dorsomorphin  (Millipore)

 
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    Name:
    Dorsomorphin
    Description:

    Catalog Number:
    P5499
    Price:
    None
    Applications:
    Dorsomorphin has been used:. as an inhibitor of adenosine monophosphate-activated protein kinase (AMPK) to find the effects of trans-resveratrol on lipid mobilization in 3T3-L1 (a murine cell line of adipocytes) cells. as an AMPK inhibitor, to indicate the involvement of AMPK/mTOR (mammalian target of rapamycin) pathway in LRG (liraglutide) -induced autophagy. in the induction step, employed in in vitro differentiation of Friedreich′s ataxia (FRDA) and induced pluripotent stem cells (iPSCs) to neurospheres and neurons using ES (embryonic stem cell) media
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    Structured Review

    Millipore dorsomorphin
    Dorsomorphin

    https://www.bioz.com/result/dorsomorphin/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dorsomorphin - by Bioz Stars, 2021-09
    97/100 stars

    Images

    1) Product Images from "Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice"

    Article Title: Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI39939

    Effects of dorsomorphin on Salmonella -induced changes in hepcidin expression and serum iron levels.
    Figure Legend Snippet: Effects of dorsomorphin on Salmonella -induced changes in hepcidin expression and serum iron levels.

    Techniques Used: Expressing

    Effects of dorsomorphin treatment on Salmonella -induced intestinal inflammation in WT mice.
    Figure Legend Snippet: Effects of dorsomorphin treatment on Salmonella -induced intestinal inflammation in WT mice.

    Techniques Used: Mouse Assay

    2) Product Images from "Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva *"

    Article Title: Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.365932

    Structure of the ALK2-FKBP12 complex. A , secondary structure elements are labeled and shown as ribbons . Disordered parts of the L45 loop and A loop are indicated by a thin dashed line . Dorsomorphin is shown in gray stick representation, and Mg 2+ ion ( cyan ) and sulfate molecules ( purple ) are displayed as spheres. Inset box shows the specific interactions of dorsomorphin ( yellow sticks ) with the ATP pocket in ALK2. Hydrogen bonds ( blue dashed lines ) are formed with the hinge amide of His-286 and via a water molecule to Glu-248 (α C ). The planarity of the phenyl ring is restricted by the close packing of Val-214 and Gly-289 ( dashed gray lines). B , FKBP12 binding is dominated by insertion of the αGS2 helix into the central FK506-binding pocket of FKBP12. Here, the ALK5 residues Leu-195–Leu-196 are replaced by ALK2 Phe-198–Leu-199 resulting in a subtle shift in the complex interface. The hydrophobic contact surface in FKBP12 is colored yellow. C , | F o | − | F c | omit map contoured at 3σ ( green mesh ) for the bound dorsomorphin ligand ( yellow sticks ).
    Figure Legend Snippet: Structure of the ALK2-FKBP12 complex. A , secondary structure elements are labeled and shown as ribbons . Disordered parts of the L45 loop and A loop are indicated by a thin dashed line . Dorsomorphin is shown in gray stick representation, and Mg 2+ ion ( cyan ) and sulfate molecules ( purple ) are displayed as spheres. Inset box shows the specific interactions of dorsomorphin ( yellow sticks ) with the ATP pocket in ALK2. Hydrogen bonds ( blue dashed lines ) are formed with the hinge amide of His-286 and via a water molecule to Glu-248 (α C ). The planarity of the phenyl ring is restricted by the close packing of Val-214 and Gly-289 ( dashed gray lines). B , FKBP12 binding is dominated by insertion of the αGS2 helix into the central FK506-binding pocket of FKBP12. Here, the ALK5 residues Leu-195–Leu-196 are replaced by ALK2 Phe-198–Leu-199 resulting in a subtle shift in the complex interface. The hydrophobic contact surface in FKBP12 is colored yellow. C , | F o | − | F c | omit map contoured at 3σ ( green mesh ) for the bound dorsomorphin ligand ( yellow sticks ).

    Techniques Used: Labeling, Binding Assay

    3) Product Images from "An Atypical Canonical Bone Morphogenetic Protein (BMP) Signaling Pathway Regulates Msh Homeobox 1 (Msx1) Expression during Odontogenesis *"

    Article Title: An Atypical Canonical Bone Morphogenetic Protein (BMP) Signaling Pathway Regulates Msh Homeobox 1 (Msx1) Expression during Odontogenesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.600064

    BMP/Smad1/5/8 signaling pathway regulates Msx1 expression. A , immunofluorescence shows expressions of pp38 ( panel a ), pERK ( panel b ), and pJNK ( panel c ) in E13.5 molar tooth germ. B , in situ hybridization shows Msx1 expression in E12.5 molar germs after 12 h or 1 day in organ culture in the presence of DMSO ( panels a and d ), dorsomorphin ( panels b and e ), or SB203580 and U0126 ( panels c and f ). Panels a–c , whole mount in situ hybridization; panels d–f , tissue section in situ hybridization. C , a Western blot assay shows attenuation of Msx1 expression by dorsomorphin but not SB203580 and U0126 in BMP-induced primary dental mesenchymal cells from E13.5 embryos. GAPDH was used as internal control. Quantitative analysis is shown in the lower part. **, p
    Figure Legend Snippet: BMP/Smad1/5/8 signaling pathway regulates Msx1 expression. A , immunofluorescence shows expressions of pp38 ( panel a ), pERK ( panel b ), and pJNK ( panel c ) in E13.5 molar tooth germ. B , in situ hybridization shows Msx1 expression in E12.5 molar germs after 12 h or 1 day in organ culture in the presence of DMSO ( panels a and d ), dorsomorphin ( panels b and e ), or SB203580 and U0126 ( panels c and f ). Panels a–c , whole mount in situ hybridization; panels d–f , tissue section in situ hybridization. C , a Western blot assay shows attenuation of Msx1 expression by dorsomorphin but not SB203580 and U0126 in BMP-induced primary dental mesenchymal cells from E13.5 embryos. GAPDH was used as internal control. Quantitative analysis is shown in the lower part. **, p

    Techniques Used: Expressing, Immunofluorescence, In Situ Hybridization, Organ Culture, Western Blot

    4) Product Images from "Serum-induced up-regulation of hepcidin expression involves the bone morphogenetic protein signaling pathway"

    Article Title: Serum-induced up-regulation of hepcidin expression involves the bone morphogenetic protein signaling pathway

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2013.10.065

    Effect of dorsomorphin on serum-induced activation of the SMAD signaling pathway in HepG2 cells. HepG2 cells were stimulated with 10% fetal calf serum for 6 hours in the presence or absence of 5 μM dorsomorphin (DM) or an equivalent volume of
    Figure Legend Snippet: Effect of dorsomorphin on serum-induced activation of the SMAD signaling pathway in HepG2 cells. HepG2 cells were stimulated with 10% fetal calf serum for 6 hours in the presence or absence of 5 μM dorsomorphin (DM) or an equivalent volume of

    Techniques Used: Activation Assay

    Effect of dorsomorphin on serum-induced hepcidin expression in HepG2 cells. HepG2 cells were stimulated with 10% fetal calf serum for 6 hours in the presence or absence of 5 μM dorsomorphin (DM) or an equivalent volume of the vehicle DMSO. As
    Figure Legend Snippet: Effect of dorsomorphin on serum-induced hepcidin expression in HepG2 cells. HepG2 cells were stimulated with 10% fetal calf serum for 6 hours in the presence or absence of 5 μM dorsomorphin (DM) or an equivalent volume of the vehicle DMSO. As

    Techniques Used: Expressing

    5) Product Images from "Admp regulates tail bending by controlling the intercalation of the ventral epidermis through myosin phosphorylation"

    Article Title: Admp regulates tail bending by controlling the intercalation of the ventral epidermis through myosin phosphorylation

    Journal: bioRxiv

    doi: 10.1101/2021.09.21.461063

    Model of embryonic tail bending in Ciona . The previous study reported that the asymmetric actomyosin contraction in the notochord is the main factor for the tail bending until st. 20 (upper blue rectangular). On the other hand, Admp/BMP signaling (the flow by red rectangles) transmits the signal to the ventral midline epidermal cells as phosphorylated Smad from neurula to initial tailbud. pSmad translocates the localization of the pMLC from the basal side (dorsal side) to the apical side (ventral side), which changes the cell polarity and promotes the cell-cell intercalation of the ventral midline epidermal cells during early to mid-tailbud stages (st. 20 to st. 23). The ongoing mediolateral intercalation at the ventral epidermis confers a resistance (orange arrows) to AP elongation force (blue arrow) that is possibly provided by the notochord, which causes the bending tail shape in the Ciona tailbud embryo at st. 22. On the other hand, the Admp MO or dorsomorphin-treatment disrupts the cell polarity and causes the no tail-bending embryo at st. 20-23 and incomplete intercalation at st. 24 (the flow by green rectangles). Inhibition of pMLC by Y27632 also causes no ventral bending. Final overall embryo shape at st. 24 is similar among WT and Admp MO embryos, but the ventral cell-cell intercalation was disrupted. Thus, Admp/BMP signaling, apart from its role of peripheral nervous system (PNS) differentiation (lower blue rectangular), regulate temporal tail bending during early to middle tailbud stages (st. 20 to st. 23).
    Figure Legend Snippet: Model of embryonic tail bending in Ciona . The previous study reported that the asymmetric actomyosin contraction in the notochord is the main factor for the tail bending until st. 20 (upper blue rectangular). On the other hand, Admp/BMP signaling (the flow by red rectangles) transmits the signal to the ventral midline epidermal cells as phosphorylated Smad from neurula to initial tailbud. pSmad translocates the localization of the pMLC from the basal side (dorsal side) to the apical side (ventral side), which changes the cell polarity and promotes the cell-cell intercalation of the ventral midline epidermal cells during early to mid-tailbud stages (st. 20 to st. 23). The ongoing mediolateral intercalation at the ventral epidermis confers a resistance (orange arrows) to AP elongation force (blue arrow) that is possibly provided by the notochord, which causes the bending tail shape in the Ciona tailbud embryo at st. 22. On the other hand, the Admp MO or dorsomorphin-treatment disrupts the cell polarity and causes the no tail-bending embryo at st. 20-23 and incomplete intercalation at st. 24 (the flow by green rectangles). Inhibition of pMLC by Y27632 also causes no ventral bending. Final overall embryo shape at st. 24 is similar among WT and Admp MO embryos, but the ventral cell-cell intercalation was disrupted. Thus, Admp/BMP signaling, apart from its role of peripheral nervous system (PNS) differentiation (lower blue rectangular), regulate temporal tail bending during early to middle tailbud stages (st. 20 to st. 23).

    Techniques Used: Inhibition

    The alignment of the tail epidermal cells of DMSO-treated and Dorsomorphin-treated embryo at st. 24. The cell-cell intercalation was completed in the DMSO-treated embryo (left), and the tail epidermal cells consist of 8 rows: dorsal (yellow), two dorsal medio-lateral (green), ventral (red), two ventral medio-lateral (orange) and two laterals (blue) rows. On the other hand, there is a specific inhibition of the intercalation of the ventral row in Dorsomorphin-treated embryos (right). (N = 2 in DMSO and 2 in Dorsomorphin)
    Figure Legend Snippet: The alignment of the tail epidermal cells of DMSO-treated and Dorsomorphin-treated embryo at st. 24. The cell-cell intercalation was completed in the DMSO-treated embryo (left), and the tail epidermal cells consist of 8 rows: dorsal (yellow), two dorsal medio-lateral (green), ventral (red), two ventral medio-lateral (orange) and two laterals (blue) rows. On the other hand, there is a specific inhibition of the intercalation of the ventral row in Dorsomorphin-treated embryos (right). (N = 2 in DMSO and 2 in Dorsomorphin)

    Techniques Used: Inhibition

    Morphants of tail bending. (A) mid-tailbud stage embryo of WT, DMSO treatment, and Dorsomorphin. DMSO and Dorsomorphin were treated after the mid neurula stage (st. 15). The dorsomorphin-treated embryo didn’t bend tail, similar to the Admp MO embryo ( Fig.1A ). (B) MOs against Msxb were injected. While Admp MO was affected the tail bending ( Fig.1A ), Msxb MO was not affected the tail bending. Scale bar, 100 μm. The number of examined embryos and the proportion of representative embryos are shown in each panel, respectively. All embryos are shown anterior to the left.
    Figure Legend Snippet: Morphants of tail bending. (A) mid-tailbud stage embryo of WT, DMSO treatment, and Dorsomorphin. DMSO and Dorsomorphin were treated after the mid neurula stage (st. 15). The dorsomorphin-treated embryo didn’t bend tail, similar to the Admp MO embryo ( Fig.1A ). (B) MOs against Msxb were injected. While Admp MO was affected the tail bending ( Fig.1A ), Msxb MO was not affected the tail bending. Scale bar, 100 μm. The number of examined embryos and the proportion of representative embryos are shown in each panel, respectively. All embryos are shown anterior to the left.

    Techniques Used: Injection

    6) Product Images from "Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP)"

    Article Title: Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP)

    Journal: Experimental & Molecular Medicine

    doi: 10.3858/emm.2010.42.3.017

    Influence of SMAD and ERK activity on HMSC differentiation. HMSCs were treated with BMP2 (200 ng/ml) to induce osteogenic differentiation. Cells were co-treated with either dorsomorphin (2 µM) or chordin (2 µg/ml), respectively. qRT-PCR revealed reduced expression of osteopontin (A) and osteocalcin (B) mRNA after treatment. Expression of aggrecan (C) and SOX9 (D) stayed unchanged after dorsomorphin treatment whereas chordin led to induced expression of these two genes. The experiments were repeated 3 times using cells from different donors. ( * : P
    Figure Legend Snippet: Influence of SMAD and ERK activity on HMSC differentiation. HMSCs were treated with BMP2 (200 ng/ml) to induce osteogenic differentiation. Cells were co-treated with either dorsomorphin (2 µM) or chordin (2 µg/ml), respectively. qRT-PCR revealed reduced expression of osteopontin (A) and osteocalcin (B) mRNA after treatment. Expression of aggrecan (C) and SOX9 (D) stayed unchanged after dorsomorphin treatment whereas chordin led to induced expression of these two genes. The experiments were repeated 3 times using cells from different donors. ( * : P

    Techniques Used: Activity Assay, Quantitative RT-PCR, Expressing

    7) Product Images from "Functional modeling in zebrafish demonstrates that the atrial-fibrillation-associated gene GREM2 regulates cardiac laterality, cardiomyocyte differentiation and atrial rhythm"

    Article Title: Functional modeling in zebrafish demonstrates that the atrial-fibrillation-associated gene GREM2 regulates cardiac laterality, cardiomyocyte differentiation and atrial rhythm

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.010488

    Loss of Grem2 leads to increased BMP signaling and cardiac defects that can be reversed by the BMP inhibitor dorsomorphin. (A) Whole mount immunohistochemistry using antibodies recognizing the phosphorylated forms of Smads1/5/8 shows stronger pSmad protein staining in grem2 morphants than in controls. Arrowhead marks somite boundaries. (B) Frontal close up views of the heart after pSmad1/5/8 antibody staining at 48 hpf shows sharply increased nuclear staining in cardiomyocytes of Grem2-depleted embryos. The heart is outlined by dotted lines; arrowheads mark pSmad-positive nuclei. (C) Wild-type (WT) embryos and grem2 morphants were incubated with dorsomorphin or its vehicle DMSO between 16 and 48 hpf and stained at 48 hpf with cmlc2 and amhc probes to visualize the ventricle (V) and atrium (A). Dorsomorphin treatment restored atrial patterning and differentiation (arrowheads point to the atrioventricular boundary in wild-type hearts; dotted lines demarcate ventricle-atrium boundary in grem2 morphants). (D) Quantification of heart size (Heart Area) and atrial bulb restoration (Presence of Atrial Bulb) in DMSO-treated controls (WT), DMSO-treated grem2 morphants (MO) and grem2 morphants treated with dorsomorphin (MO+DM). Dorsomorphin treatment restores atrial development. Error bars represent s.d. P -values and number of embryos analyzed ( n ) are indicated. b, brain; e, eye; m, mouth; y, yolk.
    Figure Legend Snippet: Loss of Grem2 leads to increased BMP signaling and cardiac defects that can be reversed by the BMP inhibitor dorsomorphin. (A) Whole mount immunohistochemistry using antibodies recognizing the phosphorylated forms of Smads1/5/8 shows stronger pSmad protein staining in grem2 morphants than in controls. Arrowhead marks somite boundaries. (B) Frontal close up views of the heart after pSmad1/5/8 antibody staining at 48 hpf shows sharply increased nuclear staining in cardiomyocytes of Grem2-depleted embryos. The heart is outlined by dotted lines; arrowheads mark pSmad-positive nuclei. (C) Wild-type (WT) embryos and grem2 morphants were incubated with dorsomorphin or its vehicle DMSO between 16 and 48 hpf and stained at 48 hpf with cmlc2 and amhc probes to visualize the ventricle (V) and atrium (A). Dorsomorphin treatment restored atrial patterning and differentiation (arrowheads point to the atrioventricular boundary in wild-type hearts; dotted lines demarcate ventricle-atrium boundary in grem2 morphants). (D) Quantification of heart size (Heart Area) and atrial bulb restoration (Presence of Atrial Bulb) in DMSO-treated controls (WT), DMSO-treated grem2 morphants (MO) and grem2 morphants treated with dorsomorphin (MO+DM). Dorsomorphin treatment restores atrial development. Error bars represent s.d. P -values and number of embryos analyzed ( n ) are indicated. b, brain; e, eye; m, mouth; y, yolk.

