalized to the GFP group. N=3 per group. All data shown as mean ± SEM. * p

    Journal: bioRxiv

    Article Title: Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis

    doi: 10.1101/2020.02.12.945790

    Figure Lengend Snippet: GATA4, JMJD3, and BRG1 form a large epigenetic complex which is disrupted by canonical TGF-β signaling A and B) Co-IPs using nuclear lysates harvested from HEK293T cells transfected with MYC-GHMT and HA-JMJD3. Following Co-IP with anti-MYC (A) or anti-HA (B), proteins were resolved by SDS-PAGE and immunoblotted for BRG1, HA, MYC and SMAD2/3. C) Co-IPs using nuclear lysates harvested from HEK293T cells co-transfected with MYC-GATA4 and either β-GAL (control) or HA-JMJD3. Following Co-IP with anti-MYC, proteins were resolved by SDS-PAGE and immunoblotted for BRG1, HA, and MYC. D) Co-IPs using nuclear lysates harvested from HEK293Ts co-transfected with HA-JMJD3, MYC-GATA4, and GFP, SMAD2, or SMAD7. Following Co-IP with anti-MYC, lysates were resolved by SDS-PAGE and immunoblotted for BRG1, HA, SMAD2/3, and MYC. E) Quantification of interactions between GATA4 and JMJD3 or BRG1 shown in panel D. N=3 per group. F) Luciferase reporter assay performed in HEK293Ts co-transfected with Myh6 promoter vector, Renilla vector, GHMT, pCMV-HA (empty vector control) or HA-JMJD3, and shGFP or sh Smarca4 (BRG1). Reporter activation was determined as a ratio of firefly luciferase to Renilla luciferase and normalized to HEK293Ts transfected with Myh6 promoter vector, Renilla vector, and pCMV-HA alone. N=3 per group. G) Luciferase reporter assay performed in HEK293Ts co-transfected with Myh6 promoter vector, Renilla vector, GHMT, HA-JMJD3, and GFP, SMAD2, or SMAD7. Reporter activation was determined as a ratio of firefly luciferase to Renilla luciferase and normalized to the GFP group. N=3 per group. All data shown as mean ± SEM. * p

    Article Snippet: The following primary antibodies were used: anti-p-Smad2 (Cell Signaling 3108S, 1:1000), anti-total Smad2/3 (Cell Signaling 8685S, 1:1000), anti-HA (Rockland 600-401-384, 1:5000), anti-Myc (Santa Cruz sc-789, 1:500), anti-Brg1 (Santa Cruz sc-17796, 1:500), and anti-GAPDH (Life Technologies AM4300, 1:5000).

    Techniques: Transfection, Co-Immunoprecipitation Assay, SDS Page, Luciferase, Reporter Assay, Plasmid Preparation, Activation Assay

    TGF-β signaling impairs demethylation of H3K27me3 and recruitment of GATA4 to target genes A) Global ChIP-seq density heatmap of H3K27me3 in Day 7 GHMT2m-reprogrammed cells with indicated TGF-β pathway manipulation and undifferentiated MEFs at ± 2kb from annotated transcription start sites (TSS). B and C) Representative IGV tracks depicting H3K27me3 intensity at Gata4 (B) and Tbx20 (C) promoters from Day 7 GHMT2m-reprogrammed cells with indicated TGF-β pathway manipulation and undifferentiated MEFs. D and E) Logarithmic transformation of H3K27me3 levels in relation to undifferentiated MEFs at ± 2kb from the annotated TSS of genes within the Muscle System Process Gene Ontology pathway (GO:0003012) that were also upregulated > 2 fold in GHMT2m + A-83-01 reprogrammed cells compared to undifferentiated MEFs as identified by RNA-seq (D) and at candidate cardiac genes selected from GO:0003012 (E). F) H3K27me3 levels at cardiac gene promoters Gata4, Tbx20, Actn2, Des, and Myh6 from Day 7 GHMT2m-reprogrammed cells co-infected with GFP, SMAD2, or SMAD7 or treated with DMSO, 5 ng/mL TGF-β1, or 0.5 µM A-83-01 analyzed by ChIP-qPCR. Undifferentiated MEFs served as a positive control for H3K27me3 levels. Data are presented as percentage of input. N=4 for MEFs, N=6 for GHMT2m + GFP + DMSO, N=6 for GHMT2m + GFP + TGF-β1, N=5 for GHMT2m + GFP + A-83-01, N=4 for GHMT2m + SMAD2, and N=5 for GHMT2m + SMAD7. G) GATA4 levels at cardiac gene promoters Nppa, Tbx20, and Myh6 from Day 5 GHMT2m-reprogrammed cells treated with DMSO, 5 ng/mL TGF-β1, or 0.5 µM A-83-01 analyzed by ChIP-qPCR. Data are presented as enrichment to IgG control. N=5 for GHMT2m + DMSO, N=4 for GHMT2m + TGF-β1, N=5 for GHMT2m + A-83-01. All data shown as mean ± SEM. *, # p

    Journal: bioRxiv

    Article Title: Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis

    doi: 10.1101/2020.02.12.945790

    Figure Lengend Snippet: TGF-β signaling impairs demethylation of H3K27me3 and recruitment of GATA4 to target genes A) Global ChIP-seq density heatmap of H3K27me3 in Day 7 GHMT2m-reprogrammed cells with indicated TGF-β pathway manipulation and undifferentiated MEFs at ± 2kb from annotated transcription start sites (TSS). B and C) Representative IGV tracks depicting H3K27me3 intensity at Gata4 (B) and Tbx20 (C) promoters from Day 7 GHMT2m-reprogrammed cells with indicated TGF-β pathway manipulation and undifferentiated MEFs. D and E) Logarithmic transformation of H3K27me3 levels in relation to undifferentiated MEFs at ± 2kb from the annotated TSS of genes within the Muscle System Process Gene Ontology pathway (GO:0003012) that were also upregulated > 2 fold in GHMT2m + A-83-01 reprogrammed cells compared to undifferentiated MEFs as identified by RNA-seq (D) and at candidate cardiac genes selected from GO:0003012 (E). F) H3K27me3 levels at cardiac gene promoters Gata4, Tbx20, Actn2, Des, and Myh6 from Day 7 GHMT2m-reprogrammed cells co-infected with GFP, SMAD2, or SMAD7 or treated with DMSO, 5 ng/mL TGF-β1, or 0.5 µM A-83-01 analyzed by ChIP-qPCR. Undifferentiated MEFs served as a positive control for H3K27me3 levels. Data are presented as percentage of input. N=4 for MEFs, N=6 for GHMT2m + GFP + DMSO, N=6 for GHMT2m + GFP + TGF-β1, N=5 for GHMT2m + GFP + A-83-01, N=4 for GHMT2m + SMAD2, and N=5 for GHMT2m + SMAD7. G) GATA4 levels at cardiac gene promoters Nppa, Tbx20, and Myh6 from Day 5 GHMT2m-reprogrammed cells treated with DMSO, 5 ng/mL TGF-β1, or 0.5 µM A-83-01 analyzed by ChIP-qPCR. Data are presented as enrichment to IgG control. N=5 for GHMT2m + DMSO, N=4 for GHMT2m + TGF-β1, N=5 for GHMT2m + A-83-01. All data shown as mean ± SEM. *, # p

    Article Snippet: The following primary antibodies were used: anti-p-Smad2 (Cell Signaling 3108S, 1:1000), anti-total Smad2/3 (Cell Signaling 8685S, 1:1000), anti-HA (Rockland 600-401-384, 1:5000), anti-Myc (Santa Cruz sc-789, 1:500), anti-Brg1 (Santa Cruz sc-17796, 1:500), and anti-GAPDH (Life Technologies AM4300, 1:5000).

