Journal: DARU Journal of Pharmaceutical Sciences

Article Title: Evaluation of antitumor activity of a TGF-beta receptor I inhibitor (SD-208) on human colon adenocarcinoma

doi: 10.1186/2008-2231-22-47

Figure Lengend Snippet: Colon adenocarcinoma (grade IV) stained immunohistochemically with anti-Ki-67. Positive immunoreactive showing brown nuclear expression of Ki-67 with a large number (~80%) of stained nuclei reflecting active cellular proliferation in the treated nude mice with SD-208 (A) and control (B) : Immunohischemical assay showed no significant difference between tests and controls in terms of cellular proliferation (P > 0.05).

Article Snippet: Chemical description and biological activity TGβRI kinase inhibitor, SD-208 SD-208 (Sigma Aldrich; Belgium) is a selective and orally active pyridopyrimidine type TGβRI kinase inhibitor with an IC50 of approximately 35 nmol/L against TβRI kinase activity in vitro .

Techniques: Staining, Expressing, Mouse Assay

Journal: DARU Journal of Pharmaceutical Sciences

Article Title: Evaluation of antitumor activity of a TGF-beta receptor I inhibitor (SD-208) on human colon adenocarcinoma

doi: 10.1186/2008-2231-22-47

Figure Lengend Snippet: Effect of SD-208 on the cell growth and proliferation of the SW-48 cells. SW-48 cells were treated by 0.5, 1 and 2 μM for 48 h. Cell proliferation was examined by MTT and BrdU assays as described in methods. A : MTT assay of SW-48 cells after treatment with SD-208 in comparison with controls (untreated and treated with DMSO). B : BrdU assay of SW-48 cells after treatment with SD-208 comparison with controls (untreated and treated with DMSO). All data are reported as the percentage change in comparison with the controls, which were arbitrarily assigned 100% cell proliferation. Analysis of one-way ANOVA was used to compare the cell proliferation of SW-48 cells in different concentrations of SD-208 to control. P value

Article Snippet: Chemical description and biological activity TGβRI kinase inhibitor, SD-208 SD-208 (Sigma Aldrich; Belgium) is a selective and orally active pyridopyrimidine type TGβRI kinase inhibitor with an IC50 of approximately 35 nmol/L against TβRI kinase activity in vitro .

Techniques: MTT Assay, BrdU Staining

Journal: DARU Journal of Pharmaceutical Sciences

Article Title: Evaluation of antitumor activity of a TGF-beta receptor I inhibitor (SD-208) on human colon adenocarcinoma

doi: 10.1186/2008-2231-22-47

Figure Lengend Snippet: A representative of colorectal adenocarcinoma model. Tumor implantation; athymic nude mice implanted by SW-48 cell line, the cells were grown as tumor xenografts after 10 days. Cancer-bearing nude mice were treated with 50 mg/kg/day of SD-208 (A) or without SD-208 (B) .

Article Snippet: Chemical description and biological activity TGβRI kinase inhibitor, SD-208 SD-208 (Sigma Aldrich; Belgium) is a selective and orally active pyridopyrimidine type TGβRI kinase inhibitor with an IC50 of approximately 35 nmol/L against TβRI kinase activity in vitro .

Techniques: Tumor Implantation, Mouse Assay

Journal: DARU Journal of Pharmaceutical Sciences

Article Title: Evaluation of antitumor activity of a TGF-beta receptor I inhibitor (SD-208) on human colon adenocarcinoma

doi: 10.1186/2008-2231-22-47

Figure Lengend Snippet: A representative results of pathological examinations of nude mice tumors with or without SD-208 treatment. A : tumors of nude mice treated with 50 mg/kg/day stained with H E. B : H E staining of tumor tissues of control. No significant difference histologically was observed (P > 0.05).

Article Snippet: Chemical description and biological activity TGβRI kinase inhibitor, SD-208 SD-208 (Sigma Aldrich; Belgium) is a selective and orally active pyridopyrimidine type TGβRI kinase inhibitor with an IC50 of approximately 35 nmol/L against TβRI kinase activity in vitro .

Techniques: Mouse Assay, Staining

Journal: DARU Journal of Pharmaceutical Sciences

Article Title: Evaluation of antitumor activity of a TGF-beta receptor I inhibitor (SD-208) on human colon adenocarcinoma

doi: 10.1186/2008-2231-22-47

Figure Lengend Snippet: Colon adenocarcinoma (grade IV) stained immunohistochemically with anti-CD34. The paraffin embedded sections of tumor tissues were stained with anti-CD34 antibody and positive immunoreactive indicating brown cytoplasmic membrane of endothelial cells and marked microvessel's proliferation. Microvessel density (MVD) was ~40 microvessel/mm 2 . SD-208-treated tumors (A) vs controls (B) revealed no significant immunohistologically differences (P > 0.05). Note the prominent vascularity. Arrows indicate CD34 staining of the cytoplasmic membrane.

Article Snippet: Chemical description and biological activity TGβRI kinase inhibitor, SD-208 SD-208 (Sigma Aldrich; Belgium) is a selective and orally active pyridopyrimidine type TGβRI kinase inhibitor with an IC50 of approximately 35 nmol/L against TβRI kinase activity in vitro .

