sb431542  (Tocris)

 
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    Name:
    SB 431542
    Description:
    Potent selective inhibitor of TGF βRI ALK4 and ALK7
    Catalog Number:
    1614
    Price:
    None
    Purity:
    ≥99% (HPLC)
    Category:
    TGF β Receptor Inhibitors TGF β Receptors Threonine Kinases RSTKs Receptor Serine Enzyme Linked Receptors Pharmacology
    Formula:
    4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide
    Buy from Supplier


    Structured Review

    Tocris sb431542
    SB 431542
    Potent selective inhibitor of TGF βRI ALK4 and ALK7
    https://www.bioz.com/result/sb431542/product/Tocris
    Average 99 stars, based on 92 article reviews
    Price from $9.99 to $1999.99
    sb431542 - by Bioz Stars, 2021-01
    99/100 stars

    Images

    1) Product Images from "MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo"

    Article Title: MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007621

    Yan/Tel controls the spatial restriction of nodal expression in the ectoderm. A, Inhibition of maternal Yan/Tel function disrupts dorsal-ventral axis formation. Note the radial arrangement of the primary mesenchymal cells (PMCs, arrowheads) at gastrula stage (24hpf) and the rounded shape of the embryo at prism stage (36hpf). (hpf), hours post-fertilization. B-C, Inhibition of yan/tel mRNA translation causes a massive ectopic expression of nodal and chordin at blastula (B) and gastrula (C) stages. Note that the expression has expanded throughout most of the ectoderm but is excluded from the animal pole region. apv, animal pole view. D, Inhibition of yan/tel mRNA translation expands the expression of the ventral ectodermal markers bmp2/4 and lefty and suppresses the expression of the dorsal ectodermal markers 29D and tbx2/3 . E, Inhibition of yan/tel mRNA translation expands the expression of the ventral mesodermal marker gata1/2/3 and suppresses the expression of dorsal mesodermal marker gcm . F, Random local inhibition of Yan/Tel function orients the dorsal-ventral axis. In all embryos injected randomly with the yan/tel morpholino into one blastomere at the 2 or 4-cell stage, nodal expression (blue) overlaps with the progeny of the injected blastomere (red). G, Ectopic expression of the Nodal downstream target gene chordin following inhibition of yan/tel mRNA translation depends on Nodal pathway activity. Note that inhibition of yan/tel function followed by treatment with the Nodal receptor inhibitor SB431542 blocks the ectopic expression of chordin observed in y an/tel morphants. SB, swimming blastula stage; LG, late gastrula stage. MB, mesenchyme blastula stage; vv, vegetal view; lv, lateral view. In the lateral views, animal is to the top, and ventral to the left.
    Figure Legend Snippet: Yan/Tel controls the spatial restriction of nodal expression in the ectoderm. A, Inhibition of maternal Yan/Tel function disrupts dorsal-ventral axis formation. Note the radial arrangement of the primary mesenchymal cells (PMCs, arrowheads) at gastrula stage (24hpf) and the rounded shape of the embryo at prism stage (36hpf). (hpf), hours post-fertilization. B-C, Inhibition of yan/tel mRNA translation causes a massive ectopic expression of nodal and chordin at blastula (B) and gastrula (C) stages. Note that the expression has expanded throughout most of the ectoderm but is excluded from the animal pole region. apv, animal pole view. D, Inhibition of yan/tel mRNA translation expands the expression of the ventral ectodermal markers bmp2/4 and lefty and suppresses the expression of the dorsal ectodermal markers 29D and tbx2/3 . E, Inhibition of yan/tel mRNA translation expands the expression of the ventral mesodermal marker gata1/2/3 and suppresses the expression of dorsal mesodermal marker gcm . F, Random local inhibition of Yan/Tel function orients the dorsal-ventral axis. In all embryos injected randomly with the yan/tel morpholino into one blastomere at the 2 or 4-cell stage, nodal expression (blue) overlaps with the progeny of the injected blastomere (red). G, Ectopic expression of the Nodal downstream target gene chordin following inhibition of yan/tel mRNA translation depends on Nodal pathway activity. Note that inhibition of yan/tel function followed by treatment with the Nodal receptor inhibitor SB431542 blocks the ectopic expression of chordin observed in y an/tel morphants. SB, swimming blastula stage; LG, late gastrula stage. MB, mesenchyme blastula stage; vv, vegetal view; lv, lateral view. In the lateral views, animal is to the top, and ventral to the left.

    Techniques Used: Expressing, Inhibition, Marker, Injection, Activity Assay

    MAP kinases phosphorylate sea urchin Yan/Tel. A, Western blot against a wild type HA-tagged version of yan/tel in the presence or absence of the p38 inhibitor BIRB-796, the ERK inhibitor U0126, the JNK inhibitor Sp600125 or a combination of all three drugs. Wild-type Yan/Tel typically migrates as multiple bands with three to four migrating isoforms. Slower migrating isoforms are absent after treatment with the BIRB-796, U0126 or SP600125 drugs while wild type Yan/Tel migrates as a unique faster migrating isoform after simultaneous inhibition of all three MAP Kinases. B, p38, JNK and ERK are required for strong expression of nodal . Treatment with the JNK inhibitor SP600125 or combined inhibition of p38, ERK and JNK reduce although do not abolish the expression of nodal . Lv, lateral view. HB, hatching blastula, EB, early blastula. C, Western blot against p38, JNK and ERK phosphorylated forms during development. Note that these MAP kinases are upregulated between the 60-cell stage and the EB stage. 2, 16 and 60 refers to the number of cells; VEB, Very Early Blastula; EB, Early Blastula; PHB, Prehatching Blastula; HB, Hatching blastula. D, Fluorescent immunostaining against phosphorylated forms of ERK and p38. At early blastula stage, nuclear P-ERK is detected in ectodermal cells (arrowheads) and in the precursors of the skeletogenic mesoderm located at the vegetal pole, while nuclear P-p38 shows a broader distribution. The dorsal clearance of nuclear P-p38 staining is pointed by arrowheads. EB, Early Blastula; PHB, Prehatching Blastula. E, Epistasis experiments with Yan/Tel, p38, JNK and ERK. Similar to the inhibition of yan/tel function, simultaneous inhibition of p38, ERK or JNK and Yan/Tel results in a massive ectopic expression of nodal . SB, Swimming blastula stage. F, Western blot against the HA-tagged form of wild type Yan/Tel. Overexpression of Nodal mRNA enriches the slowest migrating isoform of wild type Yan/Tel suggesting that Nodal can promote the phosphorylation of Yan/Tel. This effect can be reversed by the addition of the MAP kinase inhibitors of p38, ERK or JNK. vv, vegetal view. lv, lateral view. In lateral views, animal is to the top. G, Western blot against the HA-tagged form of wild type Yan/Tel. Treatment with the Nodal receptor inhibitor SB431542 enriches the fast migrating Yan/Tel isoform. Reciprocally, overexpression of the mRNA encoding the activated Nodal receptor Alk4QD or treatment with nickel chloride (a treatment that phenocopies nodal overexpression) promotes phosphorylation of Yan/Tel as indicated by the enrichment of the slowest migrating isoform of Yan/Tel.
    Figure Legend Snippet: MAP kinases phosphorylate sea urchin Yan/Tel. A, Western blot against a wild type HA-tagged version of yan/tel in the presence or absence of the p38 inhibitor BIRB-796, the ERK inhibitor U0126, the JNK inhibitor Sp600125 or a combination of all three drugs. Wild-type Yan/Tel typically migrates as multiple bands with three to four migrating isoforms. Slower migrating isoforms are absent after treatment with the BIRB-796, U0126 or SP600125 drugs while wild type Yan/Tel migrates as a unique faster migrating isoform after simultaneous inhibition of all three MAP Kinases. B, p38, JNK and ERK are required for strong expression of nodal . Treatment with the JNK inhibitor SP600125 or combined inhibition of p38, ERK and JNK reduce although do not abolish the expression of nodal . Lv, lateral view. HB, hatching blastula, EB, early blastula. C, Western blot against p38, JNK and ERK phosphorylated forms during development. Note that these MAP kinases are upregulated between the 60-cell stage and the EB stage. 2, 16 and 60 refers to the number of cells; VEB, Very Early Blastula; EB, Early Blastula; PHB, Prehatching Blastula; HB, Hatching blastula. D, Fluorescent immunostaining against phosphorylated forms of ERK and p38. At early blastula stage, nuclear P-ERK is detected in ectodermal cells (arrowheads) and in the precursors of the skeletogenic mesoderm located at the vegetal pole, while nuclear P-p38 shows a broader distribution. The dorsal clearance of nuclear P-p38 staining is pointed by arrowheads. EB, Early Blastula; PHB, Prehatching Blastula. E, Epistasis experiments with Yan/Tel, p38, JNK and ERK. Similar to the inhibition of yan/tel function, simultaneous inhibition of p38, ERK or JNK and Yan/Tel results in a massive ectopic expression of nodal . SB, Swimming blastula stage. F, Western blot against the HA-tagged form of wild type Yan/Tel. Overexpression of Nodal mRNA enriches the slowest migrating isoform of wild type Yan/Tel suggesting that Nodal can promote the phosphorylation of Yan/Tel. This effect can be reversed by the addition of the MAP kinase inhibitors of p38, ERK or JNK. vv, vegetal view. lv, lateral view. In lateral views, animal is to the top. G, Western blot against the HA-tagged form of wild type Yan/Tel. Treatment with the Nodal receptor inhibitor SB431542 enriches the fast migrating Yan/Tel isoform. Reciprocally, overexpression of the mRNA encoding the activated Nodal receptor Alk4QD or treatment with nickel chloride (a treatment that phenocopies nodal overexpression) promotes phosphorylation of Yan/Tel as indicated by the enrichment of the slowest migrating isoform of Yan/Tel.

    Techniques Used: Western Blot, Inhibition, Expressing, Immunostaining, Staining, Over Expression

    2) Product Images from "Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control. Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control"

    Article Title: Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control. Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13581

    Glyoxalase 1 (Glo1)‐dependent MG‐H1 and argpyrimidine (AP) depletion triggers epithelial‐to‐mesenchymal transition (EMT) via TGF‐β1/SMAD4 signalling pathway in PC3 cells. (A) Glo1 silencing (siGlo1) in PC3 cells significantly affected TGF‐β1 mRNA levels, evaluated by qRT‐PCR, (B) TGF‐β1 protein intracellular levels, evaluated by Western blot, and (C) TGF‐β1 secreted levels, evaluated by a specific ELISA kit. Pre‐treatment with aminoguanidine (AG) proved Glo1‐dependent Hsp40‐modified AP‐mediated role in controlling TGF‐β1 expression (A, B, C). (D) Glo1 silencing (siGlo1) and AG pre‐treatment significantly affected Smad4 activation, evaluated both in nuclear and in cytoplasmic fractions of PC3 cells by Western blot, proving Glo1‐dependent Hsp40‐modified AP‐mediated role in controlling TGF‐β1/Smad4 signalling pathway. Inhibition of TGF‐β1 signalling by SB431542 treatment demonstrated TGF‐β1 role in controlling (E) Smad4 activation, evaluated at nuclear and cytoplasmic levels by Western blot, (F) EMT, evaluated by Western blot analysis of the epithelial markers E‐cadherin (E‐cad) and zonula occludens‐1 (ZO‐1) or mesenchymal markers vimentin (Vim), N‐cadherin (N‐cad) and Snail, (G) MMP‐2 and MMP‐9 expression, evaluated by Western blotting or activity, evaluated by the gelatin zymography, and (H) migration and invasion capabilities, evaluated by specific assays. The Western blots were obtained by the appropriate Abs. The blots were stripped off the bound Abs and reprobed with anti‐β‐actin or lamin β1 to confirm equal loading. The Western blots shown are representative of three independent experiments. The histograms indicate mean ± SD of three different cultures, and each was tested in triplicate. siCtr: control (non‐specific siRNA). (−) untreated and (+) treated cells. * P
    Figure Legend Snippet: Glyoxalase 1 (Glo1)‐dependent MG‐H1 and argpyrimidine (AP) depletion triggers epithelial‐to‐mesenchymal transition (EMT) via TGF‐β1/SMAD4 signalling pathway in PC3 cells. (A) Glo1 silencing (siGlo1) in PC3 cells significantly affected TGF‐β1 mRNA levels, evaluated by qRT‐PCR, (B) TGF‐β1 protein intracellular levels, evaluated by Western blot, and (C) TGF‐β1 secreted levels, evaluated by a specific ELISA kit. Pre‐treatment with aminoguanidine (AG) proved Glo1‐dependent Hsp40‐modified AP‐mediated role in controlling TGF‐β1 expression (A, B, C). (D) Glo1 silencing (siGlo1) and AG pre‐treatment significantly affected Smad4 activation, evaluated both in nuclear and in cytoplasmic fractions of PC3 cells by Western blot, proving Glo1‐dependent Hsp40‐modified AP‐mediated role in controlling TGF‐β1/Smad4 signalling pathway. Inhibition of TGF‐β1 signalling by SB431542 treatment demonstrated TGF‐β1 role in controlling (E) Smad4 activation, evaluated at nuclear and cytoplasmic levels by Western blot, (F) EMT, evaluated by Western blot analysis of the epithelial markers E‐cadherin (E‐cad) and zonula occludens‐1 (ZO‐1) or mesenchymal markers vimentin (Vim), N‐cadherin (N‐cad) and Snail, (G) MMP‐2 and MMP‐9 expression, evaluated by Western blotting or activity, evaluated by the gelatin zymography, and (H) migration and invasion capabilities, evaluated by specific assays. The Western blots were obtained by the appropriate Abs. The blots were stripped off the bound Abs and reprobed with anti‐β‐actin or lamin β1 to confirm equal loading. The Western blots shown are representative of three independent experiments. The histograms indicate mean ± SD of three different cultures, and each was tested in triplicate. siCtr: control (non‐specific siRNA). (−) untreated and (+) treated cells. * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Modification, Expressing, Activation Assay, Inhibition, Activity Assay, Zymography, Migration

    3) Product Images from "Calcium mimics the chemotactic effect of conditioned media and is an effective inducer of bone regeneration"

    Article Title: Calcium mimics the chemotactic effect of conditioned media and is an effective inducer of bone regeneration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0210301

    (A). Primary calvarial cells were cultured with supplemented DMEM or additional 5mM calcium for 3, 7 and 10 days. The mRNA expression was evaluated by qPCR. The effect of calcium (black circles) and media alone (empty circles) on primary calvarial cells is displayed. (B) Expression of osteogenic genes treated for 48 h with 5 mM calcium, SB431542 and AG-1296 (10 μM), a selective TGFβ1 and PDGF receptor inhibitor respectively. Data is shown as mean ± SEM. Differences considered significant at *p
    Figure Legend Snippet: (A). Primary calvarial cells were cultured with supplemented DMEM or additional 5mM calcium for 3, 7 and 10 days. The mRNA expression was evaluated by qPCR. The effect of calcium (black circles) and media alone (empty circles) on primary calvarial cells is displayed. (B) Expression of osteogenic genes treated for 48 h with 5 mM calcium, SB431542 and AG-1296 (10 μM), a selective TGFβ1 and PDGF receptor inhibitor respectively. Data is shown as mean ± SEM. Differences considered significant at *p

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    4) Product Images from "A poised chromatin platform for TGF-? access to master regulators"

    Article Title: A poised chromatin platform for TGF-? access to master regulators

    Journal: Cell

    doi: 10.1016/j.cell.2011.11.032

    TRIM33 and SMAD Cooperation in Activation of Master Regulators (A) mESCs that were wild type, Smad2/3 -depleted, or Smad4-null were set to form EBs for 2.5 days, and then treated for 2h with activin or SB431542. ChIP analysis of the Gsc promoter was done using antibodies against TRIM33. Data are the mean ± S.D. of quadruplicates and representative of three independent experiments. (B) mESCs that were wild type or Smad4 -null were treated as in (A), and ChIP analysis of the Gsc and Mixl1 promoters was done using antibodies against H3K18ac, Data are the mean ± S.D. of quadruplicates and are representative of two independent experiments. (C) TRIM33 competes with HP1γ for binding to H3K9me-K18ac. Biotinylated histone H3 peptides with the indicated modifications were used in pull-down binding assays with a fixed amount of Flag-tagged HP1γ protein and an increasing amount of Flag-tagged TRIM33. Bead-bound proteins were analyzed by immunoblotting. (D) Activin-induced displacement of HP1γ from TRIM33 binding sites in the Gsc and Mixl1 promoters, but not in Trim33 -null EBs. mESC wild type and Trim33 -null EBs at day 2.5 were incubated with activin or SB for 2 h. ChIP assays were done using antibodies against HP1γ (brown bars) or no antibody (blue bars), and qRT-PCR primers amplifying the −2.3 kb region of Gsc or the −0.2 and -0.5kb kb regions of Mixl1 . Data are the mean ± S.D. of quadruplicates and are representative of two independent experiments. (E) Activin increases RNA Pol II binding to the Gsc and Mixl1 promoters, but not in Trim33 -null EBs. The EB samples used in (D) were subjected to ChIP using antibodies against RNA Pol II and qRT-PCR primers amplifying the -0.1 kb region of Gsc or the -0.2 kb region of Mixl1 . Data are the mean ± S.D. of quadruplicates and are representative of two independent experiments. (F) Schematic summary of signal-driven Smad activation of master regulators in embryonic stem cells. Gsc and Mixl1 present key features of the poised state in their promoters, Including the H3K9me3 mark of quiescent chromatin and the chromatin compacting factor HP1γ bound to this region. Two nodal-induced Smad complexes cooperate to promote switching of the master regulators Gsc and Mixl1 from poised to activated state in response to nodal-like signals, thereby committing embryo cells to mesendodermal fate. See Discussion for more details. .
    Figure Legend Snippet: TRIM33 and SMAD Cooperation in Activation of Master Regulators (A) mESCs that were wild type, Smad2/3 -depleted, or Smad4-null were set to form EBs for 2.5 days, and then treated for 2h with activin or SB431542. ChIP analysis of the Gsc promoter was done using antibodies against TRIM33. Data are the mean ± S.D. of quadruplicates and representative of three independent experiments. (B) mESCs that were wild type or Smad4 -null were treated as in (A), and ChIP analysis of the Gsc and Mixl1 promoters was done using antibodies against H3K18ac, Data are the mean ± S.D. of quadruplicates and are representative of two independent experiments. (C) TRIM33 competes with HP1γ for binding to H3K9me-K18ac. Biotinylated histone H3 peptides with the indicated modifications were used in pull-down binding assays with a fixed amount of Flag-tagged HP1γ protein and an increasing amount of Flag-tagged TRIM33. Bead-bound proteins were analyzed by immunoblotting. (D) Activin-induced displacement of HP1γ from TRIM33 binding sites in the Gsc and Mixl1 promoters, but not in Trim33 -null EBs. mESC wild type and Trim33 -null EBs at day 2.5 were incubated with activin or SB for 2 h. ChIP assays were done using antibodies against HP1γ (brown bars) or no antibody (blue bars), and qRT-PCR primers amplifying the −2.3 kb region of Gsc or the −0.2 and -0.5kb kb regions of Mixl1 . Data are the mean ± S.D. of quadruplicates and are representative of two independent experiments. (E) Activin increases RNA Pol II binding to the Gsc and Mixl1 promoters, but not in Trim33 -null EBs. The EB samples used in (D) were subjected to ChIP using antibodies against RNA Pol II and qRT-PCR primers amplifying the -0.1 kb region of Gsc or the -0.2 kb region of Mixl1 . Data are the mean ± S.D. of quadruplicates and are representative of two independent experiments. (F) Schematic summary of signal-driven Smad activation of master regulators in embryonic stem cells. Gsc and Mixl1 present key features of the poised state in their promoters, Including the H3K9me3 mark of quiescent chromatin and the chromatin compacting factor HP1γ bound to this region. Two nodal-induced Smad complexes cooperate to promote switching of the master regulators Gsc and Mixl1 from poised to activated state in response to nodal-like signals, thereby committing embryo cells to mesendodermal fate. See Discussion for more details. .

    Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Binding Assay, Incubation, Quantitative RT-PCR

    TRIM33 Recognizes a Dual Histone Mark Typical of Poised Chromatin (A) Representation of TRIM33 protein domains (top panel) and summary of H3 histone binding specificity of the PHD-Bromo cassette based on the present results (lower panel). (B-C) Requirement of the PHD finger of TRIM33 for induction of Gsc and Mixl1 by activin (B) or by nodal-like signals in the media (C). mESCs that were wild type, Trim33 -depleted or reconstituted with the indicated TRIM33 constructs, were set for EB formation for 2.5 days and then incubated with activin or SB431542 for 2h. Total RNA was analyzed by qRT-PCR using primers for the indicated genes. Data are the mean ± S.D of quadruplicates and are representative of three independent experiments. (D-E, and G) Binding assays with biotinylated H3 histone peptides of the indicated lengths and modifications. Peptides were coupled to avidin-agarose and used as bait to pull down Flag-tagged TRIM33 generated in HEK293T cells (D, G) or a bacterially expressed, GST-tagged C-terminal fragment of TRIM33 (E). Bound TRIM33 was detected by immunoblotting. (F) ITC-based measurements indicate that TRIM33 PHD finger prefers to bind histone H3(1-15) containing unmodified lysine 4 and trimethylated lysine 9. Dissociation constants are listed. (H) ITC-based measurements of binding affinities of the TRIM33 PHD-Bromo cassette for H3(1-28) peptides containing combinations of modifications. Dissociation constants are listed. .
    Figure Legend Snippet: TRIM33 Recognizes a Dual Histone Mark Typical of Poised Chromatin (A) Representation of TRIM33 protein domains (top panel) and summary of H3 histone binding specificity of the PHD-Bromo cassette based on the present results (lower panel). (B-C) Requirement of the PHD finger of TRIM33 for induction of Gsc and Mixl1 by activin (B) or by nodal-like signals in the media (C). mESCs that were wild type, Trim33 -depleted or reconstituted with the indicated TRIM33 constructs, were set for EB formation for 2.5 days and then incubated with activin or SB431542 for 2h. Total RNA was analyzed by qRT-PCR using primers for the indicated genes. Data are the mean ± S.D of quadruplicates and are representative of three independent experiments. (D-E, and G) Binding assays with biotinylated H3 histone peptides of the indicated lengths and modifications. Peptides were coupled to avidin-agarose and used as bait to pull down Flag-tagged TRIM33 generated in HEK293T cells (D, G) or a bacterially expressed, GST-tagged C-terminal fragment of TRIM33 (E). Bound TRIM33 was detected by immunoblotting. (F) ITC-based measurements indicate that TRIM33 PHD finger prefers to bind histone H3(1-15) containing unmodified lysine 4 and trimethylated lysine 9. Dissociation constants are listed. (H) ITC-based measurements of binding affinities of the TRIM33 PHD-Bromo cassette for H3(1-28) peptides containing combinations of modifications. Dissociation constants are listed. .

    Techniques Used: Binding Assay, Construct, Incubation, Quantitative RT-PCR, Avidin-Biotin Assay, Generated

    TRIM33 is Engaged in the Nodal Smad Pathway (A-C) Distribution of TRIM33 in stage E7.5 mouse embryos. Immunohistochemistry analysis of mouse embryo sections with antibodies against TRIM33. Images are (A) whole embryo (30x magnification), (B) node region (150x) and (C) primitive streak (150x) (D) Activin dependent formation of TRIM33-Smad2/3 and Smad4-Smad2/3 complexes in mESCs. mESC cultures were stimulated with activin A (50 ng/ml) for the indicated time periods or treated with SB431542 (SB) (10 μM) to block autocrine nodal like signals. Cell lysates were immunoprecipitated with anti-Smad2/3 antibody and immune complexes were analyzed by immunoblotting using antibodies against the indicated proteins. (E) Formation of the TRIM33-Smad2/3 complex in wild type and Smad4 -null mESCs. (F) Formation of Smad4-Smad2/3 complex in wild type and Trim33-depleted mESCs. (G) mESCs that were wild-type, Trim33 -depleted, reconstituted with TRIM33, Smad2/3 -depleted or Smad4 null were set for EB formation for 2.5 days and then incubated for 2h with activin, SB or no additions. RNA was analyzed by qRT-PCR using primers for the indicated genes. Data are the mean ± S.D of quadruplicates and represent three independent experiments. (H) Wild type or Trim33-null mESCs were treated and analyzed as described in (G). (I) Schematic representation of TRIM33 and Smad4 dependent nodal/activin gene responses. (J) Control, Trim33-depleted and Smad4 -null ESCs were set for EB formation for 2.5 days and processed as in (G). Total RNA was subjected to genome-wide transcriptiomic analysis with MOE 430A 2.0 microarray. The Venn diagram shows a summary of TRIM33 and Smad4-dependent and -independent activin responsive genes ( > 3 fold change, p
    Figure Legend Snippet: TRIM33 is Engaged in the Nodal Smad Pathway (A-C) Distribution of TRIM33 in stage E7.5 mouse embryos. Immunohistochemistry analysis of mouse embryo sections with antibodies against TRIM33. Images are (A) whole embryo (30x magnification), (B) node region (150x) and (C) primitive streak (150x) (D) Activin dependent formation of TRIM33-Smad2/3 and Smad4-Smad2/3 complexes in mESCs. mESC cultures were stimulated with activin A (50 ng/ml) for the indicated time periods or treated with SB431542 (SB) (10 μM) to block autocrine nodal like signals. Cell lysates were immunoprecipitated with anti-Smad2/3 antibody and immune complexes were analyzed by immunoblotting using antibodies against the indicated proteins. (E) Formation of the TRIM33-Smad2/3 complex in wild type and Smad4 -null mESCs. (F) Formation of Smad4-Smad2/3 complex in wild type and Trim33-depleted mESCs. (G) mESCs that were wild-type, Trim33 -depleted, reconstituted with TRIM33, Smad2/3 -depleted or Smad4 null were set for EB formation for 2.5 days and then incubated for 2h with activin, SB or no additions. RNA was analyzed by qRT-PCR using primers for the indicated genes. Data are the mean ± S.D of quadruplicates and represent three independent experiments. (H) Wild type or Trim33-null mESCs were treated and analyzed as described in (G). (I) Schematic representation of TRIM33 and Smad4 dependent nodal/activin gene responses. (J) Control, Trim33-depleted and Smad4 -null ESCs were set for EB formation for 2.5 days and processed as in (G). Total RNA was subjected to genome-wide transcriptiomic analysis with MOE 430A 2.0 microarray. The Venn diagram shows a summary of TRIM33 and Smad4-dependent and -independent activin responsive genes ( > 3 fold change, p

    Techniques Used: Immunohistochemistry, Blocking Assay, Immunoprecipitation, Incubation, Quantitative RT-PCR, Genome Wide, Microarray

    5) Product Images from "A Role for Fibrillar Collagen Deposition and the Collagen Internalization Receptor Endo180 in Glioma Invasion"

    Article Title: A Role for Fibrillar Collagen Deposition and the Collagen Internalization Receptor Endo180 in Glioma Invasion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009808

    Expression of Endo180 is regulated by TGF-β signaling. (A) U87MG cells were stimulated for 24 h with 5 ng/ml TGF-β1, 50 ng/ml EGF or 50 ng/ml PDGF-BB. Endo180 expression (mAb A5/158) and phosphorylation of the SMAD2 protein was monitored by western blotting. (B) Schematic diagram showing the Endo180 promoter fragments cloned into pGL3-basic. U87MG cells were transfected with Endo180 promoter constructs or control vectors, pGL3-empty and pGL3-CAGA12-Luc, and treated with or without 5 ng/ml TGF-β1 for 24 h. Bars represent mean values from three independent experiments ± SEM. p -values were generated using the student's t -test. (C) The −1146bp/0bp Endo180 promoter construct or the pGL3-CAGA12-Luc vector were transfected in U87MG cells. Cells were treated with TGF-β1 in the presence or absence of the inhibitors SIS3 or SB431542 for 24 h. Graphs show fold induction as compared to the untreated pGL3-CAGA12-Luc control. Error bars represent two independent experiments performed in triplicate (upper panel). In parallel, activation of TGF-β signaling was monitored by immunoblotting (lower panel). Dotted line indicates lanes taken from the same gel at the same exposure.
    Figure Legend Snippet: Expression of Endo180 is regulated by TGF-β signaling. (A) U87MG cells were stimulated for 24 h with 5 ng/ml TGF-β1, 50 ng/ml EGF or 50 ng/ml PDGF-BB. Endo180 expression (mAb A5/158) and phosphorylation of the SMAD2 protein was monitored by western blotting. (B) Schematic diagram showing the Endo180 promoter fragments cloned into pGL3-basic. U87MG cells were transfected with Endo180 promoter constructs or control vectors, pGL3-empty and pGL3-CAGA12-Luc, and treated with or without 5 ng/ml TGF-β1 for 24 h. Bars represent mean values from three independent experiments ± SEM. p -values were generated using the student's t -test. (C) The −1146bp/0bp Endo180 promoter construct or the pGL3-CAGA12-Luc vector were transfected in U87MG cells. Cells were treated with TGF-β1 in the presence or absence of the inhibitors SIS3 or SB431542 for 24 h. Graphs show fold induction as compared to the untreated pGL3-CAGA12-Luc control. Error bars represent two independent experiments performed in triplicate (upper panel). In parallel, activation of TGF-β signaling was monitored by immunoblotting (lower panel). Dotted line indicates lanes taken from the same gel at the same exposure.

    Techniques Used: Expressing, Western Blot, Clone Assay, Transfection, Construct, Generated, Plasmid Preparation, Activation Assay

    6) Product Images from "Critical Role of a Survivin/TGF-?/mTORC1 Axis in IGF-I-Mediated Growth of Prostate Epithelial Cells"

    Article Title: Critical Role of a Survivin/TGF-?/mTORC1 Axis in IGF-I-Mediated Growth of Prostate Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061896

    IGF-I enhances cell growth by suppressing TGF-β autocrine signaling. (A–C) TGF-β receptor kinase inhibitors stimulate the growth of NRP-152 cells. NRP-152 cells plated were treated with 5 µM of SB431542 (SB) (A) or with various concentrations of HTS466284 or TKDI and cell growth was measured 6 days later by counting cell number (A) or by crystal violet staining of fixed cells (B,C). (D) Growth of NRP-152-Sh-Smad2+3 cells versus NRP-152-Sh-LacZ cells in GM3 medium. (E) NRP-152-sh-LacZ and NRP-152-sh-Smad2+3 cell lines were incubated in the presence or absence of LR 3 -IGF-I (2 nM) for 5 days and cell growth was monitored daily for 5 days (D,E). Percent of growth inhibition by rapamycin in NRP-152-sh-LacZ and NRP-152-sh-Smad2+3 cell lines. Cell numbers were measured using Coulter Electronics Counter. Data shown are the average of triplicate determinations ± S.E. (*p
    Figure Legend Snippet: IGF-I enhances cell growth by suppressing TGF-β autocrine signaling. (A–C) TGF-β receptor kinase inhibitors stimulate the growth of NRP-152 cells. NRP-152 cells plated were treated with 5 µM of SB431542 (SB) (A) or with various concentrations of HTS466284 or TKDI and cell growth was measured 6 days later by counting cell number (A) or by crystal violet staining of fixed cells (B,C). (D) Growth of NRP-152-Sh-Smad2+3 cells versus NRP-152-Sh-LacZ cells in GM3 medium. (E) NRP-152-sh-LacZ and NRP-152-sh-Smad2+3 cell lines were incubated in the presence or absence of LR 3 -IGF-I (2 nM) for 5 days and cell growth was monitored daily for 5 days (D,E). Percent of growth inhibition by rapamycin in NRP-152-sh-LacZ and NRP-152-sh-Smad2+3 cell lines. Cell numbers were measured using Coulter Electronics Counter. Data shown are the average of triplicate determinations ± S.E. (*p

    Techniques Used: Staining, Incubation, Inhibition

    IGF-I induces Survivin through a Smad dependent mechanism. (A) NRP-152 cells plated overnight in GM3 were treated at various times with LR 3 -IGF-I for up to 72 h, and cell lysates were analyzed for Western blot expression of Survivin, and P-Smads 2 and 3. (B) NRP-152 cells stably expressing sh-Smads 2, 3, and 2+3 or lentiviral sh-LacZ (control) were treated with 2 nM LR 3 -IGF-I or vehicle for 24 h prior to Western blot analysis for Survivin and Smads 2 and 3. (C) NRP-152 cells stably expressing sh-Smads 2+3 or lentiviral sh-LacZ (control) were treated with DMSO vehicle or 10 µM SB431542 for 2 h prior to treatment with 2 nM LR 3 -IGF-I or vehicle for 24 h, and changes in Survivin expression was assessed by Western blot analysis. (D) NRP-152 cells were treated with either 10 µM HTS466284 or 10 µM SB43152 for 2 h prior to treatment with 2 nM LR 3 -IGF-I or vehicle for 24 h, and changes in Survivin expression were assessed by Western blot analysis. (E,F) RWPE-1 and VCaP cells plated in GM3 were treated with LR 3 -IGF-I and the TGF-β receptor kinase inhibitors HTS466284 (HTS) or TKDI for 24 h prior to lysing cells for Western blot analysis of Survivin expression. Results are representative of two to three separate experiments.
    Figure Legend Snippet: IGF-I induces Survivin through a Smad dependent mechanism. (A) NRP-152 cells plated overnight in GM3 were treated at various times with LR 3 -IGF-I for up to 72 h, and cell lysates were analyzed for Western blot expression of Survivin, and P-Smads 2 and 3. (B) NRP-152 cells stably expressing sh-Smads 2, 3, and 2+3 or lentiviral sh-LacZ (control) were treated with 2 nM LR 3 -IGF-I or vehicle for 24 h prior to Western blot analysis for Survivin and Smads 2 and 3. (C) NRP-152 cells stably expressing sh-Smads 2+3 or lentiviral sh-LacZ (control) were treated with DMSO vehicle or 10 µM SB431542 for 2 h prior to treatment with 2 nM LR 3 -IGF-I or vehicle for 24 h, and changes in Survivin expression was assessed by Western blot analysis. (D) NRP-152 cells were treated with either 10 µM HTS466284 or 10 µM SB43152 for 2 h prior to treatment with 2 nM LR 3 -IGF-I or vehicle for 24 h, and changes in Survivin expression were assessed by Western blot analysis. (E,F) RWPE-1 and VCaP cells plated in GM3 were treated with LR 3 -IGF-I and the TGF-β receptor kinase inhibitors HTS466284 (HTS) or TKDI for 24 h prior to lysing cells for Western blot analysis of Survivin expression. Results are representative of two to three separate experiments.

    Techniques Used: Western Blot, Expressing, Stable Transfection

    Transcriptional regulation by IGF-I of Survivin. (A) NRP-152 cells were co-transfected with full length (FL) or truncations (Trunc-1 to -4) of rat Survivin promoter-Firefly luciferase reporter constructs, along with CMV-Renilla control reporter one day before a 24 h treatment with LR 3 -IGF-I or vehicle, and cells were then analyzed for dual luciferase reporter activity. (B) NRP-152 cells were co-transfected with Trunc-2 Survivin promoter-luciferase construct (rSur-pro-Luc#2) and CMV-Renilla as in A, and next day cells were treated with various kinase inhibitors (LY: 10 µM LY294004; Rap: 200 pM rapamycin; 10 µM of either SB431542, SB202190, SP600125 or U0126) or DMSO vehicle for 2 h before 24 h treatment with 2 nM LR 3 -IGF-I or vehicle. (C) NRP-152 cells were co-transfected with rSur-pro-Luc#2 (WT) or rSur-pro-Luc#2 mutated at CDE and CHR (CDE/CHR Mut) along with CMV-Renilla, next day cells were treated with 2 nM LR 3 -IGF-I or vehicle, and harvested for dual luciferase activity. Data shown are relative values of Firefly luciferase normalized to Renilla luciferase, and expressed as relative luciferase units (R.L.U.). Each bar represents the average of triplicate determinations ± S.E. Statistical significance (*p
    Figure Legend Snippet: Transcriptional regulation by IGF-I of Survivin. (A) NRP-152 cells were co-transfected with full length (FL) or truncations (Trunc-1 to -4) of rat Survivin promoter-Firefly luciferase reporter constructs, along with CMV-Renilla control reporter one day before a 24 h treatment with LR 3 -IGF-I or vehicle, and cells were then analyzed for dual luciferase reporter activity. (B) NRP-152 cells were co-transfected with Trunc-2 Survivin promoter-luciferase construct (rSur-pro-Luc#2) and CMV-Renilla as in A, and next day cells were treated with various kinase inhibitors (LY: 10 µM LY294004; Rap: 200 pM rapamycin; 10 µM of either SB431542, SB202190, SP600125 or U0126) or DMSO vehicle for 2 h before 24 h treatment with 2 nM LR 3 -IGF-I or vehicle. (C) NRP-152 cells were co-transfected with rSur-pro-Luc#2 (WT) or rSur-pro-Luc#2 mutated at CDE and CHR (CDE/CHR Mut) along with CMV-Renilla, next day cells were treated with 2 nM LR 3 -IGF-I or vehicle, and harvested for dual luciferase activity. Data shown are relative values of Firefly luciferase normalized to Renilla luciferase, and expressed as relative luciferase units (R.L.U.). Each bar represents the average of triplicate determinations ± S.E. Statistical significance (*p

    Techniques Used: Transfection, Luciferase, Construct, Activity Assay

    7) Product Images from "Combinatorial Signals of Activin/Nodal and Bone Morphogenic Protein Regulate the Early Lineage Segregation of Human Embryonic Stem Cells *Combinatorial Signals of Activin/Nodal and Bone Morphogenic Protein Regulate the Early Lineage Segregation of Human Embryonic Stem Cells * S⃞"

    Article Title: Combinatorial Signals of Activin/Nodal and Bone Morphogenic Protein Regulate the Early Lineage Segregation of Human Embryonic Stem Cells *Combinatorial Signals of Activin/Nodal and Bone Morphogenic Protein Regulate the Early Lineage Segregation of Human Embryonic Stem Cells * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M803893200

    Inhibition of Activin/Nodal signaling induces trophoblast differentiation. The H1 hES cells were cultured under a feeder-free condition and treated with SB 431542 for 6 ( A and B ) or 12 days ( C ). Then cells were harvested for analyses. A , real-time polymerase chain reaction analysis of endoderm (α1- AT ), mesoderm ( cACT ), and ectoderm ( NFH ) markers. B , real-time polymerase chain reaction analysis of multiple trophoblast markers. SB431542 up-regulates trophoblast marker expression in a dose-dependent manner. C , differentiated cells form syncytial cells after incubation in CM plus 10 μmol/liter SB 431542 for 12 days. The expression level of each gene in H1 hESCs maintained on murine embryonic fibroblast feeder cells is arbitrarily defined as 1 unit. α 1-AT , α 1 -antitrypsin; cACT , cardiac actin; NFH , neurofilament heavy chain; DAPI , 4′,6-diamidino-2-phenylindole.
    Figure Legend Snippet: Inhibition of Activin/Nodal signaling induces trophoblast differentiation. The H1 hES cells were cultured under a feeder-free condition and treated with SB 431542 for 6 ( A and B ) or 12 days ( C ). Then cells were harvested for analyses. A , real-time polymerase chain reaction analysis of endoderm (α1- AT ), mesoderm ( cACT ), and ectoderm ( NFH ) markers. B , real-time polymerase chain reaction analysis of multiple trophoblast markers. SB431542 up-regulates trophoblast marker expression in a dose-dependent manner. C , differentiated cells form syncytial cells after incubation in CM plus 10 μmol/liter SB 431542 for 12 days. The expression level of each gene in H1 hESCs maintained on murine embryonic fibroblast feeder cells is arbitrarily defined as 1 unit. α 1-AT , α 1 -antitrypsin; cACT , cardiac actin; NFH , neurofilament heavy chain; DAPI , 4′,6-diamidino-2-phenylindole.

    Techniques Used: Inhibition, Cell Culture, Real-time Polymerase Chain Reaction, Marker, Expressing, Incubation

    Trophoblast differentiation in a dose- and time-dependent manner. The H1 hES cells were cultured under a feeder-free condition and treated with SB431542 for 12 days. Real-time PCR analyses of the downstream targets of Activin/Nodal signaling ( A ), the pluripotent markers ( B ), and the trophoblast markers ( C ), during differentiation of H1 cells to trophoblast cells following induction by SB431542. Relative expression levels of each gene were analyzed at 0, 2, 4, 6, 8, 10, and 12 days, respectively, after addition of SB431542. The expression level of each gene at day 0 (prior to the addition of SB431542) is arbitrarily defined as 1 unit.
    Figure Legend Snippet: Trophoblast differentiation in a dose- and time-dependent manner. The H1 hES cells were cultured under a feeder-free condition and treated with SB431542 for 12 days. Real-time PCR analyses of the downstream targets of Activin/Nodal signaling ( A ), the pluripotent markers ( B ), and the trophoblast markers ( C ), during differentiation of H1 cells to trophoblast cells following induction by SB431542. Relative expression levels of each gene were analyzed at 0, 2, 4, 6, 8, 10, and 12 days, respectively, after addition of SB431542. The expression level of each gene at day 0 (prior to the addition of SB431542) is arbitrarily defined as 1 unit.