    Techniques: Chromatin Immunoprecipitation, Transformation Assay, RNA Sequencing Assay, Infection, Real-time Polymerase Chain Reaction, Positive Control

    TGF-β signaling regulates the DR5 expression in HDFs though Smad2/3-SP1 complexes. a Measurements of DR5 promoter activity with the dual-luciferase-reporter system in HDFs treated with SB203580 (10 μM, Smad2 inhibitor) and SIS3 (5 μM, Smad3 inhibitor) for 2 h followed by TGF-β1 (10 ng/mL) treatment for 54 h ( n = 6 biologically independent experiments). b , c HDFs were transfected with SP1 and/or Smad 2/3 siRNA for 24 h. The cells were then exposed to TGF-β1 for 54 h. b mRNA DR5 levels in siRNA transfected HDFs with and without TGF-β1 treatment ( n = 4 biologically independent experiments). c Effects of siRNA-mediated knockdowns of SP1 and Smad2/3 on TGF-β1-induced DR5 levels in HDFs by western blot. Relative DR5 protein levels normalized to GAPDH ( n = 4 biologically independent experiments). d Western blot analysis of Smad2/3 immunoprecipitates (IP) of HDFs with or without TGF-β1 treatment ( n = 3 biologically independent experiments). e Binding assessment with ChIP assays of the two potential SP1 sites (site 1: −195 to −190, site 2: −159 to −154) and non-target site (NT) to the DR5 promoter in HDFs treated with TGF-β1 ( n = 4). f A representation of TGF-β pathway including Smad2/3, Smad4, and SP1 complexes in the translation mechanism of DR5. Data are shown as median ± interquartile range. The Mann–Whitney test was used. * P

    Journal: Nature Communications

    Article Title: Targeting of dermal myofibroblasts through death receptor 5 arrests fibrosis in mouse models of scleroderma

    doi: 10.1038/s41467-019-09101-4

    Figure Lengend Snippet: TGF-β signaling regulates the DR5 expression in HDFs though Smad2/3-SP1 complexes. a Measurements of DR5 promoter activity with the dual-luciferase-reporter system in HDFs treated with SB203580 (10 μM, Smad2 inhibitor) and SIS3 (5 μM, Smad3 inhibitor) for 2 h followed by TGF-β1 (10 ng/mL) treatment for 54 h ( n = 6 biologically independent experiments). b , c HDFs were transfected with SP1 and/or Smad 2/3 siRNA for 24 h. The cells were then exposed to TGF-β1 for 54 h. b mRNA DR5 levels in siRNA transfected HDFs with and without TGF-β1 treatment ( n = 4 biologically independent experiments). c Effects of siRNA-mediated knockdowns of SP1 and Smad2/3 on TGF-β1-induced DR5 levels in HDFs by western blot. Relative DR5 protein levels normalized to GAPDH ( n = 4 biologically independent experiments). d Western blot analysis of Smad2/3 immunoprecipitates (IP) of HDFs with or without TGF-β1 treatment ( n = 3 biologically independent experiments). e Binding assessment with ChIP assays of the two potential SP1 sites (site 1: −195 to −190, site 2: −159 to −154) and non-target site (NT) to the DR5 promoter in HDFs treated with TGF-β1 ( n = 4). f A representation of TGF-β pathway including Smad2/3, Smad4, and SP1 complexes in the translation mechanism of DR5. Data are shown as median ± interquartile range. The Mann–Whitney test was used. * P

    Article Snippet: Membranes were blocked in 3% BSA for 1 h at RT and incubated with primary antibodies against DR4 (Abcam), DR5 (Abcam), α-SMA (Sigma-Aldrich), PDGFR-β (Santa Cruz Biotechnology, Inc.), cleaved Caspase-8 (Cell Signaling Technology), cleaved Caspase-3 (Cell Signaling Technology), cleaved PARP-1 (Cell Signaling Technology), SP1 (Cell Signaling Technology), Smad2/3 (Cell Signaling Technology), Smad4 (Cell signaling Technology), p-Smad2/3 (Cell signaling Technology), p-JNK (Cell signaling Technology), p-p38 MAPK (Cell signaling Technology), p-p44/42 MAPK (Cell signaling Technology) and GAPDH (Santa Cruz Biotechnology, Inc.) overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at RT.

    Techniques: Expressing, Activity Assay, Luciferase, Transfection, Western Blot, Binding Assay, Chromatin Immunoprecipitation, MANN-WHITNEY

    Kindlin-2 modulates TGF-β1 signalling during chondrocyte differentiation. ( a ) Protein extracts from E11.5–13.5 Cre-negative Kindlin-2 fl/fl (WT) and Kindlin-2 Prx1 cKO (cKO) forelimbs were used for western blotting for the indicated proteins. ( b ) Immunohistochemistry. Sections of E12.5 WT and Kindlin-2 Prx1 cKO forelimbs stained with antibodies against pSmad2 antibody (top) or total Smad2/3 (bottom). Scale bar, 40 μm. ( c ) Total RNA from E15.5 WT and Kindlin-2 Prx1 cKO forelimbs were subjected to qPCR analysis for Tgf -β 1 mRNA, which was normalized to Gapdh mRNA. Results were expressed as mean±s.d. N =4. ( d ) Primary cells isolated from E12.5 WT and Kindlin-2 Prx1 cKO forelimbs were treated with or without 2 ng ml −1 TGF-β1 for 30 min, followed by western blotting for the indicated proteins. ( e ) Quantitative data of pSmad2 from four independent experiments. * P

    Journal: Nature Communications

    Article Title: Kindlin-2 controls TGF-β signalling and Sox9 expression to regulate chondrogenesis

    doi: 10.1038/ncomms8531

    Figure Lengend Snippet: Kindlin-2 modulates TGF-β1 signalling during chondrocyte differentiation. ( a ) Protein extracts from E11.5–13.5 Cre-negative Kindlin-2 fl/fl (WT) and Kindlin-2 Prx1 cKO (cKO) forelimbs were used for western blotting for the indicated proteins. ( b ) Immunohistochemistry. Sections of E12.5 WT and Kindlin-2 Prx1 cKO forelimbs stained with antibodies against pSmad2 antibody (top) or total Smad2/3 (bottom). Scale bar, 40 μm. ( c ) Total RNA from E15.5 WT and Kindlin-2 Prx1 cKO forelimbs were subjected to qPCR analysis for Tgf -β 1 mRNA, which was normalized to Gapdh mRNA. Results were expressed as mean±s.d. N =4. ( d ) Primary cells isolated from E12.5 WT and Kindlin-2 Prx1 cKO forelimbs were treated with or without 2 ng ml −1 TGF-β1 for 30 min, followed by western blotting for the indicated proteins. ( e ) Quantitative data of pSmad2 from four independent experiments. * P