Techniques: Staining

Journal: DARU Journal of Pharmaceutical Sciences

Article Title: Evaluation of antitumor activity of a TGF-beta receptor I inhibitor (SD-208) on human colon adenocarcinoma

doi: 10.1186/2008-2231-22-47

Figure Lengend Snippet: Continuous culture of SW-48 cells and treatment with SD-208. The cells were grown as monolayer epithelial-like morphology. SW-48 cells after treatment by DMSO alone as control (A) , SD-208 concentrations 0.5 μM (B) , 1 μM (C) and 2 μM (D) .

Article Snippet: Chemical description and biological activity TGβRI kinase inhibitor, SD-208 SD-208 (Sigma Aldrich; Belgium) is a selective and orally active pyridopyrimidine type TGβRI kinase inhibitor with an IC50 of approximately 35 nmol/L against TβRI kinase activity in vitro .

Techniques:

Journal: Journal of Cell Science

Article Title: Canonical Notch signaling in the developing lung is required for determination of arterial smooth muscle cells and selection of Clara versus ciliated cell fate

doi: 10.1242/jcs.058669

Figure Lengend Snippet: Notch signaling contributes to mesothelial EMT, and Notch deficiency can be rescued by TGF β in this process. Lung mesothelium in explants from E14.5 embryos was labeled with CCFSE and visualized on frozen sections using an EGFP filter as indicated. ( A ) At day 0 of culture, only surface cells were positive for CCFSE. ( B ) After two days in culture, some CCFSE-positive cells were observed in the mesenchyme. ( C , D ) Culture in 5 ng/ml TGFβ increased the migration of CCFSE-labeled cells (white arrowheads). ( E ) Migration was inhibited by 5 μM SD208, a TGFβ inhibitor. ( F-I ) Culture in DAPT containing medium decreased CCFSE-labeled cells that migrated from the surface in a dose dependent manner (G-H, white arrowheads). TGFβ allowed some migration in the presence of 5 μM DAPT (I). ( J , K ) TAT-Cre protein treatment for E14.5 Rosa YFP (J) and Rosa N1ICD-GFP (K) lungs activated the expression of N1ICD-GFP or YFP reporter in mesothelial cells and was followed by in vitro culture for 3 days. Migrated mesothelial cells were detected by staining with anti-GFP antibody on frozen sections. ( L ) The number of migrated mesothelial cells was counted and classified by distance from the surface for each genotype. The number of cells and their percentage in the total population are shown. Scale bars: 50 μm.

Article Snippet: TGFβ (2 μg/ml; R & D Systems), SD208 (5 mM; Calbiochem) or DAPT (1 mM; Calbiochem) was added to culture medium to appropriate final concentrations (described in each figure legend).

Techniques: Labeling, Migration, Expressing, In Vitro, Staining

Journal: Acta neuropathologica

Article Title: Changes in proteome solubility indicate widespread proteostatic disruption in mouse models of neurodegenerative disease

doi: 10.1007/s00401-018-1895-y

Figure Lengend Snippet: Analysis of cellular protein co-sedimentation with tau aggregates in mouse N2a cell models. (a) Average DOC-insoluble spectral counts in the seeded N2a cells for a subset of proteins that were identified as aberrantly fractionating to SDS-insoluble fractions from the brains of rTg4510 mice. (b) Average PBS-soluble spectral counts for the same proteins displayed in panel (a), indicating the level of detectability of the proteins in PBS-soluble fractions. (c) Spectral count data for the small number of proteins that meet criteria for over-representation in DOC-insoluble fractions from N2a cells seeded for tau aggregation. Proteins were accepted if they (i) achieved at least a 3-fold increase in DOC-insoluble spectra from untransfected to tau seeded cells for at least 2 out of 3 experimental replicates and (ii) during instances where untransfected cells yielded 0 peptides for any given proteins, the tau seeded condition yielded a significant G-test (p

Article Snippet: 30 μL of SDS-insoluble or 5 μL of PBS-soluble protein fractions from each animal were loaded onto a Criterion 4–20% tris-glycine gel (Bio-Rad, Hercules, CA).

Techniques: Sedimentation, Mouse Assay

Journal: Journal of Cell Science

Article Title: Dynamic remodeling of the actin cytoskeleton by FMNL1? is required for structural maintenance of the Golgi complex

doi: 10.1242/jcs.083725

Figure Lengend Snippet: Expression of diaphanous and FMNL formins in a panel of cancer cell lines. Clarified cell lysates were prepared from Jurkat, HeLa and pancreatic cancer cell lines. Equivalent amounts of protein were separated by SDS-PAGE and immunoblotted using ( A ) anti-FMNL1,

Article Snippet: For experiments in which we immunoblotted for β-actin, lysates were prepared in 1% NP40, 0.1% SDS (Bio-Rad) and 0.5% deoxychocolate (Sigma), supplemented as described above.

Techniques: Expressing, SDS Page

Journal: Bioengineering

Article Title: Decellularized Human Umbilical Artery Used as Nerve Conduit

doi: 10.3390/bioengineering5040100

Figure Lengend Snippet: Contact cytotoxicity assay. ( A ) MSCs cultured under normal conditions (negative control group); ( B ) MSCs were co-cultured with decellularized hUA, ( C ) MSCs with SDS (positive control group). Black arrows indicate the contact of MSCs with the segments of decellularized hUA. Original magnification 10×, scale bars 100 μm.