    Techniques Used: Immunohistochemistry, Staining, Incubation

    8) Product Images from "Human iPSC-derived astrocytes from ALS patients with mutated C9ORF72 show increased oxidative stress and neurotoxicity"

    Article Title: Human iPSC-derived astrocytes from ALS patients with mutated C9ORF72 show increased oxidative stress and neurotoxicity

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.11.026

    Generation of functional astrocytes from C9-mutated and control iPSC lines. (a) Schematic presentation of the protocol for induction of astrocyte differentiation. iPSC colonies cultured on feeders were detached for further differentiation as floating spheres in non-adherent plates. Initial neuralization was induced by dual SMAD inhibitors (2SMADi) SB431542 and Dorsomorphin. Further caudalization of the neural progenitor spheres was promoted by retinoic acid (RA) and ventralization by Purmorphamine (Pur). Enrichment for glial progenitors was induced by culture in the presence of epidermal growth factor (EGF) and leukemia inhibitory factor (LIF). The neural spheres were then dissociated, and the cells were plated and further cultured as a monolayer in the presence of EGF and basic fibroblast growth factor (bFGF). Final differentiation to astrocytes was induced in the presence of ciliary neurotrophic factor (CNTF). Please see details in the Methods. (b) Representative immunofluorescence images of C9-mutated (C9-L5, C9-L8) and control (Contr-L3, Contr-L9) astrocytes decorated with anti-Vimentin (red), glial fibrillary acid protein, (GFAP; green), S100β (green) at day 30 of final differentiation (dFD). Nuclei (blue) are counterstained with DAPI. Scale bars: 100 µm. These experiments were repeated 5 independent times with similar results. c. Histogram presentation of glutamate uptake assay showing that control (Contr-L3, Contr-L9, green bars) and mutated (C9-L5, C9-L8, orange bars) astrocytes uptake l -glutamate from the media at the same rate after 30 and 60 min. The results are normalized to the initial concentration of l -glutamate (50 µM). Data are represented as mean ± SEM of 3 independent experiments with astrocytes at 30–40 dFD. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: Generation of functional astrocytes from C9-mutated and control iPSC lines. (a) Schematic presentation of the protocol for induction of astrocyte differentiation. iPSC colonies cultured on feeders were detached for further differentiation as floating spheres in non-adherent plates. Initial neuralization was induced by dual SMAD inhibitors (2SMADi) SB431542 and Dorsomorphin. Further caudalization of the neural progenitor spheres was promoted by retinoic acid (RA) and ventralization by Purmorphamine (Pur). Enrichment for glial progenitors was induced by culture in the presence of epidermal growth factor (EGF) and leukemia inhibitory factor (LIF). The neural spheres were then dissociated, and the cells were plated and further cultured as a monolayer in the presence of EGF and basic fibroblast growth factor (bFGF). Final differentiation to astrocytes was induced in the presence of ciliary neurotrophic factor (CNTF). Please see details in the Methods. (b) Representative immunofluorescence images of C9-mutated (C9-L5, C9-L8) and control (Contr-L3, Contr-L9) astrocytes decorated with anti-Vimentin (red), glial fibrillary acid protein, (GFAP; green), S100β (green) at day 30 of final differentiation (dFD). Nuclei (blue) are counterstained with DAPI. Scale bars: 100 µm. These experiments were repeated 5 independent times with similar results. c. Histogram presentation of glutamate uptake assay showing that control (Contr-L3, Contr-L9, green bars) and mutated (C9-L5, C9-L8, orange bars) astrocytes uptake l -glutamate from the media at the same rate after 30 and 60 min. The results are normalized to the initial concentration of l -glutamate (50 µM). Data are represented as mean ± SEM of 3 independent experiments with astrocytes at 30–40 dFD. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Functional Assay, Cell Culture, Immunofluorescence, Concentration Assay

    9) Product Images from "The BMP ligand Pinhead together with Admp supports the robustness of embryonic patterning"

    Article Title: The BMP ligand Pinhead together with Admp supports the robustness of embryonic patterning

    Journal: Science Advances

    doi: 10.1126/sciadv.aau6455

    Expression of pinhead and admp is repressed by BMP signaling. ( A and B ) The expression of admp and pinhead was analyzed at the shield stage by in situ hybridization (A) and real-time qPCR in bmp2b morphants and embryos treated with 10 μM dorsomorphin or 5 μM DMH1 from the 1K cell stage. Error bars indicated SD. * P
    Figure Legend Snippet: Expression of pinhead and admp is repressed by BMP signaling. ( A and B ) The expression of admp and pinhead was analyzed at the shield stage by in situ hybridization (A) and real-time qPCR in bmp2b morphants and embryos treated with 10 μM dorsomorphin or 5 μM DMH1 from the 1K cell stage. Error bars indicated SD. * P

    Techniques Used: Expressing, In Situ Hybridization, Real-time Polymerase Chain Reaction

    10) Product Images from "Role of heparanase 2 (Hpa2) in gastric cancer"

    Article Title: Role of heparanase 2 (Hpa2) in gastric cancer

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2021.07.010

    Hpa2 induction by MG132 involves AMPK and HSF1. (A) Immunoblotting. MKN-45 (left) and SFC-7901 (right) gastric carcinoma cells were left untreated (0) or were treated with MG132 (20 µM) for the time indicated. Cell extracts were then prepared and subjected to immunoblotting applying antibodies directed to phospho-AMPK (pAMPK; upper panels), AMPK (second panels), phospho-HSF1 (pHSF1; third panels), HSF1 (fourth panels), phospho-JNK (fifth panels), JNK (sixth panels), phospho-p70S6K (pS6K; seventh panels), S6K (8 panels), and tubulin (lower panels). (B) Inhibitors. MKN-45 cells were similarly treated with vehicle (DMSO; Con) or MG132 without (MG) or with the indicated concentrations of 17-AAG (AAG) (HSP90 inhibitor), KRIBB 11 (KRBB; HSF1 inhibitor), rapamycin (Rap; mTOR inhibitor), and dorsomorphin (Dor; AMPK inhibitor). Total RNA was extracted after 24 hours and subjected to qPCR applying primers specific for Hpa2. Note that Hpa2 induction by MG132 is attenuated by inhibitors of AMPK and HSF1 (red arrows).* P = 0.0005 for MG vs Con; ** P = 0.0007 and P = 0.0004 for MG+KRBB vs MG and MG+Dor vs MG, respectively. (C) Quantitation of Hpa2 induction by MG132 without or with KRIBB 11 (upper panel) and dorsomorphin (middle panel). Hpa2 expression is similarly decreased following silencing of HSF and AMPK-beta (C, lower panel). (D) Hpa2 cells are more sensitive to stress conditions. Control (Vo) and Hpa2 overexpressing MKN-45 cells were treated with MG132 (20 µM) for the time indicated. Cell extracts were then prepared and subjected to immunoblotting applying antibodies directed against cleaved caspase 3 (upper panel), cleaved PARP (second panel) and tubulin (lower panel). Note increased levels of the apoptotic markers in Hpa2 cells. Numbers underneath each blot specify band intensity in Hpa2 cells in relation to the same time point in control (Vo) cells. (color version of figure is available online). AMPK, activated protein kinase; Hpa2, heparanase 2; HSF1, heat shock factor-1.
    Figure Legend Snippet: Hpa2 induction by MG132 involves AMPK and HSF1. (A) Immunoblotting. MKN-45 (left) and SFC-7901 (right) gastric carcinoma cells were left untreated (0) or were treated with MG132 (20 µM) for the time indicated. Cell extracts were then prepared and subjected to immunoblotting applying antibodies directed to phospho-AMPK (pAMPK; upper panels), AMPK (second panels), phospho-HSF1 (pHSF1; third panels), HSF1 (fourth panels), phospho-JNK (fifth panels), JNK (sixth panels), phospho-p70S6K (pS6K; seventh panels), S6K (8 panels), and tubulin (lower panels). (B) Inhibitors. MKN-45 cells were similarly treated with vehicle (DMSO; Con) or MG132 without (MG) or with the indicated concentrations of 17-AAG (AAG) (HSP90 inhibitor), KRIBB 11 (KRBB; HSF1 inhibitor), rapamycin (Rap; mTOR inhibitor), and dorsomorphin (Dor; AMPK inhibitor). Total RNA was extracted after 24 hours and subjected to qPCR applying primers specific for Hpa2. Note that Hpa2 induction by MG132 is attenuated by inhibitors of AMPK and HSF1 (red arrows).* P = 0.0005 for MG vs Con; ** P = 0.0007 and P = 0.0004 for MG+KRBB vs MG and MG+Dor vs MG, respectively. (C) Quantitation of Hpa2 induction by MG132 without or with KRIBB 11 (upper panel) and dorsomorphin (middle panel). Hpa2 expression is similarly decreased following silencing of HSF and AMPK-beta (C, lower panel). (D) Hpa2 cells are more sensitive to stress conditions. Control (Vo) and Hpa2 overexpressing MKN-45 cells were treated with MG132 (20 µM) for the time indicated. Cell extracts were then prepared and subjected to immunoblotting applying antibodies directed against cleaved caspase 3 (upper panel), cleaved PARP (second panel) and tubulin (lower panel). Note increased levels of the apoptotic markers in Hpa2 cells. Numbers underneath each blot specify band intensity in Hpa2 cells in relation to the same time point in control (Vo) cells. (color version of figure is available online). AMPK, activated protein kinase; Hpa2, heparanase 2; HSF1, heat shock factor-1.

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitation Assay, Expressing

    11) Product Images from "Bursted BMP Triggered Receptor Kinase Activity Drives Smad1 Mediated Long-Term Target Gene Oscillation in c2c12 Cells"

    Article Title: Bursted BMP Triggered Receptor Kinase Activity Drives Smad1 Mediated Long-Term Target Gene Oscillation in c2c12 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059442

    Gene expression analysis of short-time receptor stimulus and short-time Smad-phosphorylation. (A) 30 h experiment using the stable reporter cell line. The cells were stimulated with 0.1 nM (green), 1 nM (red) 10 nM (blue) BMP2 or non-stimulated (black) and after 15 minutes the stimulation medium was removed and fresh starvation medium were given to the cells. Then every hour 50 µl medium was withdrawn and the Luciferase activity was measured. The relative fold change to the unstimulated control is depicted. (B) Quantitative real-time PCR on id1 and smad6. The cells were stimulated with 0.1 nM (green), 1 nM (red) BMP2 or non-stimulated (black) for 15 minutes and then cultivated in starvation medium until cell lysis. (C) 30 h experiment using the stable reporter cell line. The cells were stimulated with the indicated ligand concentrations and after 15 minutes Dorsomorphin was added to the cells. 50 µl medium were withdrawn every hour and the Luciferase activity was measured. The relative fold change to the unstimulated control was calculated and assigned. (D) qRT-PCR analysis of id1 and smad6. The cells were stimulated with the indicated concentrations for 15 minutes and Dorsomorphin was given to the cells. Every hour one sample was lysed and frozen at −80°C until the further processing.
    Figure Legend Snippet: Gene expression analysis of short-time receptor stimulus and short-time Smad-phosphorylation. (A) 30 h experiment using the stable reporter cell line. The cells were stimulated with 0.1 nM (green), 1 nM (red) 10 nM (blue) BMP2 or non-stimulated (black) and after 15 minutes the stimulation medium was removed and fresh starvation medium were given to the cells. Then every hour 50 µl medium was withdrawn and the Luciferase activity was measured. The relative fold change to the unstimulated control is depicted. (B) Quantitative real-time PCR on id1 and smad6. The cells were stimulated with 0.1 nM (green), 1 nM (red) BMP2 or non-stimulated (black) for 15 minutes and then cultivated in starvation medium until cell lysis. (C) 30 h experiment using the stable reporter cell line. The cells were stimulated with the indicated ligand concentrations and after 15 minutes Dorsomorphin was added to the cells. 50 µl medium were withdrawn every hour and the Luciferase activity was measured. The relative fold change to the unstimulated control was calculated and assigned. (D) qRT-PCR analysis of id1 and smad6. The cells were stimulated with the indicated concentrations for 15 minutes and Dorsomorphin was given to the cells. Every hour one sample was lysed and frozen at −80°C until the further processing.

    Techniques Used: Expressing, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Lysis, Quantitative RT-PCR

    12) Product Images from "Dynamic Smad-mediated BMP signaling revealed through transgenic zebrafish"

    Article Title: Dynamic Smad-mediated BMP signaling revealed through transgenic zebrafish

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    doi: 10.1002/dvdy.22567

    d2GFP expression driven by the BRE promoter is down-regulated by dorsomorphin. Larvae expressing BRE:d2GFP were treated with DMSO as a control (B, D) or with 50 μM dorsomorphin (A, C) at 24 hpf for 3 hours. d2GFP expression was reduced, particularly
    Figure Legend Snippet: d2GFP expression driven by the BRE promoter is down-regulated by dorsomorphin. Larvae expressing BRE:d2GFP were treated with DMSO as a control (B, D) or with 50 μM dorsomorphin (A, C) at 24 hpf for 3 hours. d2GFP expression was reduced, particularly

    Techniques Used: Expressing

    13) 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

    Ascorbic acid induction of cardiomyogenesis involves the BMP-signaling cascade. A. Cardiomyogenesis assessment by quantification of RFP + -CMs by flow cytometry analysis after treatment with BMP2 (10 ng/mL), added at Day 0, Ascorbic acid, added at Day 2, and their combination (Day 0 + Day 2, n = 5). B. Western Blots of the cardiac markers GATA4 and T following treatment with BMP2 (10 ng/mL, Day 0), AA (Day 2), and their combination (Day 0 + Day 2, n = 6). C. Cardiomyogenesis assessment by flow cytometry quantification of RFP + -CMs after dorsomorphin treatment (2 μM), an inhibitor of SMAD1-activation, added at Day 0 or Day 2, alone or in combination with AA (Day 2) (n = 3). D. Western Blots of the cardiac markers GATA4 and T following dorsomorphin inhibition (Day 2, 2 μM, n = 5), alone or in combination with AA (Day 2). *p
    Figure Legend Snippet: Ascorbic acid induction of cardiomyogenesis involves the BMP-signaling cascade. A. Cardiomyogenesis assessment by quantification of RFP + -CMs by flow cytometry analysis after treatment with BMP2 (10 ng/mL), added at Day 0, Ascorbic acid, added at Day 2, and their combination (Day 0 + Day 2, n = 5). B. Western Blots of the cardiac markers GATA4 and T following treatment with BMP2 (10 ng/mL, Day 0), AA (Day 2), and their combination (Day 0 + Day 2, n = 6). C. Cardiomyogenesis assessment by flow cytometry quantification of RFP + -CMs after dorsomorphin treatment (2 μM), an inhibitor of SMAD1-activation, added at Day 0 or Day 2, alone or in combination with AA (Day 2) (n = 3). D. Western Blots of the cardiac markers GATA4 and T following dorsomorphin inhibition (Day 2, 2 μM, n = 5), alone or in combination with AA (Day 2). *p

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

    14) Product Images from "Apcdd1 is a dual BMP/Wnt inhibitor in the developing nervous system and skin"

    Article Title: Apcdd1 is a dual BMP/Wnt inhibitor in the developing nervous system and skin

    Journal: bioRxiv

    doi: 10.1101/843714

    Role of APCDD1 in zebrafish axis elongation and dorso-ventral patterning. A. Partial sequence of exon 1 surrounding the initiation codon (red font) showing the binding sites for apcdd1l- mo (red font) and TALEN left and right arms (blue font) used to induce double strand breaks. The induced x60 mutant allele has a 7-nucleotide deletion, leading to a frame shift followed by 18 premature stops in exon 1 (a presumptive null). B. Expression of the organizer gene goosecoid ( gsc ) at 6 hpf (early gastrula stage). Knockdown of apcdd1l , or loss of apcdd1l in MZmutants, disinhibits maternal Wnt, resulting in expansion of the organizer. Animal pole views with dorsal to the right. C, D. Expression of neural plate marker sox19b at the end of gastrulation (C) and at the 6-somite stage (D). The early expansion of gsc in apcdd1- morpants and MZmutants results in a dorsalized phenotype that persists through the end of gastrulation, but dorsalization is rapidly reversed by early somitogenesis stages. Dorsal views with anterior to the top. E. Expression of the Bmp feedback inhibitor sizzled ( szl ) in ventral ectoderm (anterior up) at the end of gastrulation. Expression of szl marks cells experiencing active Bmp signaling. Injection of apcdd1l mRNA into wild-type embryos at the one-cell stage results in downregulation of szl by the end of gastrulation. F. Expression of szl in ventral ectoderm (anterior up) at the 10-somite stage. apcdd1l- morphants and MZmutants exhibit an expanded ventral domain of szl , indicating that Bmp signaling is now higher than normal. Treatment of embryos from 10 hpf with 75 µM dorsomorphin (DM) abolishes szl expression in control embryos and partially reverses expansion of szl expression in apcdd1l- morphants. G, H. Expression of gata3 in ventral epidermis ionocytes (arrows) at 24 hpf (G) and gata1 in blood progenitors (arrows) at 27 hpf (H). Ventral ionocytes and blood progenitors are expanded in apcdd1l -morphants, indicating ventralization of caudal structures. MZapcdd1l mutants also show an expanded domain of gata1 . Treatment of apcdd1l- morphants with 75 µM DM from the end of gastrulation partially reverses ventralization. Images show lateral views with anterior to the left.
    Figure Legend Snippet: Role of APCDD1 in zebrafish axis elongation and dorso-ventral patterning. A. Partial sequence of exon 1 surrounding the initiation codon (red font) showing the binding sites for apcdd1l- mo (red font) and TALEN left and right arms (blue font) used to induce double strand breaks. The induced x60 mutant allele has a 7-nucleotide deletion, leading to a frame shift followed by 18 premature stops in exon 1 (a presumptive null). B. Expression of the organizer gene goosecoid ( gsc ) at 6 hpf (early gastrula stage). Knockdown of apcdd1l , or loss of apcdd1l in MZmutants, disinhibits maternal Wnt, resulting in expansion of the organizer. Animal pole views with dorsal to the right. C, D. Expression of neural plate marker sox19b at the end of gastrulation (C) and at the 6-somite stage (D). The early expansion of gsc in apcdd1- morpants and MZmutants results in a dorsalized phenotype that persists through the end of gastrulation, but dorsalization is rapidly reversed by early somitogenesis stages. Dorsal views with anterior to the top. E. Expression of the Bmp feedback inhibitor sizzled ( szl ) in ventral ectoderm (anterior up) at the end of gastrulation. Expression of szl marks cells experiencing active Bmp signaling. Injection of apcdd1l mRNA into wild-type embryos at the one-cell stage results in downregulation of szl by the end of gastrulation. F. Expression of szl in ventral ectoderm (anterior up) at the 10-somite stage. apcdd1l- morphants and MZmutants exhibit an expanded ventral domain of szl , indicating that Bmp signaling is now higher than normal. Treatment of embryos from 10 hpf with 75 µM dorsomorphin (DM) abolishes szl expression in control embryos and partially reverses expansion of szl expression in apcdd1l- morphants. G, H. Expression of gata3 in ventral epidermis ionocytes (arrows) at 24 hpf (G) and gata1 in blood progenitors (arrows) at 27 hpf (H). Ventral ionocytes and blood progenitors are expanded in apcdd1l -morphants, indicating ventralization of caudal structures. MZapcdd1l mutants also show an expanded domain of gata1 . Treatment of apcdd1l- morphants with 75 µM DM from the end of gastrulation partially reverses ventralization. Images show lateral views with anterior to the left.