    Article Snippet: Antibodies used for western blotting in this study were Kindlin-1 (Sigma, SAB4200465, 1:1,000), Kindlin-3 (Cell Signaling, Cat# 13,843, 1:300), TGFβ R1 (Santa Cruz, sc-9048, 1:500), TGFβ R2 (Santa Cruz, sc-400, 1:500), pFAK (Santa Cruz, sc-11765, 1:300), Sox9 (Santa Cruz Biotechnology, sc-20095, 1:500), pSmad2/3 (Cell Signaling Technology, cat#: 8828S, 1:500), total Smad2/3 (Cell Signaling Technology, cat#: 8685S, 1:500), integrin β1 (BD, cat# 610468, 1:3,000), ILK (Cell Signaling, cat# I0783, 1:1,000), p38 MAPK (Cell Signaling, cat# 9,212, 1:1,000), phospho-p38 MAPK (Cell Signaling, cat# 9,216, 1:1,000), (Sigma-Aldrich, cat#: T-7451, 1:1,000), tubulin (Sigma-Aldrich, cat#: T-7451, 1:5,000), β-actin (Sigma-Aldrich, cat#: A5316, 1:4,000).

    Techniques: Western Blot, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Isolation

    GDF1 is not an active ligand but enhances Nodal activity. ( A – C ) The activity of the Nodal-responsive reporter (n2) 7 luc in the Xenopus animal cap assay was determined after injection of mRNAs for Nodal (10 pg), GDF1 (1000 pg), or the Nodal coreceptor Cripto (20 pg) ( A ); of mRNAs for Nodal (2 pg) or GDF1 (40 pg) ( B ); or of mRNAs for Nodal (2 pg), GDF1 (40 pg), Lefty1 (50 pg), or Lefty2 (50 pg) ( C ). All embryos in B and C were also injected with 100 pg of the mRNA for the Nodal coreceptor Cryptic. ( D ) Xenopus embryos were injected with mRNAs for Nodal (++, 50 pg; +, 10 pg), GDF1 (40 pg), or Cryptic (100 pg), as indicated, after which animal caps were subjected to immunoblot analysis with antibodies to phospho-Smad2 (p-Smad2) or to α-tubulin (loading control). ( E , F ) The animal cap assay was also performed with mRNAs for zDVR1, Squint (Sqt), Cyclops (Cyc), or Flag-tagged OEP (OEP), as indicated. Injected mRNA amounts are shown in picograms (in parentheses).

    Journal: Genes & Development

    Article Title: Long-range action of Nodal requires interaction with GDF1

    doi: 10.1101/gad.1623907

    Figure Lengend Snippet: GDF1 is not an active ligand but enhances Nodal activity. ( A – C ) The activity of the Nodal-responsive reporter (n2) 7 luc in the Xenopus animal cap assay was determined after injection of mRNAs for Nodal (10 pg), GDF1 (1000 pg), or the Nodal coreceptor Cripto (20 pg) ( A ); of mRNAs for Nodal (2 pg) or GDF1 (40 pg) ( B ); or of mRNAs for Nodal (2 pg), GDF1 (40 pg), Lefty1 (50 pg), or Lefty2 (50 pg) ( C ). All embryos in B and C were also injected with 100 pg of the mRNA for the Nodal coreceptor Cryptic. ( D ) Xenopus embryos were injected with mRNAs for Nodal (++, 50 pg; +, 10 pg), GDF1 (40 pg), or Cryptic (100 pg), as indicated, after which animal caps were subjected to immunoblot analysis with antibodies to phospho-Smad2 (p-Smad2) or to α-tubulin (loading control). ( E , F ) The animal cap assay was also performed with mRNAs for zDVR1, Squint (Sqt), Cyclops (Cyc), or Flag-tagged OEP (OEP), as indicated. Injected mRNA amounts are shown in picograms (in parentheses).

    Article Snippet: For immunoblot analysis of phospho-Smad2, four animal caps were loaded per lane and probed with rabbit polyclonal antibodies to phospho-Smad2 (Cell Signaling Technology) and a mouse monoclonal antibody to α-tubulin (clone DM1A, Sigma).

    Techniques: Activity Assay, Injection

    Radiation activates the TGF-βRI/ALK5 pathway and ALK5 inhibition prevents radiation-induced suppression of migration and sprouting. (a) Increased Smad2 phosphorylation in irradiated cultured HUVEC and mouse skin (15 Gy and 20 Gy respectively) demonstrated by Western blotting analysis. Induction of p-Smad2 was biphasic with early peaks at 2–6 hours and late peaks at 24–96 hours. (n = 3). (b) Radiation induces the TGF-β pathway target PAI-1 gene in HUVEC. HUVEC were treated with the ALK5 inhibitor SB431542 at 10 µM one day before radiation. RNA was extracted before and 2 hours after irradiation and PAI-1 mRNA quantified by real time RT-PCR. (c) The ALK5 inhibitor SB431542 (SB) rescued the migration defects caused by radiation. Left panel: migration speed; right panel: migration distance. (n = 10) * P

    Journal: PLoS ONE

    Article Title: Radiotherapy Suppresses Angiogenesis in Mice through TGF-?RI/ALK5-Dependent Inhibition of Endothelial Cell Sprouting

    doi: 10.1371/journal.pone.0011084

    Figure Lengend Snippet: Radiation activates the TGF-βRI/ALK5 pathway and ALK5 inhibition prevents radiation-induced suppression of migration and sprouting. (a) Increased Smad2 phosphorylation in irradiated cultured HUVEC and mouse skin (15 Gy and 20 Gy respectively) demonstrated by Western blotting analysis. Induction of p-Smad2 was biphasic with early peaks at 2–6 hours and late peaks at 24–96 hours. (n = 3). (b) Radiation induces the TGF-β pathway target PAI-1 gene in HUVEC. HUVEC were treated with the ALK5 inhibitor SB431542 at 10 µM one day before radiation. RNA was extracted before and 2 hours after irradiation and PAI-1 mRNA quantified by real time RT-PCR. (c) The ALK5 inhibitor SB431542 (SB) rescued the migration defects caused by radiation. Left panel: migration speed; right panel: migration distance. (n = 10) * P

    Article Snippet: Antibodies and reagents Primary antibodies for staining were purchased from the following companies; anti-CD31 (Pharmingen, San Diego, CA, USA), anti-αSMA cy3-conjugated (Sigma, Buchs, Switzerland), anti-phalloidin Alexa Fluor 568 conjugated (Molecular Probes, Eugene, OR, USA), anti-Paxillin (Transduction laboratories, Lexington, KY, USA), anti-phospho-H2AX (Upstate, Billerica, MA, USA), anti-P21, anti-P53, anti-P16, anti-Smad 2/3 and anti-phospho-Smad2 (Cell Signaling technology Inc. Danvers, MA, USA).