Article Snippet: The hUA were further incubated in SDS solution (1.8 mM SDS (Sigma-Aldrich, Darmstadt, Germany), 1 M NaCl, and 25 mM EDTA in PBS 1×) at pH 7.5 for 24 h, followed by 3 washes for 5 min in PBS 1×, to completely remove the detergent.

Techniques: Cytotoxicity Assay, Cell Culture, Negative Control, Positive Control

Journal: Journal of Neuroinflammation

Article Title: TGFβRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment

doi: 10.1186/s12974-019-1664-4

Figure Lengend Snippet: Blocking TGFβR1 with SD208 decreases GFAP expression in rats infused with astrocytes expressing Nef. a Representative tissue sections immunostained for astrocyte activation marker, GFAP. Nef+placebo (left panel), n = 5, and Nef+SD208 (right panel), n = 3. b Densitometric analysis was used to quantify the intensity of GFAP staining, * p

Article Snippet: Similarly, we found that neither SD208 nor the methylcellulose vehicle cause changes in weight, distance traveled, velocity, or anxiety levels, thus eliminating these variables as possible explanations for differences in learning after SD208 treatment.

Techniques: Blocking Assay, Expressing, Activation Assay, Marker, Staining

Journal: Journal of Neuroinflammation

Article Title: TGFβRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment

doi: 10.1186/s12974-019-1664-4

Figure Lengend Snippet: SD208 does not affect weight, motor performance, or anxiety levels. a Percent of weight change during the administration of the treatment. b Distance traveled during the testing phase. c Velocity of the rats during the learning phase. d Time spent in the center of the area during the testing phase (an indicator of anxiety). p > 0.05, n = 7;13

Article Snippet: Similarly, we found that neither SD208 nor the methylcellulose vehicle cause changes in weight, distance traveled, velocity, or anxiety levels, thus eliminating these variables as possible explanations for differences in learning after SD208 treatment.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: TGFβRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment

doi: 10.1186/s12974-019-1664-4

Figure Lengend Snippet: Blocking TGFβR1 with SD208 decreases CD163 expression in rats infused with astrocytes expressing Nef. a Representative tissue sections immunostained for macrophage marker, CD163. Nef+placebo (upper panel), n = 4 and Nef+SD208 (lower panel), n = 5. b Densitometric analysis was used to quantify the intensity of CD163 staining, ** p

Article Snippet: Similarly, we found that neither SD208 nor the methylcellulose vehicle cause changes in weight, distance traveled, velocity, or anxiety levels, thus eliminating these variables as possible explanations for differences in learning after SD208 treatment.

Techniques: Blocking Assay, Expressing, Marker, Staining

Journal: Journal of Neuroinflammation

Article Title: TGFβRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment

doi: 10.1186/s12974-019-1664-4

Figure Lengend Snippet: Blocking TGFβR1 with SD208 decreases CCL2 expression in rats infused with astrocytes expressing Nef. Mean fluorescence intensity and representative rat brain tissue sections immunostained for macrophage chemoattractant marker CCL2 (green): cortex ( a ), dentate gyrus ( b ), CA3 ( c ), and CA1 ( d ). *** p

Article Snippet: Similarly, we found that neither SD208 nor the methylcellulose vehicle cause changes in weight, distance traveled, velocity, or anxiety levels, thus eliminating these variables as possible explanations for differences in learning after SD208 treatment.

Techniques: Blocking Assay, Expressing, Fluorescence, Marker

Journal: Journal of Neuroinflammation

Article Title: TGFβRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment

doi: 10.1186/s12974-019-1664-4

Figure Lengend Snippet: Blocking TGFβR1 with SD208 decreases phospho-SMAD2 in rats infused with astrocytes expressing Nef. a Representative tissue sections immunostained for phospho-SMAD2; Nef+placebo (upper panel) and Nef+SD208 (lower panel). b Densitometric analysis of the hippocampus was used to quantify SMAD2 phosphorylation (right hemisphere: * p

Article Snippet: Similarly, we found that neither SD208 nor the methylcellulose vehicle cause changes in weight, distance traveled, velocity, or anxiety levels, thus eliminating these variables as possible explanations for differences in learning after SD208 treatment.

Techniques: Blocking Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: TGFβRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment

doi: 10.1186/s12974-019-1664-4

Figure Lengend Snippet: Nef-dependent upregulation of CCL2 at 48 h requires TGFβ-1 signaling. a GFP and Nef are expressed strongly for 48 h after transfection of rat primary astrocytes in vitro regardless of treatment with SD208, n = 3. b Quantitative PCR of TGFβ-1 expression in astrocytes expressing GFP or Nef at 24 and 48 h. GFP n = 10; Nef n = 9; * p = 0.03. c Quantitative PCR of CCL2 expression in astrocytes expressing GFP or Nef with or without SD208 at 24 and 48 h: GFP (white bar) n = 10, Nef (black bar) n = 8, and Nef+ SD208 (shaded bar) n = 3; *** p

Article Snippet: Similarly, we found that neither SD208 nor the methylcellulose vehicle cause changes in weight, distance traveled, velocity, or anxiety levels, thus eliminating these variables as possible explanations for differences in learning after SD208 treatment.