    Techniques Used: Sequencing, Binding Assay, Mutagenesis, Expressing, Marker, Injection

    15) Product Images from "Metformin promotes tau aggregation and exacerbates abnormal behavior in a mouse model of tauopathy"

    Article Title: Metformin promotes tau aggregation and exacerbates abnormal behavior in a mouse model of tauopathy

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-016-0082-7

    Metformin reduces tau phosphorylation in mouse cortical neurons via the AMPK-mTOR pathway. Primary cortical neurons were incubated for 6 h with or without 2.5 mM metformin (Met), 10 nM okadaic acid (OA), 10 μM rapamycin (Rapa), and 10 μM dorsomorphin (Dorso) alone or in combination. All treatments were performed in the absence of insulin, except when specifically indicated. a Quantitative analysis of phospho-tau (pTau) in cortical neurons treated with or without metformin in the absence or presence of insulin. b Quantitative analysis of pTau in cortical neurons treated with or without metformin in the absence or presence of okadaic acid. c - d Quantitative analysis of pAMPK ( c ) and pTau ( d ) in cortical neurons treated with or without metformin and dorsomorphin alone or in combination. e Quantitative analysis of pS6 in cortical neurons treated with or without metformin in the absence or presence of insulin. f - g . Quantitative analysis of pTau ( f ) and pS6 ( g ) in cortical neurons treated with or without metformin and rapamycin. h - i . Quantitative analysis of pS6 ( h ) and pTau ( i ) in neurons non-infected (NI), infected with GFP and S16H-Rheb or GFP alone and treated with metformin. Levels of phosphorylated protein were normalized to levels of respective proteins. In panels a, b, d, f, and i, pTau and total tau were analyzed using AT8 and Tau5 antibodies, respectively. In all graphs, bars represent the average ratio ± SEM of 3 independent experiments. * p
    Figure Legend Snippet: Metformin reduces tau phosphorylation in mouse cortical neurons via the AMPK-mTOR pathway. Primary cortical neurons were incubated for 6 h with or without 2.5 mM metformin (Met), 10 nM okadaic acid (OA), 10 μM rapamycin (Rapa), and 10 μM dorsomorphin (Dorso) alone or in combination. All treatments were performed in the absence of insulin, except when specifically indicated. a Quantitative analysis of phospho-tau (pTau) in cortical neurons treated with or without metformin in the absence or presence of insulin. b Quantitative analysis of pTau in cortical neurons treated with or without metformin in the absence or presence of okadaic acid. c - d Quantitative analysis of pAMPK ( c ) and pTau ( d ) in cortical neurons treated with or without metformin and dorsomorphin alone or in combination. e Quantitative analysis of pS6 in cortical neurons treated with or without metformin in the absence or presence of insulin. f - g . Quantitative analysis of pTau ( f ) and pS6 ( g ) in cortical neurons treated with or without metformin and rapamycin. h - i . Quantitative analysis of pS6 ( h ) and pTau ( i ) in neurons non-infected (NI), infected with GFP and S16H-Rheb or GFP alone and treated with metformin. Levels of phosphorylated protein were normalized to levels of respective proteins. In panels a, b, d, f, and i, pTau and total tau were analyzed using AT8 and Tau5 antibodies, respectively. In all graphs, bars represent the average ratio ± SEM of 3 independent experiments. * p

    Techniques Used: Incubation, Infection

    16) Product Images from "Small Molecules Dorsomorphin and LDN-193189 Inhibit Myostatin/GDF8 Signaling and Promote Functional Myoblast Differentiation *"

    Article Title: Small Molecules Dorsomorphin and LDN-193189 Inhibit Myostatin/GDF8 Signaling and Promote Functional Myoblast Differentiation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.604397

    Inhibitor binding to ActRII. A , overview of the ActRIIA crystal structure showing dorsomorphin bound in the ATP pocket. B , interactions of dorsomorphin with ActRIIA. The inhibitor forms direct hydrogen bonds with the kinase domain hinge residue His-270 as well as with the catalytic lysine residue Lys-219, which in turn forms a salt bridge with the catalytic glutamate Glu-232. C , binding measurements using ITC show that dorsomorphin binds ActRIIA with a K D value of 58 n m . D , superposition of the ActRIIA and ALK2 ( 53 ) structures reveals a similar inhibitor binding pocket. For clarity, only dorsomorphin from the ActRIIA structure is shown. Residue numbers are given for ActRIIA, and any substitutions in ALK2 are shown in parentheses . In ActRIIA, the catalytic lysine (Lys-219) forms a bridging hydrogen bonding interaction between dorsomorphin and the catalytic glutamate (Glu-232), whereas in ALK2, the catalytic lysine is displaced, and this interaction is instead mediated by a water molecule. E , superposition of the ActRIIA and ActRIIB structures ( 23 ) reveals the strict conservation of the residues lining the respective ATP binding pockets. The inhibitor LDN-193189 ( magenta stick representation ) was modeled based on the bound LDN-193189 in ALK2 ( 26 ) and the bound dorsomorphin ( gray stick representation ) in this ActRIIA structure. Residue numbers are given for ActRIIA, and any substitutions in ActRIIB are shown in parentheses .
    Figure Legend Snippet: Inhibitor binding to ActRII. A , overview of the ActRIIA crystal structure showing dorsomorphin bound in the ATP pocket. B , interactions of dorsomorphin with ActRIIA. The inhibitor forms direct hydrogen bonds with the kinase domain hinge residue His-270 as well as with the catalytic lysine residue Lys-219, which in turn forms a salt bridge with the catalytic glutamate Glu-232. C , binding measurements using ITC show that dorsomorphin binds ActRIIA with a K D value of 58 n m . D , superposition of the ActRIIA and ALK2 ( 53 ) structures reveals a similar inhibitor binding pocket. For clarity, only dorsomorphin from the ActRIIA structure is shown. Residue numbers are given for ActRIIA, and any substitutions in ALK2 are shown in parentheses . In ActRIIA, the catalytic lysine (Lys-219) forms a bridging hydrogen bonding interaction between dorsomorphin and the catalytic glutamate (Glu-232), whereas in ALK2, the catalytic lysine is displaced, and this interaction is instead mediated by a water molecule. E , superposition of the ActRIIA and ActRIIB structures ( 23 ) reveals the strict conservation of the residues lining the respective ATP binding pockets. The inhibitor LDN-193189 ( magenta stick representation ) was modeled based on the bound LDN-193189 in ALK2 ( 26 ) and the bound dorsomorphin ( gray stick representation ) in this ActRIIA structure. Residue numbers are given for ActRIIA, and any substitutions in ActRIIB are shown in parentheses .

    Techniques Used: Binding Assay

    Ligand-specific effects of kinase inhibitors on Smad2/3 and Smad1/5 phosphorylation. Serum-starved C2C12 cells were pretreated for 30 min with 0.5 μ m ( left panels ) or 5 μ m ( right panels ) dorsomorphin, LDN-193189, or SB-431542, before stimulation for 45 min with 5 n m BMP2, 8 n m GDF8, or 100 p m TGF-β1. Phosphorylated Smad2/3 ( A ) or Smad1/5 ( B ) was detected by immunoblotting and quantified densitometrically.
    Figure Legend Snippet: Ligand-specific effects of kinase inhibitors on Smad2/3 and Smad1/5 phosphorylation. Serum-starved C2C12 cells were pretreated for 30 min with 0.5 μ m ( left panels ) or 5 μ m ( right panels ) dorsomorphin, LDN-193189, or SB-431542, before stimulation for 45 min with 5 n m BMP2, 8 n m GDF8, or 100 p m TGF-β1. Phosphorylated Smad2/3 ( A ) or Smad1/5 ( B ) was detected by immunoblotting and quantified densitometrically.

    Techniques Used:

    Dorsomorphin and LDN-193189 counteract GDF8-induced repression of myogenic differentiation. A , confluent C2C12 cells were switched from growth medium ( GM ) to differentiation medium ( Diff M ; DMEM containing 2% horse serum) with inhibitors ( Inh ), dorsomorphin or LDN-193189, and differentiated for 1 day in the presence or absence of ligand ( Lig ), GDF8. Myogenic transcription factors MyoD and myogenin were detected by immunoblotting. The concentration of DMSO was 0.05% in all samples. B , C2C12 cells were transfected with a myogenin promoter-dependent firefly luciferase reporter construct ( Myg-luc ) 1 day before the medium was replaced by differentiation medium (DMEM containing 2% horse serum) supplemented with the respective inhibitor. After pretreatment for 30 min, 8 n m GDF8, 5 n m BMP2, or 100 p m TGF-β1 was added. DMSO concentration was 0.05% in all samples. Luciferase activities in lysates were determined after 3 days of differentiation. Bars , mean ± S.D. ( error bars ) of Myg-luciferase activities, normalized to unstimulated DMSO controls of four independent experiments. C , confluent C2C12 cells were allowed to differentiate for 3 days in differentiation medium (DMEM containing 2% horse serum) in the presence or absence of 5 μ m dorsomorphin or 0.5 μ m LDN-193189 and stimulated or not with 8 n m GDF8. DMSO concentration was 0.05% in all samples. At day 3, cells were immunostained for skeletal MHC ( green ). Nuclei were stained with DAPI ( blue ). Scale bars , 0.5 mm. Myogenic differentiation was quantified using digital image analysis of the fractions of nuclei that were in MHC-positive areas and by the relative MHC-positive area in each picture. Asterisks indicate statistical significance (*, p
    Figure Legend Snippet: Dorsomorphin and LDN-193189 counteract GDF8-induced repression of myogenic differentiation. A , confluent C2C12 cells were switched from growth medium ( GM ) to differentiation medium ( Diff M ; DMEM containing 2% horse serum) with inhibitors ( Inh ), dorsomorphin or LDN-193189, and differentiated for 1 day in the presence or absence of ligand ( Lig ), GDF8. Myogenic transcription factors MyoD and myogenin were detected by immunoblotting. The concentration of DMSO was 0.05% in all samples. B , C2C12 cells were transfected with a myogenin promoter-dependent firefly luciferase reporter construct ( Myg-luc ) 1 day before the medium was replaced by differentiation medium (DMEM containing 2% horse serum) supplemented with the respective inhibitor. After pretreatment for 30 min, 8 n m GDF8, 5 n m BMP2, or 100 p m TGF-β1 was added. DMSO concentration was 0.05% in all samples. Luciferase activities in lysates were determined after 3 days of differentiation. Bars , mean ± S.D. ( error bars ) of Myg-luciferase activities, normalized to unstimulated DMSO controls of four independent experiments. C , confluent C2C12 cells were allowed to differentiate for 3 days in differentiation medium (DMEM containing 2% horse serum) in the presence or absence of 5 μ m dorsomorphin or 0.5 μ m LDN-193189 and stimulated or not with 8 n m GDF8. DMSO concentration was 0.05% in all samples. At day 3, cells were immunostained for skeletal MHC ( green ). Nuclei were stained with DAPI ( blue ). Scale bars , 0.5 mm. Myogenic differentiation was quantified using digital image analysis of the fractions of nuclei that were in MHC-positive areas and by the relative MHC-positive area in each picture. Asterisks indicate statistical significance (*, p

    Techniques Used: Concentration Assay, Transfection, Luciferase, Construct, Staining

    Dorsomorphin and LDN-193189 inhibit GDF8-induced signaling pathways in undifferentiated and in differentiated primary human myoblasts and in C2C12 premyoblasts. A , primary human myoblasts were allowed to differentiate to myotubes for 6 days, as indicated by expression of MHC. Cells were then serum-starved and pretreated for 30 min with 5 μ m dorsomorphin or 0.5 μ m LDN-193189, before they were stimulated for 45 min with 8 n m GDF8. GDF8-induced phosphorylation of Smad2/3 and p38 was detected by immunoblotting and quantified densitometrically. B , undifferentiated primary human myoblasts were serum-starved, preincubated for 30 min with 5 μ m dorsomorphin or 0.5 μ m LDN-193189, and stimulated for 45 min with 8 n m GDF8. The absence of MHC expression indicates the undifferentiated status. GDF8-induced phosphorylation of Smad2/3 and p38 was detected by immunoblotting and quantified densitometrically. C , serum-starved C2C12 premyoblasts were pretreated for 30 min with 5 μ m dorsomorphin or 0.5 μ m LDN-193189 before stimulation for 45 min with GDF8. GDF8-induced phosphorylation of Smad2/3 and p38 was detected by immunoblotting and quantified densitometrically. D , serum-starved C2C12 premyoblast cells were pretreated for 30 min with increasing concentrations of dorsomorphin or LDN-193189 before stimulation for 45 min with 4 n m GDF8. GDF8-induced phosphorylation of Smad2/3 was detected by immunoblotting and quantified densitometrically.
    Figure Legend Snippet: Dorsomorphin and LDN-193189 inhibit GDF8-induced signaling pathways in undifferentiated and in differentiated primary human myoblasts and in C2C12 premyoblasts. A , primary human myoblasts were allowed to differentiate to myotubes for 6 days, as indicated by expression of MHC. Cells were then serum-starved and pretreated for 30 min with 5 μ m dorsomorphin or 0.5 μ m LDN-193189, before they were stimulated for 45 min with 8 n m GDF8. GDF8-induced phosphorylation of Smad2/3 and p38 was detected by immunoblotting and quantified densitometrically. B , undifferentiated primary human myoblasts were serum-starved, preincubated for 30 min with 5 μ m dorsomorphin or 0.5 μ m LDN-193189, and stimulated for 45 min with 8 n m GDF8. The absence of MHC expression indicates the undifferentiated status. GDF8-induced phosphorylation of Smad2/3 and p38 was detected by immunoblotting and quantified densitometrically. C , serum-starved C2C12 premyoblasts were pretreated for 30 min with 5 μ m dorsomorphin or 0.5 μ m LDN-193189 before stimulation for 45 min with GDF8. GDF8-induced phosphorylation of Smad2/3 and p38 was detected by immunoblotting and quantified densitometrically. D , serum-starved C2C12 premyoblast cells were pretreated for 30 min with increasing concentrations of dorsomorphin or LDN-193189 before stimulation for 45 min with 4 n m GDF8. GDF8-induced phosphorylation of Smad2/3 was detected by immunoblotting and quantified densitometrically.

    Techniques Used: Expressing

    Dorsomorphin and LDN-193189 promote the formation of a contractile myotube network. Confluent C2C12 cells were switched to differentiation medium (DMEM containing 2% horse serum). At day 4 of differentiation, cells were treated with 5 μ m DM, 0.5 μ m LDN-193189, or vehicle (0.05% DMSO). At day 6, contractile activities were recorded by time lapse DIC microscopy (3.3 frames/s, 30 s). A , visualization of contractile areas and activities. Top panels , contracting areas with color coding for the mean activities. Only active areas are visible , whereas inactive areas appear black. Bottom panels , time course of activity for the respective movies. For each treatment, three representative data sets are shown: samples with activities at the lower 25% ( left ), median ( middle ), or 75% percentiles ( right ) of the mean activities of all samples with the respective treatment. B , median activities in movies of DMSO ( n = 28)-, dorsomorphin ( n = 32)-, and LDN-193189 ( n = 28)-treated cells. AU , arbitrary units. Asterisks indicate statistical significance (*, p
    Figure Legend Snippet: Dorsomorphin and LDN-193189 promote the formation of a contractile myotube network. Confluent C2C12 cells were switched to differentiation medium (DMEM containing 2% horse serum). At day 4 of differentiation, cells were treated with 5 μ m DM, 0.5 μ m LDN-193189, or vehicle (0.05% DMSO). At day 6, contractile activities were recorded by time lapse DIC microscopy (3.3 frames/s, 30 s). A , visualization of contractile areas and activities. Top panels , contracting areas with color coding for the mean activities. Only active areas are visible , whereas inactive areas appear black. Bottom panels , time course of activity for the respective movies. For each treatment, three representative data sets are shown: samples with activities at the lower 25% ( left ), median ( middle ), or 75% percentiles ( right ) of the mean activities of all samples with the respective treatment. B , median activities in movies of DMSO ( n = 28)-, dorsomorphin ( n = 32)-, and LDN-193189 ( n = 28)-treated cells. AU , arbitrary units. Asterisks indicate statistical significance (*, p

    Techniques Used: Microscopy, Activity Assay

    Dorsomorphin treatment facilitates myotube formation. A , confluent C2C12 cells were switched to differentiation medium (DMEM containing 2% horse serum). At day 4 of differentiation, cells were treated with 5 μ m dorsomorphin ( DM ), 4.4 n m noggin, both in combination, or with vehicle (0.05% DMSO). At day 6, cells were immunostained for skeletal MHC ( green ). Nuclei were stained with DAPI ( blue ). Scale bars , 0.5 mm. Myogenic differentiation was quantified as in Fig. 5 . B , confluent primary human myoblasts on Matrigel-coated Permanox slides were switched to differentiation medium (DMEM containing 2% horse serum). At day 4 of differentiation, cells were treated with 5 μ m dorsomorphin or vehicle (0.05% DMSO). At day 6, myogenesis was visualized and quantified as in Fig. 5 . C , dorsomorphin-treated C2C12 cells displayed a typical intermittent MHC staining of A-bands and unstained Z-discs. Confluent C2C12 cells were differentiated for 4 days in differentiation medium (DMEM containing 2% horse serum). At days 4 and 5 of differentiation, cells were treated with 5 μ m dorsomorphin. At day 6, cells were immunostained for skeletal MHC ( green ). Nuclei were stained with DAPI ( blue ). Scale bar , 0.5 mm. Right panel , enlarged area of the MHC displayed in black and white to depict the striated muscle phenotype.
    Figure Legend Snippet: Dorsomorphin treatment facilitates myotube formation. A , confluent C2C12 cells were switched to differentiation medium (DMEM containing 2% horse serum). At day 4 of differentiation, cells were treated with 5 μ m dorsomorphin ( DM ), 4.4 n m noggin, both in combination, or with vehicle (0.05% DMSO). At day 6, cells were immunostained for skeletal MHC ( green ). Nuclei were stained with DAPI ( blue ). Scale bars , 0.5 mm. Myogenic differentiation was quantified as in Fig. 5 . B , confluent primary human myoblasts on Matrigel-coated Permanox slides were switched to differentiation medium (DMEM containing 2% horse serum). At day 4 of differentiation, cells were treated with 5 μ m dorsomorphin or vehicle (0.05% DMSO). At day 6, myogenesis was visualized and quantified as in Fig. 5 . C , dorsomorphin-treated C2C12 cells displayed a typical intermittent MHC staining of A-bands and unstained Z-discs. Confluent C2C12 cells were differentiated for 4 days in differentiation medium (DMEM containing 2% horse serum). At days 4 and 5 of differentiation, cells were treated with 5 μ m dorsomorphin. At day 6, cells were immunostained for skeletal MHC ( green ). Nuclei were stained with DAPI ( blue ). Scale bar , 0.5 mm. Right panel , enlarged area of the MHC displayed in black and white to depict the striated muscle phenotype.