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    Activation of BMP4 is required for trophoblast differentiation. The parental ( G-GFP ) and AR1-c1 hES cells were cultured under a feeder-free condition and treated with BMP4 (50 ng/ml) or SB431542 (10 μ m ) for 10 days. A , cells were harvested for real-time PCR analyses. B , immunofluorescent staining for trophoectoderm markers TROMA-I ( red ) from the differentiated G-GFP and AR1-c1 cells.
    Figure Legend Snippet: Activation of BMP4 is required for trophoblast differentiation. The parental ( G-GFP ) and AR1-c1 hES cells were cultured under a feeder-free condition and treated with BMP4 (50 ng/ml) or SB431542 (10 μ m ) for 10 days. A , cells were harvested for real-time PCR analyses. B , immunofluorescent staining for trophoectoderm markers TROMA-I ( red ) from the differentiated G-GFP and AR1-c1 cells.

    Techniques Used: Activation Assay, Cell Culture, Real-time Polymerase Chain Reaction, Staining

    Inhibition of Activin/Nodal signaling down-regulates FGF and Wnt signals, but up-regulates BMP signals. The H1 hES cells were cultured under a feeder-free condition and treated with SB431542 for 6 days. Then cells were harvested for real-time polymerase chain reaction analysis of ligands of the FGF ( A ), Wnt ( A ), and BMP ( B ) signaling pathways and Western analysis of Oct4, Smad2, p -Smad2, Smad1, and p -Smad1 ( C ).
    Figure Legend Snippet: Inhibition of Activin/Nodal signaling down-regulates FGF and Wnt signals, but up-regulates BMP signals. The H1 hES cells were cultured under a feeder-free condition and treated with SB431542 for 6 days. Then cells were harvested for real-time polymerase chain reaction analysis of ligands of the FGF ( A ), Wnt ( A ), and BMP ( B ) signaling pathways and Western analysis of Oct4, Smad2, p -Smad2, Smad1, and p -Smad1 ( C ).

    Techniques Used: Inhibition, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

    hESC-derived trophoblast cells secrete placental hormones. A and B , Immunofluorescence for CG-α and CG-β. H1 cells were treated with CM, or CM plus 10 μmol/liter SB431542 or 300 ng/ml Follistatin for 12 days. C , immunoassays of placental hormones. Conditioned culture medium from H1 cells cultured in CM; CM + 1 SB; CM + 10 SB; CM + 30 FS; or CM + 300 FS were collected at the indicated times and subjected to immunoassays for hCG, estradiol ( E2 ), and progesterone ( Prog ). CM + 1SB , CM plus 1 μmol/liter SB431542; CM + 10SB , CM plus 10 μmol/liter SB431542; CM + 3FS , CM plus 3 ng/ml Follistatin; CM + 30FS , CM plus 30 ng/ml Follistatin; CM + 300FS , CM plus 300 ng/ml Follistatin.
    Figure Legend Snippet: hESC-derived trophoblast cells secrete placental hormones. A and B , Immunofluorescence for CG-α and CG-β. H1 cells were treated with CM, or CM plus 10 μmol/liter SB431542 or 300 ng/ml Follistatin for 12 days. C , immunoassays of placental hormones. Conditioned culture medium from H1 cells cultured in CM; CM + 1 SB; CM + 10 SB; CM + 30 FS; or CM + 300 FS were collected at the indicated times and subjected to immunoassays for hCG, estradiol ( E2 ), and progesterone ( Prog ). CM + 1SB , CM plus 1 μmol/liter SB431542; CM + 10SB , CM plus 10 μmol/liter SB431542; CM + 3FS , CM plus 3 ng/ml Follistatin; CM + 30FS , CM plus 30 ng/ml Follistatin; CM + 300FS , CM plus 300 ng/ml Follistatin.

    Techniques Used: Derivative Assay, Immunofluorescence, Cell Culture

    Inhibition of Activin/Nodal signaling induces differentiation of hESCs. The H1 hES cells were cultured under a feeder-free condition and treated with SB431542 or Follistatin for 6 days. Then cells were harvested for analyses. A , real-time polymerase chain reaction analysis of the downstream targets of Activin/Nodal signaling. H1 human embryonic stem cells were maintained in CM supplemented with varying concentrations of SB431542 ( upper ) or Follistatin ( lower ) for 6 days. B , morphological changes of SB431542-treated or Follistatin-treated H1 cells. C , SSEA4 immunofluorescence of H1 cells treated with CM, or CM plus 10 μmol/liter SB431542 or 300 ng/ml Follistatin for 6 days. D , real-time polymerase chain reaction analysis of the pluripotent markers, Oct4 and Nanog. The expression level of each gene in H1 hESCs maintained on murine embryonic fibroblast feeder cells is arbitrarily defined as 1 unit. MEF , hESCs maintained on murine embryonic fibroblast feeder; SB , SB431542; FST , Follistatin.
    Figure Legend Snippet: Inhibition of Activin/Nodal signaling induces differentiation of hESCs. The H1 hES cells were cultured under a feeder-free condition and treated with SB431542 or Follistatin for 6 days. Then cells were harvested for analyses. A , real-time polymerase chain reaction analysis of the downstream targets of Activin/Nodal signaling. H1 human embryonic stem cells were maintained in CM supplemented with varying concentrations of SB431542 ( upper ) or Follistatin ( lower ) for 6 days. B , morphological changes of SB431542-treated or Follistatin-treated H1 cells. C , SSEA4 immunofluorescence of H1 cells treated with CM, or CM plus 10 μmol/liter SB431542 or 300 ng/ml Follistatin for 6 days. D , real-time polymerase chain reaction analysis of the pluripotent markers, Oct4 and Nanog. The expression level of each gene in H1 hESCs maintained on murine embryonic fibroblast feeder cells is arbitrarily defined as 1 unit. MEF , hESCs maintained on murine embryonic fibroblast feeder; SB , SB431542; FST , Follistatin.

    Techniques Used: Inhibition, Cell Culture, Real-time Polymerase Chain Reaction, Immunofluorescence, Expressing

    8) Product Images from "Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression"

    Article Title: Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025021

    TGF-β induced Mmp9 expression levels are increased by exogenous wild-type Smad3 and some Smad3 variants in C2C12 cells. RNA was isolated from cell lysates prepared from two independent sets (A and B) of C2C12 cell populations independently infected with retrovirus without the myc-Smad3 gene (EV) or retrovirus encoding wild-type or mutant Smad3s. The C2C12 cell populations had been exposed to either 1 µM SB431542 or 100 pM TGF-β for 24 hours before RNA isolation. Mmp9 and beta-actin mRNA expression levels were detected by quantitative RT-PCR in three replicate wells and the MMP9 levels were normalized to the level of beta-actin in each cell lysate. TGF-β increased the expression levels of MMP9 in all of the cell lysates. Expression of wild-type Smad3 increased the basal expression and induced expression levels of Mmp9 five-ten fold over the levels in the cells infected with the EV retrovirus. Expression of Smad3 D408H did not increase the basal expression or induced levels compared to cells infected with the EV retrovirus. Some other Smad3 mutants affected either the basal or induced expression levels (summarized in Table 5 ). The numerical values indicating the heights of the bars are only shown if the value is > 1. The error bars indicate the standard deviations of the three replicate wells of Q-RT-PCR reactions run on each RNA preparation. While the levels of Mmp9 expression differed between the A and B cell populations for some of the mutants, the rank orders across the set of mutants were highly correlated. For SB431542 treated cells, Kendall's rank correlation coefficient (tau) for the A and B cell populations was 0.58 (P = 0.006), and that for TGF-beta treated cells was 0.61 (P = 0.006).
    Figure Legend Snippet: TGF-β induced Mmp9 expression levels are increased by exogenous wild-type Smad3 and some Smad3 variants in C2C12 cells. RNA was isolated from cell lysates prepared from two independent sets (A and B) of C2C12 cell populations independently infected with retrovirus without the myc-Smad3 gene (EV) or retrovirus encoding wild-type or mutant Smad3s. The C2C12 cell populations had been exposed to either 1 µM SB431542 or 100 pM TGF-β for 24 hours before RNA isolation. Mmp9 and beta-actin mRNA expression levels were detected by quantitative RT-PCR in three replicate wells and the MMP9 levels were normalized to the level of beta-actin in each cell lysate. TGF-β increased the expression levels of MMP9 in all of the cell lysates. Expression of wild-type Smad3 increased the basal expression and induced expression levels of Mmp9 five-ten fold over the levels in the cells infected with the EV retrovirus. Expression of Smad3 D408H did not increase the basal expression or induced levels compared to cells infected with the EV retrovirus. Some other Smad3 mutants affected either the basal or induced expression levels (summarized in Table 5 ). The numerical values indicating the heights of the bars are only shown if the value is > 1. The error bars indicate the standard deviations of the three replicate wells of Q-RT-PCR reactions run on each RNA preparation. While the levels of Mmp9 expression differed between the A and B cell populations for some of the mutants, the rank orders across the set of mutants were highly correlated. For SB431542 treated cells, Kendall's rank correlation coefficient (tau) for the A and B cell populations was 0.58 (P = 0.006), and that for TGF-beta treated cells was 0.61 (P = 0.006).

    Techniques Used: Expressing, Isolation, Infection, Mutagenesis, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    Smad3 and TGF-β alter the levels of gene expression in C2C12 cells. C2C12 cell populations infected with retrovirus without the myc-Smad3 gene (Empty Vector, EV) or retrovirus encoding wild-type Smad3 were treated with either 1 µM SB431542 or 100 pM TGF-β for 24 hours before RNA isolation. The 16 genes were amplified by RT-PCR using the primer sets shown in Table S3 . The annealing temperature and number of cycles of PCR were optimized for each primer set. The overexpression of Smad3 had modest effects on the levels of some genes (Adamts4, Cst6, Pdgfb, Pai1 and Wnt11) but had detectable effects on the levels of other genes (Fermt1, Gfpt2, Il11, Mmp9, Myl1, Npy, Olfm2, Sgcg). The expression levels of three genes, Cst6, Myl1 and Sgcg, were decreased by TGF-βtreatment. Overexpression of Smad3 caused further reductions in the levels of Myl1 and Sgcg.
    Figure Legend Snippet: Smad3 and TGF-β alter the levels of gene expression in C2C12 cells. C2C12 cell populations infected with retrovirus without the myc-Smad3 gene (Empty Vector, EV) or retrovirus encoding wild-type Smad3 were treated with either 1 µM SB431542 or 100 pM TGF-β for 24 hours before RNA isolation. The 16 genes were amplified by RT-PCR using the primer sets shown in Table S3 . The annealing temperature and number of cycles of PCR were optimized for each primer set. The overexpression of Smad3 had modest effects on the levels of some genes (Adamts4, Cst6, Pdgfb, Pai1 and Wnt11) but had detectable effects on the levels of other genes (Fermt1, Gfpt2, Il11, Mmp9, Myl1, Npy, Olfm2, Sgcg). The expression levels of three genes, Cst6, Myl1 and Sgcg, were decreased by TGF-βtreatment. Overexpression of Smad3 caused further reductions in the levels of Myl1 and Sgcg.

    Techniques Used: Expressing, Infection, Plasmid Preparation, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Over Expression

    9) Product Images from "Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial‐Like Cells"

    Article Title: Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial‐Like Cells

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.2750

    SB431542 exposure during early differentiation enhanced the hiPSC differentiation to corneal epithelial progenitors. Schematic outline for optimization experiment using SB431542 (A) . Quantitative real‐time polymerase chain reaction analysis of the putative limbal stem cells ( ΔNp63 ) gene for SB‐Ad3 cells from G5 subgroups on day 20 of optimization experiment following different durations of SB431542 exposure (B) . Colony forming efficiency assays for the SB431542 exposed and unexposed G5 subgroups on day 20 (C) . Data presented as mean ± SEM, n = 3. *, statistically different compared with untreated group. **, p
    Figure Legend Snippet: SB431542 exposure during early differentiation enhanced the hiPSC differentiation to corneal epithelial progenitors. Schematic outline for optimization experiment using SB431542 (A) . Quantitative real‐time polymerase chain reaction analysis of the putative limbal stem cells ( ΔNp63 ) gene for SB‐Ad3 cells from G5 subgroups on day 20 of optimization experiment following different durations of SB431542 exposure (B) . Colony forming efficiency assays for the SB431542 exposed and unexposed G5 subgroups on day 20 (C) . Data presented as mean ± SEM, n = 3. *, statistically different compared with untreated group. **, p

    Techniques Used: Real-time Polymerase Chain Reaction

    10) Product Images from "Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells"

    Article Title: Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2014.08.137

    LCD shows direct differentiation outcomes of H9 cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with KSR and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P
    Figure Legend Snippet: LCD shows direct differentiation outcomes of H9 cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with KSR and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P

    Techniques Used: Derivative Assay, Software

    11) Product Images from "miR-373 is regulated by TGFβ signaling and promotes mesendoderm differentiation in human Embryonic Stem Cells"

    Article Title: miR-373 is regulated by TGFβ signaling and promotes mesendoderm differentiation in human Embryonic Stem Cells

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2014.03.020

    Inhibition of TGFβ signaling in hESCs results in deregulated miRNA expression (A) RUES2 cells were cultured under pluripotency conditions in the presence of either 10μM SB431542 (SB) or DMSO, and were harvested at the indicated time points. (B) Western blot showing that SB treatment leads to complete inhibition of C-terminal Smad2/3 phosphorylation (P-SMAD2), without affecting total Smad2/3 protein (SMAD2) levels. β-Tubulin was used as a loading control. (C) Real-Time qPCR analysis of Nodal and Lefty2 ] is depicted. The numbers are relative to the first nucleotide of pre-miR-371. (F) Real-Time qPCR analysis of the chromatin immunoprecipitated with the indicated antibodies. Fold change is relative to a negative control genomic region on chromosome 4. Data points represent average from triplicates (*p
    Figure Legend Snippet: Inhibition of TGFβ signaling in hESCs results in deregulated miRNA expression (A) RUES2 cells were cultured under pluripotency conditions in the presence of either 10μM SB431542 (SB) or DMSO, and were harvested at the indicated time points. (B) Western blot showing that SB treatment leads to complete inhibition of C-terminal Smad2/3 phosphorylation (P-SMAD2), without affecting total Smad2/3 protein (SMAD2) levels. β-Tubulin was used as a loading control. (C) Real-Time qPCR analysis of Nodal and Lefty2 ] is depicted. The numbers are relative to the first nucleotide of pre-miR-371. (F) Real-Time qPCR analysis of the chromatin immunoprecipitated with the indicated antibodies. Fold change is relative to a negative control genomic region on chromosome 4. Data points represent average from triplicates (*p

    Techniques Used: Inhibition, Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control

    12) Product Images from "Betaglycan Alters NFκB-TGFβ2 Cross Talk to Reduce Survival of Human Granulosa Tumor Cells"

    Article Title: Betaglycan Alters NFκB-TGFβ2 Cross Talk to Reduce Survival of Human Granulosa Tumor Cells

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2012-1239

    GCT cell viability is dependent upon the combined activities of NFκB, SMAD2, and SMAD3. A, Assessment of KGN cell viability by MTT assay, after chemical inhibition of ALK4/5/7 using SB431542 (2.5μM) in DMSO (vehicle) for 10 hours. Cells
    Figure Legend Snippet: GCT cell viability is dependent upon the combined activities of NFκB, SMAD2, and SMAD3. A, Assessment of KGN cell viability by MTT assay, after chemical inhibition of ALK4/5/7 using SB431542 (2.5μM) in DMSO (vehicle) for 10 hours. Cells

    Techniques Used: MTT Assay, Inhibition

    13) Product Images from "Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix"

    Article Title: Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1306562110

    EVC maturation. ( A ) Sorted VEcad + from hiPSC-BC1–derived EVCs subcultured for an additional 6 d in 50 ng/mL VEGF and SB431542-supplemented conditions and analyzed for the expression of VEcad, CD31, and CD146 (representative flow cytometry plots;
    Figure Legend Snippet: EVC maturation. ( A ) Sorted VEcad + from hiPSC-BC1–derived EVCs subcultured for an additional 6 d in 50 ng/mL VEGF and SB431542-supplemented conditions and analyzed for the expression of VEcad, CD31, and CD146 (representative flow cytometry plots;

    Techniques Used: Derivative Assay, Expressing, Flow Cytometry, Cytometry

    14) Product Images from "Nodal signalling imposes left-right asymmetry upon neurogenesis in the habenular nuclei"

    Article Title: Nodal signalling imposes left-right asymmetry upon neurogenesis in the habenular nuclei

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.034793

    Nodal signalling promotes early asymmetric neurogenesis. ( A-D ) Confocal projections of Tg( huC:gfp ) transgenic zebrafish embryos incubated with DMSO (A,C) or with SB431542 (B,D) from 10 hpf and fixed at 34 (A,B) or 38 (C,D) hpf. Arrows indicate that the left pools of HuC + neurons are bigger than those on the right in DMSO-treated embryos (A,C), whereas the left and right pools are symmetric in embryos incubated with SB431542 (B,D). The epiphysis is indicated by a blue line. Habenular neurogenesis is also delayed in SB431542-treated embryos. ( E-H ) Numbers of left and right HuC + habenular neurons (E,G) and corresponding AI (F,H) in embryos treated with DMSO or SB431542 at 10 (E,F) or 16 (G,H) hpf. Habenular neurons were detected in Tg( huC:gfp ) transgenic embryos using an anti-GFP (E) or HuC/D (G) antibody. A symmetry line (black) is included as a reference in E and G. Dotted lines represent best-fit linear regressions. Horizontal black lines in F and H indicate the median AI value for the group of like-treated embryos.
    Figure Legend Snippet: Nodal signalling promotes early asymmetric neurogenesis. ( A-D ) Confocal projections of Tg( huC:gfp ) transgenic zebrafish embryos incubated with DMSO (A,C) or with SB431542 (B,D) from 10 hpf and fixed at 34 (A,B) or 38 (C,D) hpf. Arrows indicate that the left pools of HuC + neurons are bigger than those on the right in DMSO-treated embryos (A,C), whereas the left and right pools are symmetric in embryos incubated with SB431542 (B,D). The epiphysis is indicated by a blue line. Habenular neurogenesis is also delayed in SB431542-treated embryos. ( E-H ) Numbers of left and right HuC + habenular neurons (E,G) and corresponding AI (F,H) in embryos treated with DMSO or SB431542 at 10 (E,F) or 16 (G,H) hpf. Habenular neurons were detected in Tg( huC:gfp ) transgenic embryos using an anti-GFP (E) or HuC/D (G) antibody. A symmetry line (black) is included as a reference in E and G. Dotted lines represent best-fit linear regressions. Horizontal black lines in F and H indicate the median AI value for the group of like-treated embryos.

    Techniques Used: Transgenic Assay, Incubation

    15) Product Images from "Nodal signalling imposes left-right asymmetry upon neurogenesis in the habenular nuclei"

    Article Title: Nodal signalling imposes left-right asymmetry upon neurogenesis in the habenular nuclei

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.034793

    Nodal signalling promotes early asymmetric neurogenesis. ( A-D ) Confocal projections of Tg( huC:gfp ) transgenic zebrafish embryos incubated with DMSO (A,C) or with SB431542 (B,D) from 10 hpf and fixed at 34 (A,B) or 38 (C,D) hpf. Arrows indicate that the left pools of HuC + neurons are bigger than those on the right in DMSO-treated embryos (A,C), whereas the left and right pools are symmetric in embryos incubated with SB431542 (B,D). The epiphysis is indicated by a blue line. Habenular neurogenesis is also delayed in SB431542-treated embryos. ( E-H ) Numbers of left and right HuC + habenular neurons (E,G) and corresponding AI (F,H) in embryos treated with DMSO or SB431542 at 10 (E,F) or 16 (G,H) hpf. Habenular neurons were detected in Tg( huC:gfp ) transgenic embryos using an anti-GFP (E) or HuC/D (G) antibody. A symmetry line (black) is included as a reference in E and G. Dotted lines represent best-fit linear regressions. Horizontal black lines in F and H indicate the median AI value for the group of like-treated embryos.
    Figure Legend Snippet: Nodal signalling promotes early asymmetric neurogenesis. ( A-D ) Confocal projections of Tg( huC:gfp ) transgenic zebrafish embryos incubated with DMSO (A,C) or with SB431542 (B,D) from 10 hpf and fixed at 34 (A,B) or 38 (C,D) hpf. Arrows indicate that the left pools of HuC + neurons are bigger than those on the right in DMSO-treated embryos (A,C), whereas the left and right pools are symmetric in embryos incubated with SB431542 (B,D). The epiphysis is indicated by a blue line. Habenular neurogenesis is also delayed in SB431542-treated embryos. ( E-H ) Numbers of left and right HuC + habenular neurons (E,G) and corresponding AI (F,H) in embryos treated with DMSO or SB431542 at 10 (E,F) or 16 (G,H) hpf. Habenular neurons were detected in Tg( huC:gfp ) transgenic embryos using an anti-GFP (E) or HuC/D (G) antibody. A symmetry line (black) is included as a reference in E and G. Dotted lines represent best-fit linear regressions. Horizontal black lines in F and H indicate the median AI value for the group of like-treated embryos.