Techniques: Transfection, In Vitro, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Neuroinflammation

Article Title: TGFβRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment

doi: 10.1186/s12974-019-1664-4

Figure Lengend Snippet: Nef increases TGFβR1 expression in astrocytes. a Representative images of TGFβR1 expression in astrocytes transfected with GFP or Nef (TGFβRI in red, GFP or Nef in green, DAPI in blue). Additional groups of the transfected cells were treated with SD208. Ten images per treatment from three individual experiments were obtained. b Quantification of mean fluorescent intensity in the nucleus ( p = 0.02) and cytoplasm ( p

Article Snippet: Similarly, we found that neither SD208 nor the methylcellulose vehicle cause changes in weight, distance traveled, velocity, or anxiety levels, thus eliminating these variables as possible explanations for differences in learning after SD208 treatment.

Techniques: Expressing, Transfection

Journal: Journal of Neuroinflammation

Article Title: TGFβRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment

doi: 10.1186/s12974-019-1664-4

Figure Lengend Snippet: SD208 rescues the spatial learning impairment in rats treated with Nef. a A schematic of behavioral model. b Average exploration time of rats from two groups (Nef + placebo, black bar, n = 7; Nef + SD208, white bar, n = 13) for each object during learning phase; c percent exploration time for the test object prior to (open bars) and after (closed bars) moving the location. All error bars represent standard error. * p

Article Snippet: Similarly, we found that neither SD208 nor the methylcellulose vehicle cause changes in weight, distance traveled, velocity, or anxiety levels, thus eliminating these variables as possible explanations for differences in learning after SD208 treatment.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: TGFβRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment

doi: 10.1186/s12974-019-1664-4

Figure Lengend Snippet: Nef does not change the amount of total SMAD4 in astrocytes and neurons. a – d Total SMAD4 expression in astrocytes and neurons show no significant difference between Nef and GFP samples; neither when treated with SD208. e Representative immunoblot of total SMAD4 in astrocytes expressing Nef or GFP confirms total SMAD4 protein expression

Article Snippet: Similarly, we found that neither SD208 nor the methylcellulose vehicle cause changes in weight, distance traveled, velocity, or anxiety levels, thus eliminating these variables as possible explanations for differences in learning after SD208 treatment.

Techniques: Expressing

Journal: The EMBO Journal

Article Title: Tyrosine dephosphorylation is required for Bak activation in apoptosis

doi: 10.1038/emboj.2010.244

Figure Lengend Snippet: Bak undergoes tyrosine dephosphorylated during the initiation of apoptosis. ( A ) 2D gel analysis of Bak from HT1080 cells using narrow-range linear pI gradient IPG-focusing strips and 15% Tris-glycine SDS–PAGE showed partial dephosphorylation after UV treatment, complete dephosphorylation was achieved by λ-phosphatase treatment. ( B ) 2D gel analysis of Bak of mitochondrial extracts from HT1080 cells treated either with ser/thr (PP1) or tyr (YOP) phosphatase, which resulted in new Bak species (arrowed). ( C ) Histograms of FACS analysis of Bak Ab-1-specific fluorescence in control untreated HT1080 cells, HT1080 cells±pre-treatment for 30 min before UV treatment with phophatase inhibitors sodium stibogluconate (SS; 110 μM), phenylarsine oxide (PAO; 5 μM), Cyclosporin A (75 μM) and Calyculin A (2 nM). Samples were analysed 4 h after UV damage. ( D ) Quantification of the increase in Bak Ab-1-specific fluorescence±4 h apoptotic stimuli camptothecin (CPT; 6 μM), Etoposide (EP; 10 μM), Staurosporine (STS; 100 nM) or 5 mJ/cm 2 UV in the presence and absence of tyrosine phoshatase inhibitors 110 μM SS or 5 μM PAO with the y -axis showing levels of Bak-specific fluorescence and the x -axis showing treatment conditions.

Article Snippet: Bound proteins were eluted in denaturing SDS buffer and electrophoresed on 4–12% NuPAGE bis-Tris gels (Invitrogen).

Techniques: Two-Dimensional Gel Electrophoresis, SDS Page, De-Phosphorylation Assay, FACS, Fluorescence, Cycling Probe Technology

Journal: Mediators of Inflammation

Article Title: Costimulation of Murine Osteoblasts with Interferon-γ and Tumor Necrosis Factor-α Induces Apoptosis through Downregulation of Bcl-2 and Release of Cytochrome c from Mitochondria

doi: 10.1155/2018/3979606

Figure Lengend Snippet: Effect of IFN γ and TNF α costimulation on cytochrome c , Bcl-2, and Bax levels in MC3T3-E1 cells. Cells were cultured as described in Figure 4 and treated with 10 ng/ml IFN γ and 5 ng/ml TNF α . Samples were collected at various time points and fractionated into cytosolic and mitochondrial (Mito) fractions, of which 20 μ g protein was subjected to 12% SDS-PAGE and immunoblotted with antibodies to cytochrome c . The mitochondrial fraction was also analyzed by western blot for Bcl-2 and Bax. Results are representative of three independent experiments.

Article Snippet: Western Blot Protein samples obtained as described were subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) using a semidry transfer cell (Bio-Rad).