    Techniques Used: Staining

    Dorsomorphin and LDN-193189 efficiently inhibit GDF8 induced Smad3/4 reporter gene activity. Undifferentiated C2C12 cells were transfected with Smad3/4-responsive (CAGA) 12 -luciferase or Smad1/5-responsive BRE-luciferase reporter constructs together with constitutively expressed Renilla luciferase overnight. Cells were serum-starved and stimulated for 6 h with 20 n m GDF8, 100 p m TGF-β, or 10 n m BMP2, together with the receptor kinase inhibitors dorsomorphin, LDN-193189, and SB-431542, as indicated. Luciferase activities are presented as relative luciferase units ( RLU ; firefly luciferase activity normalized to Renilla activity). Bars , mean ± S.D. ( error bars ) of triplicates. A , dorsomorphin and LDN-193189 repressed GDF8-induced (CAGA) 12 -luciferase activity from 0.5 and 0.05 μ m , respectively. B , BMP2-induced BRE-luciferase activity was efficiently repressed by 0.5 μ m dorsomorphin or 0.05 μ m LDN-193189. C , TGF-β-induced (CAGA) 12 -luciferase activity was repressed by 5 μ m dorsomorphin or 0.5 μ m LDN-193189.
    Figure Legend Snippet: Dorsomorphin and LDN-193189 efficiently inhibit GDF8 induced Smad3/4 reporter gene activity. Undifferentiated C2C12 cells were transfected with Smad3/4-responsive (CAGA) 12 -luciferase or Smad1/5-responsive BRE-luciferase reporter constructs together with constitutively expressed Renilla luciferase overnight. Cells were serum-starved and stimulated for 6 h with 20 n m GDF8, 100 p m TGF-β, or 10 n m BMP2, together with the receptor kinase inhibitors dorsomorphin, LDN-193189, and SB-431542, as indicated. Luciferase activities are presented as relative luciferase units ( RLU ; firefly luciferase activity normalized to Renilla activity). Bars , mean ± S.D. ( error bars ) of triplicates. A , dorsomorphin and LDN-193189 repressed GDF8-induced (CAGA) 12 -luciferase activity from 0.5 and 0.05 μ m , respectively. B , BMP2-induced BRE-luciferase activity was efficiently repressed by 0.5 μ m dorsomorphin or 0.05 μ m LDN-193189. C , TGF-β-induced (CAGA) 12 -luciferase activity was repressed by 5 μ m dorsomorphin or 0.5 μ m LDN-193189.

    Techniques Used: Activity Assay, Transfection, Luciferase, Construct

    17) Product Images from "The immunophilin FKBP12 inhibits hepcidin expression by binding the BMP type I receptor ALK2 in hepatocytes"

    Article Title: The immunophilin FKBP12 inhibits hepcidin expression by binding the BMP type I receptor ALK2 in hepatocytes

    Journal: Blood

    doi: 10.1182/blood-2017-04-780692

    Displacement of FKBP12 from ALK2 increases hepcidin through BMP-SMAD pathway activation. (A) HuH7 cells were transiently transfected with FKBP12 MYC-FLAG and ALK2 wt-MYC and treated with TAC (1 μg/mL), rapamycin (RAPA, 100 nM), GPI-1046 (100 μg/mL), or vehicle for 15 hours. Protein extracts were immunoprecipitated with an anti-FLAG M2 affinity gel (Sigma-Aldrich). Total extract and immunoprecipitated proteins were loaded onto a 12% SDS-PAGE and analyzed by western blot. ALK2 and FKBP12 were detected by using anti-MYC and anti-FKBP12 antibodies, respectively. Molecular weight markers are indicated on the right. (B) HuH7 cells were transfected with FKBP12 MYC-FLAG in the presence of ALK2 wt-MYC , ALK2 R206H-MYC or ALK2 Q207E-MYC , ALK2 R258S-MYC , or empty vector. When indicated, cells were treated for 15 hours with BMP6 (100 ng/mL). Whole-cell extract was immunoprecipitated and analyzed as described in panel A. Molecular weight markers are indicated on the right. (C) HuH7 cells were transfected with the Smad1 FLAG expressing vector in the presence of ALK2 wt-MYC , ALK2 R206H-MYC , ALK2 Q207E-MYC , ALK2 R258S-MYC , or empty vector (mock). When indicated, transfected cells were treated with BMP6 (50 ng/mL) for 30 minutes or 1.5 hours. Cells were lysed, loaded onto 10% SDS-PAGE, and analyzed by western blot. Activation of the BMP-SMAD pathway was detected by using an antibody recognizing phospho-SMAD1/5/8 and total SMAD1. ALK2 was detected by using anti-MYC antibody. Molecular weight markers are indicated on the right. (D,E) RNA was isolated from HuH7 cells transfected with ALK2 wt-MYC -, ALK2 R206H-MYC -, ALK2 Q207E-MYC -, or ALK2 R258S-MYC -expressing vector. Hepcidin ( HAMP ) (D) and ID1 (E) expression levels were quantified by qRT-PCR and normalized to the housekeeping gene GAPDH . Mean ΔCt values in each group were subjected to a change of origin by subtracting the mean ΔCt of ALK2 wt -transfected cells (D: −1.2; E: −3.9). Estimates of the fold changes in gene expression (2 −ΔΔCt ) are shown on the graphs. (F) Hep3B cells were transfected with hepcidin promoter firefly luciferase reporter ( HAMP -Luc) and increasing concentration of ALK2 wt-MYC-FLAG -, ALK2 R206H-MYC-FLAG -, ALK2 Q207E-MYC-FLAG -, or ALK2 R258S-MYC-FLAG -expressing vector. Cells transfected with the highest concentration of ALK2 cDNA were treated with dorsomorphin (DM, 10 μM). Cells were lysed and analyzed for the luciferase activity that was normalized to an untreated mean value of 1. (G) Hep3B cells were transfected with hepcidin promoter firefly luciferase reporter ( HAMP -Luc), ALK2 wt-MYC , ALK2 R206H-MYC , ALK2 R258S-MYC , or empty vector (mock) and increasing concentration of FKBP12. Luciferase activity was normalized to an untreated mean value of 1. (A-F) Representative results of experiments performed in triplicate. Error bars indicate SD. The 2-way ANOVA was used in panel F (ALK2 wt vs ALK2 mutants). * P
    Figure Legend Snippet: Displacement of FKBP12 from ALK2 increases hepcidin through BMP-SMAD pathway activation. (A) HuH7 cells were transiently transfected with FKBP12 MYC-FLAG and ALK2 wt-MYC and treated with TAC (1 μg/mL), rapamycin (RAPA, 100 nM), GPI-1046 (100 μg/mL), or vehicle for 15 hours. Protein extracts were immunoprecipitated with an anti-FLAG M2 affinity gel (Sigma-Aldrich). Total extract and immunoprecipitated proteins were loaded onto a 12% SDS-PAGE and analyzed by western blot. ALK2 and FKBP12 were detected by using anti-MYC and anti-FKBP12 antibodies, respectively. Molecular weight markers are indicated on the right. (B) HuH7 cells were transfected with FKBP12 MYC-FLAG in the presence of ALK2 wt-MYC , ALK2 R206H-MYC or ALK2 Q207E-MYC , ALK2 R258S-MYC , or empty vector. When indicated, cells were treated for 15 hours with BMP6 (100 ng/mL). Whole-cell extract was immunoprecipitated and analyzed as described in panel A. Molecular weight markers are indicated on the right. (C) HuH7 cells were transfected with the Smad1 FLAG expressing vector in the presence of ALK2 wt-MYC , ALK2 R206H-MYC , ALK2 Q207E-MYC , ALK2 R258S-MYC , or empty vector (mock). When indicated, transfected cells were treated with BMP6 (50 ng/mL) for 30 minutes or 1.5 hours. Cells were lysed, loaded onto 10% SDS-PAGE, and analyzed by western blot. Activation of the BMP-SMAD pathway was detected by using an antibody recognizing phospho-SMAD1/5/8 and total SMAD1. ALK2 was detected by using anti-MYC antibody. Molecular weight markers are indicated on the right. (D,E) RNA was isolated from HuH7 cells transfected with ALK2 wt-MYC -, ALK2 R206H-MYC -, ALK2 Q207E-MYC -, or ALK2 R258S-MYC -expressing vector. Hepcidin ( HAMP ) (D) and ID1 (E) expression levels were quantified by qRT-PCR and normalized to the housekeeping gene GAPDH . Mean ΔCt values in each group were subjected to a change of origin by subtracting the mean ΔCt of ALK2 wt -transfected cells (D: −1.2; E: −3.9). Estimates of the fold changes in gene expression (2 −ΔΔCt ) are shown on the graphs. (F) Hep3B cells were transfected with hepcidin promoter firefly luciferase reporter ( HAMP -Luc) and increasing concentration of ALK2 wt-MYC-FLAG -, ALK2 R206H-MYC-FLAG -, ALK2 Q207E-MYC-FLAG -, or ALK2 R258S-MYC-FLAG -expressing vector. Cells transfected with the highest concentration of ALK2 cDNA were treated with dorsomorphin (DM, 10 μM). Cells were lysed and analyzed for the luciferase activity that was normalized to an untreated mean value of 1. (G) Hep3B cells were transfected with hepcidin promoter firefly luciferase reporter ( HAMP -Luc), ALK2 wt-MYC , ALK2 R206H-MYC , ALK2 R258S-MYC , or empty vector (mock) and increasing concentration of FKBP12. Luciferase activity was normalized to an untreated mean value of 1. (A-F) Representative results of experiments performed in triplicate. Error bars indicate SD. The 2-way ANOVA was used in panel F (ALK2 wt vs ALK2 mutants). * P

    Techniques Used: Activation Assay, Transfection, Immunoprecipitation, SDS Page, Western Blot, Molecular Weight, Plasmid Preparation, Expressing, Isolation, Quantitative RT-PCR, Luciferase, Concentration Assay, Activity Assay

    18) Product Images from "Effects of Genistein on Differentiation and Viability of Human Visceral Adipocytes"

    Article Title: Effects of Genistein on Differentiation and Viability of Human Visceral Adipocytes

    Journal: Nutrients

    doi: 10.3390/nu10080978

    Effects of genistein in pre-adipocytes, cultured in physiological condition, on cell viability ( A ) and mitochondrial membrane potential ( B ): In A and B, the effects of genistein (G) 10 pM, 1 µM, 50 µM and 200 µM, adiponectin (Adipo; 100 µM), wortmannin (W; 200 µM), dorsomorphin (D; 200 µM). C = control. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: # p
    Figure Legend Snippet: Effects of genistein in pre-adipocytes, cultured in physiological condition, on cell viability ( A ) and mitochondrial membrane potential ( B ): In A and B, the effects of genistein (G) 10 pM, 1 µM, 50 µM and 200 µM, adiponectin (Adipo; 100 µM), wortmannin (W; 200 µM), dorsomorphin (D; 200 µM). C = control. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: # p

    Techniques Used: Cell Culture

    19) Product Images from "Apcdd1 is a dual BMP/Wnt inhibitor in the developing nervous system and skin"

    Article Title: Apcdd1 is a dual BMP/Wnt inhibitor in the developing nervous system and skin

    Journal: bioRxiv

    doi: 10.1101/843714

    Role of APCDD1 in zebrafish axis elongation and dorso-ventral patterning. A. Partial sequence of exon 1 surrounding the initiation codon (red font) showing the binding sites for apcdd1l- mo (red font) and TALEN left and right arms (blue font) used to induce double strand breaks. The induced x60 mutant allele has a 7-nucleotide deletion, leading to a frame shift followed by 18 premature stops in exon 1 (a presumptive null). B. Expression of the organizer gene goosecoid ( gsc ) at 6 hpf (early gastrula stage). Knockdown of apcdd1l , or loss of apcdd1l in MZmutants, disinhibits maternal Wnt, resulting in expansion of the organizer. Animal pole views with dorsal to the right. C, D. Expression of neural plate marker sox19b at the end of gastrulation (C) and at the 6-somite stage (D). The early expansion of gsc in apcdd1- morpants and MZmutants results in a dorsalized phenotype that persists through the end of gastrulation, but dorsalization is rapidly reversed by early somitogenesis stages. Dorsal views with anterior to the top. E. Expression of the Bmp feedback inhibitor sizzled ( szl ) in ventral ectoderm (anterior up) at the end of gastrulation. Expression of szl marks cells experiencing active Bmp signaling. Injection of apcdd1l mRNA into wild-type embryos at the one-cell stage results in downregulation of szl by the end of gastrulation. F. Expression of szl in ventral ectoderm (anterior up) at the 10-somite stage. apcdd1l- morphants and MZmutants exhibit an expanded ventral domain of szl , indicating that Bmp signaling is now higher than normal. Treatment of embryos from 10 hpf with 75 µM dorsomorphin (DM) abolishes szl expression in control embryos and partially reverses expansion of szl expression in apcdd1l- morphants. G, H. Expression of gata3 in ventral epidermis ionocytes (arrows) at 24 hpf (G) and gata1 in blood progenitors (arrows) at 27 hpf (H). Ventral ionocytes and blood progenitors are expanded in apcdd1l -morphants, indicating ventralization of caudal structures. MZapcdd1l mutants also show an expanded domain of gata1 . Treatment of apcdd1l- morphants with 75 µM DM from the end of gastrulation partially reverses ventralization. Images show lateral views with anterior to the left.
    Figure Legend Snippet: Role of APCDD1 in zebrafish axis elongation and dorso-ventral patterning. A. Partial sequence of exon 1 surrounding the initiation codon (red font) showing the binding sites for apcdd1l- mo (red font) and TALEN left and right arms (blue font) used to induce double strand breaks. The induced x60 mutant allele has a 7-nucleotide deletion, leading to a frame shift followed by 18 premature stops in exon 1 (a presumptive null). B. Expression of the organizer gene goosecoid ( gsc ) at 6 hpf (early gastrula stage). Knockdown of apcdd1l , or loss of apcdd1l in MZmutants, disinhibits maternal Wnt, resulting in expansion of the organizer. Animal pole views with dorsal to the right. C, D. Expression of neural plate marker sox19b at the end of gastrulation (C) and at the 6-somite stage (D). The early expansion of gsc in apcdd1- morpants and MZmutants results in a dorsalized phenotype that persists through the end of gastrulation, but dorsalization is rapidly reversed by early somitogenesis stages. Dorsal views with anterior to the top. E. Expression of the Bmp feedback inhibitor sizzled ( szl ) in ventral ectoderm (anterior up) at the end of gastrulation. Expression of szl marks cells experiencing active Bmp signaling. Injection of apcdd1l mRNA into wild-type embryos at the one-cell stage results in downregulation of szl by the end of gastrulation. F. Expression of szl in ventral ectoderm (anterior up) at the 10-somite stage. apcdd1l- morphants and MZmutants exhibit an expanded ventral domain of szl , indicating that Bmp signaling is now higher than normal. Treatment of embryos from 10 hpf with 75 µM dorsomorphin (DM) abolishes szl expression in control embryos and partially reverses expansion of szl expression in apcdd1l- morphants. G, H. Expression of gata3 in ventral epidermis ionocytes (arrows) at 24 hpf (G) and gata1 in blood progenitors (arrows) at 27 hpf (H). Ventral ionocytes and blood progenitors are expanded in apcdd1l -morphants, indicating ventralization of caudal structures. MZapcdd1l mutants also show an expanded domain of gata1 . Treatment of apcdd1l- morphants with 75 µM DM from the end of gastrulation partially reverses ventralization. Images show lateral views with anterior to the left.

    Techniques Used: Sequencing, Binding Assay, Mutagenesis, Expressing, Marker, Injection

    20) Product Images from "Emdogain-Regulated Gene Expression in Palatal Fibroblasts Requires TGF-βRI Kinase Signaling"

    Article Title: Emdogain-Regulated Gene Expression in Palatal Fibroblasts Requires TGF-βRI Kinase Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105672

    BMP receptors ALK2, ALK3, and ALK6 do not mediate the effect of Emdogain on IL-11 expression. Palatal fibroblasts were incubated with (A) 100 µg/ml Emdogain or serum-free medium alone and BMP type I receptor inhibitors dorsomorphin (DORSO; 10 µM) and LDN193189 (LDN; 10 µM). Palatal fibroblasts were also incubated with (B) recombinant human BMP-2 and BMP-7 (both 100 ng/ml). RT-PCR was performed for IL-11 **P
    Figure Legend Snippet: BMP receptors ALK2, ALK3, and ALK6 do not mediate the effect of Emdogain on IL-11 expression. Palatal fibroblasts were incubated with (A) 100 µg/ml Emdogain or serum-free medium alone and BMP type I receptor inhibitors dorsomorphin (DORSO; 10 µM) and LDN193189 (LDN; 10 µM). Palatal fibroblasts were also incubated with (B) recombinant human BMP-2 and BMP-7 (both 100 ng/ml). RT-PCR was performed for IL-11 **P

    Techniques Used: Expressing, Incubation, Recombinant, Reverse Transcription Polymerase Chain Reaction

    21) Product Images from "Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia"

    Article Title: Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2015.11.003

    ALK3 and ALK6 are required for GDF2-induced SMAD1/5 phosphorylation. (A-B) Western blotting for pSMAD1/5 activation in PA1 and MCF10A cells in the presence and absence of dorsomorphin 1 μM (+) or 3 μM (++), SB431542 3 μM (+) or 5 μM (++), or ML347 500 nM (+) or 1 mM (++) as indicated with and without GDF2 (10 ng/ml) as indicated (quantification of pSMAD1/5 levels presented in Supplementary Figure S2 C ). (C) Immunoblotting of pSMAD1/5 in PA1 cells in the presence of shRNAs to ALK2, ALK3, ALK6, and BMPRII without and with GDF2 treatment (10 ng/ml) for 30 minutes. (D) QRT-PCR analyses of (C) to confirm reduced expression of ALK2, ALK3, ALK6, and BMPRII expression as indicated. (E) Kinase inactive ALK3 and ALK6 inhibit SMAD1/5 phosphorylation. Western blotting as indicated in MCF10A and PA1 cells in the presence of either mock transfected or HA-tagged kinase inactive ALK3 (ALK3 K-R) or ALK6 (ALK6 K-R) and treated with GDF2 for the time points indicated. Actin was the loading control. (F) Dorsomorphin inhibits SMAD1/5 transcriptional activation. BRE-luciferase reporter activity in indicated cells in the absence (GDF2 alone) or presence of 1 μM dorsomorphin (GDF2+DM). Fold induction of luciferase activity compared with DMSO-treated control cells is presented.
    Figure Legend Snippet: ALK3 and ALK6 are required for GDF2-induced SMAD1/5 phosphorylation. (A-B) Western blotting for pSMAD1/5 activation in PA1 and MCF10A cells in the presence and absence of dorsomorphin 1 μM (+) or 3 μM (++), SB431542 3 μM (+) or 5 μM (++), or ML347 500 nM (+) or 1 mM (++) as indicated with and without GDF2 (10 ng/ml) as indicated (quantification of pSMAD1/5 levels presented in Supplementary Figure S2 C ). (C) Immunoblotting of pSMAD1/5 in PA1 cells in the presence of shRNAs to ALK2, ALK3, ALK6, and BMPRII without and with GDF2 treatment (10 ng/ml) for 30 minutes. (D) QRT-PCR analyses of (C) to confirm reduced expression of ALK2, ALK3, ALK6, and BMPRII expression as indicated. (E) Kinase inactive ALK3 and ALK6 inhibit SMAD1/5 phosphorylation. Western blotting as indicated in MCF10A and PA1 cells in the presence of either mock transfected or HA-tagged kinase inactive ALK3 (ALK3 K-R) or ALK6 (ALK6 K-R) and treated with GDF2 for the time points indicated. Actin was the loading control. (F) Dorsomorphin inhibits SMAD1/5 transcriptional activation. BRE-luciferase reporter activity in indicated cells in the absence (GDF2 alone) or presence of 1 μM dorsomorphin (GDF2+DM). Fold induction of luciferase activity compared with DMSO-treated control cells is presented.