    Techniques Used: Transgenic Assay, Incubation

    16) Product Images from "Cell cycle regulators control mesoderm specification in human pluripotent stem cells"

    Article Title: Cell cycle regulators control mesoderm specification in human pluripotent stem cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.008251

    Production of mesoderm subtypes from hPSCs. A , schematic overview of the differentiation protocol. LPM was induced by FGF2, the PI3K inhibitor LY294002, and BMP4, and then SMCs were generated using TGFβ and PDGF-BB. CM was induced by Activin, FGF2, the PI3K inhibitor LY29400, and BMP4, and cardiac cells were obtained by inhibiting WNT signaling in the presence of BMP4, FGF2, and retinoic acid. PSM was induced by FGF2 and the WNT signaling agonist CHIR99021. PSM was then generated using FGF2, retinoic acid, and dual inhibition of TGFβ and BMP4 signaling using the small molecules SB431542 and LDN193189, respectively. Chondrocyte differentiation was induced by FGF2 and BMP4. B , RT-qPCR analysis for expression of pluripotency ( NANOG ) and mesoderm markers ( T , NKX2.5 , TAGLN , and CNN1 ) during SMC differentiation. C , immunostaining analysis for the expression of early mesoderm markers BRACHYURY, NKX2.5, and SMC marker TAGLN. Scale bar , 200 μm. D , RT-qPCR analysis for the expression of pluripotency ( NANOG ) and mesoderm markers ( EOMES , NKX2.5 , TNNT2 , and ACTN2 ) during cardiomyocyte differentiation. E , immunostaining analysis for the anterior primitive streak marker EOMES, CM marker NKX2.5, and cardiomyocyte marker troponin T. Scale bar , 200 μm. F , RT-qPCR analysis for expression of pluripotency ( NANOG ) and mesoderm ( T , CDX2 , COL2A1 , and ACAN ) markers during chondrogenic differentiation. G , immunostaining analysis for the expression of early mesoderm marker BRACHYURY, PSM marker PAX3, and Alcian blue staining of chondrocytes differentiation. Scale bar , 200 μm. Error bars represent ± S.D. ( n = 6). Ordinary one-way analysis of variance test followed by Dunnett's test for multiple comparisons was performed. *, p
    Figure Legend Snippet: Production of mesoderm subtypes from hPSCs. A , schematic overview of the differentiation protocol. LPM was induced by FGF2, the PI3K inhibitor LY294002, and BMP4, and then SMCs were generated using TGFβ and PDGF-BB. CM was induced by Activin, FGF2, the PI3K inhibitor LY29400, and BMP4, and cardiac cells were obtained by inhibiting WNT signaling in the presence of BMP4, FGF2, and retinoic acid. PSM was induced by FGF2 and the WNT signaling agonist CHIR99021. PSM was then generated using FGF2, retinoic acid, and dual inhibition of TGFβ and BMP4 signaling using the small molecules SB431542 and LDN193189, respectively. Chondrocyte differentiation was induced by FGF2 and BMP4. B , RT-qPCR analysis for expression of pluripotency ( NANOG ) and mesoderm markers ( T , NKX2.5 , TAGLN , and CNN1 ) during SMC differentiation. C , immunostaining analysis for the expression of early mesoderm markers BRACHYURY, NKX2.5, and SMC marker TAGLN. Scale bar , 200 μm. D , RT-qPCR analysis for the expression of pluripotency ( NANOG ) and mesoderm markers ( EOMES , NKX2.5 , TNNT2 , and ACTN2 ) during cardiomyocyte differentiation. E , immunostaining analysis for the anterior primitive streak marker EOMES, CM marker NKX2.5, and cardiomyocyte marker troponin T. Scale bar , 200 μm. F , RT-qPCR analysis for expression of pluripotency ( NANOG ) and mesoderm ( T , CDX2 , COL2A1 , and ACAN ) markers during chondrogenic differentiation. G , immunostaining analysis for the expression of early mesoderm marker BRACHYURY, PSM marker PAX3, and Alcian blue staining of chondrocytes differentiation. Scale bar , 200 μm. Error bars represent ± S.D. ( n = 6). Ordinary one-way analysis of variance test followed by Dunnett's test for multiple comparisons was performed. *, p

    Techniques Used: Generated, Inhibition, Quantitative RT-PCR, Expressing, Immunostaining, Marker, Staining

    17) Product Images from "H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling"

    Article Title: H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2019.08.016

    TRIM33 and Smad2/3 Colocalize at Chromatin of Mesendodermal Genes in EBs, but Not in ESCs (A) TRIM33 and Smad2/3 form complex upon nodal signaling both in ESCs and EBs. GFP-TRIM33 overexpressing ESCs or day-2.5 EBs were treated with activin A (hereafter, EB-AC) or SB431542 (hereafter, EB-SB) for 2 h. Cell lysates were immunoprecipitated with anti-GFP-Trap affinity beads, and immune complexes were analyzed by immunoblotting using antibodies against the indicated proteins. (B and C) Venn diagrams show the overlap of genes with both TRIM33 and Smad2/3 peaks at their promoters in ESCs (B), EB-AC, and EB-SB (C). (D) Wild-type (WT) and Trim33 null (KO) EBs at day 2.5 were treated with activin A (AC) or SB431542 (SB) for 2 h, then the total RNA was analyzed using RNA-seq. We identified a group of genes whose expression depends on TRIM33-directed nodal signaling, namely genes depending on nodal signaling and TRIM33 (GDNT) (fold change > 1.5 for AC versus SB in WT EBs, or > 1.5 for WT versus KO in EB-AC, false discovery rate
    Figure Legend Snippet: TRIM33 and Smad2/3 Colocalize at Chromatin of Mesendodermal Genes in EBs, but Not in ESCs (A) TRIM33 and Smad2/3 form complex upon nodal signaling both in ESCs and EBs. GFP-TRIM33 overexpressing ESCs or day-2.5 EBs were treated with activin A (hereafter, EB-AC) or SB431542 (hereafter, EB-SB) for 2 h. Cell lysates were immunoprecipitated with anti-GFP-Trap affinity beads, and immune complexes were analyzed by immunoblotting using antibodies against the indicated proteins. (B and C) Venn diagrams show the overlap of genes with both TRIM33 and Smad2/3 peaks at their promoters in ESCs (B), EB-AC, and EB-SB (C). (D) Wild-type (WT) and Trim33 null (KO) EBs at day 2.5 were treated with activin A (AC) or SB431542 (SB) for 2 h, then the total RNA was analyzed using RNA-seq. We identified a group of genes whose expression depends on TRIM33-directed nodal signaling, namely genes depending on nodal signaling and TRIM33 (GDNT) (fold change > 1.5 for AC versus SB in WT EBs, or > 1.5 for WT versus KO in EB-AC, false discovery rate

    Techniques Used: Immunoprecipitation, RNA Sequencing Assay, Expressing

    18) Product Images from "Tissue-Engineered Vascular Grafts Created From Human Induced Pluripotent Stem Cells"

    Article Title: Tissue-Engineered Vascular Grafts Created From Human Induced Pluripotent Stem Cells

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2014-0065

    Derivation of mesenchymal progenitor cells from human iPS cells. (A): Strategy for feeder-free serum-free differentiation using key growth factors: FGF2, SB431542, and Wnt3a. (B): Phase contrast images showing distinct morphological changes from hiPS
    Figure Legend Snippet: Derivation of mesenchymal progenitor cells from human iPS cells. (A): Strategy for feeder-free serum-free differentiation using key growth factors: FGF2, SB431542, and Wnt3a. (B): Phase contrast images showing distinct morphological changes from hiPS

    Techniques Used:

    19) Product Images from "Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *"

    Article Title: Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.502492

    In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast
    Figure Legend Snippet: In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast

    Techniques Used: In Vitro

    20) Product Images from "Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *"

    Article Title: Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.502492

    In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast
    Figure Legend Snippet: In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast

    Techniques Used: In Vitro

    21) Product Images from "Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *"

    Article Title: Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.502492

    In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast
    Figure Legend Snippet: In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast

    Techniques Used: In Vitro

    22) Product Images from "Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *"

    Article Title: Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.502492

    In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast
    Figure Legend Snippet: In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast

    Techniques Used: In Vitro

    23) Product Images from "Fluoxetine Prevents Aβ1-42-Induced Toxicity via a Paracrine Signaling Mediated by Transforming-Growth-Factor-β1"

    Article Title: Fluoxetine Prevents Aβ1-42-Induced Toxicity via a Paracrine Signaling Mediated by Transforming-Growth-Factor-β1

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2016.00389

    The neuroprotective effects of fluoxetine against Aβ 1-42 -induced toxicity are mediated by TGF-β1. Mixed cortical cultures were challenged with Aβ 1-42 oligomers (1 μM) for 48 h in the absence or presence of fluoxetine (1 μM) applied alone or combined with the selective inhibitor of Smad-dependent TGF-β1 signaling, SB431542 (SB; 10 μM) or with a neutralizing antibody specific for TGF-β1 (anti-TGF-β1) applied at a concentration of 2 μg/ml. Aβ toxicity in mixed neuronal cultures was assessed by cell counting after trypan blue staining. Cell counts was performed in three random microscopic fields/well. Values are expressed as percentage of Aβ 1-42 toxicity and are means ± SEM of nine determinations ∗ p
    Figure Legend Snippet: The neuroprotective effects of fluoxetine against Aβ 1-42 -induced toxicity are mediated by TGF-β1. Mixed cortical cultures were challenged with Aβ 1-42 oligomers (1 μM) for 48 h in the absence or presence of fluoxetine (1 μM) applied alone or combined with the selective inhibitor of Smad-dependent TGF-β1 signaling, SB431542 (SB; 10 μM) or with a neutralizing antibody specific for TGF-β1 (anti-TGF-β1) applied at a concentration of 2 μg/ml. Aβ toxicity in mixed neuronal cultures was assessed by cell counting after trypan blue staining. Cell counts was performed in three random microscopic fields/well. Values are expressed as percentage of Aβ 1-42 toxicity and are means ± SEM of nine determinations ∗ p

    Techniques Used: Concentration Assay, Cell Counting, Staining

    24) Product Images from "Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts"

    Article Title: Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts

    Journal: Scars, Burns & Healing

    doi: 10.1177/2059513120908857

    Western blot analysis and immunofluorescence of αSMA expression in HS-derived fibroblasts. HS- derived fibroblasts were pre-treated with hTGF-β1 (5 ng/mL) followed by ALK5 inhibition, subdivided into exon skipping (ALK5ViM [2 µM]) (a) and SB431542 (10 µM) (b). After 24 h, samples were harvested for protein analysis or fixed for immunofluorescence (c). (a, b) The mean relative band intensity of αSMA was calculated by dividing the relative αSMA band intensity by its corresponding relative β-actin (loading control) band intensity. n = 3, error bars represent standard error of the mean. (c) Immunofluorescence for αSMA (red) and merged images show αSMA DAPI nuclear staining (blue). These images are representative for two separate experiments. Scalebar: 100 µm. HS, hypertrophic scar.
    Figure Legend Snippet: Western blot analysis and immunofluorescence of αSMA expression in HS-derived fibroblasts. HS- derived fibroblasts were pre-treated with hTGF-β1 (5 ng/mL) followed by ALK5 inhibition, subdivided into exon skipping (ALK5ViM [2 µM]) (a) and SB431542 (10 µM) (b). After 24 h, samples were harvested for protein analysis or fixed for immunofluorescence (c). (a, b) The mean relative band intensity of αSMA was calculated by dividing the relative αSMA band intensity by its corresponding relative β-actin (loading control) band intensity. n = 3, error bars represent standard error of the mean. (c) Immunofluorescence for αSMA (red) and merged images show αSMA DAPI nuclear staining (blue). These images are representative for two separate experiments. Scalebar: 100 µm. HS, hypertrophic scar.

    Techniques Used: Western Blot, Immunofluorescence, Expressing, Derivative Assay, Inhibition, Staining

    Effect of exon skipping on HS-derived fibroblast migration rate. Fibroblasts were treated with ViMs, SB431542 (for 24 h) or TGF-β1 (for 4 h). (a) Images of the scratches were taken at 0 h and 20 h and show visible wound closure over time. Scale bar: 200 μm. (b) The calculated migration rates for the different conditions 20 h post-scratch. Images were analysed using TScratch software. n = 5; * P
    Figure Legend Snippet: Effect of exon skipping on HS-derived fibroblast migration rate. Fibroblasts were treated with ViMs, SB431542 (for 24 h) or TGF-β1 (for 4 h). (a) Images of the scratches were taken at 0 h and 20 h and show visible wound closure over time. Scale bar: 200 μm. (b) The calculated migration rates for the different conditions 20 h post-scratch. Images were analysed using TScratch software. n = 5; * P

    Techniques Used: Derivative Assay, Migration, Software

    Immunofluorescence and quantification of activated ALK5 in HS-derived fibroblasts. Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) ALK5 IF staining (red) for the following conditions: TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1. Nuclear staining was performed with DAPI, displayed in corresponding merged images. Scale bar: 100 µm. (b) Quantification of ALK5 activation presented as average area fraction. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Control [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) and was calculated with ImageJ. n = 3; * P
    Figure Legend Snippet: Immunofluorescence and quantification of activated ALK5 in HS-derived fibroblasts. Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) ALK5 IF staining (red) for the following conditions: TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1. Nuclear staining was performed with DAPI, displayed in corresponding merged images. Scale bar: 100 µm. (b) Quantification of ALK5 activation presented as average area fraction. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Control [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) and was calculated with ImageJ. n = 3; * P

    Techniques Used: Immunofluorescence, Derivative Assay, Staining, Activation Assay

    pSmad2 immunofluorescence together with (p)MEK immunoblot staining performed on HS-derived fibroblasts. (a–e) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) pSmad2 IF (red) images of ScrViM or ALK5ViM treated HS-derived fibroblasts. Nuclear staining was performed with DAPI. Scale bar: 50 μm. (b–d) Quantification of fold induction of pSmad2 nuclear translocation. One dot represents one donor. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Ctrl [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) with ImageJ. Positive values for pSmad2 are represented as fold induction over their control condition (at baseline level: Ctrl and TGF-β1 for hTGF-β1-post treated samples) indicated by the grey line. n = 3, error bars represent standard error of the mean. (f, g) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. The relative band intensity of total MEK and pMEK was calculated by dividing the relative band intensity of each sample by its corresponding relative GAPDH (loading control) band intensity. For quantification of pMEK activation, pMEK:total MEK ratio was calculated for each sample. n = 2.
    Figure Legend Snippet: pSmad2 immunofluorescence together with (p)MEK immunoblot staining performed on HS-derived fibroblasts. (a–e) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) pSmad2 IF (red) images of ScrViM or ALK5ViM treated HS-derived fibroblasts. Nuclear staining was performed with DAPI. Scale bar: 50 μm. (b–d) Quantification of fold induction of pSmad2 nuclear translocation. One dot represents one donor. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Ctrl [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) with ImageJ. Positive values for pSmad2 are represented as fold induction over their control condition (at baseline level: Ctrl and TGF-β1 for hTGF-β1-post treated samples) indicated by the grey line. n = 3, error bars represent standard error of the mean. (f, g) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. The relative band intensity of total MEK and pMEK was calculated by dividing the relative band intensity of each sample by its corresponding relative GAPDH (loading control) band intensity. For quantification of pMEK activation, pMEK:total MEK ratio was calculated for each sample. n = 2.

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Translocation Assay, Activation Assay

    25) Product Images from "Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts"

    Article Title: Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts

    Journal: Scars, Burns & Healing

    doi: 10.1177/2059513120908857

    Western blot analysis and immunofluorescence of αSMA expression in HS-derived fibroblasts. HS- derived fibroblasts were pre-treated with hTGF-β1 (5 ng/mL) followed by ALK5 inhibition, subdivided into exon skipping (ALK5ViM [2 µM]) (a) and SB431542 (10 µM) (b). After 24 h, samples were harvested for protein analysis or fixed for immunofluorescence (c). (a, b) The mean relative band intensity of αSMA was calculated by dividing the relative αSMA band intensity by its corresponding relative β-actin (loading control) band intensity. n = 3, error bars represent standard error of the mean. (c) Immunofluorescence for αSMA (red) and merged images show αSMA DAPI nuclear staining (blue). These images are representative for two separate experiments. Scalebar: 100 µm. HS, hypertrophic scar.
    Figure Legend Snippet: Western blot analysis and immunofluorescence of αSMA expression in HS-derived fibroblasts. HS- derived fibroblasts were pre-treated with hTGF-β1 (5 ng/mL) followed by ALK5 inhibition, subdivided into exon skipping (ALK5ViM [2 µM]) (a) and SB431542 (10 µM) (b). After 24 h, samples were harvested for protein analysis or fixed for immunofluorescence (c). (a, b) The mean relative band intensity of αSMA was calculated by dividing the relative αSMA band intensity by its corresponding relative β-actin (loading control) band intensity. n = 3, error bars represent standard error of the mean. (c) Immunofluorescence for αSMA (red) and merged images show αSMA DAPI nuclear staining (blue). These images are representative for two separate experiments. Scalebar: 100 µm. HS, hypertrophic scar.

    Techniques Used: Western Blot, Immunofluorescence, Expressing, Derivative Assay, Inhibition, Staining

    Effect of exon skipping on HS-derived fibroblast migration rate. Fibroblasts were treated with ViMs, SB431542 (for 24 h) or TGF-β1 (for 4 h). (a) Images of the scratches were taken at 0 h and 20 h and show visible wound closure over time. Scale bar: 200 μm. (b) The calculated migration rates for the different conditions 20 h post-scratch. Images were analysed using TScratch software. n = 5; * P
    Figure Legend Snippet: Effect of exon skipping on HS-derived fibroblast migration rate. Fibroblasts were treated with ViMs, SB431542 (for 24 h) or TGF-β1 (for 4 h). (a) Images of the scratches were taken at 0 h and 20 h and show visible wound closure over time. Scale bar: 200 μm. (b) The calculated migration rates for the different conditions 20 h post-scratch. Images were analysed using TScratch software. n = 5; * P

    Techniques Used: Derivative Assay, Migration, Software

    Immunofluorescence and quantification of activated ALK5 in HS-derived fibroblasts. Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) ALK5 IF staining (red) for the following conditions: TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1. Nuclear staining was performed with DAPI, displayed in corresponding merged images. Scale bar: 100 µm. (b) Quantification of ALK5 activation presented as average area fraction. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Control [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) and was calculated with ImageJ. n = 3; * P
    Figure Legend Snippet: Immunofluorescence and quantification of activated ALK5 in HS-derived fibroblasts. Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) ALK5 IF staining (red) for the following conditions: TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1. Nuclear staining was performed with DAPI, displayed in corresponding merged images. Scale bar: 100 µm. (b) Quantification of ALK5 activation presented as average area fraction. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Control [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) and was calculated with ImageJ. n = 3; * P

    Techniques Used: Immunofluorescence, Derivative Assay, Staining, Activation Assay

    pSmad2 immunofluorescence together with (p)MEK immunoblot staining performed on HS-derived fibroblasts. (a–e) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) pSmad2 IF (red) images of ScrViM or ALK5ViM treated HS-derived fibroblasts. Nuclear staining was performed with DAPI. Scale bar: 50 μm. (b–d) Quantification of fold induction of pSmad2 nuclear translocation. One dot represents one donor. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Ctrl [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) with ImageJ. Positive values for pSmad2 are represented as fold induction over their control condition (at baseline level: Ctrl and TGF-β1 for hTGF-β1-post treated samples) indicated by the grey line. n = 3, error bars represent standard error of the mean. (f, g) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. The relative band intensity of total MEK and pMEK was calculated by dividing the relative band intensity of each sample by its corresponding relative GAPDH (loading control) band intensity. For quantification of pMEK activation, pMEK:total MEK ratio was calculated for each sample. n = 2.
    Figure Legend Snippet: pSmad2 immunofluorescence together with (p)MEK immunoblot staining performed on HS-derived fibroblasts. (a–e) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) pSmad2 IF (red) images of ScrViM or ALK5ViM treated HS-derived fibroblasts. Nuclear staining was performed with DAPI. Scale bar: 50 μm. (b–d) Quantification of fold induction of pSmad2 nuclear translocation. One dot represents one donor. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Ctrl [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) with ImageJ. Positive values for pSmad2 are represented as fold induction over their control condition (at baseline level: Ctrl and TGF-β1 for hTGF-β1-post treated samples) indicated by the grey line. n = 3, error bars represent standard error of the mean. (f, g) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. The relative band intensity of total MEK and pMEK was calculated by dividing the relative band intensity of each sample by its corresponding relative GAPDH (loading control) band intensity. For quantification of pMEK activation, pMEK:total MEK ratio was calculated for each sample. n = 2.