Techniques: Cell Culture, SDS Page, Western Blot

Journal: Mediators of Inflammation

Article Title: Costimulation of Murine Osteoblasts with Interferon-γ and Tumor Necrosis Factor-α Induces Apoptosis through Downregulation of Bcl-2 and Release of Cytochrome c from Mitochondria

doi: 10.1155/2018/3979606

Figure Lengend Snippet: Overexpression of Bcl-2 attenuates IFN γ - and TNF α -induced cytotoxicity in MC3T3-E1 cells. (a) Western blot for Bcl-2 in cells stably expressing Bcl-2. Total cell lysates were prepared from cells stably transfected with control vector (pcDNA3) or a Bcl-2 expression vector (pCMV-Bcl-2), of which 20 μ g protein was subjected to 12% SDS-PAGE and analyzed with antibodies to Bcl-2. The blot was stripped and reprobed with anti-tubulin to confirm loading of equal amounts of total protein. (b) Effect of IFN γ and TNF α costimulation on the viability of cells stably expressing Bcl-2. Cells stably transfected with control vector or pCMV-Bcl-2 were seeded in 96-well plates, incubated for 5 days to form a confluent monolayer, and costimulated with 10 ng/ml IFN γ and 5 ng/ml TNF α . Cell viability was monitored over time. Data represent the mean ± SEM of three independent experiments. ∗ p

Article Snippet: Western Blot Protein samples obtained as described were subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) using a semidry transfer cell (Bio-Rad).

Techniques: Over Expression, Western Blot, Stable Transfection, Expressing, Transfection, Plasmid Preparation, SDS Page, Incubation

Journal: Oncotarget

Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

doi: 10.18632/oncotarget.23202

Figure Lengend Snippet: Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P

Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

Techniques: Activation Assay, SDS Page

Journal: Oncotarget

Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

doi: 10.18632/oncotarget.23202

Figure Lengend Snippet: Regulatory effect of Scutellarein on cell cycle progression of AGS and SNU484 cells AGS and SNU-484cells were treated with indicated concentrations of scutellarein for 24 h. ( A – B ) Cell cycle distribution was determined by using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).The data were analyzed using CXP Software. ( C–D ) Effect of Scutellarein on cell cycle-related proteins (cyclin B1, CDK1 and CDC25C) expression level in A549 cells. Cells were treated with Scutellarein (0, 25, 50 and 100 μM) for 24 h. Cell lysates were subjected to SDS–PAGE and analyzed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of Scutellarein on expression of cell cycle-related proteins level were represented. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗ P

Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

Techniques: Software, Expressing, SDS Page, Western Blot, Standard Deviation

Journal: Oncotarget

Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

doi: 10.18632/oncotarget.23202

Figure Lengend Snippet: Caspases activation and subsequent cleavage of PARP in scutellarein -treated AGS and SNU-484cells AGS and SNU-484 cells were treated with indicated concentrations of scutellarein for 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of Bax/Bcl-xL ratio, Cl.caspase-9, Cl.caspase-3 and Cl.PARP proteins expressions were expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗∗ P

Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

Techniques: Activation Assay, SDS Page, Standard Deviation

Journal: PLoS ONE

Article Title: Effects of CK2β subunit down-regulation on Akt signalling in HK-2 renal cells

doi: 10.1371/journal.pone.0227340

Figure Lengend Snippet: Phosphorylation of Akt by different forms of CK2. (A) Increasing amounts of CK2α (1.25, 2.5 and 5 ng), CK2α’ (40, 80 and 160 ng) or CK2α2β2 (2.5, 5 and 10 ng), corresponding to equal activity towards CK2-tide peptide, were incubated with 0.3 μg of active Akt1 or 0.3 μg of inactive Akt1, or 1 μg β-casein as a control. In the right panel, 0.3 μg of active form of Akt was incubated with CK2α (1.25 ng), CK2α2β2 (5 ng), CK2α’ (40 ng) or CK2α’2β2 (25 ng) in the presence of 150 mM NaCl. The amount of each enzyme used was equally active against the CK2 specific peptide (CK2-tide: RRRADDSDDDDD ) as determined by kinase activity assays (not shown). Upon radioactive phosphorylation, proteins were separated by SDS-PAGE. A representative digital autoradiography of the dried gel is shown. The bar graph on the right shows the relative quantitation of the bands (means ± SEM, n = 3), performed by analysis with CyclonePlus Storage Phosphor System, (PerkinElmer); the unit amounts of each isoform are indicated, and activity is reported as % of that measured with 5 units of CK2α. (B) shCV HK-2 and shCK2β HK-2 were transiently transfected with CK2β pCMV-HA vector. Representative Western blot analysis with the indicated antibodies is shown. β-actin was used as a loading control. Quantification (p-Akt(S129)/ AKT) is shown on the bar graph on the right (means ± SEM, n = 2).

Article Snippet: Equal amounts of proteins were loaded in 10% SDS polyacrylamide gels (SDS-PAGE), subjected to electrophoresis and subsequently electrotransfered to polyvinylidene fluoride membranes (PVDF, Immobilion P, Millipore).