    Techniques: Inhibition, Migration, Irradiation, Cell Culture, Western Blot, Quantitative RT-PCR

    ADPL profiling of differential enzyme activity correlated with distinct phenotypes in native contexts. a Representative ADPL images measuring endogenous NCEH1 activity in paired aggressive (SKOV3 and PC3) and non-aggressive (OVCAR3 and LNCaP) cancer cell lines from ovarian and prostate cancers, respectively. b Relative quantification of NCEH1 activity in each cell line from a . Statistical evaluations shown are comparing mean ADPL signal between non-aggressive and aggressive cells within each tissue of origin. Quantification in b : LNCaP ( n = 47), PC3 ( n = 43), OVCAR3 ( n = 60), SKOV3 ( n = 35). c α-Biotin western blot “gel-based” profiling of serine hydrolase activity in the four cell lines is shown. The two bands at ~42 and 45 kDa are glycoforms of NCEH1; the overlapped intermediate band is another enzyme family member. α-NCEH1 immunoblotting indicates protein abundance. α-GAPDH immunoblotting from the same experiment is shown as a loading control. Scale bars = 10 μm in all images. Blue channel: DAPI nuclear; red channel: ADPL signal; gray channel: DIC. *** P

    Journal: Nature Communications

    Article Title: An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells

    doi: 10.1038/s41467-017-01854-0

    Figure Lengend Snippet: ADPL profiling of differential enzyme activity correlated with distinct phenotypes in native contexts. a Representative ADPL images measuring endogenous NCEH1 activity in paired aggressive (SKOV3 and PC3) and non-aggressive (OVCAR3 and LNCaP) cancer cell lines from ovarian and prostate cancers, respectively. b Relative quantification of NCEH1 activity in each cell line from a . Statistical evaluations shown are comparing mean ADPL signal between non-aggressive and aggressive cells within each tissue of origin. Quantification in b : LNCaP ( n = 47), PC3 ( n = 43), OVCAR3 ( n = 60), SKOV3 ( n = 35). c α-Biotin western blot “gel-based” profiling of serine hydrolase activity in the four cell lines is shown. The two bands at ~42 and 45 kDa are glycoforms of NCEH1; the overlapped intermediate band is another enzyme family member. α-NCEH1 immunoblotting indicates protein abundance. α-GAPDH immunoblotting from the same experiment is shown as a loading control. Scale bars = 10 μm in all images. Blue channel: DAPI nuclear; red channel: ADPL signal; gray channel: DIC. *** P

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:2000, F1804, Sigma Aldrich), anti-NCEH1 (in-house mouse polyclonal 1:2000 from 1 mg/mL stock), anti-GAPDH (1:2000, Cell Signaling Technology, #2118 S).

    Techniques: Activity Assay, Western Blot

    Western blot analysis of the effect of Sanguinarine on CEM/ADR5000 leukemia cells. Evaluation of the P-gp, NFκB, and IκBα expressions. β-actin was used as loading control. Bands were normalized to β-actin in order to obtain numerical values (Mean ± SD ).

    Journal: Frontiers in Pharmacology

    Article Title: Molecular Determinants of Sensitivity or Resistance of Cancer Cells Toward Sanguinarine

    doi: 10.3389/fphar.2018.00136

    Figure Lengend Snippet: Western blot analysis of the effect of Sanguinarine on CEM/ADR5000 leukemia cells. Evaluation of the P-gp, NFκB, and IκBα expressions. β-actin was used as loading control. Bands were normalized to β-actin in order to obtain numerical values (Mean ± SD ).

    Article Snippet: Membranes were incubated at 4°C overnight with primary antibodies including: P-gp (1:1000) (Thermoscientific, Darmstadt, Germany), NFκB (1:1000) (Cell Signaling Technology, Frankfurt, Germany), IκBα (1:1000) (Cell Signaling Technology, Frankfurt, Germany), and β-actin (1:2000) (Cell Signaling Technology, Frankfurt, Germany).

    Techniques: Western Blot

    Expression of other IFT proteins in conditional Ift140 KO mice A. Representative Western blotting images showing testicular expression of IFT20, IFT25, IFT27, IFT74, IFT81, and IFT88 using specific antibodies in three control and three conditional Ift140 mice. β-actin was used as a loading control. B. Statistical analysis of relative expression of the IFT proteins normalized by β-actin. There is no difference in expression levels of these selective IFT proteins between the controls and the Ift140 KO mice. C. Localization of IFT20 in the germ cells of control and conditional Ift140 knockout mice. In mice with both genotypes, IFT20 is present in the Golgi bodies of spermatocytes (upper panels), acrosome of round spermatids (middle panels), and manchette of elongating spermatids (lower panels). However, in the control animal, IFT20 appears to attach closely to the nuclear membrane (white arrow) in round spermatids; in the Ift140 KO, IFT20 does not seem to attach tightly to the nuclear membrane (dashed white arrow). D. Abnormal localization of IFT27 in the conditional Ift140 knockout mice. The granule-like pattern of the IFT27 signal was never observed in the control mice. E. Abnormal localization of IFT88 in the conditional Ift140 knockout mice. In the control mouse, IFT88 is present in the cytoplasm of spermatocytes and round spermatids, manchette of elongating spermatids, and the developing tails; In the Ift140 KO mice, even though IFT88 is still present in the cytoplasm of spermatocytes and round spermatids, it accumulates abnormally in the germ cells at later stages (white arrow heads).

    Journal: Cytoskeleton (Hoboken, N.J.)

    Article Title: Intraflagellar Transporter Protein 140 (IFT140), a component of IFT-A complex, is Essential for Male Fertility and Spermiogenesis in Mice

    doi: 10.1002/cm.21427

    Figure Lengend Snippet: Expression of other IFT proteins in conditional Ift140 KO mice A. Representative Western blotting images showing testicular expression of IFT20, IFT25, IFT27, IFT74, IFT81, and IFT88 using specific antibodies in three control and three conditional Ift140 mice. β-actin was used as a loading control. B. Statistical analysis of relative expression of the IFT proteins normalized by β-actin. There is no difference in expression levels of these selective IFT proteins between the controls and the Ift140 KO mice. C. Localization of IFT20 in the germ cells of control and conditional Ift140 knockout mice. In mice with both genotypes, IFT20 is present in the Golgi bodies of spermatocytes (upper panels), acrosome of round spermatids (middle panels), and manchette of elongating spermatids (lower panels). However, in the control animal, IFT20 appears to attach closely to the nuclear membrane (white arrow) in round spermatids; in the Ift140 KO, IFT20 does not seem to attach tightly to the nuclear membrane (dashed white arrow). D. Abnormal localization of IFT27 in the conditional Ift140 knockout mice. The granule-like pattern of the IFT27 signal was never observed in the control mice. E. Abnormal localization of IFT88 in the conditional Ift140 knockout mice. In the control mouse, IFT88 is present in the cytoplasm of spermatocytes and round spermatids, manchette of elongating spermatids, and the developing tails; In the Ift140 KO mice, even though IFT88 is still present in the cytoplasm of spermatocytes and round spermatids, it accumulates abnormally in the germ cells at later stages (white arrow heads).