    Techniques Used: Western Blot, Activation Assay, Quantitative RT-PCR, Expressing, Transfection, Luciferase, Activity Assay

    Anoikis susceptibility is mediated via ALK3/ALK6. (A) Western blotting of 4T1 cells grown under anoikis conditions either with or without 1 μM dorsomorphin and GDF2 as indicated. Lysates were immunoblotted against CC3. Actin was the loading control (quantification of CC3 levels presented in Supplementary Figure S3 E ). (B) Kinase mutants ALK3 K-R and ALK6 K-R reduce anoikis. Western blotting of PA1 cells expressing control, ALK3 K-R, or ALK6 K-R was plated for anoikis (Methods) in the absence or presence of GDF2 for indicated times (quantification of CC3 levels presented in Supplementary Figure S3 F ). (C) MTT absorbance values of PA1 cells expressing shScr, shALK3 or shALK6 after 24 hours of anoikis in the absence and presence of 10 ng/ml of GDF2 are presented. Error bars indicate SEM. (D) Western blotting of same cells as in (C) harvested after 24 hours of anoikis and immunoblotted for CC3. Actin was the loading control. (E) Western blotting as indicated of PA1 cells expressing shSMAD1 or shScr at the indicated times under anoikis conditions (quantification of CC3 presented in Supplementary Figure S3 G ). SMAD1 transcript levels upon shRNA to SMAD1 relative to controls are presented in the adjacent graph. Actin was the loading control.
    Figure Legend Snippet: Anoikis susceptibility is mediated via ALK3/ALK6. (A) Western blotting of 4T1 cells grown under anoikis conditions either with or without 1 μM dorsomorphin and GDF2 as indicated. Lysates were immunoblotted against CC3. Actin was the loading control (quantification of CC3 levels presented in Supplementary Figure S3 E ). (B) Kinase mutants ALK3 K-R and ALK6 K-R reduce anoikis. Western blotting of PA1 cells expressing control, ALK3 K-R, or ALK6 K-R was plated for anoikis (Methods) in the absence or presence of GDF2 for indicated times (quantification of CC3 levels presented in Supplementary Figure S3 F ). (C) MTT absorbance values of PA1 cells expressing shScr, shALK3 or shALK6 after 24 hours of anoikis in the absence and presence of 10 ng/ml of GDF2 are presented. Error bars indicate SEM. (D) Western blotting of same cells as in (C) harvested after 24 hours of anoikis and immunoblotted for CC3. Actin was the loading control. (E) Western blotting as indicated of PA1 cells expressing shSMAD1 or shScr at the indicated times under anoikis conditions (quantification of CC3 presented in Supplementary Figure S3 G ). SMAD1 transcript levels upon shRNA to SMAD1 relative to controls are presented in the adjacent graph. Actin was the loading control.

    Techniques Used: Western Blot, Expressing, MTT Assay, shRNA

    GDF2 expression in breast and ovarian cells. (A) Indicated cell lines were lysed and immunoblotted for GDF2 (*lower band). Actin was the loading control. (B) Secreted GDF2 levels as determined using ELISAs in indicated breast and ovarian cell lines. (C) QRT-PCR levels of GDF2 transcript in the presence of increasing AZA for the indicated time points. Error bars indicate SEM. (D) Western blotting for CC3 and pSMAD1/5 in PA1 cells either untreated or treated with AZA under anoikis conditions for the indicated times. Dorsomorphin (5 μM) was added in combination during anoikis where indicated (Dm). Actin was the loading control. (E) Promoter methylation of GDF2 . GDF2 promoter methylation status of ovarian cancer patients ( N = 47; black line) plotted against normal fallopian tube epithelium (gray line) from the same experiment. Values were plotted using the GraphPad Prism software. Beta values are presented (* P
    Figure Legend Snippet: GDF2 expression in breast and ovarian cells. (A) Indicated cell lines were lysed and immunoblotted for GDF2 (*lower band). Actin was the loading control. (B) Secreted GDF2 levels as determined using ELISAs in indicated breast and ovarian cell lines. (C) QRT-PCR levels of GDF2 transcript in the presence of increasing AZA for the indicated time points. Error bars indicate SEM. (D) Western blotting for CC3 and pSMAD1/5 in PA1 cells either untreated or treated with AZA under anoikis conditions for the indicated times. Dorsomorphin (5 μM) was added in combination during anoikis where indicated (Dm). Actin was the loading control. (E) Promoter methylation of GDF2 . GDF2 promoter methylation status of ovarian cancer patients ( N = 47; black line) plotted against normal fallopian tube epithelium (gray line) from the same experiment. Values were plotted using the GraphPad Prism software. Beta values are presented (* P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Methylation, Software

    22) Product Images from "A Chemical Screen Identifies Small Molecules that Regulate Hepcidin Expression"

    Article Title: A Chemical Screen Identifies Small Molecules that Regulate Hepcidin Expression

    Journal: Blood cells, molecules & diseases

    doi: 10.1016/j.bcmd.2014.06.002

    Quantitative realtime RT-PCR for A. Hepcidin , B. ID3 , C. SOCS3 . Following 8 hours of serum starvation in α-MEM/1%FBS, HepG2 cells were treated for 24 hours in α-MEM/1%FBS with DMSO 1%, BMP6 100 ng/ml, IL-6 100 ng/ml, dorsomorphin (an inhibitor
    Figure Legend Snippet: Quantitative realtime RT-PCR for A. Hepcidin , B. ID3 , C. SOCS3 . Following 8 hours of serum starvation in α-MEM/1%FBS, HepG2 cells were treated for 24 hours in α-MEM/1%FBS with DMSO 1%, BMP6 100 ng/ml, IL-6 100 ng/ml, dorsomorphin (an inhibitor

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    23) Product Images from "Transforming Growth Factor ? Receptor Type 1 Is Essential for Female Reproductive Tract Integrity and Function"

    Article Title: Transforming Growth Factor ? Receptor Type 1 Is Essential for Female Reproductive Tract Integrity and Function

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002320

    Cellular distribution and functional characterization of TGFBR1 in mouse ovary. (A to C) β-gal staining of ovaries from immature [(A; untreated) and (C; PMSG-hCG treated)] and adult (B) Tgfbr1 +/bgal mice. (D) Suppression of Tgfbr1 mRNA in mouse granulosa cells by recombinant BMP15 or GDF9 after 5 h treatment (n = 3). (E to H) Expansion of COCs from control and Tgfbr1 cKO mice in vitro in the absence (E and G) or presence (F and H) of EGF (10 ng/ml). (I) Cumulus expansion index (CEI) of the in vitro cultured COCs from control (n = 4) and Tgfbr1 cKO (n = 5) mice. (J and K) Preovulatory follicles from Tgfbr1 cKO mice demonstrating cumulus expansion. PF, primary follicle; SF, secondary follicle; AF, antral follicle; Oo, oocyte; TC, thecal cell; GC, granulosa cell; CC, cumulus cell; CL, corpus luteum; COC, cumulus-oocyte complex. Scale bars = 50 µm (J and K); 100 µm (A and C); and 200 µm (B). (L) Ovulation and oocyte fertilization potential of Tgfbr1 cKO mice (n = 8–10). (M to P) Blastocysts or degenerated oocytes/embryos recovered from control (M) and Tgfbr1 cKO mice (N to P). (Q to T) Identification of receptor preference for GDF9 signaling in mouse granulosa cells using small molecule inhibitors and Alk6 −/− granulosa cells. Dorsomorphin (DM; 4 µM) was preincubated with granulosa cells for 1 h before BMP15 (100 ng/ml) or GDF9 (15 ng/ml) was added. DM markedly reduced Ptx3 induction by BMP15 (Q), while a similar effect was not observed on GDF9-induced Ptx3 mRNA expression (R). Induction of Ptx3 mRNA by GDF9 (100 ng/ml) was not attenuated in Alk6 −/− granulosa cells (S). However, SB-505124 (SB; 1 µM) suppressed GDF9-induced Ptx3 mRNA expression (T). Con, control. n = 3 for each group. Relative mRNA levels of Tgfbr1 and Ptx3 were normalized to Gapdh . Data are presented as mean ± SEM. Bars without a common letter are significantly different at P
    Figure Legend Snippet: Cellular distribution and functional characterization of TGFBR1 in mouse ovary. (A to C) β-gal staining of ovaries from immature [(A; untreated) and (C; PMSG-hCG treated)] and adult (B) Tgfbr1 +/bgal mice. (D) Suppression of Tgfbr1 mRNA in mouse granulosa cells by recombinant BMP15 or GDF9 after 5 h treatment (n = 3). (E to H) Expansion of COCs from control and Tgfbr1 cKO mice in vitro in the absence (E and G) or presence (F and H) of EGF (10 ng/ml). (I) Cumulus expansion index (CEI) of the in vitro cultured COCs from control (n = 4) and Tgfbr1 cKO (n = 5) mice. (J and K) Preovulatory follicles from Tgfbr1 cKO mice demonstrating cumulus expansion. PF, primary follicle; SF, secondary follicle; AF, antral follicle; Oo, oocyte; TC, thecal cell; GC, granulosa cell; CC, cumulus cell; CL, corpus luteum; COC, cumulus-oocyte complex. Scale bars = 50 µm (J and K); 100 µm (A and C); and 200 µm (B). (L) Ovulation and oocyte fertilization potential of Tgfbr1 cKO mice (n = 8–10). (M to P) Blastocysts or degenerated oocytes/embryos recovered from control (M) and Tgfbr1 cKO mice (N to P). (Q to T) Identification of receptor preference for GDF9 signaling in mouse granulosa cells using small molecule inhibitors and Alk6 −/− granulosa cells. Dorsomorphin (DM; 4 µM) was preincubated with granulosa cells for 1 h before BMP15 (100 ng/ml) or GDF9 (15 ng/ml) was added. DM markedly reduced Ptx3 induction by BMP15 (Q), while a similar effect was not observed on GDF9-induced Ptx3 mRNA expression (R). Induction of Ptx3 mRNA by GDF9 (100 ng/ml) was not attenuated in Alk6 −/− granulosa cells (S). However, SB-505124 (SB; 1 µM) suppressed GDF9-induced Ptx3 mRNA expression (T). Con, control. n = 3 for each group. Relative mRNA levels of Tgfbr1 and Ptx3 were normalized to Gapdh . Data are presented as mean ± SEM. Bars without a common letter are significantly different at P

    Techniques Used: Functional Assay, Staining, Mouse Assay, Recombinant, In Vitro, Cell Culture, Expressing

    24) Product Images from "Phenformin and metformin inhibit growth and migration of LN229 glioma cells in vitro and in vivo"

    Article Title: Phenformin and metformin inhibit growth and migration of LN229 glioma cells in vitro and in vivo

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S168981

    The effects of Phen and Met on LN229 cells in vitro. Notes: ( A ) The expression levels of AMPK and p-AMPK after treatment with Phen or Met in LN229 cells. ( B ) The cell death of LN229 after treatment with Phen, Met, or in combination with dorsomorphin. ( C ) The levels of ATP in LN229 cells after treatment with Phen or Met. ( D ) The levels of lactate in LN229 cells after treatment with Phen or Met. ( E ) The death of LN229 cells after treatment with Phen, Met, or lactate. * p
    Figure Legend Snippet: The effects of Phen and Met on LN229 cells in vitro. Notes: ( A ) The expression levels of AMPK and p-AMPK after treatment with Phen or Met in LN229 cells. ( B ) The cell death of LN229 after treatment with Phen, Met, or in combination with dorsomorphin. ( C ) The levels of ATP in LN229 cells after treatment with Phen or Met. ( D ) The levels of lactate in LN229 cells after treatment with Phen or Met. ( E ) The death of LN229 cells after treatment with Phen, Met, or lactate. * p

    Techniques Used: In Vitro, Expressing

    25) Product Images from "Live Fluorescent Staining Platform for Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization"

    Article Title: Live Fluorescent Staining Platform for Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules22122068

    Evaluation of mineralization of the vertebrate column in dorsomorphin-treated zebrafish. ( A ) The gross morphology of zebrafish aged at 7 dpf which have been treated with different concentrations of dorsomorphin (10, 20, and 30 µM, left bright-filed panel) from 3 dpf onwards. Calcein staining on control and dorsomorphin-treated embryos aged at 7 dpf (right green fluorescent panel; ( B ) Quantification of mineralization degree detecting the fluorescence intensity at the area of centrum form ring in the notochord. (values are mean ± SD; tested by one-way ANOVA pairwise comparison; N = fish number; Different letters indicate significant differences) ( C ) Chemical structure of dorsomorphin.
    Figure Legend Snippet: Evaluation of mineralization of the vertebrate column in dorsomorphin-treated zebrafish. ( A ) The gross morphology of zebrafish aged at 7 dpf which have been treated with different concentrations of dorsomorphin (10, 20, and 30 µM, left bright-filed panel) from 3 dpf onwards. Calcein staining on control and dorsomorphin-treated embryos aged at 7 dpf (right green fluorescent panel; ( B ) Quantification of mineralization degree detecting the fluorescence intensity at the area of centrum form ring in the notochord. (values are mean ± SD; tested by one-way ANOVA pairwise comparison; N = fish number; Different letters indicate significant differences) ( C ) Chemical structure of dorsomorphin.

    Techniques Used: Staining, Fluorescence, Fluorescence In Situ Hybridization

    26) Product Images from "Fructose and glucose can regulate mammalian target of rapamycin complex 1 and lipogenic gene expression via distinct pathways"

    Article Title: Fructose and glucose can regulate mammalian target of rapamycin complex 1 and lipogenic gene expression via distinct pathways

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.782557

    Fructose acutely suppresses mTORC1 in vitro . A–C , primary rat hepatocytes were stimulated with or without fructose or glucose for 10 min. Whole-cell lysates were used for the measurement of intracellular ATP levels ( A ) or immunoblotting ( B and C ). The quantified results of three independent experiments are shown ( C , right panel ). D , primary rat hepatocytes were stimulated with fructose in the presence or absence of dorsomorphin and subjected to immunoblotting ( left ); the quantified results of three independent experiments are shown ( right ). Bars and error bars correspond to the mean and S.E., respectively. *, p
    Figure Legend Snippet: Fructose acutely suppresses mTORC1 in vitro . A–C , primary rat hepatocytes were stimulated with or without fructose or glucose for 10 min. Whole-cell lysates were used for the measurement of intracellular ATP levels ( A ) or immunoblotting ( B and C ). The quantified results of three independent experiments are shown ( C , right panel ). D , primary rat hepatocytes were stimulated with fructose in the presence or absence of dorsomorphin and subjected to immunoblotting ( left ); the quantified results of three independent experiments are shown ( right ). Bars and error bars correspond to the mean and S.E., respectively. *, p

    Techniques Used: In Vitro

    27) Product Images from "A New Class of Small Molecule Inhibitor of BMP Signaling"

    Article Title: A New Class of Small Molecule Inhibitor of BMP Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062721

    K02288 induces dorsalization of zebrafish embryos. (A) Brightfield photographs of 26 hours old Tg(BRE:mRFP) transgenic embryos treated with DMSO or varying doses of K02288 from the 8- to 16-cell stage. Severity of the dorsalization correlated with the dose of K02288. Very strong dorsalized phenotypes were observed with 8–10 µM K02288. (B) The phenotypes of the embryos shown in A were classified according to Kishimoto et al. [49] . (C) Western blot for mRFP in extracts prepared from Tg(BRE:mRFP) embryos treated in a parallel experiment. Loss of mRFP protein was evident at 8–10 µM K02288. As a control, the effects of dorsomorphin (DM) and LDN-193189 (LDN) on mRFP expression are also shown. Protein loading control is shown with the MCM6 blot.
    Figure Legend Snippet: K02288 induces dorsalization of zebrafish embryos. (A) Brightfield photographs of 26 hours old Tg(BRE:mRFP) transgenic embryos treated with DMSO or varying doses of K02288 from the 8- to 16-cell stage. Severity of the dorsalization correlated with the dose of K02288. Very strong dorsalized phenotypes were observed with 8–10 µM K02288. (B) The phenotypes of the embryos shown in A were classified according to Kishimoto et al. [49] . (C) Western blot for mRFP in extracts prepared from Tg(BRE:mRFP) embryos treated in a parallel experiment. Loss of mRFP protein was evident at 8–10 µM K02288. As a control, the effects of dorsomorphin (DM) and LDN-193189 (LDN) on mRFP expression are also shown. Protein loading control is shown with the MCM6 blot.

    Techniques Used: Transgenic Assay, Western Blot, Expressing

    Identification of a novel 2-aminopyridine inhibitor of ALK2. (A) Schematic summary of a thermal shift assay screen using recombinant ALK2 kinase domain. A novel 2-aminopyridine hit K02288 was identified with an affinity for ALK2 intermediate between dorsomorphin and LDN-193189. Complete screening data are shown in supplemental Table S1 . (B) In vitro kinase assays showed K02288 specificity for ALK1,2,3,6 over ALK4,5. IC 50 measurements were performed in triplicate at the Km value of ATP. (C) ActRIIA kinase inhibition was determined using the Kinase-Glo® assay (Promega). IC 50 measurements were performed in duplicate at the Km value of ATP. (D) Summary of IC 50 measurements in all experiments.
    Figure Legend Snippet: Identification of a novel 2-aminopyridine inhibitor of ALK2. (A) Schematic summary of a thermal shift assay screen using recombinant ALK2 kinase domain. A novel 2-aminopyridine hit K02288 was identified with an affinity for ALK2 intermediate between dorsomorphin and LDN-193189. Complete screening data are shown in supplemental Table S1 . (B) In vitro kinase assays showed K02288 specificity for ALK1,2,3,6 over ALK4,5. IC 50 measurements were performed in triplicate at the Km value of ATP. (C) ActRIIA kinase inhibition was determined using the Kinase-Glo® assay (Promega). IC 50 measurements were performed in duplicate at the Km value of ATP. (D) Summary of IC 50 measurements in all experiments.