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Translocation Assay, Activation Assay

    26) Product Images from "Interaction of hepatic stellate cells with neutrophils and macrophages in the liver following oncogenic kras activation in transgenic zebrafish"

    Article Title: Interaction of hepatic stellate cells with neutrophils and macrophages in the liver following oncogenic kras activation in transgenic zebrafish

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26612-0

    Effect of inhibition of Tgfb signaling on liver tumorigenesis and pro-tumor gene expression in hepatocytes, neutrophils and macrophages. 3-dpf wildtype or kras + larvae were treated with dox and SB431542 for 4 days. ( A , B ) Gross morphology of larvae after 4 days treatments ( A ) and quantification of liver size ( B ). ( C–E ) Expression of selected genes for pro-fibrosis, EMT and anti-tumor in hepatocytes ( C ), neutrophils ( D ) and macrophages ( E ). Fold changes refer the expression levels in kras + larvae versus wildtype larvae. In all experiments, n = 20 for each group. *P
    Figure Legend Snippet: Effect of inhibition of Tgfb signaling on liver tumorigenesis and pro-tumor gene expression in hepatocytes, neutrophils and macrophages. 3-dpf wildtype or kras + larvae were treated with dox and SB431542 for 4 days. ( A , B ) Gross morphology of larvae after 4 days treatments ( A ) and quantification of liver size ( B ). ( C–E ) Expression of selected genes for pro-fibrosis, EMT and anti-tumor in hepatocytes ( C ), neutrophils ( D ) and macrophages ( E ). Fold changes refer the expression levels in kras + larvae versus wildtype larvae. In all experiments, n = 20 for each group. *P

    Techniques Used: Inhibition, Expressing

    27) Product Images from "Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *"

    Article Title: Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.502492

    In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast
    Figure Legend Snippet: In vitro differentiation of rabbit ES cells (rdES2-1) into a neural lineage, induced by RA and SB431542 ( SB ). A, schematic representation of the early neural differentiation strategy. Red type shows the additional medium components. B, phase-contrast

    Techniques Used: In Vitro

    28) Product Images from "IKK/NF-κB signaling contributes to glioblastoma stem cell maintenance"

    Article Title: IKK/NF-κB signaling contributes to glioblastoma stem cell maintenance

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12507

    TGF-β is one source of NF-κB activation in GBM A. 6969 and GBM6 explants were stimulated with 10ng/mL TGF-β for 6 or 24 hours, then analyzed by immunoblotting for phosphorylation of Smad and p65. B. 6969 and GBM6 explants were treated with 10 μM SB431542, a TGF-βR1 inhibitor, for 6 or 24 hours, then analyzed by immunoblotting for p65 phosphorylation. C. 6969 or 7030 cells were transfected with 3x-κB luciferase reporter and treated with DMSO or 10 μM SB431542 for 24 hours, then harvested and analyzed for luciferase activity ( n = 3, ** p
    Figure Legend Snippet: TGF-β is one source of NF-κB activation in GBM A. 6969 and GBM6 explants were stimulated with 10ng/mL TGF-β for 6 or 24 hours, then analyzed by immunoblotting for phosphorylation of Smad and p65. B. 6969 and GBM6 explants were treated with 10 μM SB431542, a TGF-βR1 inhibitor, for 6 or 24 hours, then analyzed by immunoblotting for p65 phosphorylation. C. 6969 or 7030 cells were transfected with 3x-κB luciferase reporter and treated with DMSO or 10 μM SB431542 for 24 hours, then harvested and analyzed for luciferase activity ( n = 3, ** p

    Techniques Used: Activation Assay, Transfection, Luciferase, Activity Assay

    29) Product Images from "USP26 regulates TGF‐β signaling by deubiquitinating and stabilizing SMAD7"

    Article Title: USP26 regulates TGF‐β signaling by deubiquitinating and stabilizing SMAD7

    Journal: EMBO Reports

    doi: 10.15252/embr.201643270

    USP 26 modulates TGF ‐β‐mediated biological responses MDA‐MB‐231 cells stably expressing a lentiviral hairpin targeting USP26 (L1) or relevant control vector were plated for scratch assay and treated with either SB431542 (5 μM) or TGF‐β (5 ng/ml); panels show migration at 0 and 24 h. Representative images are shown (scale bars, 50 μm). Percentage of migrated area within the white dotted lines was estimated with respect to control (0 h) and a graph was plotted. ***P
    Figure Legend Snippet: USP 26 modulates TGF ‐β‐mediated biological responses MDA‐MB‐231 cells stably expressing a lentiviral hairpin targeting USP26 (L1) or relevant control vector were plated for scratch assay and treated with either SB431542 (5 μM) or TGF‐β (5 ng/ml); panels show migration at 0 and 24 h. Representative images are shown (scale bars, 50 μm). Percentage of migrated area within the white dotted lines was estimated with respect to control (0 h) and a graph was plotted. ***P

    Techniques Used: Multiple Displacement Amplification, Stable Transfection, Expressing, Plasmid Preparation, Wound Healing Assay, Migration

    30) Product Images from "MicroRNA-132 promotes fibroblast migration via regulating RAS p21 protein activator 1 in skin wound healing"

    Article Title: MicroRNA-132 promotes fibroblast migration via regulating RAS p21 protein activator 1 in skin wound healing

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07513-0

    Expression and regulation of miR-132 in wounds. ( A ) The intact skin (Day 0), Day 1 and Day 7 wounds were collected from healthy donors. ( B ) MiR-132 expression was analyzed in the epidermis and dermis of the biopsies from donor 1–5, which were separated by laser capture microdissection. ( C ) QRT-PCR analysis of miR-132 expression in the cells isolated from the Day6 wounds of 5 donors, including CD45- and CD45 + cells from epidermis, and CD90 + , CD14 + , CD3 + and CD90-CD14-CD3- (Derm-) cells from dermis. ( D ) QRT-PCR analysis of miR-132 expression in dermal CD90 + cells from intact skin and Day6 wounds. V1–5 indicate 5 healthy volunteers. QRT-PCR analysis of miR-132 ( E ) or its primary precursor pri-miR-132 ( F ) in HDFs treated with 10 nM TGF-β1 for 48 hours (n = 3). ( G ) The TGF-β receptor inhibitor SB431542 was applied 15 minutes before adding TGF-β1 to HDFs, and miR-132 expression was analyzed 48 hours later (n = 3). The data are presented as mean ± s.e.m. in ( B , C ), as mean ± s.d. in ( E-G ). *P
    Figure Legend Snippet: Expression and regulation of miR-132 in wounds. ( A ) The intact skin (Day 0), Day 1 and Day 7 wounds were collected from healthy donors. ( B ) MiR-132 expression was analyzed in the epidermis and dermis of the biopsies from donor 1–5, which were separated by laser capture microdissection. ( C ) QRT-PCR analysis of miR-132 expression in the cells isolated from the Day6 wounds of 5 donors, including CD45- and CD45 + cells from epidermis, and CD90 + , CD14 + , CD3 + and CD90-CD14-CD3- (Derm-) cells from dermis. ( D ) QRT-PCR analysis of miR-132 expression in dermal CD90 + cells from intact skin and Day6 wounds. V1–5 indicate 5 healthy volunteers. QRT-PCR analysis of miR-132 ( E ) or its primary precursor pri-miR-132 ( F ) in HDFs treated with 10 nM TGF-β1 for 48 hours (n = 3). ( G ) The TGF-β receptor inhibitor SB431542 was applied 15 minutes before adding TGF-β1 to HDFs, and miR-132 expression was analyzed 48 hours later (n = 3). The data are presented as mean ± s.e.m. in ( B , C ), as mean ± s.d. in ( E-G ). *P

    Techniques Used: Expressing, Laser Capture Microdissection, Quantitative RT-PCR, Isolation

    31) Product Images from "Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts"

    Article Title: Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts

    Journal: Scars, Burns & Healing

    doi: 10.1177/2059513120908857

    Western blot analysis and immunofluorescence of αSMA expression in HS-derived fibroblasts. HS- derived fibroblasts were pre-treated with hTGF-β1 (5 ng/mL) followed by ALK5 inhibition, subdivided into exon skipping (ALK5ViM [2 µM]) (a) and SB431542 (10 µM) (b). After 24 h, samples were harvested for protein analysis or fixed for immunofluorescence (c). (a, b) The mean relative band intensity of αSMA was calculated by dividing the relative αSMA band intensity by its corresponding relative β-actin (loading control) band intensity. n = 3, error bars represent standard error of the mean. (c) Immunofluorescence for αSMA (red) and merged images show αSMA DAPI nuclear staining (blue). These images are representative for two separate experiments. Scalebar: 100 µm. HS, hypertrophic scar.
    Figure Legend Snippet: Western blot analysis and immunofluorescence of αSMA expression in HS-derived fibroblasts. HS- derived fibroblasts were pre-treated with hTGF-β1 (5 ng/mL) followed by ALK5 inhibition, subdivided into exon skipping (ALK5ViM [2 µM]) (a) and SB431542 (10 µM) (b). After 24 h, samples were harvested for protein analysis or fixed for immunofluorescence (c). (a, b) The mean relative band intensity of αSMA was calculated by dividing the relative αSMA band intensity by its corresponding relative β-actin (loading control) band intensity. n = 3, error bars represent standard error of the mean. (c) Immunofluorescence for αSMA (red) and merged images show αSMA DAPI nuclear staining (blue). These images are representative for two separate experiments. Scalebar: 100 µm. HS, hypertrophic scar.

    Techniques Used: Western Blot, Immunofluorescence, Expressing, Derivative Assay, Inhibition, Staining

    Effect of exon skipping on HS-derived fibroblast migration rate. Fibroblasts were treated with ViMs, SB431542 (for 24 h) or TGF-β1 (for 4 h). (a) Images of the scratches were taken at 0 h and 20 h and show visible wound closure over time. Scale bar: 200 μm. (b) The calculated migration rates for the different conditions 20 h post-scratch. Images were analysed using TScratch software. n = 5; * P
    Figure Legend Snippet: Effect of exon skipping on HS-derived fibroblast migration rate. Fibroblasts were treated with ViMs, SB431542 (for 24 h) or TGF-β1 (for 4 h). (a) Images of the scratches were taken at 0 h and 20 h and show visible wound closure over time. Scale bar: 200 μm. (b) The calculated migration rates for the different conditions 20 h post-scratch. Images were analysed using TScratch software. n = 5; * P

    Techniques Used: Derivative Assay, Migration, Software

    Immunofluorescence and quantification of activated ALK5 in HS-derived fibroblasts. Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) ALK5 IF staining (red) for the following conditions: TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1. Nuclear staining was performed with DAPI, displayed in corresponding merged images. Scale bar: 100 µm. (b) Quantification of ALK5 activation presented as average area fraction. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Control [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) and was calculated with ImageJ. n = 3; * P
    Figure Legend Snippet: Immunofluorescence and quantification of activated ALK5 in HS-derived fibroblasts. Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) ALK5 IF staining (red) for the following conditions: TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1. Nuclear staining was performed with DAPI, displayed in corresponding merged images. Scale bar: 100 µm. (b) Quantification of ALK5 activation presented as average area fraction. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Control [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) and was calculated with ImageJ. n = 3; * P

    Techniques Used: Immunofluorescence, Derivative Assay, Staining, Activation Assay

    pSmad2 immunofluorescence together with (p)MEK immunoblot staining performed on HS-derived fibroblasts. (a–e) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) pSmad2 IF (red) images of ScrViM or ALK5ViM treated HS-derived fibroblasts. Nuclear staining was performed with DAPI. Scale bar: 50 μm. (b–d) Quantification of fold induction of pSmad2 nuclear translocation. One dot represents one donor. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Ctrl [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) with ImageJ. Positive values for pSmad2 are represented as fold induction over their control condition (at baseline level: Ctrl and TGF-β1 for hTGF-β1-post treated samples) indicated by the grey line. n = 3, error bars represent standard error of the mean. (f, g) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. The relative band intensity of total MEK and pMEK was calculated by dividing the relative band intensity of each sample by its corresponding relative GAPDH (loading control) band intensity. For quantification of pMEK activation, pMEK:total MEK ratio was calculated for each sample. n = 2.
    Figure Legend Snippet: pSmad2 immunofluorescence together with (p)MEK immunoblot staining performed on HS-derived fibroblasts. (a–e) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) pSmad2 IF (red) images of ScrViM or ALK5ViM treated HS-derived fibroblasts. Nuclear staining was performed with DAPI. Scale bar: 50 μm. (b–d) Quantification of fold induction of pSmad2 nuclear translocation. One dot represents one donor. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Ctrl [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) with ImageJ. Positive values for pSmad2 are represented as fold induction over their control condition (at baseline level: Ctrl and TGF-β1 for hTGF-β1-post treated samples) indicated by the grey line. n = 3, error bars represent standard error of the mean. (f, g) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. The relative band intensity of total MEK and pMEK was calculated by dividing the relative band intensity of each sample by its corresponding relative GAPDH (loading control) band intensity. For quantification of pMEK activation, pMEK:total MEK ratio was calculated for each sample. n = 2.

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Translocation Assay, Activation Assay

    32) Product Images from "Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts"

    Article Title: Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts

    Journal: Scars, Burns & Healing

    doi: 10.1177/2059513120908857

    Western blot analysis and immunofluorescence of αSMA expression in HS-derived fibroblasts. HS- derived fibroblasts were pre-treated with hTGF-β1 (5 ng/mL) followed by ALK5 inhibition, subdivided into exon skipping (ALK5ViM [2 µM]) (a) and SB431542 (10 µM) (b). After 24 h, samples were harvested for protein analysis or fixed for immunofluorescence (c). (a, b) The mean relative band intensity of αSMA was calculated by dividing the relative αSMA band intensity by its corresponding relative β-actin (loading control) band intensity. n = 3, error bars represent standard error of the mean. (c) Immunofluorescence for αSMA (red) and merged images show αSMA DAPI nuclear staining (blue). These images are representative for two separate experiments. Scalebar: 100 µm. HS, hypertrophic scar.
    Figure Legend Snippet: Western blot analysis and immunofluorescence of αSMA expression in HS-derived fibroblasts. HS- derived fibroblasts were pre-treated with hTGF-β1 (5 ng/mL) followed by ALK5 inhibition, subdivided into exon skipping (ALK5ViM [2 µM]) (a) and SB431542 (10 µM) (b). After 24 h, samples were harvested for protein analysis or fixed for immunofluorescence (c). (a, b) The mean relative band intensity of αSMA was calculated by dividing the relative αSMA band intensity by its corresponding relative β-actin (loading control) band intensity. n = 3, error bars represent standard error of the mean. (c) Immunofluorescence for αSMA (red) and merged images show αSMA DAPI nuclear staining (blue). These images are representative for two separate experiments. Scalebar: 100 µm. HS, hypertrophic scar.

    Techniques Used: Western Blot, Immunofluorescence, Expressing, Derivative Assay, Inhibition, Staining

    Effect of exon skipping on HS-derived fibroblast migration rate. Fibroblasts were treated with ViMs, SB431542 (for 24 h) or TGF-β1 (for 4 h). (a) Images of the scratches were taken at 0 h and 20 h and show visible wound closure over time. Scale bar: 200 μm. (b) The calculated migration rates for the different conditions 20 h post-scratch. Images were analysed using TScratch software. n = 5; * P
    Figure Legend Snippet: Effect of exon skipping on HS-derived fibroblast migration rate. Fibroblasts were treated with ViMs, SB431542 (for 24 h) or TGF-β1 (for 4 h). (a) Images of the scratches were taken at 0 h and 20 h and show visible wound closure over time. Scale bar: 200 μm. (b) The calculated migration rates for the different conditions 20 h post-scratch. Images were analysed using TScratch software. n = 5; * P

    Techniques Used: Derivative Assay, Migration, Software

    Immunofluorescence and quantification of activated ALK5 in HS-derived fibroblasts. Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) ALK5 IF staining (red) for the following conditions: TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1. Nuclear staining was performed with DAPI, displayed in corresponding merged images. Scale bar: 100 µm. (b) Quantification of ALK5 activation presented as average area fraction. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Control [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) and was calculated with ImageJ. n = 3; * P
    Figure Legend Snippet: Immunofluorescence and quantification of activated ALK5 in HS-derived fibroblasts. Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) ALK5 IF staining (red) for the following conditions: TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1. Nuclear staining was performed with DAPI, displayed in corresponding merged images. Scale bar: 100 µm. (b) Quantification of ALK5 activation presented as average area fraction. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Control [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) and was calculated with ImageJ. n = 3; * P

    Techniques Used: Immunofluorescence, Derivative Assay, Staining, Activation Assay

    pSmad2 immunofluorescence together with (p)MEK immunoblot staining performed on HS-derived fibroblasts. (a–e) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) pSmad2 IF (red) images of ScrViM or ALK5ViM treated HS-derived fibroblasts. Nuclear staining was performed with DAPI. Scale bar: 50 μm. (b–d) Quantification of fold induction of pSmad2 nuclear translocation. One dot represents one donor. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Ctrl [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) with ImageJ. Positive values for pSmad2 are represented as fold induction over their control condition (at baseline level: Ctrl and TGF-β1 for hTGF-β1-post treated samples) indicated by the grey line. n = 3, error bars represent standard error of the mean. (f, g) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. The relative band intensity of total MEK and pMEK was calculated by dividing the relative band intensity of each sample by its corresponding relative GAPDH (loading control) band intensity. For quantification of pMEK activation, pMEK:total MEK ratio was calculated for each sample. n = 2.
    Figure Legend Snippet: pSmad2 immunofluorescence together with (p)MEK immunoblot staining performed on HS-derived fibroblasts. (a–e) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. (a) pSmad2 IF (red) images of ScrViM or ALK5ViM treated HS-derived fibroblasts. Nuclear staining was performed with DAPI. Scale bar: 50 μm. (b–d) Quantification of fold induction of pSmad2 nuclear translocation. One dot represents one donor. Fluorescent signal was calculated as area fraction for every donor in eight different conditions (Ctrl [untreated], ScrViM, ALK5ViM, SB431542, TGF-β1, ScrViM+TGF-β1, ALK5ViM+TGF-β1 and SB431542+TGF-β1) with ImageJ. Positive values for pSmad2 are represented as fold induction over their control condition (at baseline level: Ctrl and TGF-β1 for hTGF-β1-post treated samples) indicated by the grey line. n = 3, error bars represent standard error of the mean. (f, g) Fibroblasts were pre-treated with ViMs (2 μM) or SB431542 (10 μM) for 24 h. Next, fibroblasts were treated with hTGF-β1 (5 ng/mL) for 1 h. The relative band intensity of total MEK and pMEK was calculated by dividing the relative band intensity of each sample by its corresponding relative GAPDH (loading control) band intensity. For quantification of pMEK activation, pMEK:total MEK ratio was calculated for each sample. n = 2.

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Translocation Assay, Activation Assay

    33) Product Images from "Stimulation of MMP-9 of oral epithelial cells by areca nut extract is related to TGF-β/Smad2-dependent and –independent pathways and prevented by betel leaf extract, hydroxychavicol and melatonin"

    Article Title: Stimulation of MMP-9 of oral epithelial cells by areca nut extract is related to TGF-β/Smad2-dependent and –independent pathways and prevented by betel leaf extract, hydroxychavicol and melatonin

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102565

    Role of TGF-β and Smad2 signaling on ANE-induced MMP-9 expression/secretion of oral epithelial cells. ( A ) Stimulation of TGF-β protein expression of SAS cells by ANE (800 μg/ml) and its attenuation by SB431542 (1 and 5 μM), ( B ) ANE (100-800 μg/ml) stimulated Smad2 phosphorylation of SAS cells after 24-hr of exposure, ( C ) ANE (50-800 μg/ml) stimulated Smad2 phosphorylation of GK after 24-hr of exposure, ( D ) SB431542 (1 and 5 μM) attenuated the ANE (800 μg/ml)-induced p-Smad2 expression of SAS cells, ( E ) SB431542 attenuated the ANE (800 μg/ml)-induced p-Smad2 expression of GK, ( F ) SB431542 prevented the ANE (800 μg/ml)-induced MMP-9 secretion of SAS cells, ( G ) SB431542 prevented the ANE (800 μg/ml)-induced MMP-9 secretion of GK, ( H ) SB431542 showed little effect on ANE (800 μg/ml)-induced cytotoxicity of SAS cells (as % of control, 100%). *denotes statistically significant difference when compared with control. #denotes statistically significant difference when compared with ANE-treated group.
    Figure Legend Snippet: Role of TGF-β and Smad2 signaling on ANE-induced MMP-9 expression/secretion of oral epithelial cells. ( A ) Stimulation of TGF-β protein expression of SAS cells by ANE (800 μg/ml) and its attenuation by SB431542 (1 and 5 μM), ( B ) ANE (100-800 μg/ml) stimulated Smad2 phosphorylation of SAS cells after 24-hr of exposure, ( C ) ANE (50-800 μg/ml) stimulated Smad2 phosphorylation of GK after 24-hr of exposure, ( D ) SB431542 (1 and 5 μM) attenuated the ANE (800 μg/ml)-induced p-Smad2 expression of SAS cells, ( E ) SB431542 attenuated the ANE (800 μg/ml)-induced p-Smad2 expression of GK, ( F ) SB431542 prevented the ANE (800 μg/ml)-induced MMP-9 secretion of SAS cells, ( G ) SB431542 prevented the ANE (800 μg/ml)-induced MMP-9 secretion of GK, ( H ) SB431542 showed little effect on ANE (800 μg/ml)-induced cytotoxicity of SAS cells (as % of control, 100%). *denotes statistically significant difference when compared with control. #denotes statistically significant difference when compared with ANE-treated group.