    Article Snippet: The membranes were immunoblotted with indicated antibodies at 4°C overnight: anti-IFT140, anti-IFT88, anti-IFT20 and anti-IFT27 (1:2000, Dr. Pazour’s laboratory); anti-IFT25 (1:2000, Protein Tech, 15732-1-AP); anti-IFT74 (1:1000, Antibody Verify); anti-IFT81 (1:1000, Proteintech Group); anti-β-actin (1:2000, Cell Signaling).

    Techniques: Expressing, Mouse Assay, Western Blot, Knock-Out

    Establishment of iMSCs. a Scheme of establishment of iMSCs from mouse embryonic fibroblasts. MEF mouse embryonic fibroblast, Six1-MEF Six1 transduced MEFs, 4TF-MEF 4TFs (Six1, Eya1, Esrrb, and Pax3) transduced MEFs; iMSC induced myogenic stem cells. b IF of Pax7, Myf5, and MyoD. Each myogenic marker is represented in red. Nuclei are shown in blue stained by DAPI. Scale bar = 40 µm. c Co-staining of Pax7 and Myf5. Pax7 is represented in green and Myf5 is represented in red. Scale bar = 20 µm. d Genome integration test. All the size of the bands is 200 bp. The gel images were cropped and aligned. e Immunoblotting of transduced factors—Six1 (37 kDa), Eya1 (65 kDa), Esrrb (55 kDa), Pax3 (58 kDa), and GAPDH (37 kDa). The blot images were cropped and aligned. All the four factors were expressed in protein level. f Karyotype analysis of iMSCs. The iMSCs showed a normal karyotype (38, XY)

    Journal: Cell Death & Disease

    Article Title: Establishment of stably expandable induced myogenic stem cells by four transcription factors

    doi: 10.1038/s41419-018-1114-8

    Figure Lengend Snippet: Establishment of iMSCs. a Scheme of establishment of iMSCs from mouse embryonic fibroblasts. MEF mouse embryonic fibroblast, Six1-MEF Six1 transduced MEFs, 4TF-MEF 4TFs (Six1, Eya1, Esrrb, and Pax3) transduced MEFs; iMSC induced myogenic stem cells. b IF of Pax7, Myf5, and MyoD. Each myogenic marker is represented in red. Nuclei are shown in blue stained by DAPI. Scale bar = 40 µm. c Co-staining of Pax7 and Myf5. Pax7 is represented in green and Myf5 is represented in red. Scale bar = 20 µm. d Genome integration test. All the size of the bands is 200 bp. The gel images were cropped and aligned. e Immunoblotting of transduced factors—Six1 (37 kDa), Eya1 (65 kDa), Esrrb (55 kDa), Pax3 (58 kDa), and GAPDH (37 kDa). The blot images were cropped and aligned. All the four factors were expressed in protein level. f Karyotype analysis of iMSCs. The iMSCs showed a normal karyotype (38, XY)

    Article Snippet: The blot was blocked with 5% skim milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against Six1 (1:1000, Cell Signaling), Eya1 (1:500, Abcam), Pax3 (1:500, R & D Systems), Esrrb (1:1000, R & D Systems), GRB2 (1:1000, Santa Cruz), and GAPDH (1:2000, Cell Signaling).

    Techniques: Marker, Staining

    Ube3A is decreased in Tg2576 mice hippocampus. a – c Representative western blot showing levels of total and active RhoA ( c ), and levels of Ube3A, Arc, Ephexin-5, and EphB2 in wild-type (WT) and Tg2576 mice. b – g Quantification of western blot data showing elevated levels of active RhoA ( b ), Arc ( d ), Ephexin-5 ( e ), and EphB2 ( g ) in Tg2576 relative to WT mice. f Quantification of western blot data showing decreased levels of Ube3A in Tg2576 relative to WT mice. Protein quantifications were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping gene. Data presented as mean ± s.d. of N = 5 WT and N = 5 Tg2576 mice. Note that statistical analyses were performed using unpaired Student’s t -test for all experiments

    Journal: Communications Biology

    Article Title: Dysfunction of the ubiquitin ligase E3A Ube3A/E6-AP contributes to synaptic pathology in Alzheimer’s disease

    doi: 10.1038/s42003-019-0350-5

    Figure Lengend Snippet: Ube3A is decreased in Tg2576 mice hippocampus. a – c Representative western blot showing levels of total and active RhoA ( c ), and levels of Ube3A, Arc, Ephexin-5, and EphB2 in wild-type (WT) and Tg2576 mice. b – g Quantification of western blot data showing elevated levels of active RhoA ( b ), Arc ( d ), Ephexin-5 ( e ), and EphB2 ( g ) in Tg2576 relative to WT mice. f Quantification of western blot data showing decreased levels of Ube3A in Tg2576 relative to WT mice. Protein quantifications were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping gene. Data presented as mean ± s.d. of N = 5 WT and N = 5 Tg2576 mice. Note that statistical analyses were performed using unpaired Student’s t -test for all experiments

    Article Snippet: Antibodies used in this study include the following (Supplementary Table , Supplementary References ): rabbit anti-Ube3A (Cell Signaling, 1:1000), mouse anti-RhoA (Santa Cruz, 1:1000; Cell Signaling, 1:1000), mouse anti-Arc (Santa Cruz, 1:1000), mouse anti-GluR1 (Millipore, 1:1000 for immunofluorescence), rabbit rabbit anti-GluR1 (Cell Signaling, 1:1000 for western blot), anti-Ephexin-5 (Pierce and Abcam, 1:1000), rabbit anti-Lfc (Cell Signaling, 1:1000), rabbit anti-Ephexin-1 (ECM, 1:500), rabbit anti-phospho-c-Abl (Cell Signaling, 1:1000), rabbit anti-GAPDH (Cell Signaling, 1:2000), rabbit anti-HA (Cell Signaling, 1:1000 for western blot, 1:100 for immunprecipitation), rabbit anti-PSD95 (Cell Signaling, 1:1000), rabbit anti-EphB2 (Millipore, 1:1000), Mouse anti-P53 (Santa Cruz, 1:1000), and mouse anti-APP (Millipore, 1:2000).

    Techniques: Mouse Assay, Western Blot

    Ampkα1 sensitive up-regulation of Tgf-β-activated protein kinase 1 (Tak1) following unilateral ureteral obstruction. A. Representative original Western blots and arithmetic means ± SEM (n = 9) of normalized Tgf-β precursor/Gapdh protein ratio in renal tissue from non-obstructed control kidney (CTR) and obstructed kidney (UUO) of Ampkα1 knockout mice (black bars, Ampkα1 -/- ) and respective wild-type mice (white bars, Ampkα1 +/+ ) following 7 days of unilateral ureteral obstruction. B. Representative original Western blots and arithmetic means ± SEM (n = 9) of normalized phospho-Tak1 (Ser 412 )/ Gapdh protein ratio and total Tak1/ Gapdh protein ratio in renal tissue from non-obstructed control kidney (CTR) and obstructed kidney (UUO) of Ampkα1 knockout mice (black bars, Ampkα1 -/- ) and respective wild-type mice (white bars, Ampkα1 +/+ ) following 7 days of unilateral ureteral obstruction. C. Representative original Western blots and arithmetic means ± SEM (n = 9) of normalized phospho-Smad2 (Ser 465/467 )/ Gapdh protein ratio and total Smad2/ Gapdh protein ratio in renal tissue from non-obstructed control kidney (CTR) and obstructed kidney (UUO) of Ampkα1 knockout mice (black bars, Ampkα1 -/- ) and respective wild-type mice (white bars, Ampkα1 +/+ ) following 7 days of unilateral ureteral obstruction. *(p