    Techniques Used: Thermal Shift Assay, Recombinant, In Vitro, Inhibition, Glo Assay

    K02288 does not inhibit vasculature development. (Top panels) Brightfield photographs of 48 hours old Tg ( fli1a : eGFP) embryos treated with DMSO or chemical inhibitors from 12 hours post fertilization. Embryos were manually dechorionated after bud stage before treatment. (Center panels) Same view under UV light for visualization of eGFP expression in the vasculature. Dorsomorphin and LDN-193189 treatment resulted in intersomitic vessel (ISV) formation defects, consistent with their known inhibition of VEGF signaling. (Lower panels) Higher magnification views of representative embryos and phenotype summary. The most severe phenotypes were observed with dorsomorphin. No effects on ISV formation were observed with K02288 treatment.
    Figure Legend Snippet: K02288 does not inhibit vasculature development. (Top panels) Brightfield photographs of 48 hours old Tg ( fli1a : eGFP) embryos treated with DMSO or chemical inhibitors from 12 hours post fertilization. Embryos were manually dechorionated after bud stage before treatment. (Center panels) Same view under UV light for visualization of eGFP expression in the vasculature. Dorsomorphin and LDN-193189 treatment resulted in intersomitic vessel (ISV) formation defects, consistent with their known inhibition of VEGF signaling. (Lower panels) Higher magnification views of representative embryos and phenotype summary. The most severe phenotypes were observed with dorsomorphin. No effects on ISV formation were observed with K02288 treatment.

    Techniques Used: Expressing, Inhibition

    28) Product Images from "Ezetimibe Attenuates Oxidative Stress and Neuroinflammation via the AMPK/Nrf2/TXNIP Pathway after MCAO in Rats"

    Article Title: Ezetimibe Attenuates Oxidative Stress and Neuroinflammation via the AMPK/Nrf2/TXNIP Pathway after MCAO in Rats

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2020/4717258

    Experimental design and animal groups. DHE: dihydroethidium; DMSO: dimethyl sulfoxide; dorsomorphin: AMPK inhibitor; Eze: Ezetimibe; IHC: immunohistochemistry; MCAO: middle cerebral artery occlusion; MDA: malonaldehyde; Scr siRNA: scramble siRNA; TTC: 2,3,5-triphenyltetrazolium chloride; WB: western blot.
    Figure Legend Snippet: Experimental design and animal groups. DHE: dihydroethidium; DMSO: dimethyl sulfoxide; dorsomorphin: AMPK inhibitor; Eze: Ezetimibe; IHC: immunohistochemistry; MCAO: middle cerebral artery occlusion; MDA: malonaldehyde; Scr siRNA: scramble siRNA; TTC: 2,3,5-triphenyltetrazolium chloride; WB: western blot.

    Techniques Used: Immunohistochemistry, Multiple Displacement Amplification, Western Blot

    Dorsomorphin and Nrf2 siRNA reversed the neuroprotective effects of Eze after MCAO. (a) Representative image of TTC-staining brain slices, (b) quantified infarction volumes, (c) modified Garcia scores, and (d) beam walking scores at 24 h after MCAO. ∗ p
    Figure Legend Snippet: Dorsomorphin and Nrf2 siRNA reversed the neuroprotective effects of Eze after MCAO. (a) Representative image of TTC-staining brain slices, (b) quantified infarction volumes, (c) modified Garcia scores, and (d) beam walking scores at 24 h after MCAO. ∗ p

    Techniques Used: Staining, Modification

    29) Product Images from "The positional identity of mouse ES cell-generated neurons is affected by BMP signaling"

    Article Title: The positional identity of mouse ES cell-generated neurons is affected by BMP signaling

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-012-1182-3

    Effects of BMP inhibition on ESCs neural conversion and cell fate acquisition: a–c double immunocytodetection of Nestin ( green ) and FoxG1 ( red ) at the end of step II in ESCs cultured in CDMM ( a ) or in CDMM + Noggin (150 nM, b ). FoxG1-positive cells were always co-labeled by Nestin. Numbers in ( c ) show ratios of Nestin-positive cells among total cells ( light blue bars ), or ratios of FoxG1-positive cells among Nestin-positive cells ( red bars ). d–f VGlut2 ( red in e ), β-III-Tubulin ( green in f ), and Gad65 ( red in f ) immunocytodetection and cell counts in Noggin-treated ESCs at step III + 4 days. Arrow in ( f ) indicates a β-III-Tubulin/Gad65 double positive cell. d The ratios of cells positive for the markers in ( e and f ). g–o Immunocytodetection of FoxG1 ( red in g–j ), Tbr1 ( red in k–n ) and acetylated-Tubulin ( green in g–n ) in ESCs cells at the end of step III after differentiation in CDMM (control; g , k ), CDMM plus Noggin (400 nM; h , l , j , n ) and Dorsomorphin (5 uM; i , m ). o Cell ratios of FoxG1-positive and Tbr1-positive cells from culture conditions as in ( g–n ), and for ESCs treated with SAG, RA or 150 nM Noggin (not shown). p A group of neurons almost all positive for Satb2 nuclear staining. q Numbers show the ratios of Tbr1 or Satb2 positive cells over time in 150 nM Noggin-treated ESC cultures. Error bars standard error; * p
    Figure Legend Snippet: Effects of BMP inhibition on ESCs neural conversion and cell fate acquisition: a–c double immunocytodetection of Nestin ( green ) and FoxG1 ( red ) at the end of step II in ESCs cultured in CDMM ( a ) or in CDMM + Noggin (150 nM, b ). FoxG1-positive cells were always co-labeled by Nestin. Numbers in ( c ) show ratios of Nestin-positive cells among total cells ( light blue bars ), or ratios of FoxG1-positive cells among Nestin-positive cells ( red bars ). d–f VGlut2 ( red in e ), β-III-Tubulin ( green in f ), and Gad65 ( red in f ) immunocytodetection and cell counts in Noggin-treated ESCs at step III + 4 days. Arrow in ( f ) indicates a β-III-Tubulin/Gad65 double positive cell. d The ratios of cells positive for the markers in ( e and f ). g–o Immunocytodetection of FoxG1 ( red in g–j ), Tbr1 ( red in k–n ) and acetylated-Tubulin ( green in g–n ) in ESCs cells at the end of step III after differentiation in CDMM (control; g , k ), CDMM plus Noggin (400 nM; h , l , j , n ) and Dorsomorphin (5 uM; i , m ). o Cell ratios of FoxG1-positive and Tbr1-positive cells from culture conditions as in ( g–n ), and for ESCs treated with SAG, RA or 150 nM Noggin (not shown). p A group of neurons almost all positive for Satb2 nuclear staining. q Numbers show the ratios of Tbr1 or Satb2 positive cells over time in 150 nM Noggin-treated ESC cultures. Error bars standard error; * p

    Techniques Used: Inhibition, Cell Culture, Labeling, Staining

    30) Product Images from "BMP4 Regulates Vascular Progenitor Development in Human Embryonic Stem Cells Through a Smad-Dependent Pathway"

    Article Title: BMP4 Regulates Vascular Progenitor Development in Human Embryonic Stem Cells Through a Smad-Dependent Pathway

    Journal: Journal of cellular biochemistry

    doi: 10.1002/jcb.22410

    Smad-dependent pathway mediates BMP4 effect on CD34+CD31+ cell generation. A: The hESCs were stimulated by BMP4 (50 ng/ml) with or without dorsomorphin (5 μM) for 30 min. The phosphorylation of Smad1/5/8 was examined by Western blot analysis.
    Figure Legend Snippet: Smad-dependent pathway mediates BMP4 effect on CD34+CD31+ cell generation. A: The hESCs were stimulated by BMP4 (50 ng/ml) with or without dorsomorphin (5 μM) for 30 min. The phosphorylation of Smad1/5/8 was examined by Western blot analysis.

    Techniques Used: Western Blot

    31) Product Images from "Activation of melanocortin receptor 4 with RO27-3225 attenuates neuroinflammation through AMPK/JNK/p38 MAPK pathway after intracerebral hemorrhage in mice"

    Article Title: Activation of melanocortin receptor 4 with RO27-3225 attenuates neuroinflammation through AMPK/JNK/p38 MAPK pathway after intracerebral hemorrhage in mice

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1140-6

    The effects of RO27-3225 and AMPK inhibitor dorsomorphin on the expression of MC4R and its downstream signaling proteins. a Representative western blot bands. b – g Quantitative analyses of MC4R, phosphorylated AMPK, phosphorylated JNK, phosphorylated p38 MAPK, TNF-α, and IL-1β in the ipsilateral hemisphere at 24 h after ICH. * p
    Figure Legend Snippet: The effects of RO27-3225 and AMPK inhibitor dorsomorphin on the expression of MC4R and its downstream signaling proteins. a Representative western blot bands. b – g Quantitative analyses of MC4R, phosphorylated AMPK, phosphorylated JNK, phosphorylated p38 MAPK, TNF-α, and IL-1β in the ipsilateral hemisphere at 24 h after ICH. * p

    Techniques Used: Expressing, Western Blot

    32) Product Images from "Liver Specification in the Absence of Cardiac Differentiation Revealed by Differential Sensitivity to Wnt/β Catenin Pathway Activation"

    Article Title: Liver Specification in the Absence of Cardiac Differentiation Revealed by Differential Sensitivity to Wnt/β Catenin Pathway Activation

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.00155

    BMP signaling is required for liver development. (A) Design of the experiment. Lineage tracer was injected (B) alone or (C–F) with ∼30 pg/blastomere of truncated BMP Receptor (tBR) mRNA in dorso-vegetal blastomere D1 at the 32-cell stage. Heart and liver were revealed by double WMISH of myl7 (turquoise) and nr1h5 (purple). (C–F) four examples showing attenuation of liver fate specification in vivo following localized BMP inhibition by tBR (red-pink; pointed by arrows). N = 11, all showing effect on nr1h5 expression. Ventral views are shown, anterior is up. BMP signaling inhibition attenuates liver cell fate specification in Gata4 injected AC. (G) qPCR analyses of st. 34 explants show downregulation of nr1h5 as a consequence of BMP inhibition via tBR. At st. 10, tBR has no effect on the ability of Gata4 to induce sox17 but reduces hhex induction. (H) Treatment of Gata4-expressing AC explants with 30 μM dorsomorphin (DM) leads to downregulation of nr1h5 .
    Figure Legend Snippet: BMP signaling is required for liver development. (A) Design of the experiment. Lineage tracer was injected (B) alone or (C–F) with ∼30 pg/blastomere of truncated BMP Receptor (tBR) mRNA in dorso-vegetal blastomere D1 at the 32-cell stage. Heart and liver were revealed by double WMISH of myl7 (turquoise) and nr1h5 (purple). (C–F) four examples showing attenuation of liver fate specification in vivo following localized BMP inhibition by tBR (red-pink; pointed by arrows). N = 11, all showing effect on nr1h5 expression. Ventral views are shown, anterior is up. BMP signaling inhibition attenuates liver cell fate specification in Gata4 injected AC. (G) qPCR analyses of st. 34 explants show downregulation of nr1h5 as a consequence of BMP inhibition via tBR. At st. 10, tBR has no effect on the ability of Gata4 to induce sox17 but reduces hhex induction. (H) Treatment of Gata4-expressing AC explants with 30 μM dorsomorphin (DM) leads to downregulation of nr1h5 .

    Techniques Used: Injection, In Vivo, Inhibition, Expressing, Real-time Polymerase Chain Reaction

    33) Product Images from "Upregulated ethanolamine phospholipid synthesis via selenoprotein I is required for effective metabolic reprogramming during T cell activation"

    Article Title: Upregulated ethanolamine phospholipid synthesis via selenoprotein I is required for effective metabolic reprogramming during T cell activation

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2021.101170

    Dorsomorphin treatment inhibited T cell proliferation. Activated WT T cells were treated with different doses of dorsomorphin during CSFE assays. (A) Images of cultured cells at 18 h post-TCR-stimulation with clusters of proliferating cells decreasing with increasing doses of dorsomorphin. Scale bar = 125 mm. (B) Flow cytometry data used to calculate the Proliferation Index. (C) CSFE assays using different concentrations of the AMPK inhibitor, dorsomorphin, decreased the Proliferation Index from the data shown in Figure S5 . (D) Western blot analysis showed that 0.75 mM of dorsomorphin treatment led to decreased phosphorylation of AMPK after 5 h of TCR stimulation similar to SELENOI KO. Means of replicates (n = 3) were compared using Student's t test and means in panel I (n = 4) were analyzed using one-way ANOVA with Tukey's post-test and expressed as mean ± SEM with ∗p
    Figure Legend Snippet: Dorsomorphin treatment inhibited T cell proliferation. Activated WT T cells were treated with different doses of dorsomorphin during CSFE assays. (A) Images of cultured cells at 18 h post-TCR-stimulation with clusters of proliferating cells decreasing with increasing doses of dorsomorphin. Scale bar = 125 mm. (B) Flow cytometry data used to calculate the Proliferation Index. (C) CSFE assays using different concentrations of the AMPK inhibitor, dorsomorphin, decreased the Proliferation Index from the data shown in Figure S5 . (D) Western blot analysis showed that 0.75 mM of dorsomorphin treatment led to decreased phosphorylation of AMPK after 5 h of TCR stimulation similar to SELENOI KO. Means of replicates (n = 3) were compared using Student's t test and means in panel I (n = 4) were analyzed using one-way ANOVA with Tukey's post-test and expressed as mean ± SEM with ∗p

    Techniques Used: Cell Culture, Flow Cytometry, Western Blot

    34) Product Images from "SIRT3 Protects Rotenone-induced Injury in SH-SY5Y Cells by Promoting Autophagy through the LKB1-AMPK-mTOR Pathway"

    Article Title: SIRT3 Protects Rotenone-induced Injury in SH-SY5Y Cells by Promoting Autophagy through the LKB1-AMPK-mTOR Pathway

    Journal: Aging and Disease

    doi: 10.14336/AD.2017.0517

    SIRT3 induces autophagy through the LKB1-AMPK-mTOR pathway. (A) The SH-SY5Y cells with or without SIRT3 overexpression were treated with 40 μM Dorsomorphin or control for 100 min. The cell lysates were analyzed by western blotting with the indicated antibodies. Data show the quantification of the p-LKB1/LKB1 ratio (B), p-AMPK/AMPK ratio (C), p-mTOR/mTOR levels (D) and LC3II (E). β-actin is used as a loading control. #: P
    Figure Legend Snippet: SIRT3 induces autophagy through the LKB1-AMPK-mTOR pathway. (A) The SH-SY5Y cells with or without SIRT3 overexpression were treated with 40 μM Dorsomorphin or control for 100 min. The cell lysates were analyzed by western blotting with the indicated antibodies. Data show the quantification of the p-LKB1/LKB1 ratio (B), p-AMPK/AMPK ratio (C), p-mTOR/mTOR levels (D) and LC3II (E). β-actin is used as a loading control. #: P

    Techniques Used: Over Expression, Western Blot

    35) Product Images from "Apcdd1 is a dual BMP/Wnt inhibitor in the developing nervous system and skin"

    Article Title: Apcdd1 is a dual BMP/Wnt inhibitor in the developing nervous system and skin

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2020.03.015

    Role of APCDD1 in zebrafish axis elongation and dorso-ventral patterning. A. Expression of apcdd1l in zebrafish embryos at the indicated times. The presence of maternal apcdd1l mRNA, confirmed by RT-PCR (not shown), is seen in all cells in the early blastula (animal pole views). By early gastrulation (6 hpf), apcdd1l is only weakly expressed with preferential staining in dorsal tissues (lateral view, dorsal to the right). By the end of gastrulation (10 hpf), apcdd1l is expressed throughout to the dorsal axis (dorsal view and lateral view, anterior up). At 24 and30 hpf, apcdd1l is expressed in the eyes, restricted regions in the brain, and dorsal tissues in the trunk and tail. B. Partial sequence of exon 1 surrounding the initiation codon (red font) showing the binding sites for apcdd1l- mo (red font) and TALEN left and right arms (blue font) used to induce double strand breaks. The induced x60 mutant allele has a 7 nucleotide deletion, leading to a frame shift followed by 18 premature stops in exon 1 (a presumptive null). C. Expression of the organizer gene goosecoid ( gsc ) at 6 hpf (early gastrula stage). Knockdown of apcdd1l , or loss of apcdd1l in MZmutants, disinhibits maternal Wnt, resulting in expansion of the organizer. Animal pole views with dorsal to the right. D - E. Expression of neural plate marker sox19b at the end of gastrulation (C) and at the 6-somite stage (D). The early expansion of gsc in apcdd1- morpants and MZmutants results in a dorsalized phenotype that persists through the end of gastrulation, but dorsalization is rapidly reversed by early somitogenesis stages. Dorsal views with anterior to the top. F. Expression of the Bmp feedback inhibitor sizzled ( szl ) in ventral ectoderm (anterior up) at the end of gastrulation. Expression of szl marks cells experiencing active Bmp signaling. Injection of apcdd1l mRNA into wild-type embryos at the one-cell stage results in downregulation of szl by the end of gastrulation. G. Expression of szl in ventral ectoderm (anterior up) at the 10-somite stage. apcdd1l- morphants and MZmutants exhibit an expanded ventral domain of szl , indicating that Bmp signaling is now higher than normal. Treatment of embryos from 10 hpf with 75 μM dorsomorphin (DM) abolishes szl expression in control embryos and partially reverses expansion of szl expression in apcdd1l- morphants. H-I . Expression of gata3 in ventral epidermis ionocytes (arrows) at 24 hpf (G) and gata1 in blood progenitors (arrows) at 27 hpf (H). Ventral ionocytes and blood progenitors are expanded in apcdd1l -morphants, indicating ventralization of caudal structures. MZapcdd1l mutants also show an expanded domain of gata1 . Treatment of apcdd1l- morphants with 75 μM DM from the end of gastrulation partially reverses ventralization. Images show lateral views with anterior to the left. At least n =15 zebrafish embryos were examined for each time point and experimental condition.
    Figure Legend Snippet: Role of APCDD1 in zebrafish axis elongation and dorso-ventral patterning. A. Expression of apcdd1l in zebrafish embryos at the indicated times. The presence of maternal apcdd1l mRNA, confirmed by RT-PCR (not shown), is seen in all cells in the early blastula (animal pole views). By early gastrulation (6 hpf), apcdd1l is only weakly expressed with preferential staining in dorsal tissues (lateral view, dorsal to the right). By the end of gastrulation (10 hpf), apcdd1l is expressed throughout to the dorsal axis (dorsal view and lateral view, anterior up). At 24 and30 hpf, apcdd1l is expressed in the eyes, restricted regions in the brain, and dorsal tissues in the trunk and tail. B. Partial sequence of exon 1 surrounding the initiation codon (red font) showing the binding sites for apcdd1l- mo (red font) and TALEN left and right arms (blue font) used to induce double strand breaks. The induced x60 mutant allele has a 7 nucleotide deletion, leading to a frame shift followed by 18 premature stops in exon 1 (a presumptive null). C. Expression of the organizer gene goosecoid ( gsc ) at 6 hpf (early gastrula stage). Knockdown of apcdd1l , or loss of apcdd1l in MZmutants, disinhibits maternal Wnt, resulting in expansion of the organizer. Animal pole views with dorsal to the right. D - E. Expression of neural plate marker sox19b at the end of gastrulation (C) and at the 6-somite stage (D). The early expansion of gsc in apcdd1- morpants and MZmutants results in a dorsalized phenotype that persists through the end of gastrulation, but dorsalization is rapidly reversed by early somitogenesis stages. Dorsal views with anterior to the top. F. Expression of the Bmp feedback inhibitor sizzled ( szl ) in ventral ectoderm (anterior up) at the end of gastrulation. Expression of szl marks cells experiencing active Bmp signaling. Injection of apcdd1l mRNA into wild-type embryos at the one-cell stage results in downregulation of szl by the end of gastrulation. G. Expression of szl in ventral ectoderm (anterior up) at the 10-somite stage. apcdd1l- morphants and MZmutants exhibit an expanded ventral domain of szl , indicating that Bmp signaling is now higher than normal. Treatment of embryos from 10 hpf with 75 μM dorsomorphin (DM) abolishes szl expression in control embryos and partially reverses expansion of szl expression in apcdd1l- morphants. H-I . Expression of gata3 in ventral epidermis ionocytes (arrows) at 24 hpf (G) and gata1 in blood progenitors (arrows) at 27 hpf (H). Ventral ionocytes and blood progenitors are expanded in apcdd1l -morphants, indicating ventralization of caudal structures. MZapcdd1l mutants also show an expanded domain of gata1 . Treatment of apcdd1l- morphants with 75 μM DM from the end of gastrulation partially reverses ventralization. Images show lateral views with anterior to the left. At least n =15 zebrafish embryos were examined for each time point and experimental condition.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Sequencing, Binding Assay, Mutagenesis, Marker, Injection