    Techniques Used: Expressing

    34) Product Images from "TGFβ2-induced formation of lipid droplets supports acidosis-driven EMT and the metastatic spreading of cancer cells"

    Article Title: TGFβ2-induced formation of lipid droplets supports acidosis-driven EMT and the metastatic spreading of cancer cells

    Journal: Nature Communications

    doi: 10.1038/s41467-019-14262-3

    EMT in acidosis-adapted cancer cells is driven by TGF-β2. a Representative immunofluorescence pictures for EMT-related protein markers in native and acidosis-adapted SiHa cells. Scale bar: 20 µm. b mRNA expression for epithelial ( CDH1 ) and mesenchymal ( CDH2 , SNAIL1 , SNAIL2 , and ZEB1 ) genes in native and acidosis-adapted SiHa cells. c Representative immunoblotting for some epithelial (ZO-1 and E-cadherin) and mesenchymal (N-cadherin, vimentin, ZEB1, Snail) protein markers in native and acidosis-adapted cancer cell lines. d , e mRNA expression for SNAIL1 ( d ) and ZEB1 ( e ) in native and acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h. f Representative immunoblotting for mesenchymal protein markers in native and acidosis-adapted SiHa cells following treatment as above. g , h mRNA expression for SNAIL1 ( g ) and ZEB1 ( h ) in native and acidosis-adapted SiHa cells following transfection of Smad2/3-targeting (or control) siRNA for 72 h. i LD content in native and acidosis-adapted SiHa cells following transfection with ZEB1-targeting (or control) siRNA for 72 h. j , k Palmitate-dependent oxygen-consumption rate (OCR) in native and acidosis-adapted SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h ( j ) or following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( k ). l Levels of cellular acetyl-CoA in native and acidosis-adapted SiHa cells. m , n Representative immunoblotting for acetylated Smad2 in native and acidosis-adapted cancer cells (with or without treatment with 4 ng/ml TGF-β2 for 6 h) ( m ) and following treatments with 30 µM etomoxir (CPT1 inhibitor), 2 µg/ml JC63.1 or 1 µg/ml FA6-152 (anti-CD36 blocking antibodies) for 24 h ( n ). Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by Student’s t -test ( l ) or two-way ANOVA ( b , d , e , g – k ) with Bonferroni multiple-comparison analysis. ** p
    Figure Legend Snippet: EMT in acidosis-adapted cancer cells is driven by TGF-β2. a Representative immunofluorescence pictures for EMT-related protein markers in native and acidosis-adapted SiHa cells. Scale bar: 20 µm. b mRNA expression for epithelial ( CDH1 ) and mesenchymal ( CDH2 , SNAIL1 , SNAIL2 , and ZEB1 ) genes in native and acidosis-adapted SiHa cells. c Representative immunoblotting for some epithelial (ZO-1 and E-cadherin) and mesenchymal (N-cadherin, vimentin, ZEB1, Snail) protein markers in native and acidosis-adapted cancer cell lines. d , e mRNA expression for SNAIL1 ( d ) and ZEB1 ( e ) in native and acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h. f Representative immunoblotting for mesenchymal protein markers in native and acidosis-adapted SiHa cells following treatment as above. g , h mRNA expression for SNAIL1 ( g ) and ZEB1 ( h ) in native and acidosis-adapted SiHa cells following transfection of Smad2/3-targeting (or control) siRNA for 72 h. i LD content in native and acidosis-adapted SiHa cells following transfection with ZEB1-targeting (or control) siRNA for 72 h. j , k Palmitate-dependent oxygen-consumption rate (OCR) in native and acidosis-adapted SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h ( j ) or following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( k ). l Levels of cellular acetyl-CoA in native and acidosis-adapted SiHa cells. m , n Representative immunoblotting for acetylated Smad2 in native and acidosis-adapted cancer cells (with or without treatment with 4 ng/ml TGF-β2 for 6 h) ( m ) and following treatments with 30 µM etomoxir (CPT1 inhibitor), 2 µg/ml JC63.1 or 1 µg/ml FA6-152 (anti-CD36 blocking antibodies) for 24 h ( n ). Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by Student’s t -test ( l ) or two-way ANOVA ( b , d , e , g – k ) with Bonferroni multiple-comparison analysis. ** p

    Techniques Used: Immunofluorescence, Expressing, Transfection, Blocking Assay

    TGFβ2-driven LD formation under acidic conditions fosters in vivo cancer cell invasiveness. a Representative immunofluorescent pictures of Alexa Fluor 568-conjugated pHLIP and BODIPY 493/503 staining in 10-day-old HT-29 spheroids. Scale bar: 100 µm. b Time-course accumulation of pHLIP during HT-29 spheroid growth. c – e Distribution (from the periphery to the core of the spheroids) of pHLIP ( c ), BODIPY 493/503 ( d ), and Ki67 ( e ) staining. f , g Representative immunofluorescent pictures ( f ) and quantification ( g ) of BODIPY 493/503 staining in 10-day-old HT-29 spheroids treated or not with 20 µM TGF-βRI inhibitor SB431542. h Time course of the growth inhibitory effects of 20 µM SB431542 on 3D tumor spheroid growth. I , j Representative immunohistochemical pictures ( i ) and quantification ( j ) of GFP- or mCherry-positive lung metastases in mice 5 weeks after i.v. injection of GFP-expressing native and mCherry-expressing acidosis-adapted HT-29 cancer cells ( n = 5–8 mice per group). Scale bar: 1 mm. k , l Representative H E staining ( k ) and quantification of metastasis area ( l ) in lungs from mice 5 weeks after i.v. injection of acidosis-adapted HT-29 cells and i.p. treatment with 40 mg/kg etomoxir ( n = 6–7 mice per group). Scale bar: 1 mm. m , n Kaplan–Meier curves depicting mouse survival after i.v. injection of native and acidosis-adapted FaDu ( m ) and HCT-116 ( n ) cancer cells. o , p Representative H E staining ( o ) and quantification of metastasis area ( p ) in lungs from mice 5 weeks after i.v. injection of lipid droplet-deprived (forskolin-treated; or not) acidosis-adapted HCT-116 cells ( n = 5 mice per group). Scale bar: 250 µm. Data are represented as mean ± SEM of three independent experiments (with ≥ 6 technical replicates). Significance was determined by Student’s t -test ( b , g , j , l , p ). * p
    Figure Legend Snippet: TGFβ2-driven LD formation under acidic conditions fosters in vivo cancer cell invasiveness. a Representative immunofluorescent pictures of Alexa Fluor 568-conjugated pHLIP and BODIPY 493/503 staining in 10-day-old HT-29 spheroids. Scale bar: 100 µm. b Time-course accumulation of pHLIP during HT-29 spheroid growth. c – e Distribution (from the periphery to the core of the spheroids) of pHLIP ( c ), BODIPY 493/503 ( d ), and Ki67 ( e ) staining. f , g Representative immunofluorescent pictures ( f ) and quantification ( g ) of BODIPY 493/503 staining in 10-day-old HT-29 spheroids treated or not with 20 µM TGF-βRI inhibitor SB431542. h Time course of the growth inhibitory effects of 20 µM SB431542 on 3D tumor spheroid growth. I , j Representative immunohistochemical pictures ( i ) and quantification ( j ) of GFP- or mCherry-positive lung metastases in mice 5 weeks after i.v. injection of GFP-expressing native and mCherry-expressing acidosis-adapted HT-29 cancer cells ( n = 5–8 mice per group). Scale bar: 1 mm. k , l Representative H E staining ( k ) and quantification of metastasis area ( l ) in lungs from mice 5 weeks after i.v. injection of acidosis-adapted HT-29 cells and i.p. treatment with 40 mg/kg etomoxir ( n = 6–7 mice per group). Scale bar: 1 mm. m , n Kaplan–Meier curves depicting mouse survival after i.v. injection of native and acidosis-adapted FaDu ( m ) and HCT-116 ( n ) cancer cells. o , p Representative H E staining ( o ) and quantification of metastasis area ( p ) in lungs from mice 5 weeks after i.v. injection of lipid droplet-deprived (forskolin-treated; or not) acidosis-adapted HCT-116 cells ( n = 5 mice per group). Scale bar: 250 µm. Data are represented as mean ± SEM of three independent experiments (with ≥ 6 technical replicates). Significance was determined by Student’s t -test ( b , g , j , l , p ). * p

    Techniques Used: In Vivo, Staining, Immunohistochemistry, Mouse Assay, Injection, Expressing

    TGF-β2 promotes FA uptake and TG accumulation into LD. a – c Abundance of neutral lipids (NL), phospholipids (PL) and free fatty acids (FFA) ( a ), abundance of saturated and monounsaturated fatty acids (SFA and MUFA, respectively) in the neutral lipid fraction ( b ), and LD content ( c ) in native SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h. d , e 14 C-palmitate uptake for 10 min in SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h ( d ) and in acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days in absence or presence of 4 ng/ml TGF-β2 for 24 h ( e ). f – h Representative immunoblotting for cell surface-localized CD36 and total biotinylated proteins in native and acidosis-adapted SiHa cells following treatment with 4 ng/ml TGF-β2 for 6 h and 24 h ( f ), following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( g ) or with 4 ng/ml TGF-β2 and 10 µM PKC-ζ pseudo-substrate inhibitor for 24 h ( h ). i , j Quantification of surface-localized CD36 in native and acidosis-adapted SiHa cells treated as indicated in ( h ). k , l mRNA ( k ) and protein expression of DGAT1 ( l ) in native and acidosis-adapted tumor cells. m – o Representative immunoblotting for DGAT1 in native and acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( m ), with 10 µM PKC-ζ pseudo-substrate inhibitor for 24 h ( n ) or with 10 µM GW6471 for 48 h ( o ). p mRNA expression of PLIN2 in native and acidosis-adapted SiHa cells following treatment with 10 µM TGFβ2-specific antisense oligonucleotide Trabedersen for 7 days. q Co-expression analysis of TGFB2 , PLIN1 , PLIN2 , and PLIN3 genes in human healthy volunteers and colorectal cancer patient samples. Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by Student’s t -test ( c , d , j ), by one-way ANOVA ( e – i ) or two-way ANOVA ( a , k , p ) with Bonferroni multiple-comparison analysis. * p
    Figure Legend Snippet: TGF-β2 promotes FA uptake and TG accumulation into LD. a – c Abundance of neutral lipids (NL), phospholipids (PL) and free fatty acids (FFA) ( a ), abundance of saturated and monounsaturated fatty acids (SFA and MUFA, respectively) in the neutral lipid fraction ( b ), and LD content ( c ) in native SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h. d , e 14 C-palmitate uptake for 10 min in SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h ( d ) and in acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days in absence or presence of 4 ng/ml TGF-β2 for 24 h ( e ). f – h Representative immunoblotting for cell surface-localized CD36 and total biotinylated proteins in native and acidosis-adapted SiHa cells following treatment with 4 ng/ml TGF-β2 for 6 h and 24 h ( f ), following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( g ) or with 4 ng/ml TGF-β2 and 10 µM PKC-ζ pseudo-substrate inhibitor for 24 h ( h ). i , j Quantification of surface-localized CD36 in native and acidosis-adapted SiHa cells treated as indicated in ( h ). k , l mRNA ( k ) and protein expression of DGAT1 ( l ) in native and acidosis-adapted tumor cells. m – o Representative immunoblotting for DGAT1 in native and acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( m ), with 10 µM PKC-ζ pseudo-substrate inhibitor for 24 h ( n ) or with 10 µM GW6471 for 48 h ( o ). p mRNA expression of PLIN2 in native and acidosis-adapted SiHa cells following treatment with 10 µM TGFβ2-specific antisense oligonucleotide Trabedersen for 7 days. q Co-expression analysis of TGFB2 , PLIN1 , PLIN2 , and PLIN3 genes in human healthy volunteers and colorectal cancer patient samples. Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by Student’s t -test ( c , d , j ), by one-way ANOVA ( e – i ) or two-way ANOVA ( a , k , p ) with Bonferroni multiple-comparison analysis. * p

    Techniques Used: Expressing

    TGF-β2 supports invasiveness and LD formation in acidosis-adapted cancer cells. a Heatmap representation of the top 20 genes upregulated in the indicated acidosis-adapted cancer cells. b mRNA expression for TGFB2 in native and acidosis-adapted tumor cells. c , d Levels of active form of TGF-β2 in crude extracts (membrane-bound) from native and acidosis-adapted tumor cells ( c ) or in conditioned media (secreted) from native SiHa cells exposed to acidic pH 6.5 for 24 h ( d ). e Representative immunoblotting for phosphorylated and total forms of Smad2 (Ser465/467) and Smad3 (Ser423/425) in native and acidosis-adapted SiHa cells (with or without treatment with 4 ng/ml TGF-β2 for 6 h). f mRNA expression for TGFB2 in native SiHa cells following treatment with 4 ng/ml TGF-β2 for 24 h. g Representative immunoblotting for TSP-1 in native and acidosis-adapted tumor cells. h Levels of active form of TGF-β2 in conditioned media from native and acidosis-adapted SiHa cells following transfection of TSP1-targeting (or control) siRNA for 72 h. i – k Representative immunoblotting for phosphorylated and total forms of Smad3 (Ser423/425) ( i ), invasion capacity in Matrigel-coated Boyden chambers for 24 h ( j ) and LD content ( k ) for native and acidosis-adapted SiHa cells following treatment with 10 µM TGFβ2-specific antisense oligonucleotide Trabedersen for 7 days or 2 µM TGF-βRI inhibitor SB431542 for 24 h. l LD content in acidosis-adapted SiHa following treatment with 10 µM TGFβ2-specific antisense oligonucleotide Trabedersen for 7 days in absence or presence of 4 ng/ml TGF-β2 for 24 h. Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by Student’s t -test ( d , f ), one-way ANOVA ( l ) or two-way ANOVA ( b – h , j , k ) with Bonferroni multiple-comparison analysis. * p
    Figure Legend Snippet: TGF-β2 supports invasiveness and LD formation in acidosis-adapted cancer cells. a Heatmap representation of the top 20 genes upregulated in the indicated acidosis-adapted cancer cells. b mRNA expression for TGFB2 in native and acidosis-adapted tumor cells. c , d Levels of active form of TGF-β2 in crude extracts (membrane-bound) from native and acidosis-adapted tumor cells ( c ) or in conditioned media (secreted) from native SiHa cells exposed to acidic pH 6.5 for 24 h ( d ). e Representative immunoblotting for phosphorylated and total forms of Smad2 (Ser465/467) and Smad3 (Ser423/425) in native and acidosis-adapted SiHa cells (with or without treatment with 4 ng/ml TGF-β2 for 6 h). f mRNA expression for TGFB2 in native SiHa cells following treatment with 4 ng/ml TGF-β2 for 24 h. g Representative immunoblotting for TSP-1 in native and acidosis-adapted tumor cells. h Levels of active form of TGF-β2 in conditioned media from native and acidosis-adapted SiHa cells following transfection of TSP1-targeting (or control) siRNA for 72 h. i – k Representative immunoblotting for phosphorylated and total forms of Smad3 (Ser423/425) ( i ), invasion capacity in Matrigel-coated Boyden chambers for 24 h ( j ) and LD content ( k ) for native and acidosis-adapted SiHa cells following treatment with 10 µM TGFβ2-specific antisense oligonucleotide Trabedersen for 7 days or 2 µM TGF-βRI inhibitor SB431542 for 24 h. l LD content in acidosis-adapted SiHa following treatment with 10 µM TGFβ2-specific antisense oligonucleotide Trabedersen for 7 days in absence or presence of 4 ng/ml TGF-β2 for 24 h. Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by Student’s t -test ( d , f ), one-way ANOVA ( l ) or two-way ANOVA ( b – h , j , k ) with Bonferroni multiple-comparison analysis. * p

    Techniques Used: Expressing, Transfection

    35) Product Images from "GLIS3 Transcriptionally Activates WNT Genes to Promote Differentiation of Human Embryonic Stem Cells into Posterior Neural Progenitors"

    Article Title: GLIS3 Transcriptionally Activates WNT Genes to Promote Differentiation of Human Embryonic Stem Cells into Posterior Neural Progenitors

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.2941

    Differentiation of G3-hESC into neural progenitor cells. (A) Schematic of the pINDUCER20 system encoding Flag-GLIS3-HA under control of a doxycycline (DOX)-inducible promoter used to generate G3-hESCs. (B) Differentiation protocol using the activin/TGFβ/BMP pathways inhibitors, dorsomorphin and SB431542, to induce differentiation of hESCs into NPCs. (C) Time course of GLIS3 mRNA induction in G3-hESCs treated with or without DOX as determined by qRT-PCR analysis. Data are normalized to GAPDH . Error bars represent SEM of three biological replicates. * p
    Figure Legend Snippet: Differentiation of G3-hESC into neural progenitor cells. (A) Schematic of the pINDUCER20 system encoding Flag-GLIS3-HA under control of a doxycycline (DOX)-inducible promoter used to generate G3-hESCs. (B) Differentiation protocol using the activin/TGFβ/BMP pathways inhibitors, dorsomorphin and SB431542, to induce differentiation of hESCs into NPCs. (C) Time course of GLIS3 mRNA induction in G3-hESCs treated with or without DOX as determined by qRT-PCR analysis. Data are normalized to GAPDH . Error bars represent SEM of three biological replicates. * p

    Techniques Used: Quantitative RT-PCR

    36) Product Images from "TGF-Beta Negatively Regulates the BMP2-Dependent Early Commitment of Periodontal Ligament Cells into Hard Tissue Forming Cells"

    Article Title: TGF-Beta Negatively Regulates the BMP2-Dependent Early Commitment of Periodontal Ligament Cells into Hard Tissue Forming Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125590

    Expression of TGF-β/BMP receptor and Smads in MPDL22 cells. (A) Semiquantitative RT-PCR analysis of the expression of TGF-β receptor genes ALK-1 , -4 , -5 , -7 , and TβRII , and BMP receptor genes ALK-2 , -3 , -6 , BMPR2 , and Smad1–7 . Human glycerralaldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as an internal control. (B) Western blotting analysis of TGF-β/BMP receptor induced by TGF-β (4 ng/mL) or BMP-2 (50 ng/mL) in the presence or absence of SB431542 (10 μM). Protein levels of TGF-β receptor I (TGF-βRI), TGF-β receptor II (TGF-βRII), BMP receptor I (BMPRI), and BMP receptor II (BMPRII) were measured. β-actin was used as a protein loading control. Quantitative analysis is shown as the relative ratios of TGF-β or BMP receptors I/II and β-actin by densitometric analysis. Values represent the mean ± SD of 3 independent assays. (-): control; B: BMP-2; T: TGF-β. (C) The relative quantification of TGF-β1 , TGF-β2 , and TGF-β3 mRNAs in MPDL22 cells by RT-qPCR. Quantitative mRNA values were normalized to the amount of GAPDH mRNA. (D) TGF-β production from MPDL22 cells. Protein expression levels of TGF-β were examined by ELISA. Culture supernatants of MPDL22 cells were aspirated after 24 h of culture with or without BMP-2 (50 ng/mL) and SB431542 (10 μM). B: BMP-2; SB: SB431542. **: p
    Figure Legend Snippet: Expression of TGF-β/BMP receptor and Smads in MPDL22 cells. (A) Semiquantitative RT-PCR analysis of the expression of TGF-β receptor genes ALK-1 , -4 , -5 , -7 , and TβRII , and BMP receptor genes ALK-2 , -3 , -6 , BMPR2 , and Smad1–7 . Human glycerralaldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as an internal control. (B) Western blotting analysis of TGF-β/BMP receptor induced by TGF-β (4 ng/mL) or BMP-2 (50 ng/mL) in the presence or absence of SB431542 (10 μM). Protein levels of TGF-β receptor I (TGF-βRI), TGF-β receptor II (TGF-βRII), BMP receptor I (BMPRI), and BMP receptor II (BMPRII) were measured. β-actin was used as a protein loading control. Quantitative analysis is shown as the relative ratios of TGF-β or BMP receptors I/II and β-actin by densitometric analysis. Values represent the mean ± SD of 3 independent assays. (-): control; B: BMP-2; T: TGF-β. (C) The relative quantification of TGF-β1 , TGF-β2 , and TGF-β3 mRNAs in MPDL22 cells by RT-qPCR. Quantitative mRNA values were normalized to the amount of GAPDH mRNA. (D) TGF-β production from MPDL22 cells. Protein expression levels of TGF-β were examined by ELISA. Culture supernatants of MPDL22 cells were aspirated after 24 h of culture with or without BMP-2 (50 ng/mL) and SB431542 (10 μM). B: BMP-2; SB: SB431542. **: p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    SB431542 treatment on ALP activity and expression of osteoblastic differentiation-related genes in MPDL22 cells. (A) MPDL22 cells were cultured in mineralization-inducing medium in the presence or absence of BMP-2 (50 ng/mL), TGF-β (4 ng/mL) and SB431542 (10 μM). MPDL22 cells were harvested at the indicated time points. ALPase activity was determined as described in the methods section. Activity in U/mg protein for the cell lysates is shown. **: p
    Figure Legend Snippet: SB431542 treatment on ALP activity and expression of osteoblastic differentiation-related genes in MPDL22 cells. (A) MPDL22 cells were cultured in mineralization-inducing medium in the presence or absence of BMP-2 (50 ng/mL), TGF-β (4 ng/mL) and SB431542 (10 μM). MPDL22 cells were harvested at the indicated time points. ALPase activity was determined as described in the methods section. Activity in U/mg protein for the cell lysates is shown. **: p