    Journal: PLoS ONE

    Article Title: Impact of AMP-Activated Protein Kinase α1 Deficiency on Tissue Injury following Unilateral Ureteral Obstruction

    doi: 10.1371/journal.pone.0135235

    Figure Lengend Snippet: Ampkα1 sensitive up-regulation of Tgf-β-activated protein kinase 1 (Tak1) following unilateral ureteral obstruction. A. Representative original Western blots and arithmetic means ± SEM (n = 9) of normalized Tgf-β precursor/Gapdh protein ratio in renal tissue from non-obstructed control kidney (CTR) and obstructed kidney (UUO) of Ampkα1 knockout mice (black bars, Ampkα1 -/- ) and respective wild-type mice (white bars, Ampkα1 +/+ ) following 7 days of unilateral ureteral obstruction. B. Representative original Western blots and arithmetic means ± SEM (n = 9) of normalized phospho-Tak1 (Ser 412 )/ Gapdh protein ratio and total Tak1/ Gapdh protein ratio in renal tissue from non-obstructed control kidney (CTR) and obstructed kidney (UUO) of Ampkα1 knockout mice (black bars, Ampkα1 -/- ) and respective wild-type mice (white bars, Ampkα1 +/+ ) following 7 days of unilateral ureteral obstruction. C. Representative original Western blots and arithmetic means ± SEM (n = 9) of normalized phospho-Smad2 (Ser 465/467 )/ Gapdh protein ratio and total Smad2/ Gapdh protein ratio in renal tissue from non-obstructed control kidney (CTR) and obstructed kidney (UUO) of Ampkα1 knockout mice (black bars, Ampkα1 -/- ) and respective wild-type mice (white bars, Ampkα1 +/+ ) following 7 days of unilateral ureteral obstruction. *(p

    Article Snippet: The membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-α-smooth muscle actin, rabbit anti-collagen I (diluted 1:1000, Abcam), rabbit anti-phospho-AMPKα Thr172 , rabbit anti-AMPKα, rabbit anti-phospho-ACC (Ser79 ), rabbit anti-ACC, rabbit anti-phospho-TAK1 Ser412 , rabbit anti-TAK1, rabbit anti-phospho-Smad2 (Ser465/467 ), rabbit anti-Smad2, rabbit anti-GAPDH, rabbit anti-Tgf-β (diluted 1:1000, Cell Signaling), goat anti-Ampka2 (used at a 1:2000 dilution, Santa Cruz), rabbit anti-AMPKα1 (diluted 1:1000, Novus Biologicals) and then with secondary goat anti-rabbit HRP-conjugated antibody (diluted 1:1000, Cell Signaling) or donkey anti-goat HRP-conjugated antibody (diluted 1:1000, Santa Cruz) for 1 hour at room temperature.

    Techniques: Western Blot, Knock-Out, Mouse Assay

    Transient activation of TGFβ signaling induced Snail up-regulation, which was required for anastasis, and caused increased migration in the late stage. (A) Cleavage of PARP1 and Snail protein in HeLa cells stably expressing scrambled shRNA or Snail shRNA treated with or without EtOH for 3 h. The white dotted line divides the lanes that were cropped from the same blot. (B) Recovery rate of HeLa-scrambled shRNA and HeLa-Snail shRNA cells after mock treatment or EtOH treatment ( n = 3). (C) Recovery rate of HeLa-scrambled shRNA and HeLa-Snail shRNA cells after control treatment or STS treatment ( n = 3). In all bar graphs, error bars represent the standard error of the mean. (D) Western blots of full-length PARP1 (FL PARP1) and cleaved PARP1 in HeLa-scrambled shRNA and HeLa-Snail shRNA cells treated with STS or control. The white dotted line divides the lanes that were cropped from the same blot. (E) Western blots of phosphor-Smad2/3 (pSmad2/3), total Smad2/3, Snail in cells after mock treatment, EtOH treatment, or starvation (T) and in cells recovering from these treatments (recovery). (F) The level of pSmad2/3 and Smad2/3 in HeLa cells treated with STS or vehicle control (T) and in cells recovering from STS or control treatment (recovery). (G) The level of pSmad2/3, Smad2/3, and Snail in apoptotic cells (treatment); cells after 1 h of recovery (recovery 1 h); and cells after 4 h of recovery (recovery 4 h). The addition of LY364497 and EtOH is indicated. The numbers under the blots are the intensity of the bands or the indicated ratio relative to the mock-treated sample. (H) The mRNA expression of Snail (SNAI1) after 1 h of recovery. The addition of LY364947 and EtOH is indicated. (I) Mean migration speed of the indicated group of cells during wound-healing assay ( n = 8). Before wound-healing assay, cells were treated with or without EtOH together with 0.1% DMSO or 5 µM LY364947 for 3 h, followed by 4 h of recovery with 0.1% DMSO or 5 µM LY364947 and an additional 16 h of recovery without any inhibitor. Error bars represent 95% confidence interval. *, P

    Journal: The Journal of Cell Biology

    Article Title: A molecular signature for anastasis, recovery from the brink of apoptotic cell death

    doi: 10.1083/jcb.201706134

    Figure Lengend Snippet: Transient activation of TGFβ signaling induced Snail up-regulation, which was required for anastasis, and caused increased migration in the late stage. (A) Cleavage of PARP1 and Snail protein in HeLa cells stably expressing scrambled shRNA or Snail shRNA treated with or without EtOH for 3 h. The white dotted line divides the lanes that were cropped from the same blot. (B) Recovery rate of HeLa-scrambled shRNA and HeLa-Snail shRNA cells after mock treatment or EtOH treatment ( n = 3). (C) Recovery rate of HeLa-scrambled shRNA and HeLa-Snail shRNA cells after control treatment or STS treatment ( n = 3). In all bar graphs, error bars represent the standard error of the mean. (D) Western blots of full-length PARP1 (FL PARP1) and cleaved PARP1 in HeLa-scrambled shRNA and HeLa-Snail shRNA cells treated with STS or control. The white dotted line divides the lanes that were cropped from the same blot. (E) Western blots of phosphor-Smad2/3 (pSmad2/3), total Smad2/3, Snail in cells after mock treatment, EtOH treatment, or starvation (T) and in cells recovering from these treatments (recovery). (F) The level of pSmad2/3 and Smad2/3 in HeLa cells treated with STS or vehicle control (T) and in cells recovering from STS or control treatment (recovery). (G) The level of pSmad2/3, Smad2/3, and Snail in apoptotic cells (treatment); cells after 1 h of recovery (recovery 1 h); and cells after 4 h of recovery (recovery 4 h). The addition of LY364497 and EtOH is indicated. The numbers under the blots are the intensity of the bands or the indicated ratio relative to the mock-treated sample. (H) The mRNA expression of Snail (SNAI1) after 1 h of recovery. The addition of LY364947 and EtOH is indicated. (I) Mean migration speed of the indicated group of cells during wound-healing assay ( n = 8). Before wound-healing assay, cells were treated with or without EtOH together with 0.1% DMSO or 5 µM LY364947 for 3 h, followed by 4 h of recovery with 0.1% DMSO or 5 µM LY364947 and an additional 16 h of recovery without any inhibitor. Error bars represent 95% confidence interval. *, P