    36) Product Images from "Efficient Derivation of Multipotent Neural Stem/Progenitor Cells from Non-Human Primate Embryonic Stem Cells"

    Article Title: Efficient Derivation of Multipotent Neural Stem/Progenitor Cells from Non-Human Primate Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049469

    Neurosphere formation of marmoset ESCs. ( A ) Protocol to derive neurospheres from marmoset ESCs by EB formation. EBs were cultured in suspension in ultra-low cluster dishes for 2 weeks in the presence of 3 µM dorsomorphin or 1×10 −6 M RA. Dorsomorphin or RA were added on day 1 or 5 of EB formation, respectively. EBs were then dissociated and cultured in suspension for 12–14 days to form neurospheres in MHM medium containing 2% B27 and 20 ng/ml FGF-2. Primary neurospheres were dissociated and cultured in suspension again with FGF-2 to form secondary neurospheres. ( B ) Neurosphere formation rates are presented as the percentages of neurospheres among total cells plated. EBs treated with 3 µM dorsomorphin or 1×10 −6 M RA were dissociated and cultured in MHM medium containing 2% B27 and 20 ng/ml FGF-2 at a density of 2.5×10 4 cells/ml in an ultra-low cluster 96-well plate for 1 week, and then neurospheres larger than 50 µm in diameter were counted. Data are presented as the means ± SEM ( n = 3). ( C ) Representative morphologies of EBs, primary neurospheres and secondary neurospheres under each condition. Scale bars, 100 µm for EBs, 200 µm for neurospheres.
    Figure Legend Snippet: Neurosphere formation of marmoset ESCs. ( A ) Protocol to derive neurospheres from marmoset ESCs by EB formation. EBs were cultured in suspension in ultra-low cluster dishes for 2 weeks in the presence of 3 µM dorsomorphin or 1×10 −6 M RA. Dorsomorphin or RA were added on day 1 or 5 of EB formation, respectively. EBs were then dissociated and cultured in suspension for 12–14 days to form neurospheres in MHM medium containing 2% B27 and 20 ng/ml FGF-2. Primary neurospheres were dissociated and cultured in suspension again with FGF-2 to form secondary neurospheres. ( B ) Neurosphere formation rates are presented as the percentages of neurospheres among total cells plated. EBs treated with 3 µM dorsomorphin or 1×10 −6 M RA were dissociated and cultured in MHM medium containing 2% B27 and 20 ng/ml FGF-2 at a density of 2.5×10 4 cells/ml in an ultra-low cluster 96-well plate for 1 week, and then neurospheres larger than 50 µm in diameter were counted. Data are presented as the means ± SEM ( n = 3). ( C ) Representative morphologies of EBs, primary neurospheres and secondary neurospheres under each condition. Scale bars, 100 µm for EBs, 200 µm for neurospheres.

    Techniques Used: Cell Culture

    37) Product Images from "Zebrafish cardiac development requires a conserved secondary heart field"

    Article Title: Zebrafish cardiac development requires a conserved secondary heart field

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.061473

    The absence of Bmp signaling induces an expansion of the bulbus arteriosus at the expense of the ventricle. ( A-C ) Ventral view of 72 hpf zebrafish treated with dorsomorphin (B,C) or DMSO carrier (A) from 24 to 72 hpf. ( D ) The bulbus arteriosus can be
    Figure Legend Snippet: The absence of Bmp signaling induces an expansion of the bulbus arteriosus at the expense of the ventricle. ( A-C ) Ventral view of 72 hpf zebrafish treated with dorsomorphin (B,C) or DMSO carrier (A) from 24 to 72 hpf. ( D ) The bulbus arteriosus can be

    Techniques Used:

    38) Product Images from "Mesendogen, a novel inhibitor of TRPM6, promotes mesoderm and definitive endoderm differentiation of human embryonic stem cells through alteration of magnesium homeostasis"

    Article Title: Mesendogen, a novel inhibitor of TRPM6, promotes mesoderm and definitive endoderm differentiation of human embryonic stem cells through alteration of magnesium homeostasis

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2015.e00046

    A working model for a potential role of magnesium during cell fate specification. (A) Western blotting of neural progenitor markers SOX1, PAX6, and SOX2 in H1 hESCs cultured in neural differentiation medium for 7 days untreated (Control) or treated with Dorsomorphin (1 μM). The Dorsomorphin treated samples were also incubated with DMSO, 2-APB (50 μM), Ruthenium Red (RR; 50 μM), and MEG (10 μM), respectively. α-TUBULIN was used as a loading control. n = 3 independent experiments. (B) Schematic representation of a working model summarizing the findings of this study and suggesting a potential role of magnesium during cell fate specification.
    Figure Legend Snippet: A working model for a potential role of magnesium during cell fate specification. (A) Western blotting of neural progenitor markers SOX1, PAX6, and SOX2 in H1 hESCs cultured in neural differentiation medium for 7 days untreated (Control) or treated with Dorsomorphin (1 μM). The Dorsomorphin treated samples were also incubated with DMSO, 2-APB (50 μM), Ruthenium Red (RR; 50 μM), and MEG (10 μM), respectively. α-TUBULIN was used as a loading control. n = 3 independent experiments. (B) Schematic representation of a working model summarizing the findings of this study and suggesting a potential role of magnesium during cell fate specification.

    Techniques Used: Western Blot, Cell Culture, Incubation

    39) Product Images from "Quercetin, a Lead Compound against Type 2 Diabetes Ameliorates Glucose Uptake via AMPK Pathway in Skeletal Muscle Cell Line"

    Article Title: Quercetin, a Lead Compound against Type 2 Diabetes Ameliorates Glucose Uptake via AMPK Pathway in Skeletal Muscle Cell Line

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00336

    Effect of inhibitors on 2-NBDG uptake. (A) Effect of PI3K inhibitor, wortmannin, on 2-NBDG uptake in L6 myotubes. L6 myotubes pretreated with wortmannin followed by co-incubation with Rozi: rosiglitazone (100 nM); Insulin (100 nM); Qn (2, 3): quercetin (10 and 100 μM) for 24 h. Each value represents mean ± SD (standard deviation) from triplicate measurements ( n = 3) of three different experiments. Significance test between different groups was determined by using one way ANOVA followed by Duncan’s multiple range test the significance accepted at P ≤ 0.05. ∗ P ≤ 0.05 versus same groups with wortmannin. (B) Effect of AMPK inhibitor, Dorsomorphin, on 2-NBDG uptake in L6 myotubes. L6 myotubes pretreated with dorsomorphin followed by co-incubation with Rozi: rosiglitazone (100 nM); Qn (2, 3): quercetin (10 and 100 μM) for 24 h. Each value represents mean ± SD (standard deviation) from triplicate measurements ( n = 3) of three different experiments. Significance test between different groups was determined by using one way ANOVA followed by Duncan’s multiple range test the significance accepted at P ≤ 0.05. ∗ P ≤ 0.05 versus same groups with dorsomorphin.
    Figure Legend Snippet: Effect of inhibitors on 2-NBDG uptake. (A) Effect of PI3K inhibitor, wortmannin, on 2-NBDG uptake in L6 myotubes. L6 myotubes pretreated with wortmannin followed by co-incubation with Rozi: rosiglitazone (100 nM); Insulin (100 nM); Qn (2, 3): quercetin (10 and 100 μM) for 24 h. Each value represents mean ± SD (standard deviation) from triplicate measurements ( n = 3) of three different experiments. Significance test between different groups was determined by using one way ANOVA followed by Duncan’s multiple range test the significance accepted at P ≤ 0.05. ∗ P ≤ 0.05 versus same groups with wortmannin. (B) Effect of AMPK inhibitor, Dorsomorphin, on 2-NBDG uptake in L6 myotubes. L6 myotubes pretreated with dorsomorphin followed by co-incubation with Rozi: rosiglitazone (100 nM); Qn (2, 3): quercetin (10 and 100 μM) for 24 h. Each value represents mean ± SD (standard deviation) from triplicate measurements ( n = 3) of three different experiments. Significance test between different groups was determined by using one way ANOVA followed by Duncan’s multiple range test the significance accepted at P ≤ 0.05. ∗ P ≤ 0.05 versus same groups with dorsomorphin.

    Techniques Used: Incubation, Standard Deviation

    40) Product Images from "Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity"

    Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

    Journal: Journal of Cerebral Blood Flow & Metabolism

    doi: 10.1177/0271678X17720816

    Occludin influences energetic processes by controlling AMPK activation . (a) SPRICE quantitation of active (phospho) AMPK in occludin-deficient (OCC−), occludin overexpressing (OCC+), and control (SCR) pericytes. Values represent the ratio of phosphorylated vs. the respective total AMPK expression, normalized against DRAQ-5. (b) ATP content in pericytes treated with AICAR (AMPK activator, AMPK+) or dorsomorphin (AMPK inhibitor, AMPK−). (c) ATP quantitation in occludin-deficient (OCC−) or control (SCR) pericytes treated or not with AICAR or dorsomorphin as in (c). (d) Fluorospectrometric quantitation of 2-NBDG taken up by WT-pericytes treated with dorsomorphin (AMPK-) and/or insulin. (e) Similar 2-NBDG quantitation in occludin overexpressing (OCC+) pericytes treated with dorsomorphin and/or insulin. Graphs show average percentual difference over WT pericytes treated with PBS (Vh), represented by the horizontal axis at 0, SEM, n =6, p vs. WT. (f) Representative confocal microscopy images of WT pericytes immunostained for the lysosomal marker LAMP1 (green) and occludin (red). Nu: Cell nucleus. (g) Representative confocal microscopy images of HIV-infected pericytes 24 h post-infection (occludin depletion phase), immunostained as in (f). (h) Representative confocal microscopy images of HIV-infected pericytes 96 h post-infection (occludin recovery phase) and immunostained as in (f) and (g). Non-infected (NI) and HIV-infected pericytes at different time points post infection were analyzed by SPRICE to quantify (i) 2-NBDG uptake and (j) active AMPK (graph shows the ratio between phosphorylated and total AMPK levels). (k) Fluorospectrometric quantitation of stoichiometric DRAQ-5 uptake as an index of cell proliferation in non-infected pericytes treated with vehicle (Vh), insulin, and dorsomorphin (AMPK-). (i to k), average ± SEM, n = 6, p vs. WT.
    Figure Legend Snippet: Occludin influences energetic processes by controlling AMPK activation . (a) SPRICE quantitation of active (phospho) AMPK in occludin-deficient (OCC−), occludin overexpressing (OCC+), and control (SCR) pericytes. Values represent the ratio of phosphorylated vs. the respective total AMPK expression, normalized against DRAQ-5. (b) ATP content in pericytes treated with AICAR (AMPK activator, AMPK+) or dorsomorphin (AMPK inhibitor, AMPK−). (c) ATP quantitation in occludin-deficient (OCC−) or control (SCR) pericytes treated or not with AICAR or dorsomorphin as in (c). (d) Fluorospectrometric quantitation of 2-NBDG taken up by WT-pericytes treated with dorsomorphin (AMPK-) and/or insulin. (e) Similar 2-NBDG quantitation in occludin overexpressing (OCC+) pericytes treated with dorsomorphin and/or insulin. Graphs show average percentual difference over WT pericytes treated with PBS (Vh), represented by the horizontal axis at 0, SEM, n =6, p vs. WT. (f) Representative confocal microscopy images of WT pericytes immunostained for the lysosomal marker LAMP1 (green) and occludin (red). Nu: Cell nucleus. (g) Representative confocal microscopy images of HIV-infected pericytes 24 h post-infection (occludin depletion phase), immunostained as in (f). (h) Representative confocal microscopy images of HIV-infected pericytes 96 h post-infection (occludin recovery phase) and immunostained as in (f) and (g). Non-infected (NI) and HIV-infected pericytes at different time points post infection were analyzed by SPRICE to quantify (i) 2-NBDG uptake and (j) active AMPK (graph shows the ratio between phosphorylated and total AMPK levels). (k) Fluorospectrometric quantitation of stoichiometric DRAQ-5 uptake as an index of cell proliferation in non-infected pericytes treated with vehicle (Vh), insulin, and dorsomorphin (AMPK-). (i to k), average ± SEM, n = 6, p vs. WT.

    Techniques Used: Activation Assay, Quantitation Assay, Expressing, Confocal Microscopy, Marker, Infection

    Related Articles

    Cell Culture:

    Article Title: Electrophysiological maturation and increased excitability of human iPSC-derived neurons in HTR2A variant-related sleep bruxism
    Article Snippet: .. Neuronal differentiation was performed as previously described ( ) but with minor modifications. hiPSC lines were dissociated to form embryoid bodies and cultured on mitomycin C-treated SNL murine fibroblast feeder cells in standard human embryonic stem cell culture (hESC) medium (DMEM/F12; Sigma Aldrich, St. Louis, MO, USA) containing 20% knockout serum replacement (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine, 0.1 mM non-essential amino acids (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Sigma Aldrich), 0.5% penicillin/streptomycin, and 4 ng/mL fibroblast growth factor 2 (FGF-2; PeproTech, Rocky Hill, NJ, USA) in an atmosphere containing 3% CO2 ; the medium was changed every other day. hiPSCs were pretreated for 5 days with 3 μM SB431542 (Tocris, Bristol, UK), 3 μM dorsomorphin (Sigma Aldrich), and 3 μM CHIR99021 (ReproCELL, Kanagawa, Japan). ..

    Stem Cell Culture:

    Article Title: Electrophysiological maturation and increased excitability of human iPSC-derived neurons in HTR2A variant-related sleep bruxism
    Article Snippet: .. Neuronal differentiation was performed as previously described ( ) but with minor modifications. hiPSC lines were dissociated to form embryoid bodies and cultured on mitomycin C-treated SNL murine fibroblast feeder cells in standard human embryonic stem cell culture (hESC) medium (DMEM/F12; Sigma Aldrich, St. Louis, MO, USA) containing 20% knockout serum replacement (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine, 0.1 mM non-essential amino acids (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Sigma Aldrich), 0.5% penicillin/streptomycin, and 4 ng/mL fibroblast growth factor 2 (FGF-2; PeproTech, Rocky Hill, NJ, USA) in an atmosphere containing 3% CO2 ; the medium was changed every other day. hiPSCs were pretreated for 5 days with 3 μM SB431542 (Tocris, Bristol, UK), 3 μM dorsomorphin (Sigma Aldrich), and 3 μM CHIR99021 (ReproCELL, Kanagawa, Japan). ..

    Knock-Out:

    Article Title: Electrophysiological maturation and increased excitability of human iPSC-derived neurons in HTR2A variant-related sleep bruxism
    Article Snippet: .. Neuronal differentiation was performed as previously described ( ) but with minor modifications. hiPSC lines were dissociated to form embryoid bodies and cultured on mitomycin C-treated SNL murine fibroblast feeder cells in standard human embryonic stem cell culture (hESC) medium (DMEM/F12; Sigma Aldrich, St. Louis, MO, USA) containing 20% knockout serum replacement (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine, 0.1 mM non-essential amino acids (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Sigma Aldrich), 0.5% penicillin/streptomycin, and 4 ng/mL fibroblast growth factor 2 (FGF-2; PeproTech, Rocky Hill, NJ, USA) in an atmosphere containing 3% CO2 ; the medium was changed every other day. hiPSCs were pretreated for 5 days with 3 μM SB431542 (Tocris, Bristol, UK), 3 μM dorsomorphin (Sigma Aldrich), and 3 μM CHIR99021 (ReproCELL, Kanagawa, Japan). ..

    Incubation:

    Article Title: An AMP-activated protein kinase complex with two distinctive alpha subunits is involved in nutritional stress responses in Trypanosoma cruzi
    Article Snippet: .. For Dorsomorphin (CC) (#P5499, Sigma Aldrich, St. Louis, Missouri, United States or ab120843, Abcam, Cambridge, United Kingdom) treatment protein extracts were incubated in an ice bath with the 1 μM final concentration of the inhibitor for 10 min previous to the addition of the mix containing the SAMS. ..