    Techniques Used: ALP Assay, Activity Assay, Expressing, Cell Culture

    Effects of BMP-2 and SB431542 on collagen synthesis during osteoblastic differentiation of MPDL22 cells. (A) SB431542 (10 µM) was added to MPDL22 cells during osteogenic differentiation with or without BMP-2 (50 ng/mL) at different times as indicated in the left panel. The right panel shows the van Gieson staining, which stains collagen pink. (B) The relative quantification of Col1A1 mRNA in BMP-2-induced MPDL22 cells was assessed during osteogenic differentiation in the presence or absence of SB431542 (10 μM). MPDL22 cells were harvested every 3 days and isolated mRNA was assessed by RT-qPCR. Quantitative mRNA values were normalized to the amount of GAPDH mRNA. B: BMP-2; SB: SB431542, **: p
    Figure Legend Snippet: Effects of BMP-2 and SB431542 on collagen synthesis during osteoblastic differentiation of MPDL22 cells. (A) SB431542 (10 µM) was added to MPDL22 cells during osteogenic differentiation with or without BMP-2 (50 ng/mL) at different times as indicated in the left panel. The right panel shows the van Gieson staining, which stains collagen pink. (B) The relative quantification of Col1A1 mRNA in BMP-2-induced MPDL22 cells was assessed during osteogenic differentiation in the presence or absence of SB431542 (10 μM). MPDL22 cells were harvested every 3 days and isolated mRNA was assessed by RT-qPCR. Quantitative mRNA values were normalized to the amount of GAPDH mRNA. B: BMP-2; SB: SB431542, **: p

    Techniques Used: Staining, Isolation, Quantitative RT-PCR

    Effects of BMP-2 and SB431542 treatment at different periods during osteoblastic differentiation of MPDL22 cells. (A) MPDL22 cells were cultured in mineralization-inducing medium in the presence or absence of BMP-2 (50 ng/mL). SB431542 (10 μM) was also added to the culture at different times as indicated in the left panel. The middle panel shows Alizarin red staining at day 12. The right panel shows the quantification by densitometric analysis of the Alizarin red staining. **: p
    Figure Legend Snippet: Effects of BMP-2 and SB431542 treatment at different periods during osteoblastic differentiation of MPDL22 cells. (A) MPDL22 cells were cultured in mineralization-inducing medium in the presence or absence of BMP-2 (50 ng/mL). SB431542 (10 μM) was also added to the culture at different times as indicated in the left panel. The middle panel shows Alizarin red staining at day 12. The right panel shows the quantification by densitometric analysis of the Alizarin red staining. **: p

    Techniques Used: Cell Culture, Staining

    Effects of SB431542 on the TGF-β/Smad transcriptional responses in MPDL22 cells. (A) Activation of Smad3, Erk, and p38 induced by TGF-β (4 ng/mL) with or without pretreatment with SB431542 (10 μM). Phosphorylation levels and protein levels were determined by western blotting. (B) Promoter activity of TGF-β responsive gene PAI-1 . MPDL22 cells were transfected with ( CAGA ) 12 -Luc reporter plasmid as indicated. Twenty-four hours after transfection, cells were treated with TGF-β (4 ng/mL), SB431542 (10 μM) or both overnight. (-): control; B: BMP-2; T: TGF-β; SB: SB431542. **: p
    Figure Legend Snippet: Effects of SB431542 on the TGF-β/Smad transcriptional responses in MPDL22 cells. (A) Activation of Smad3, Erk, and p38 induced by TGF-β (4 ng/mL) with or without pretreatment with SB431542 (10 μM). Phosphorylation levels and protein levels were determined by western blotting. (B) Promoter activity of TGF-β responsive gene PAI-1 . MPDL22 cells were transfected with ( CAGA ) 12 -Luc reporter plasmid as indicated. Twenty-four hours after transfection, cells were treated with TGF-β (4 ng/mL), SB431542 (10 μM) or both overnight. (-): control; B: BMP-2; T: TGF-β; SB: SB431542. **: p

    Techniques Used: Activation Assay, Western Blot, Activity Assay, Transfection, Plasmid Preparation

    Effects of SB431542 on mineralized nodule formation by MPDL22 cells. (A) Osteogenic differentiation of MPDL22 cells was induced by culture in mineralization inducing medium with or without BMP-2 (50 ng/mL), FGF-2 (50 ng/mL) and PDGF-BB (20 ng/mL) in the presence or absence of TGF-β (4 ng/mL) and SB431542 (10 μM). Calcified nodule formation was determined at day 12 by Alizarin red staining. (B) Quantification of calcified nodule formation by MPDL22 cells induced by BMP-2 in the presence or absence of AA (50 mg/mL) plus β-GP (50 mM), BMP-2 (50 ng/mL) and SB431542 (10 μM). Densitometric analysis was applied to the scanned culture plate images at day 12. Positive scores were calculated by multiplying the stained area by its Alizarin red staining color density. B: BMP-2; SB: SB431542. **: p
    Figure Legend Snippet: Effects of SB431542 on mineralized nodule formation by MPDL22 cells. (A) Osteogenic differentiation of MPDL22 cells was induced by culture in mineralization inducing medium with or without BMP-2 (50 ng/mL), FGF-2 (50 ng/mL) and PDGF-BB (20 ng/mL) in the presence or absence of TGF-β (4 ng/mL) and SB431542 (10 μM). Calcified nodule formation was determined at day 12 by Alizarin red staining. (B) Quantification of calcified nodule formation by MPDL22 cells induced by BMP-2 in the presence or absence of AA (50 mg/mL) plus β-GP (50 mM), BMP-2 (50 ng/mL) and SB431542 (10 μM). Densitometric analysis was applied to the scanned culture plate images at day 12. Positive scores were calculated by multiplying the stained area by its Alizarin red staining color density. B: BMP-2; SB: SB431542. **: p

    Techniques Used: Staining

    37) Product Images from "Activation of the TGFβ pathway impairs endothelial to haematopoietic transition"

    Article Title: Activation of the TGFβ pathway impairs endothelial to haematopoietic transition

    Journal: Scientific Reports

    doi: 10.1038/srep21518

    TGFβ treatment blocks blood cell formation, reduces the frequency of haematopoietic cells and increases the frequency of endothelial cells. ( A ) Images of the haemogenic endothelium (HE) culture taken after 24, 48 and 60 hours in different conditions. The scale bar corresponds to 200 μm. ( B ) Quantification of the number of round cells generated during time-lapse imaging in the 4 indicated conditions. Each value represents the mean of the number of round cells for 9 areas of the same well. Error bars represent standard deviation. ( C ) Bar graphs representing the average cell number of the 3 indicated conditions from 3 independent experiments. Error bars correspond to standard deviation. The p-values were calculated with Student’s t-test (2 tails, type 3). *DMSO versus TGFβ2 p-value = 0.035 (n = 3); *SB431542 versus TGFβ2 p-value = 0.03 (n = 3). ( D ) Flow cytometry analysis of VE-Cad and CD41 expression after 3 days of culture following the addition of TGFβ2 compared to control condition for one representative experiment. ( E ) Bar graphs representing the average frequency of the 2 indicated populations from 3 independent experiments. Error bars correspond to standard deviation. The p-values were calculated with Student’s t-test (2 tails, type 3). VE-Cad − CD41 + population p-values: **Medium only versus TGFβ2 p-value = 0.004 (n = 3); **DMSO versus TGFβ2 p-value = 0.004 (n = 3); **SB431542 versus TGFβ2 p-value = 0.0001 (n = 3). VE-Cad + CD41 − population p-values: **Medium only versus TGFβ2 p-value = 0.00018 (n = 3); **DMSO versus TGFβ2 p-value = 0.0011 (n = 3); **SB431542 versus TGFβ2 p-value = 0.0015 (n = 3).
    Figure Legend Snippet: TGFβ treatment blocks blood cell formation, reduces the frequency of haematopoietic cells and increases the frequency of endothelial cells. ( A ) Images of the haemogenic endothelium (HE) culture taken after 24, 48 and 60 hours in different conditions. The scale bar corresponds to 200 μm. ( B ) Quantification of the number of round cells generated during time-lapse imaging in the 4 indicated conditions. Each value represents the mean of the number of round cells for 9 areas of the same well. Error bars represent standard deviation. ( C ) Bar graphs representing the average cell number of the 3 indicated conditions from 3 independent experiments. Error bars correspond to standard deviation. The p-values were calculated with Student’s t-test (2 tails, type 3). *DMSO versus TGFβ2 p-value = 0.035 (n = 3); *SB431542 versus TGFβ2 p-value = 0.03 (n = 3). ( D ) Flow cytometry analysis of VE-Cad and CD41 expression after 3 days of culture following the addition of TGFβ2 compared to control condition for one representative experiment. ( E ) Bar graphs representing the average frequency of the 2 indicated populations from 3 independent experiments. Error bars correspond to standard deviation. The p-values were calculated with Student’s t-test (2 tails, type 3). VE-Cad − CD41 + population p-values: **Medium only versus TGFβ2 p-value = 0.004 (n = 3); **DMSO versus TGFβ2 p-value = 0.004 (n = 3); **SB431542 versus TGFβ2 p-value = 0.0001 (n = 3). VE-Cad + CD41 − population p-values: **Medium only versus TGFβ2 p-value = 0.00018 (n = 3); **DMSO versus TGFβ2 p-value = 0.0011 (n = 3); **SB431542 versus TGFβ2 p-value = 0.0015 (n = 3).

    Techniques Used: Generated, Imaging, Standard Deviation, Flow Cytometry, Cytometry, Expressing

    TGFβ signalling activation decreases haematopoietic gene expression and increases EMT and vascular genes transcription. ( A ) Box plots comparing the expression of vascular specific genes between the control, SB431542 and TGFβ2 conditions. ( B ) Box plots comparing the expression of haematopoietic genes between the 3 conditions. ( C ) Box plots comparing the expression of EMT specific genes between the 3 conditions. The box plots were generated from 4 independent experiments. For each plot, the top and bottom box edges correspond to the first and third quartiles. The black line inside the box represents the median. The top and bottom whisker lines mark the maximum and minimum values of the data set, respectively. The corresponding p-values were calculated with Student’s t-test ( Supplementary Table S7 ).
    Figure Legend Snippet: TGFβ signalling activation decreases haematopoietic gene expression and increases EMT and vascular genes transcription. ( A ) Box plots comparing the expression of vascular specific genes between the control, SB431542 and TGFβ2 conditions. ( B ) Box plots comparing the expression of haematopoietic genes between the 3 conditions. ( C ) Box plots comparing the expression of EMT specific genes between the 3 conditions. The box plots were generated from 4 independent experiments. For each plot, the top and bottom box edges correspond to the first and third quartiles. The black line inside the box represents the median. The top and bottom whisker lines mark the maximum and minimum values of the data set, respectively. The corresponding p-values were calculated with Student’s t-test ( Supplementary Table S7 ).

    Techniques Used: Activation Assay, Expressing, Generated, Whisker Assay

    38) Product Images from "Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial‐Like Cells"

    Article Title: Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial‐Like Cells

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.2750

    SB431542 exposure during early differentiation enhanced the hiPSC differentiation to corneal epithelial progenitors. Schematic outline for optimization experiment using SB431542 (A) . Quantitative real‐time polymerase chain reaction analysis of the putative limbal stem cells ( ΔNp63 ) gene for SB‐Ad3 cells from G5 subgroups on day 20 of optimization experiment following different durations of SB431542 exposure (B) . Colony forming efficiency assays for the SB431542 exposed and unexposed G5 subgroups on day 20 (C) . Data presented as mean ± SEM, n = 3. *, statistically different compared with untreated group. **, p
    Figure Legend Snippet: SB431542 exposure during early differentiation enhanced the hiPSC differentiation to corneal epithelial progenitors. Schematic outline for optimization experiment using SB431542 (A) . Quantitative real‐time polymerase chain reaction analysis of the putative limbal stem cells ( ΔNp63 ) gene for SB‐Ad3 cells from G5 subgroups on day 20 of optimization experiment following different durations of SB431542 exposure (B) . Colony forming efficiency assays for the SB431542 exposed and unexposed G5 subgroups on day 20 (C) . Data presented as mean ± SEM, n = 3. *, statistically different compared with untreated group. **, p

    Techniques Used: Real-time Polymerase Chain Reaction

    39) Product Images from "Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial‐Like Cells"

    Article Title: Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial‐Like Cells

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.2750

    SB431542 exposure during early differentiation enhanced the hiPSC differentiation to corneal epithelial progenitors. Schematic outline for optimization experiment using SB431542 (A) . Quantitative real‐time polymerase chain reaction analysis of the putative limbal stem cells ( ΔNp63 ) gene for SB‐Ad3 cells from G5 subgroups on day 20 of optimization experiment following different durations of SB431542 exposure (B) . Colony forming efficiency assays for the SB431542 exposed and unexposed G5 subgroups on day 20 (C) . Data presented as mean ± SEM, n = 3. *, statistically different compared with untreated group. **, p
    Figure Legend Snippet: SB431542 exposure during early differentiation enhanced the hiPSC differentiation to corneal epithelial progenitors. Schematic outline for optimization experiment using SB431542 (A) . Quantitative real‐time polymerase chain reaction analysis of the putative limbal stem cells ( ΔNp63 ) gene for SB‐Ad3 cells from G5 subgroups on day 20 of optimization experiment following different durations of SB431542 exposure (B) . Colony forming efficiency assays for the SB431542 exposed and unexposed G5 subgroups on day 20 (C) . Data presented as mean ± SEM, n = 3. *, statistically different compared with untreated group. **, p

    Techniques Used: Real-time Polymerase Chain Reaction

    40) Product Images from "Ascorbic acid promotes cardiomyogenesis through SMAD1 signaling in differentiating mouse embryonic stem cells"

    Article Title: Ascorbic acid promotes cardiomyogenesis through SMAD1 signaling in differentiating mouse embryonic stem cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0188569

    SMADs unidirectional cross-talk either promotes or impairs cardiomyogenesis. The inhibition of the TGFβ-signaling by SB431542 decreases the activation (phosphorylation) of the TGFβ-dependent SMAD2, as expected, but also downregulates the activation (phosphorylation) of the BMP-dependent SMAD1. SMAD2 negative feedback on SMAD1 impairs cardiomyogenesis. In contrast, the induction of the BMP-cascade by conditional expression of SMAD1, BMP2-stimulation or AA treatment, increases the activation (phosphorylation) of the BMP-dependent SMAD1, as expected, but also upregulates the activation (phosphorylation) of the TGFβ-dependent SMAD2. SMAD1 positive feedback on SMAD2 promotes cardiomyogenesis. These results show that SMAD1 is critical for cardiomyogenesis, and that AA acts in part through this positive unidirectional cross-talk.
    Figure Legend Snippet: SMADs unidirectional cross-talk either promotes or impairs cardiomyogenesis. The inhibition of the TGFβ-signaling by SB431542 decreases the activation (phosphorylation) of the TGFβ-dependent SMAD2, as expected, but also downregulates the activation (phosphorylation) of the BMP-dependent SMAD1. SMAD2 negative feedback on SMAD1 impairs cardiomyogenesis. In contrast, the induction of the BMP-cascade by conditional expression of SMAD1, BMP2-stimulation or AA treatment, increases the activation (phosphorylation) of the BMP-dependent SMAD1, as expected, but also upregulates the activation (phosphorylation) of the TGFβ-dependent SMAD2. SMAD1 positive feedback on SMAD2 promotes cardiomyogenesis. These results show that SMAD1 is critical for cardiomyogenesis, and that AA acts in part through this positive unidirectional cross-talk.

    Techniques Used: Inhibition, Activation Assay, Expressing

    Ascorbic acid induction of cardiomyogenesis involves the TGFβ-signaling pathway. A-B. Cardiomyocyte-induction assessed by flow cytometry of RFP + -CMs at Day 7+3 of differentiation following treatments with A. Activin A (100 ng/mL) added at Day 2 (n = 12) and B. SB431542 (10 μM), an inhibitor of SMAD2-activation, added at Day 0 (n = 3) or Day 2 (n = 3), performed alone or in combination with AA (Day 2). C. Western Blot of the cardiac markers GATA4 and T assessed at Day 3 of differentiation following treatments with SB431542 (10 μM) and AA, both performed at Day 2 (n = 4). D. Flow cytometry assessment of cardiomyogenesis (RFP + -CMs) at Day 7+3 of differentiation following treatments with TDGF1 (100 ng/mL) added at Day 2 (n = 3), alone or in combination with AA (Day 2).*p
    Figure Legend Snippet: Ascorbic acid induction of cardiomyogenesis involves the TGFβ-signaling pathway. A-B. Cardiomyocyte-induction assessed by flow cytometry of RFP + -CMs at Day 7+3 of differentiation following treatments with A. Activin A (100 ng/mL) added at Day 2 (n = 12) and B. SB431542 (10 μM), an inhibitor of SMAD2-activation, added at Day 0 (n = 3) or Day 2 (n = 3), performed alone or in combination with AA (Day 2). C. Western Blot of the cardiac markers GATA4 and T assessed at Day 3 of differentiation following treatments with SB431542 (10 μM) and AA, both performed at Day 2 (n = 4). D. Flow cytometry assessment of cardiomyogenesis (RFP + -CMs) at Day 7+3 of differentiation following treatments with TDGF1 (100 ng/mL) added at Day 2 (n = 3), alone or in combination with AA (Day 2).*p

    Techniques Used: Flow Cytometry, Cytometry, Activation Assay, Western Blot

    Related Articles

    Concentration Assay:

    Article Title: Calcium mimics the chemotactic effect of conditioned media and is an effective inducer of bone regeneration
    Article Snippet: .. SB431542, a selective TGFB type I receptor inhibitor (Tocris), was solubilized using DMSO at a 10 mM stock concentration, and a final concentration of 10 μM. .. AG-1296, a selective PDGF receptor inhibitor (Abcam), was used at a final 10 μM concentration.

    RNA Extraction:

    Article Title: A poised chromatin platform for TGF-? access to master regulators
    Article Snippet: .. Prior to total RNA extraction cells were treated with activin A (50 ng/ml, R & D Systems) or SB431542 (10 μM, TOCRIS). .. Human embryonic stem cells (hESCs) (WA-09; passage 35-45) were cultured on MEFs.

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    Tocris sb431542
    Yan/Tel controls the spatial restriction of nodal expression in the ectoderm. A, Inhibition of maternal Yan/Tel function disrupts dorsal-ventral axis formation. Note the radial arrangement of the primary mesenchymal cells (PMCs, arrowheads) at gastrula stage (24hpf) and the rounded shape of the embryo at prism stage (36hpf). (hpf), hours post-fertilization. B-C, Inhibition of yan/tel mRNA translation causes a massive ectopic expression of nodal and chordin at blastula (B) and gastrula (C) stages. Note that the expression has expanded throughout most of the ectoderm but is excluded from the animal pole region. apv, animal pole view. D, Inhibition of yan/tel mRNA translation expands the expression of the ventral ectodermal markers bmp2/4 and lefty and suppresses the expression of the dorsal ectodermal markers 29D and tbx2/3 . E, Inhibition of yan/tel mRNA translation expands the expression of the ventral mesodermal marker gata1/2/3 and suppresses the expression of dorsal mesodermal marker gcm . F, Random local inhibition of Yan/Tel function orients the dorsal-ventral axis. In all embryos injected randomly with the yan/tel morpholino into one blastomere at the 2 or 4-cell stage, nodal expression (blue) overlaps with the progeny of the injected blastomere (red). G, Ectopic expression of the Nodal downstream target gene chordin following inhibition of yan/tel mRNA translation depends on Nodal pathway activity. Note that inhibition of yan/tel function followed by treatment with the Nodal receptor inhibitor <t>SB431542</t> blocks the ectopic expression of chordin observed in y an/tel morphants. SB, swimming blastula stage; LG, late gastrula stage. MB, mesenchyme blastula stage; vv, vegetal view; lv, lateral view. In the lateral views, animal is to the top, and ventral to the left.
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    Yan/Tel controls the spatial restriction of nodal expression in the ectoderm. A, Inhibition of maternal Yan/Tel function disrupts dorsal-ventral axis formation. Note the radial arrangement of the primary mesenchymal cells (PMCs, arrowheads) at gastrula stage (24hpf) and the rounded shape of the embryo at prism stage (36hpf). (hpf), hours post-fertilization. B-C, Inhibition of yan/tel mRNA translation causes a massive ectopic expression of nodal and chordin at blastula (B) and gastrula (C) stages. Note that the expression has expanded throughout most of the ectoderm but is excluded from the animal pole region. apv, animal pole view. D, Inhibition of yan/tel mRNA translation expands the expression of the ventral ectodermal markers bmp2/4 and lefty and suppresses the expression of the dorsal ectodermal markers 29D and tbx2/3 . E, Inhibition of yan/tel mRNA translation expands the expression of the ventral mesodermal marker gata1/2/3 and suppresses the expression of dorsal mesodermal marker gcm . F, Random local inhibition of Yan/Tel function orients the dorsal-ventral axis. In all embryos injected randomly with the yan/tel morpholino into one blastomere at the 2 or 4-cell stage, nodal expression (blue) overlaps with the progeny of the injected blastomere (red). G, Ectopic expression of the Nodal downstream target gene chordin following inhibition of yan/tel mRNA translation depends on Nodal pathway activity. Note that inhibition of yan/tel function followed by treatment with the Nodal receptor inhibitor SB431542 blocks the ectopic expression of chordin observed in y an/tel morphants. SB, swimming blastula stage; LG, late gastrula stage. MB, mesenchyme blastula stage; vv, vegetal view; lv, lateral view. In the lateral views, animal is to the top, and ventral to the left.