    Article Snippet: The primary antibodies used were rabbit anti-Egr1 (no. 4154; Cell Signaling), rabbit anti–c-Fos (no. 2250; Cell Signaling), rabbit anti–c-Jun (no. 9165; Cell Signaling), mouse anti-Snail (no. 3895; Cell Signaling), rabbit anti-PARP1 (no. 9532; Cell Signaling), rabbit anti-Smad2/3 (no. 8685; Cell Signaling), rabbit anti-pSmad2/3 (no. 8828; Cell Signaling), mouse anti–cytochrome c (no. sc-13560; Santa Cruz Biotechnology), mouse anti-Cox4 (no. 11967; Cell Signaling), rabbit anti–caspase 9 (no. 9502; Cell Signaling), mouse anti–caspase 8 (no. 9746; Cell Signaling), rabbit anti-Tubulin (no. 2128; Cell Signaling), and mouse anti–α-Tubulin (T6199; Sigma).

    Techniques: Activation Assay, Migration, Stable Transfection, Expressing, shRNA, Western Blot, Wound Healing Assay

    Protein expression levels of Smad2, Smad3, Smad4, and Smad7 among the different groups.

    Journal: Drug Design, Development and Therapy

    Article Title: Edaravone inhibits pressure overload-induced cardiac fibrosis and dysfunction by reducing expression of angiotensin II AT1 receptor

    doi: 10.2147/DDDT.S144807

    Figure Lengend Snippet: Protein expression levels of Smad2, Smad3, Smad4, and Smad7 among the different groups.

    Article Snippet: In brief, proteins (60 μg) were separated by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified with the following antibodies to quantify their protein levels: rabbit anti-AT1 and AT2 receptor polyclonal antibodies (Santa Cruz Biotechnology Inc, Dallas, TX, USA), a mouse anti-TGFβ1 monoclonal antibody (Abcam, Cambridge, UK), rabbit anti-Smad2, 3, 4, and 7 monoclonal antibodies (Cell Signaling Technology, Danvers, MA, USA), a mouse anti-collagen type III monoclonal antibody (Sigma-Aldrich Co, St Louis, MO, USA), and a mouse anti-beta actin monoclonal antibody (Sigma-Aldrich Co), respectively.

    Techniques: Expressing

    Inhibition of Smad2/3 enhances reprogramming, but reduces Oct4 expression

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Polycomb determines responses to Smad2/3 signaling in embryonic stem cell differentiation and in reprogramming

    doi: 10.1002/stem.1417

    Figure Lengend Snippet: Inhibition of Smad2/3 enhances reprogramming, but reduces Oct4 expression

    Article Snippet: Anti-Smad2/3 was from BD biosciences.

    Techniques: Inhibition, Expressing

    Polycomb and Smad2/3 are essential for the exit from ground state

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Polycomb determines responses to Smad2/3 signaling in embryonic stem cell differentiation and in reprogramming

    doi: 10.1002/stem.1417

    Figure Lengend Snippet: Polycomb and Smad2/3 are essential for the exit from ground state

    Article Snippet: Anti-Smad2/3 was from BD biosciences.

    Techniques:

    Smad2/3 maintains Oct4 during differentiation by counteracting Polycomb

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Polycomb determines responses to Smad2/3 signaling in embryonic stem cell differentiation and in reprogramming

    doi: 10.1002/stem.1417

    Figure Lengend Snippet: Smad2/3 maintains Oct4 during differentiation by counteracting Polycomb

    Article Snippet: Anti-Smad2/3 was from BD biosciences.

    Techniques:

    Inhibition of Smad2/3 does not affect cell division rate or promote MET

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Polycomb determines responses to Smad2/3 signaling in embryonic stem cell differentiation and in reprogramming

    doi: 10.1002/stem.1417

    Figure Lengend Snippet: Inhibition of Smad2/3 does not affect cell division rate or promote MET

    Article Snippet: Anti-Smad2/3 was from BD biosciences.

    Techniques: Inhibition

    Inhibiting Smad2/3 enhances reprogramming dependent on Polycomb

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Polycomb determines responses to Smad2/3 signaling in embryonic stem cell differentiation and in reprogramming

    doi: 10.1002/stem.1417

    Figure Lengend Snippet: Inhibiting Smad2/3 enhances reprogramming dependent on Polycomb

    Article Snippet: Anti-Smad2/3 was from BD biosciences.

    Techniques:

    Chromatin accessible regions bound by Smad2/3 are enriched for cell cycle regulators in quiescent TPCs A, Schematic representation of integrated ChIP, ATAC, and RNA-seq analyses. B, Global distribution of Smad2/3 bound regulatory elements. C, Venn diagram shows overlap of genes with Smad2/3 bound open chromatin in close proximity and transcripts that are differentially expressed between Q- and P-TPCs. D, Scatter plot visualizes GO data of Smad2/3 regulated genes with transcripts that are differentially expressed between Q- and P-TPCs. Color labels denote statistical significance and circle sizes visualize GO-term frequency (more general terms are larger). E, Bar graphs show differential fold change of Cdc25b expression between Q- and P-TPCs in two independent SCCs. F, Histograms visualizing Smad2/3 binding and chromatin accessibility around the Cdc25b locus in SCC TPCs. G, ChIP-qPCR of Smad2/3 at the Cdc25b enhancer after 1hr TGFβ1 (blue). Bar graphs show mean fold change. Error bars denote ±s.e.m. (n=3, p

    Journal: Cell stem cell

    Article Title: TGFβ-induced quiescence mediates chemoresistance of tumor propagating cells in squamous cell carcinoma

    doi: 10.1016/j.stem.2017.10.001

    Figure Lengend Snippet: Chromatin accessible regions bound by Smad2/3 are enriched for cell cycle regulators in quiescent TPCs A, Schematic representation of integrated ChIP, ATAC, and RNA-seq analyses. B, Global distribution of Smad2/3 bound regulatory elements. C, Venn diagram shows overlap of genes with Smad2/3 bound open chromatin in close proximity and transcripts that are differentially expressed between Q- and P-TPCs. D, Scatter plot visualizes GO data of Smad2/3 regulated genes with transcripts that are differentially expressed between Q- and P-TPCs. Color labels denote statistical significance and circle sizes visualize GO-term frequency (more general terms are larger). E, Bar graphs show differential fold change of Cdc25b expression between Q- and P-TPCs in two independent SCCs. F, Histograms visualizing Smad2/3 binding and chromatin accessibility around the Cdc25b locus in SCC TPCs. G, ChIP-qPCR of Smad2/3 at the Cdc25b enhancer after 1hr TGFβ1 (blue). Bar graphs show mean fold change. Error bars denote ±s.e.m. (n=3, p

    Article Snippet: Antibodies used for western blotting were pSmad2/3 (1:1000; Cell Signaling, 8828S), Smad2/3 (1:1000; BD Biosciences, 610842), p53 (1:500; Cell Signaling, 2524S), phospho-p53 (Ser15) (1:1000; Cell Signaling, 1608), p21 (1:500; Santa Cruz, Sc-6246), Cyclin D1 (1:1000; Cell Signaling, 2978S), Vinculin (1:10000; Sigma, V9131), γH2AX (1:2000; Abcam, Ab11174), Caspase-3 (1:1000; Cell Signaling, 9664S), Bcl2 (1:1000; Cell Signaling, 50E3), Nrf2 (1:1000; Cell Signaling, 12721), GAPDH (1:10000; Santa Cruz, SC-32233), HRP donkey anti-rabbit IgG (H+L) (1:2000; Jackson, 711-035-152), HRP donkey anti-mouse IgG (H+L) (1:2000; Jackson, 711-035-151).