    Concentration Assay:

    Article Title: An AMP-activated protein kinase complex with two distinctive alpha subunits is involved in nutritional stress responses in Trypanosoma cruzi
    Article Snippet: .. For Dorsomorphin (CC) (#P5499, Sigma Aldrich, St. Louis, Missouri, United States or ab120843, Abcam, Cambridge, United Kingdom) treatment protein extracts were incubated in an ice bath with the 1 μM final concentration of the inhibitor for 10 min previous to the addition of the mix containing the SAMS. ..

    Blocking Assay:

    Article Title: Pharyngeal pouches provide a niche microenvironment for arch artery progenitor specification
    Article Snippet: .. To block BMP signaling, embryos were treated with DMH1 (10 μM; D8946, Sigma) or dorsomorphin (10 μM; P5499, Sigma) under dark conditions. ..

    Recombinant:

    Article Title: TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy.
    Article Snippet: .. Bafilomycin A1 (Alfa Aesar, J61835), TNF Recombinant Mouse Protein (Thermo Scientific, PMC3014), active GST-HsRIPK3 (Sigma-Aldrich, SRP5316), necrostatin-1 (Enzo Life Sciences, BML-AP309-0020), GSK’872 (Calbiochem, 530389), MK8722 (MedChemExpress, HY111363), PF739 (AOBIOUS, AOB33584), Alkaline Phosphatase (Thermo Fisher, EF0651), Q-VD-OPh (Selleck Chemicals, S7311), z-VAD-FMK (Selleck Chemicals, S7023), ProLong® Gold Antifade Reagent with DAPI (Cell Signaling Technology, 8961), LipofectamineTM RNAiMAX Transfection Reagent (Thermo Fisher, 13778150; for siRNA transfection), FuGENE® 6 (Promega, E2692; for transfection of Plat-E and HEK293), Lipofectamine™2000 (Thermo Fisher, 11668027; for transfection of L929), Magic Red Cathepsin-B/L Assay (ImmunoChemistry Technologies, 937/941), Dorsomorphin (PRKAA1/2 inhibitor; Sigma-Aldrich, P5499), SAR405 (Selleck Chemicals, S7682). ..

    Transfection:

    Article Title: TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy.
    Article Snippet: .. Bafilomycin A1 (Alfa Aesar, J61835), TNF Recombinant Mouse Protein (Thermo Scientific, PMC3014), active GST-HsRIPK3 (Sigma-Aldrich, SRP5316), necrostatin-1 (Enzo Life Sciences, BML-AP309-0020), GSK’872 (Calbiochem, 530389), MK8722 (MedChemExpress, HY111363), PF739 (AOBIOUS, AOB33584), Alkaline Phosphatase (Thermo Fisher, EF0651), Q-VD-OPh (Selleck Chemicals, S7311), z-VAD-FMK (Selleck Chemicals, S7023), ProLong® Gold Antifade Reagent with DAPI (Cell Signaling Technology, 8961), LipofectamineTM RNAiMAX Transfection Reagent (Thermo Fisher, 13778150; for siRNA transfection), FuGENE® 6 (Promega, E2692; for transfection of Plat-E and HEK293), Lipofectamine™2000 (Thermo Fisher, 11668027; for transfection of L929), Magic Red Cathepsin-B/L Assay (ImmunoChemistry Technologies, 937/941), Dorsomorphin (PRKAA1/2 inhibitor; Sigma-Aldrich, P5499), SAR405 (Selleck Chemicals, S7682). ..

    Transferring:

    Article Title: The Amyloid Precursor Protein regulates human cortical neurogenesis
    Article Snippet: .. After 24 hr, spheroids from each microwell were collected by firmly pipetting medium (with a cut end of a P1000 tip) in the well up and down and transferring it into Corning® non-treated culture dishes (Merck, CLS430591-500EA) in TeSR™-E6 (StemCell Technologies, #05946) supplemented with two inhibitors of the SMAD signalling pathway, dorsomorphin (2.5 µM, Sigma-Aldrich, P5499) and SB-431542 (10 µM, Abcam, ab120163). ..

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  • 97
    Millipore dorsomorphin
    Effects of <t>dorsomorphin</t> on Salmonella -induced changes in hepcidin expression and serum iron levels.
    Dorsomorphin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dorsomorphin/product/Millipore
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    99
    Millipore ampk inhibitors
    AMP-activated protein kinase <t>(AMPK)</t> is involved in resistin-induced matrix metalloproteinase (MMP-2) expression and cell migration JJ012 and SW1353 cells were pre-treated with Ara A (0.5 mM) and compound C (10 μM) for 30 min or pre-transfected with control, AMPKα1, or AMPKα2 siRNA for 24 h, and subsequently stimulated with resistin (3 ng/ml) for 24 h. In vitro migration, MMP-2 protein expression (JJ012 cells), and MMP-2 mRNA expression were measured by (A) Transwell assays, (B) enzyme-linked immunosorbent assay (ELISA), and (C) real-time quantitative polymerase chain reaction (RT-qPCR), respectively. Next, the JJ012 and SW1353 cells were pre-treated with SB203580 (10 μM) for 30 min or pre-transfected with control or <t>p38</t> siRNA for 24 h, and subsequently stimulated with resistin (3 ng/ml) for 24 h. In vitro migration, MMP-2 protein expression (JJ012 cells), and MMP-2 mRNA expression were measured by (D) Transwell assays, (E) ELISA, and (F) RT-qPCR, respectively. (G) The JJ012 cells were incubated with resistin for the indicated time intervals, and the p-AMPK and p38 expression were examined by western blot. (H) The JJ012 cells were pre-treated for 30 min with Ara A and compound C, or SB203580 followed by stimulation with resistin. The p-p38 and p-AMPK expression were measured by western blot. The results are expressed as mean ± SEM. *, P
    Ampk Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of dorsomorphin on Salmonella -induced changes in hepcidin expression and serum iron levels.

    Journal: The Journal of Clinical Investigation

    Article Title: Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice

    doi: 10.1172/JCI39939

    Figure Lengend Snippet: Effects of dorsomorphin on Salmonella -induced changes in hepcidin expression and serum iron levels.

    Article Snippet: Treatment with dorsomorphin (12.5 μg/g BW i.p.; Calbiochem) or an equivalent volume of the vehicle DMSO was started at the same time as the streptomycin was administered and was continued twice daily for the duration of the experiment.

    Techniques: Expressing

    Effects of dorsomorphin treatment on Salmonella -induced intestinal inflammation in WT mice.

    Journal: The Journal of Clinical Investigation

    Article Title: Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice

    doi: 10.1172/JCI39939

    Figure Lengend Snippet: Effects of dorsomorphin treatment on Salmonella -induced intestinal inflammation in WT mice.

    Article Snippet: Treatment with dorsomorphin (12.5 μg/g BW i.p.; Calbiochem) or an equivalent volume of the vehicle DMSO was started at the same time as the streptomycin was administered and was continued twice daily for the duration of the experiment.

    Techniques: Mouse Assay

    Structure of the ALK2-FKBP12 complex. A , secondary structure elements are labeled and shown as ribbons . Disordered parts of the L45 loop and A loop are indicated by a thin dashed line . Dorsomorphin is shown in gray stick representation, and Mg 2+ ion ( cyan ) and sulfate molecules ( purple ) are displayed as spheres. Inset box shows the specific interactions of dorsomorphin ( yellow sticks ) with the ATP pocket in ALK2. Hydrogen bonds ( blue dashed lines ) are formed with the hinge amide of His-286 and via a water molecule to Glu-248 (α C ). The planarity of the phenyl ring is restricted by the close packing of Val-214 and Gly-289 ( dashed gray lines). B , FKBP12 binding is dominated by insertion of the αGS2 helix into the central FK506-binding pocket of FKBP12. Here, the ALK5 residues Leu-195–Leu-196 are replaced by ALK2 Phe-198–Leu-199 resulting in a subtle shift in the complex interface. The hydrophobic contact surface in FKBP12 is colored yellow. C , | F o | − | F c | omit map contoured at 3σ ( green mesh ) for the bound dorsomorphin ligand ( yellow sticks ).

    Journal: The Journal of Biological Chemistry

    Article Title: Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva *

    doi: 10.1074/jbc.M112.365932

    Figure Lengend Snippet: Structure of the ALK2-FKBP12 complex. A , secondary structure elements are labeled and shown as ribbons . Disordered parts of the L45 loop and A loop are indicated by a thin dashed line . Dorsomorphin is shown in gray stick representation, and Mg 2+ ion ( cyan ) and sulfate molecules ( purple ) are displayed as spheres. Inset box shows the specific interactions of dorsomorphin ( yellow sticks ) with the ATP pocket in ALK2. Hydrogen bonds ( blue dashed lines ) are formed with the hinge amide of His-286 and via a water molecule to Glu-248 (α C ). The planarity of the phenyl ring is restricted by the close packing of Val-214 and Gly-289 ( dashed gray lines). B , FKBP12 binding is dominated by insertion of the αGS2 helix into the central FK506-binding pocket of FKBP12. Here, the ALK5 residues Leu-195–Leu-196 are replaced by ALK2 Phe-198–Leu-199 resulting in a subtle shift in the complex interface. The hydrophobic contact surface in FKBP12 is colored yellow. C , | F o | − | F c | omit map contoured at 3σ ( green mesh ) for the bound dorsomorphin ligand ( yellow sticks ).

    Article Snippet: The ALK2-FKBP12 complex was preincubated with 1 mm dorsomorphin (Calbiochem) at a protein concentration of 10 mg/ml and crystallized using a precipitant containing 30% PEG3350, 0.25 m ammonium sulfate, and 0.1 m BisTris, pH 6.0.

    Techniques: Labeling, Binding Assay

    BMP/Smad1/5/8 signaling pathway regulates Msx1 expression. A , immunofluorescence shows expressions of pp38 ( panel a ), pERK ( panel b ), and pJNK ( panel c ) in E13.5 molar tooth germ. B , in situ hybridization shows Msx1 expression in E12.5 molar germs after 12 h or 1 day in organ culture in the presence of DMSO ( panels a and d ), dorsomorphin ( panels b and e ), or SB203580 and U0126 ( panels c and f ). Panels a–c , whole mount in situ hybridization; panels d–f , tissue section in situ hybridization. C , a Western blot assay shows attenuation of Msx1 expression by dorsomorphin but not SB203580 and U0126 in BMP-induced primary dental mesenchymal cells from E13.5 embryos. GAPDH was used as internal control. Quantitative analysis is shown in the lower part. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: An Atypical Canonical Bone Morphogenetic Protein (BMP) Signaling Pathway Regulates Msh Homeobox 1 (Msx1) Expression during Odontogenesis *

    doi: 10.1074/jbc.M114.600064

    Figure Lengend Snippet: BMP/Smad1/5/8 signaling pathway regulates Msx1 expression. A , immunofluorescence shows expressions of pp38 ( panel a ), pERK ( panel b ), and pJNK ( panel c ) in E13.5 molar tooth germ. B , in situ hybridization shows Msx1 expression in E12.5 molar germs after 12 h or 1 day in organ culture in the presence of DMSO ( panels a and d ), dorsomorphin ( panels b and e ), or SB203580 and U0126 ( panels c and f ). Panels a–c , whole mount in situ hybridization; panels d–f , tissue section in situ hybridization. C , a Western blot assay shows attenuation of Msx1 expression by dorsomorphin but not SB203580 and U0126 in BMP-induced primary dental mesenchymal cells from E13.5 embryos. GAPDH was used as internal control. Quantitative analysis is shown in the lower part. **, p

    Article Snippet: For small molecule inhibition experiments, dorsomorphin (Sigma) or SB203580 (Cell Signaling) and U0126 (Cell Signaling) were added into the medium, respectively, at a final concentration of 20 μ m .

    Techniques: Expressing, Immunofluorescence, In Situ Hybridization, Organ Culture, Western Blot

    AMP-activated protein kinase (AMPK) is involved in resistin-induced matrix metalloproteinase (MMP-2) expression and cell migration JJ012 and SW1353 cells were pre-treated with Ara A (0.5 mM) and compound C (10 μM) for 30 min or pre-transfected with control, AMPKα1, or AMPKα2 siRNA for 24 h, and subsequently stimulated with resistin (3 ng/ml) for 24 h. In vitro migration, MMP-2 protein expression (JJ012 cells), and MMP-2 mRNA expression were measured by (A) Transwell assays, (B) enzyme-linked immunosorbent assay (ELISA), and (C) real-time quantitative polymerase chain reaction (RT-qPCR), respectively. Next, the JJ012 and SW1353 cells were pre-treated with SB203580 (10 μM) for 30 min or pre-transfected with control or p38 siRNA for 24 h, and subsequently stimulated with resistin (3 ng/ml) for 24 h. In vitro migration, MMP-2 protein expression (JJ012 cells), and MMP-2 mRNA expression were measured by (D) Transwell assays, (E) ELISA, and (F) RT-qPCR, respectively. (G) The JJ012 cells were incubated with resistin for the indicated time intervals, and the p-AMPK and p38 expression were examined by western blot. (H) The JJ012 cells were pre-treated for 30 min with Ara A and compound C, or SB203580 followed by stimulation with resistin. The p-p38 and p-AMPK expression were measured by western blot. The results are expressed as mean ± SEM. *, P

    Journal: Oncotarget

    Article Title: Resistin promotes tumor metastasis by down-regulation of miR-519d through the AMPK/p38 signaling pathway in human chondrosarcoma cells

    doi:

    Figure Lengend Snippet: AMP-activated protein kinase (AMPK) is involved in resistin-induced matrix metalloproteinase (MMP-2) expression and cell migration JJ012 and SW1353 cells were pre-treated with Ara A (0.5 mM) and compound C (10 μM) for 30 min or pre-transfected with control, AMPKα1, or AMPKα2 siRNA for 24 h, and subsequently stimulated with resistin (3 ng/ml) for 24 h. In vitro migration, MMP-2 protein expression (JJ012 cells), and MMP-2 mRNA expression were measured by (A) Transwell assays, (B) enzyme-linked immunosorbent assay (ELISA), and (C) real-time quantitative polymerase chain reaction (RT-qPCR), respectively. Next, the JJ012 and SW1353 cells were pre-treated with SB203580 (10 μM) for 30 min or pre-transfected with control or p38 siRNA for 24 h, and subsequently stimulated with resistin (3 ng/ml) for 24 h. In vitro migration, MMP-2 protein expression (JJ012 cells), and MMP-2 mRNA expression were measured by (D) Transwell assays, (E) ELISA, and (F) RT-qPCR, respectively. (G) The JJ012 cells were incubated with resistin for the indicated time intervals, and the p-AMPK and p38 expression were examined by western blot. (H) The JJ012 cells were pre-treated for 30 min with Ara A and compound C, or SB203580 followed by stimulation with resistin. The p-p38 and p-AMPK expression were measured by western blot. The results are expressed as mean ± SEM. *, P

    Article Snippet: AMPK inhibitors (Ara A and compound C), p38 inhibitor (SB203580), MMP-2 inhibitor, and human MMP-2 ELISA kits were purchased from Calbiochem (San Diego, CA).

    Techniques: Expressing, Migration, Acetylene Reduction Assay, Transfection, In Vitro, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Incubation, Western Blot

    Resistin promotes cell migration and matrix metalloproteinase (MMP-2) expression by down-regulating microRNA (miR)-519d expression (A) The JJ012 and SW1353 cells were transfected with miR-519d mimic or inhibitor for 24 h, and cell migration ability was examined by Transwell assay. (B) The JJ012 cells were transfected with an miR-519d mimic or inhibitor for 24 h, and MMP-2 expression was examined by western blot (upper panel), and enzyme-linked immunosorbent assay (lower panel). (C) The JJ012 and SW1353 cells were incubated with resistin (0.3–3 ng/ml) for 24 h, and miR-519d expression was detected by real-time quantitative polymerase chain reaction. (D) Sequences of miR-519d and the potential miR-519d binding site at the MMP-2 3′ untranslated region (3′ UTR; WT-MMP-2 3′ UTR). Also shown are the nucleotides mutated in the MMP-2 3′UTR mutant (MUT-MMP2 3′ UTR). (E) The JJ012 and SW1353 cells were transfected with a wild-type or mutant MMP-2 3′ UTR luciferase plasmid for 24 h followed by stimulation with resistin (0.3-3 ng/ml) for 24 h, and the relative luciferase activity was measured. The JJ012 cells were pre-treated with AMPK and p38 inhibitors for 30 min or pre-transfected with specific siRNAs for 24 h followed by stimulation with resistin (3 ng/ml) for 24 h; and the (F, G) miR-519d expression and (H) MMP-2 3′ UTR activity were examined. The results are expressed as mean ± SEM. *, P

    Journal: Oncotarget

    Article Title: Resistin promotes tumor metastasis by down-regulation of miR-519d through the AMPK/p38 signaling pathway in human chondrosarcoma cells

    doi:

    Figure Lengend Snippet: Resistin promotes cell migration and matrix metalloproteinase (MMP-2) expression by down-regulating microRNA (miR)-519d expression (A) The JJ012 and SW1353 cells were transfected with miR-519d mimic or inhibitor for 24 h, and cell migration ability was examined by Transwell assay. (B) The JJ012 cells were transfected with an miR-519d mimic or inhibitor for 24 h, and MMP-2 expression was examined by western blot (upper panel), and enzyme-linked immunosorbent assay (lower panel). (C) The JJ012 and SW1353 cells were incubated with resistin (0.3–3 ng/ml) for 24 h, and miR-519d expression was detected by real-time quantitative polymerase chain reaction. (D) Sequences of miR-519d and the potential miR-519d binding site at the MMP-2 3′ untranslated region (3′ UTR; WT-MMP-2 3′ UTR). Also shown are the nucleotides mutated in the MMP-2 3′UTR mutant (MUT-MMP2 3′ UTR). (E) The JJ012 and SW1353 cells were transfected with a wild-type or mutant MMP-2 3′ UTR luciferase plasmid for 24 h followed by stimulation with resistin (0.3-3 ng/ml) for 24 h, and the relative luciferase activity was measured. The JJ012 cells were pre-treated with AMPK and p38 inhibitors for 30 min or pre-transfected with specific siRNAs for 24 h followed by stimulation with resistin (3 ng/ml) for 24 h; and the (F, G) miR-519d expression and (H) MMP-2 3′ UTR activity were examined. The results are expressed as mean ± SEM. *, P

    Article Snippet: AMPK inhibitors (Ara A and compound C), p38 inhibitor (SB203580), MMP-2 inhibitor, and human MMP-2 ELISA kits were purchased from Calbiochem (San Diego, CA).

    Techniques: Migration, Expressing, Transfection, Transwell Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Real-time Polymerase Chain Reaction, Binding Assay, Mutagenesis, Luciferase, Plasmid Preparation, Activity Assay