Techniques: Activity Assay, Incubation, SDS Page, Autoradiography, Quantitation Assay, Transfection, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Gene Activation through the Modulation of Nucleoid Structures by a Horizontally Transferred Regulator, Pch, in Enterohemorrhagic Escherichia coli

doi: 10.1371/journal.pone.0149718

Figure Lengend Snippet: Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by SDS-PAGE and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.

Article Snippet: The proteins were separated by SDS-polyacrylamide (12% or 10%) gel electrophoresis (SDS-PAGE) and were transferred onto an Immobilon membrane (Millipore).

Techniques: Incubation, Magnetic Beads, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Journal: Molecular Biology of the Cell

Article Title: The Roles of Two Distinct Regions of PINCH-1 in the Regulation of Cell Attachment and Spreading

doi: 10.1091/mbc.E10-05-0459

Figure Lengend Snippet: Association of Rsu-1 and PINCH-1. (A) 293T and HA-PINCH-1-expressing 293T cells were lysed and affinity precipitated using anti-HA affinity matrix. The precipitates were separated by SDS-PAGE and silver stained. Each band was excised and identified by LC-MS/MS. (B) 293T cells were transfected with HA-tagged FL, ΔLIM1, ΔC, and LIM1 and immunoprecipitated with anti-HA antibody. Immunoprecipitates were probed for Rsu-1 and HA. (C) 293T cells were transfected with myc-tagged Rsu-1, and cell lysates were affinity precipitated with GST and fusion proteins of GST and the indicated fragments. Precipitates were subjected to immunoblotting with anti-myc antibody. The lower panel shows Coomassie blue staining of GST fusion proteins. LIM5: aa251–304, LIM5+C: aa251–325, C: aa305–325. (D) Schematic representation of Rsu-1 deletion mutants that were used in the experiments: FL, aa1–277; LRR, aa1–202; C, aa203–277. (E) 293T cells were transfected with myc-tagged full-length or mutant Rsu-1, and cell lysates were affinity precipitated with a fusion protein of GST and LIM5+C. Precipitates were subjected to immunoblotting with anti-myc antibody. (F) FL cells were seeded onto fibronectin-coated coverslips and fixed 20 min, 60 min, or 8 h later to visualize the localization of GFP-PINCH-1 and Rsu-1 during cell spreading (scale bar = 20 μm): 20 min, attached and round; 60 min, initial spread; 8 h, stable spread. (G) shCtrl and shPINCH cells were immunostained with anti-vinculin and anti–Rsu-1 antibodies (scale bar = 20 μm).

Article Snippet: Equal protein quantities were separated on SDS-polyacrylamide electrophoresis (SDS-PAGE) gels and transferred to PVDF membrane (Millipore, Billerica, MA).

Techniques: Expressing, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Transfection, Immunoprecipitation, Mutagenesis

Journal: Scientific Reports

Article Title: Protein and DNA synthesis demonstrated in cell-free Ehrlichia chaffeensis organisms in axenic medium

doi: 10.1038/s41598-018-27574-z

Figure Lengend Snippet: Protein biosynthesis assessed by protein fractionation and Western blot analysis. ( A ) Silver stained SDS containing polyacrylamide gel-resolved protein fractions were assessed for the protein abundance variations in RCs incubated in the axenic media with different carbon sources as in Fig. 5 . ( B ) As in panel A, except that the resolved proteins were transferred to a nylon membrane and assessed by Western blot analysis using mouse polyclonal serum against E . chaffeensis .

Article Snippet: For normalization of bacterial total protein content, the suspensions of E . chaffeensis cell-free fractions were lysed in 1% SDS solution for 5 min at 100 °C and the total protein concentration was determined using Protein Assay kit (Bio-Rad, Hercules, CA).

Techniques: Fractionation, Western Blot, Staining, Incubation

Journal: Biochemical Journal

Article Title: Ube2W conjugates ubiquitin to ?-amino groups of protein N-termini

doi: 10.1042/BJ20130244

Figure Lengend Snippet: The Ubc13–Ube2V heterodimer catalyses RNF4-dependent ubiquitylation of N-terminally mono-ubiquitylated poly-SUMO-2 ( A ) Screen for E2 enzymes that recognize N-terminally mono-ubiquitylated poly-SUMO-2 as a substrate. The indicated E2 enzymes were incubated for 60 min together with ubiquitin, UBE1, Ube2W and RNF4, with mono-ubiquitylated Pep.6his-SUMO-2 ×4 as a substrate (see Experimental section). The upper panels show the Coomassie Blue-stained gel images, and the lower panels show anti-ubiquitin antibody Western blots. The asterisks indicate the position of unanchored ubiquitin chains. ( B ) In vitro ubiquitylation reactions using purified mono-ubiquitylated Pep.6His-SUMO-2 ×4 as a substrate and Ubc13–Ube2V as the E2 conjugating enzyme. Dependence on RNF4 and incubation time is shown. ( C ) Schematic presentation of the recombinantly expressed ubiquitin–poly-SUMO-2 construct used in ( D ). ( D ) Ubc13, Ube2V2, UBE1 and ubiquitin [either wild-type (WT) or mutant] were incubated with RNF4 and substrate [either Pep.6His-Ub-SUMO-2 ×1 –SUMO-2-(12–92) ×3 (see Figure 3 A) or Pep.6His-SUMO-2 ×1 –SUMO-2-(12–92) ×3 ] as indicated. Reaction time points were taken at 0, 10, 30 and 100 min. The reactions were analysed by SDS/PAGE, followed by Coomassie Blue staining.