    Techniques: Chromatin Immunoprecipitation, RNA Sequencing Assay, Expressing, Binding Assay, Real-time Polymerase Chain Reaction

    NIT1 recruits Smad2/3 and then activates the TGFβ-Smad2/3 pathway by interacting with SARA and SMAD2/3 in CRC. a Protein interactions among NIT1, SARA and SMAD2/3 using coimmunoprecipitation (co-IP) assays. b Western blot analysis of Smad3, p-Smad3, Smad2 and p-Smad2 after increasing or decreasing the expression of NIT1 in CRC cells as indicated. GAPDH served as the loading control. c Negative control or si-SARA was transfected into over-expressed NIT1 CRC cells to detect the expression levels of SARA, Smad3, p-Smad3, Smad2 and p-Smad2 using western blot assays. GAPDH served as the loading control

    Journal: Cell Death & Disease

    Article Title: NIT1 suppresses tumour proliferation by activating the TGFβ1–Smad2/3 signalling pathway in colorectal cancer

    doi: 10.1038/s41419-018-0333-3

    Figure Lengend Snippet: NIT1 recruits Smad2/3 and then activates the TGFβ-Smad2/3 pathway by interacting with SARA and SMAD2/3 in CRC. a Protein interactions among NIT1, SARA and SMAD2/3 using coimmunoprecipitation (co-IP) assays. b Western blot analysis of Smad3, p-Smad3, Smad2 and p-Smad2 after increasing or decreasing the expression of NIT1 in CRC cells as indicated. GAPDH served as the loading control. c Negative control or si-SARA was transfected into over-expressed NIT1 CRC cells to detect the expression levels of SARA, Smad3, p-Smad3, Smad2 and p-Smad2 using western blot assays. GAPDH served as the loading control

    Article Snippet: The membrane was then blocked in PBST solution containing 5% non-fat milk and was incubated at 4 °C overnight with the specific primary antibodies anti-NIT1 (1:500 dilution; Proteintech, USA), anti-β-tubulin (1:2000 dilution; zsgb-bio, Beijing), anti-GAPDH (1:5000 dilution; Proteintech, USA), and anti-p53 (1:500 dilution; Proteintech, USA), anti-p21 (1:1000 dilution; Abcam, USA), anti-p27 (1:1000 dilution; Abcam, USA), anti-cyclinD1 (1:1000 dilution; Abcam, USA), anti-CDK4 (1:500 dilution; Proteintech, USA), anti-CDK6 (1:500 dilution; Proteintech, USA), anti-myc (1:500 dilution; Proteintech, USA), anti-bax (1:1000 dilution; Abcam, USA), anti-bcl2 (1:1000 dilution; Abcam, USA), anti-caspase3 (1:1000 dilution; CST, USA), anti-PARP (1:1000 dilution; CST, USA), anti-SARA (1:500 dilution; Abcam, USA), anti-Smad2/3 (1:500 dilution; Abcam, USA), anti-Smad2 (1:1000 dilution; Abcam, USA), anti-Smad3 (1:1000 dilution; Abcam, USA), anti-p-Smad2 (1:500 dilution; CST, USA), and anti-p-Smad3 (1:1000 dilution; CST, USA), followed by incubation with their respective appropriate second antibodies.

    Techniques: Co-Immunoprecipitation Assay, Western Blot, Expressing, Negative Control, Transfection

    A model of NIT1 involvement in CRC. NIT1 activates the TGFβ-Smad2/3 signalling pathway by interacting with SARA and SMAD3. In addition, the induction of TGFβ1 or overexpression of SMAD3 increases the expression levels of NIT1. We demonstrated that there is a positive feedback loop involving the regulation of CRC cell proliferation between NIT1 and activation of the TGFβ–Smad pathway. We also presented that NIT1 induces cell cycle arrest and apoptosis, which also contribute to the growth inhibition of CRC

    Journal: Cell Death & Disease

    Article Title: NIT1 suppresses tumour proliferation by activating the TGFβ1–Smad2/3 signalling pathway in colorectal cancer

    doi: 10.1038/s41419-018-0333-3

    Figure Lengend Snippet: A model of NIT1 involvement in CRC. NIT1 activates the TGFβ-Smad2/3 signalling pathway by interacting with SARA and SMAD3. In addition, the induction of TGFβ1 or overexpression of SMAD3 increases the expression levels of NIT1. We demonstrated that there is a positive feedback loop involving the regulation of CRC cell proliferation between NIT1 and activation of the TGFβ–Smad pathway. We also presented that NIT1 induces cell cycle arrest and apoptosis, which also contribute to the growth inhibition of CRC

    Article Snippet: The membrane was then blocked in PBST solution containing 5% non-fat milk and was incubated at 4 °C overnight with the specific primary antibodies anti-NIT1 (1:500 dilution; Proteintech, USA), anti-β-tubulin (1:2000 dilution; zsgb-bio, Beijing), anti-GAPDH (1:5000 dilution; Proteintech, USA), and anti-p53 (1:500 dilution; Proteintech, USA), anti-p21 (1:1000 dilution; Abcam, USA), anti-p27 (1:1000 dilution; Abcam, USA), anti-cyclinD1 (1:1000 dilution; Abcam, USA), anti-CDK4 (1:500 dilution; Proteintech, USA), anti-CDK6 (1:500 dilution; Proteintech, USA), anti-myc (1:500 dilution; Proteintech, USA), anti-bax (1:1000 dilution; Abcam, USA), anti-bcl2 (1:1000 dilution; Abcam, USA), anti-caspase3 (1:1000 dilution; CST, USA), anti-PARP (1:1000 dilution; CST, USA), anti-SARA (1:500 dilution; Abcam, USA), anti-Smad2/3 (1:500 dilution; Abcam, USA), anti-Smad2 (1:1000 dilution; Abcam, USA), anti-Smad3 (1:1000 dilution; Abcam, USA), anti-p-Smad2 (1:500 dilution; CST, USA), and anti-p-Smad3 (1:1000 dilution; CST, USA), followed by incubation with their respective appropriate second antibodies.

    Techniques: Over Expression, Expressing, Activation Assay, Inhibition