    Journal: PLoS Genetics

    Article Title: MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo

    doi: 10.1371/journal.pgen.1007621

    Figure Lengend Snippet: Yan/Tel controls the spatial restriction of nodal expression in the ectoderm. A, Inhibition of maternal Yan/Tel function disrupts dorsal-ventral axis formation. Note the radial arrangement of the primary mesenchymal cells (PMCs, arrowheads) at gastrula stage (24hpf) and the rounded shape of the embryo at prism stage (36hpf). (hpf), hours post-fertilization. B-C, Inhibition of yan/tel mRNA translation causes a massive ectopic expression of nodal and chordin at blastula (B) and gastrula (C) stages. Note that the expression has expanded throughout most of the ectoderm but is excluded from the animal pole region. apv, animal pole view. D, Inhibition of yan/tel mRNA translation expands the expression of the ventral ectodermal markers bmp2/4 and lefty and suppresses the expression of the dorsal ectodermal markers 29D and tbx2/3 . E, Inhibition of yan/tel mRNA translation expands the expression of the ventral mesodermal marker gata1/2/3 and suppresses the expression of dorsal mesodermal marker gcm . F, Random local inhibition of Yan/Tel function orients the dorsal-ventral axis. In all embryos injected randomly with the yan/tel morpholino into one blastomere at the 2 or 4-cell stage, nodal expression (blue) overlaps with the progeny of the injected blastomere (red). G, Ectopic expression of the Nodal downstream target gene chordin following inhibition of yan/tel mRNA translation depends on Nodal pathway activity. Note that inhibition of yan/tel function followed by treatment with the Nodal receptor inhibitor SB431542 blocks the ectopic expression of chordin observed in y an/tel morphants. SB, swimming blastula stage; LG, late gastrula stage. MB, mesenchyme blastula stage; vv, vegetal view; lv, lateral view. In the lateral views, animal is to the top, and ventral to the left.

    Article Snippet: Treatments with U0126 (Calbiochem, 10 μM) BIRB796 (Selleckchem, S1574, 3 μM), SP600125 (Tocris, 0,5 μM), SB431542 (Tocris, 1614, 10 μM) and MG132 (Tocris 10 μM) were performed by adding the drug from stocks prepared in DMSO.

    Techniques: Expressing, Inhibition, Marker, Injection, Activity Assay

    MAP kinases phosphorylate sea urchin Yan/Tel. A, Western blot against a wild type HA-tagged version of yan/tel in the presence or absence of the p38 inhibitor BIRB-796, the ERK inhibitor U0126, the JNK inhibitor Sp600125 or a combination of all three drugs. Wild-type Yan/Tel typically migrates as multiple bands with three to four migrating isoforms. Slower migrating isoforms are absent after treatment with the BIRB-796, U0126 or SP600125 drugs while wild type Yan/Tel migrates as a unique faster migrating isoform after simultaneous inhibition of all three MAP Kinases. B, p38, JNK and ERK are required for strong expression of nodal . Treatment with the JNK inhibitor SP600125 or combined inhibition of p38, ERK and JNK reduce although do not abolish the expression of nodal . Lv, lateral view. HB, hatching blastula, EB, early blastula. C, Western blot against p38, JNK and ERK phosphorylated forms during development. Note that these MAP kinases are upregulated between the 60-cell stage and the EB stage. 2, 16 and 60 refers to the number of cells; VEB, Very Early Blastula; EB, Early Blastula; PHB, Prehatching Blastula; HB, Hatching blastula. D, Fluorescent immunostaining against phosphorylated forms of ERK and p38. At early blastula stage, nuclear P-ERK is detected in ectodermal cells (arrowheads) and in the precursors of the skeletogenic mesoderm located at the vegetal pole, while nuclear P-p38 shows a broader distribution. The dorsal clearance of nuclear P-p38 staining is pointed by arrowheads. EB, Early Blastula; PHB, Prehatching Blastula. E, Epistasis experiments with Yan/Tel, p38, JNK and ERK. Similar to the inhibition of yan/tel function, simultaneous inhibition of p38, ERK or JNK and Yan/Tel results in a massive ectopic expression of nodal . SB, Swimming blastula stage. F, Western blot against the HA-tagged form of wild type Yan/Tel. Overexpression of Nodal mRNA enriches the slowest migrating isoform of wild type Yan/Tel suggesting that Nodal can promote the phosphorylation of Yan/Tel. This effect can be reversed by the addition of the MAP kinase inhibitors of p38, ERK or JNK. vv, vegetal view. lv, lateral view. In lateral views, animal is to the top. G, Western blot against the HA-tagged form of wild type Yan/Tel. Treatment with the Nodal receptor inhibitor SB431542 enriches the fast migrating Yan/Tel isoform. Reciprocally, overexpression of the mRNA encoding the activated Nodal receptor Alk4QD or treatment with nickel chloride (a treatment that phenocopies nodal overexpression) promotes phosphorylation of Yan/Tel as indicated by the enrichment of the slowest migrating isoform of Yan/Tel.

    Journal: PLoS Genetics

    Article Title: MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo

    doi: 10.1371/journal.pgen.1007621

    Figure Lengend Snippet: MAP kinases phosphorylate sea urchin Yan/Tel. A, Western blot against a wild type HA-tagged version of yan/tel in the presence or absence of the p38 inhibitor BIRB-796, the ERK inhibitor U0126, the JNK inhibitor Sp600125 or a combination of all three drugs. Wild-type Yan/Tel typically migrates as multiple bands with three to four migrating isoforms. Slower migrating isoforms are absent after treatment with the BIRB-796, U0126 or SP600125 drugs while wild type Yan/Tel migrates as a unique faster migrating isoform after simultaneous inhibition of all three MAP Kinases. B, p38, JNK and ERK are required for strong expression of nodal . Treatment with the JNK inhibitor SP600125 or combined inhibition of p38, ERK and JNK reduce although do not abolish the expression of nodal . Lv, lateral view. HB, hatching blastula, EB, early blastula. C, Western blot against p38, JNK and ERK phosphorylated forms during development. Note that these MAP kinases are upregulated between the 60-cell stage and the EB stage. 2, 16 and 60 refers to the number of cells; VEB, Very Early Blastula; EB, Early Blastula; PHB, Prehatching Blastula; HB, Hatching blastula. D, Fluorescent immunostaining against phosphorylated forms of ERK and p38. At early blastula stage, nuclear P-ERK is detected in ectodermal cells (arrowheads) and in the precursors of the skeletogenic mesoderm located at the vegetal pole, while nuclear P-p38 shows a broader distribution. The dorsal clearance of nuclear P-p38 staining is pointed by arrowheads. EB, Early Blastula; PHB, Prehatching Blastula. E, Epistasis experiments with Yan/Tel, p38, JNK and ERK. Similar to the inhibition of yan/tel function, simultaneous inhibition of p38, ERK or JNK and Yan/Tel results in a massive ectopic expression of nodal . SB, Swimming blastula stage. F, Western blot against the HA-tagged form of wild type Yan/Tel. Overexpression of Nodal mRNA enriches the slowest migrating isoform of wild type Yan/Tel suggesting that Nodal can promote the phosphorylation of Yan/Tel. This effect can be reversed by the addition of the MAP kinase inhibitors of p38, ERK or JNK. vv, vegetal view. lv, lateral view. In lateral views, animal is to the top. G, Western blot against the HA-tagged form of wild type Yan/Tel. Treatment with the Nodal receptor inhibitor SB431542 enriches the fast migrating Yan/Tel isoform. Reciprocally, overexpression of the mRNA encoding the activated Nodal receptor Alk4QD or treatment with nickel chloride (a treatment that phenocopies nodal overexpression) promotes phosphorylation of Yan/Tel as indicated by the enrichment of the slowest migrating isoform of Yan/Tel.

    Article Snippet: Treatments with U0126 (Calbiochem, 10 μM) BIRB796 (Selleckchem, S1574, 3 μM), SP600125 (Tocris, 0,5 μM), SB431542 (Tocris, 1614, 10 μM) and MG132 (Tocris 10 μM) were performed by adding the drug from stocks prepared in DMSO.

    Techniques: Western Blot, Inhibition, Expressing, Immunostaining, Staining, Over Expression

    Glyoxalase 1 (Glo1)‐dependent MG‐H1 and argpyrimidine (AP) depletion triggers epithelial‐to‐mesenchymal transition (EMT) via TGF‐β1/SMAD4 signalling pathway in PC3 cells. (A) Glo1 silencing (siGlo1) in PC3 cells significantly affected TGF‐β1 mRNA levels, evaluated by qRT‐PCR, (B) TGF‐β1 protein intracellular levels, evaluated by Western blot, and (C) TGF‐β1 secreted levels, evaluated by a specific ELISA kit. Pre‐treatment with aminoguanidine (AG) proved Glo1‐dependent Hsp40‐modified AP‐mediated role in controlling TGF‐β1 expression (A, B, C). (D) Glo1 silencing (siGlo1) and AG pre‐treatment significantly affected Smad4 activation, evaluated both in nuclear and in cytoplasmic fractions of PC3 cells by Western blot, proving Glo1‐dependent Hsp40‐modified AP‐mediated role in controlling TGF‐β1/Smad4 signalling pathway. Inhibition of TGF‐β1 signalling by SB431542 treatment demonstrated TGF‐β1 role in controlling (E) Smad4 activation, evaluated at nuclear and cytoplasmic levels by Western blot, (F) EMT, evaluated by Western blot analysis of the epithelial markers E‐cadherin (E‐cad) and zonula occludens‐1 (ZO‐1) or mesenchymal markers vimentin (Vim), N‐cadherin (N‐cad) and Snail, (G) MMP‐2 and MMP‐9 expression, evaluated by Western blotting or activity, evaluated by the gelatin zymography, and (H) migration and invasion capabilities, evaluated by specific assays. The Western blots were obtained by the appropriate Abs. The blots were stripped off the bound Abs and reprobed with anti‐β‐actin or lamin β1 to confirm equal loading. The Western blots shown are representative of three independent experiments. The histograms indicate mean ± SD of three different cultures, and each was tested in triplicate. siCtr: control (non‐specific siRNA). (−) untreated and (+) treated cells. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control. Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control

    doi: 10.1111/jcmm.13581

    Figure Lengend Snippet: Glyoxalase 1 (Glo1)‐dependent MG‐H1 and argpyrimidine (AP) depletion triggers epithelial‐to‐mesenchymal transition (EMT) via TGF‐β1/SMAD4 signalling pathway in PC3 cells. (A) Glo1 silencing (siGlo1) in PC3 cells significantly affected TGF‐β1 mRNA levels, evaluated by qRT‐PCR, (B) TGF‐β1 protein intracellular levels, evaluated by Western blot, and (C) TGF‐β1 secreted levels, evaluated by a specific ELISA kit. Pre‐treatment with aminoguanidine (AG) proved Glo1‐dependent Hsp40‐modified AP‐mediated role in controlling TGF‐β1 expression (A, B, C). (D) Glo1 silencing (siGlo1) and AG pre‐treatment significantly affected Smad4 activation, evaluated both in nuclear and in cytoplasmic fractions of PC3 cells by Western blot, proving Glo1‐dependent Hsp40‐modified AP‐mediated role in controlling TGF‐β1/Smad4 signalling pathway. Inhibition of TGF‐β1 signalling by SB431542 treatment demonstrated TGF‐β1 role in controlling (E) Smad4 activation, evaluated at nuclear and cytoplasmic levels by Western blot, (F) EMT, evaluated by Western blot analysis of the epithelial markers E‐cadherin (E‐cad) and zonula occludens‐1 (ZO‐1) or mesenchymal markers vimentin (Vim), N‐cadherin (N‐cad) and Snail, (G) MMP‐2 and MMP‐9 expression, evaluated by Western blotting or activity, evaluated by the gelatin zymography, and (H) migration and invasion capabilities, evaluated by specific assays. The Western blots were obtained by the appropriate Abs. The blots were stripped off the bound Abs and reprobed with anti‐β‐actin or lamin β1 to confirm equal loading. The Western blots shown are representative of three independent experiments. The histograms indicate mean ± SD of three different cultures, and each was tested in triplicate. siCtr: control (non‐specific siRNA). (−) untreated and (+) treated cells. * P

    Article Snippet: The TGF‐β type I receptor inhibitor , SB431542 (0.5 μmol/L for 1 hour) was from Tocris (Milan, Italy).

    Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Modification, Expressing, Activation Assay, Inhibition, Activity Assay, Zymography, Migration

    (A). Primary calvarial cells were cultured with supplemented DMEM or additional 5mM calcium for 3, 7 and 10 days. The mRNA expression was evaluated by qPCR. The effect of calcium (black circles) and media alone (empty circles) on primary calvarial cells is displayed. (B) Expression of osteogenic genes treated for 48 h with 5 mM calcium, SB431542 and AG-1296 (10 μM), a selective TGFβ1 and PDGF receptor inhibitor respectively. Data is shown as mean ± SEM. Differences considered significant at *p

    Journal: PLoS ONE

    Article Title: Calcium mimics the chemotactic effect of conditioned media and is an effective inducer of bone regeneration

    doi: 10.1371/journal.pone.0210301

    Figure Lengend Snippet: (A). Primary calvarial cells were cultured with supplemented DMEM or additional 5mM calcium for 3, 7 and 10 days. The mRNA expression was evaluated by qPCR. The effect of calcium (black circles) and media alone (empty circles) on primary calvarial cells is displayed. (B) Expression of osteogenic genes treated for 48 h with 5 mM calcium, SB431542 and AG-1296 (10 μM), a selective TGFβ1 and PDGF receptor inhibitor respectively. Data is shown as mean ± SEM. Differences considered significant at *p

    Article Snippet: SB431542, a selective TGFB type I receptor inhibitor (Tocris), was solubilized using DMSO at a 10 mM stock concentration, and a final concentration of 10 μM.

    Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    Functional testing of Heligmosomoides polygyrus TGM transforming growth factor (TGF) -β mimic (TGM) family members. Proteins were tested functionally in the MFB-F11 bioassay (fibroblast cell line isolated from mouse embryos lacking TGF-β). (A–C) or with murine T cell induction of Foxp3 expression (D). TGM-5 is omitted from the assays as it was not possible to express recombinant protein in the same system as the other family member proteins. (A) Activity detected in the MFB-F11 bioassay after 24 h of culture with 100 ng/ml of TGM family members TGM-1, -2, -4 and -6, and 10 ug/ml of TGM-3 in the absence or presence of the type I transforming growth factor (TGF)-β receptor kinase inhibitor, SB431542. (B) Activity in the same bioassay of the family members found positive in A, in the absence or presence of the type II TGF-β receptor inhibitor, inducer of type II TGF-β receptor (TGFBR2) degradation-1 (ITD-1) (10 µM). (C) Activity in the bioassay of family members in the absence or presence of anti-TGM-1 antibody (10 µg/ml) or rat IgG control (10 µg/ml). (D) Percentage of Foxp3 induction induced by TGM-1 and TGM-2 compared with IL2 only (no induction control) and TGF-β (positive control).

    Journal: International Journal for Parasitology

    Article Title: TGF-β mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus

    doi: 10.1016/j.ijpara.2017.12.004

    Figure Lengend Snippet: Functional testing of Heligmosomoides polygyrus TGM transforming growth factor (TGF) -β mimic (TGM) family members. Proteins were tested functionally in the MFB-F11 bioassay (fibroblast cell line isolated from mouse embryos lacking TGF-β). (A–C) or with murine T cell induction of Foxp3 expression (D). TGM-5 is omitted from the assays as it was not possible to express recombinant protein in the same system as the other family member proteins. (A) Activity detected in the MFB-F11 bioassay after 24 h of culture with 100 ng/ml of TGM family members TGM-1, -2, -4 and -6, and 10 ug/ml of TGM-3 in the absence or presence of the type I transforming growth factor (TGF)-β receptor kinase inhibitor, SB431542. (B) Activity in the same bioassay of the family members found positive in A, in the absence or presence of the type II TGF-β receptor inhibitor, inducer of type II TGF-β receptor (TGFBR2) degradation-1 (ITD-1) (10 µM). (C) Activity in the bioassay of family members in the absence or presence of anti-TGM-1 antibody (10 µg/ml) or rat IgG control (10 µg/ml). (D) Percentage of Foxp3 induction induced by TGM-1 and TGM-2 compared with IL2 only (no induction control) and TGF-β (positive control).

    Article Snippet: For TGF-β receptor 1 and 2 inhibitor assays, either 5 µM SB431542 (Tocris Bioscience, UK) or 10 µM ITD-1 (Tocris Bioscience) were added to each well with DMSO added to control wells.

    Techniques: Functional Assay, Isolation, Expressing, Recombinant, Activity Assay, Positive Control

    Functional testing of recombinant truncated Heligmosomoides polygyrus transforming growth factor (TGF) -β mimic (TGM)Δ4,5. Recombinant truncated TGMΔ4,5, comprising domains 1–3 alone, was tested functionally in the MFB-F11 bioassay (fibroblast cell line isolated from mouse embryos lacking TGF-β). (A–C) and for murine T cell induction of Foxp3 expression (D). (A) Activity detected in the MFB-F11 bioassay after 24 h of culture with log fold dilutions of TGM Δ4,5 starting at 1000 ng/ml, in the absence or presence of the type I transforming growth factor (TGF)-β receptor inhibitor, SB431542. (B) As (A) but in the absence or presence of the type II TGF-β receptor inhibitor, ITD-1 (10 µM).(C). As (A) but in the presence of either polyclonal rat anti-TGM antibody (10 µg/ml) or rat IgG control (10 µg/ml). (D) Percentage of Foxp3 induction induced in naive Foxp3-GFP negative splenic CD4 + T cells in response to TGM Δ4,5 compared with TGF-β and TGM (positive induction controls).

    Journal: International Journal for Parasitology

    Article Title: TGF-β mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus

    doi: 10.1016/j.ijpara.2017.12.004

    Figure Lengend Snippet: Functional testing of recombinant truncated Heligmosomoides polygyrus transforming growth factor (TGF) -β mimic (TGM)Δ4,5. Recombinant truncated TGMΔ4,5, comprising domains 1–3 alone, was tested functionally in the MFB-F11 bioassay (fibroblast cell line isolated from mouse embryos lacking TGF-β). (A–C) and for murine T cell induction of Foxp3 expression (D). (A) Activity detected in the MFB-F11 bioassay after 24 h of culture with log fold dilutions of TGM Δ4,5 starting at 1000 ng/ml, in the absence or presence of the type I transforming growth factor (TGF)-β receptor inhibitor, SB431542. (B) As (A) but in the absence or presence of the type II TGF-β receptor inhibitor, ITD-1 (10 µM).(C). As (A) but in the presence of either polyclonal rat anti-TGM antibody (10 µg/ml) or rat IgG control (10 µg/ml). (D) Percentage of Foxp3 induction induced in naive Foxp3-GFP negative splenic CD4 + T cells in response to TGM Δ4,5 compared with TGF-β and TGM (positive induction controls).

    Article Snippet: For TGF-β receptor 1 and 2 inhibitor assays, either 5 µM SB431542 (Tocris Bioscience, UK) or 10 µM ITD-1 (Tocris Bioscience) were added to each well with DMSO added to control wells.

    Techniques: Functional Assay, Recombinant, Isolation, Expressing, Activity Assay