Article Snippet: For in-gel analyses, reaction products from in vitro ubiquitin conjugation assays, halted by the addition of SDS sample buffer containing reducing agent, were fractionated by SDS/PAGE (Novex NuPAGE 10% Bis-Tris gel; Life Technologies) using Mes SDS running buffer.

Techniques: Incubation, Staining, Western Blot, In Vitro, Purification, Construct, Mutagenesis, SDS Page

Journal: Biochemical Journal

Article Title: Ube2W conjugates ubiquitin to ?-amino groups of protein N-termini

doi: 10.1042/BJ20130244

Figure Lengend Snippet: Ube2W conjugates multiple copies of ubiquitin to isopeptide-linked SUMO-2 polymers, but only mono-ubiquitinates peptide-linked SUMO-2 polymers Coomassie Blue-stained SDS/PAGE showing the reaction products of two sets of in vitro ubiquitylation assays using either Ube2W ( A ) or UbcH5a ( B ). Reactions were incubated for 30 min and following completion of assays, half of the reaction volumes were treated with the SUMO protease SENP1 to depolymerize all SUMO-2. Sites of lysine residue ubiquitylation, as identified from the gel areas (broken-lined boxes marked 1–6), are summarized in Supplementary Table S1 (at http://www.biochemj.org/bj/453/bj4530137add.htm ). Pep (peptide) and Isopep (isopeptide) refer to the two different poly-SUMO-2 constructs shown in Figure 1 (A). Ub, ubiquitin.

Article Snippet: For in-gel analyses, reaction products from in vitro ubiquitin conjugation assays, halted by the addition of SDS sample buffer containing reducing agent, were fractionated by SDS/PAGE (Novex NuPAGE 10% Bis-Tris gel; Life Technologies) using Mes SDS running buffer.

Techniques: Staining, SDS Page, In Vitro, Incubation, Construct

Journal: Biochemical Journal

Article Title: Ube2W conjugates ubiquitin to ?-amino groups of protein N-termini

doi: 10.1042/BJ20130244

Figure Lengend Snippet: Mutations to Lys 5 , Lys 7 and Lys 11 of the N-terminal SUMO-2 do not affect ubiquitylation by Ube2W and RNF4 ( A ) Schematic presentation of isopeptide linked-poly-SUMO-2 with the first SUMO unit being full length, and the remaining three units lacking 11 N-terminal amino acids [Pep.6His-SUMO-2 ×1 –SUMO-2-(12–92) ×3 ]. This was used to create lysine residue mutants for biochemical analysis. ( B ) Four mutants of the first SUMO-2 were created, and the constructs analysed by in vitro ubiquitylation. Reactions were incubated for 30 min and products were visualized by Coomassie Blue-stained SDS/PAGE. MW, molecular mass; Ub, ubiquitin; wt, wild-type.

Article Snippet: For in-gel analyses, reaction products from in vitro ubiquitin conjugation assays, halted by the addition of SDS sample buffer containing reducing agent, were fractionated by SDS/PAGE (Novex NuPAGE 10% Bis-Tris gel; Life Technologies) using Mes SDS running buffer.

Techniques: Construct, In Vitro, Incubation, Staining, SDS Page

Journal: Biochemical Journal

Article Title: Ube2W conjugates ubiquitin to ?-amino groups of protein N-termini

doi: 10.1042/BJ20130244

Figure Lengend Snippet: Ube2W conjugates ubiquitin to SUMO-2 protein in N-termini ( A ) Re-analysis of the MS data derived from Figure 2 revealed the presence of the peptide characteristic of N-terminally ubiquitylated Pep.6His-SUMO-2 ×4 . It is noteworthy that this peptide was only detected in slice 1 of Figure 2 . The N-terminal methionine residue of the 6His-poly-SUMO-2 construct is not present. ( B ) Coomassie Blue-stained SDS/PAGE analysis of in vitro ubiquitylation reactions using Isopep.SUMO-2 ×4 as substrate. ( C ) Partial tryptic digestion was used to identify a peptide characteristic of N-terminally ubiquitylated SUMO-2 molecules. The reaction mixture contained Ube2W and RNF4, and SENP1 was used to depolymerize the SUMO construct. MW, molecular mass.

Article Snippet: For in-gel analyses, reaction products from in vitro ubiquitin conjugation assays, halted by the addition of SDS sample buffer containing reducing agent, were fractionated by SDS/PAGE (Novex NuPAGE 10% Bis-Tris gel; Life Technologies) using Mes SDS running buffer.

Techniques: Mass Spectrometry, Derivative Assay, Construct, Staining, SDS Page, In Vitro

Journal: Biochemical Journal

Article Title: Ube2W conjugates ubiquitin to ?-amino groups of protein N-termini

doi: 10.1042/BJ20130244

Figure Lengend Snippet: The general function of Ube2W is as a protein N-terminal ubiquitin-conjugating enzyme ( A ) Coomassie Blue-stained SDS/PAGE analysis of the products of in vitro ubiquitylation assays containing Ube2W and CHIP (as indicated by the broken-lined boxes). ( B ) MS/MS spectrum of the peptide diagnostic of N-terminal ubiquitylation of CHIP. ( C ) Consensus phylogenetic tree depicting the protein sequence-derived relationship between 32 proteins with sequence homology to ubiquitin E2-conjugating enzymes. The red numbers indicate consensus tree bootstrap percentages for nodes. The enzymes are colour-coded by known modifier specificity: black, ubiquitin/putative ubiquitin; red, SUMO; blue, NEDD8 (neural precursor cell expressed, developmentally down-regulated 8); green, ISG15 (ubiquitin-like protein ISG15); orange, FAT10 (also known as UBD; ubiquitin D); cyan, catalytically inactive. *Ube2E2 and Ube2L6 have both been known as UbcH8. Phylogenies created by PHYLIP ( http://cmgm.stanford.edu/phylip/ ). ( D ) Sequence alignment of domains surrounding the catalytic cysteine residues (red) of human E2 enzymes. The position of Asn 77 of Ubch5A (Ube2D1) is marked with a green circle, and the UbcH5a Asp 117 is marked with a green triangle. The alignment was created in JalView ( http://www.jalview.org/ ) [ 29 ].

Article Snippet: For in-gel analyses, reaction products from in vitro ubiquitin conjugation assays, halted by the addition of SDS sample buffer containing reducing agent, were fractionated by SDS/PAGE (Novex NuPAGE 10% Bis-Tris gel; Life Technologies) using Mes SDS running buffer.

Techniques: Staining, SDS Page, In Vitro, Chromatin Immunoprecipitation, Mass Spectrometry, Diagnostic Assay, Sequencing, Derivative Assay

Journal: Biochemical Journal

Article Title: Ube2W conjugates ubiquitin to ?-amino groups of protein N-termini

doi: 10.1042/BJ20130244

Figure Lengend Snippet: A screen for E2 enzymes that partner RNF4 in poly-SUMO ubiquitylation ( A ) Schematic representations of the native Lys 11 isopeptide bond-linked poly-SUMO-2 construct (Isopep.SUMO-2 ×4 , left-hand panel) and linear peptide bond-linked His 6 -tag poly-SUMO-2 construct (Pep.6His-SUMO-2 ×4 , right-hand panel) used as substrates in the present study. ( B ) An in vitro screen of 29 different E2 ubiquitin-conjugation enzymes ( http://www.ubiquigent.com ) was undertaken using recombinant ubiquitin, UBE1 and RNF4, with Pep.6His-SUMO-2 ×4 as the substrate (see the Experimental section for details). Reactions were incubated for 60 min and products are shown on Coomassie Blue-stained SDS/PAGE. Only five E2 enzymes showed activity in this assay (as indicated by the asterisks). The § symbol marks the position of E2 enzymes. Anti-ubiquitin antibody Western blots of these reactions can be seen in Supplementary Figure S1(C) (at http://www.biochemj.org/bj/453/bj4530137add.htm ). Coomassie Blue-stained SDS/PAGE of in vitro ubiquitylation reactions using the indicated combinations of constituents and 60-min incubation (see the Experimental section for details). Ub, ubiquitin.

Article Snippet: For in-gel analyses, reaction products from in vitro ubiquitin conjugation assays, halted by the addition of SDS sample buffer containing reducing agent, were fractionated by SDS/PAGE (Novex NuPAGE 10% Bis-Tris gel; Life Technologies) using Mes SDS running buffer.

Techniques: Construct, In Vitro, Conjugation Assay, Recombinant, Incubation, Staining, SDS Page, Activity Assay, Western Blot

Journal: Reviews in Urology

Article Title: Beyond Prostate-Specific Antigen: New Serologic Biomarkers for Improved Diagnosis and Management of Prostate Cancer

doi:

Figure Lengend Snippet: Comparison of the enzymatically active prostate-specific antigen (PSA) in tissues and seminal plasma with the inactive forms of free PSA found in serum: Active PSA contains no internal peptide bond cleavages and forms a complex with α 1 -antichymotrypsin (PSA-ACT) in serum. proPSA is a precursor form of PSA that is expressed with a 7-amino acid N-terminus leader peptide but is found in serum containing from 1 to 7 amino acids. “Benign” PSA (BPSA) contains 2 internal peptide bond cleavages. The remainder of the inactive PSA in serum (iPSA) appears to be composed largely of intact, denatured PSA, although it may contain lesser amounts of internal or N-terminus cleavages. BPH, benign prostatic hyperplasia. Adapted, with permission, from Mikolajczyk SD et al.

Article Snippet: Mikolajczyk SD, Millar LS, Kumar A, Saedi MS. Human glandular kallikrein, hK2, shows argi-nine-restricted specificity and forms complexes with plasma protease inhibitors.

Techniques: Activated Clotting Time Assay

Journal: Journal of Ovarian Research

Article Title: Curcumin induces chemo/radio-sensitization in ovarian cancer cells and curcumin nanoparticles inhibit ovarian cancer cell growth

doi: 10.1186/1757-2215-3-11

Figure Lengend Snippet: Characterization of PLGA-nanoparticle (NP) containing curcumin (Nano-CUR) and its in vitro therapeutic efficacy . (A and B) Nano-CUR particles are an appropriate size of ~70 nm . Nano-CUR size was determined by (A) dynamic light scattering (DLS) and (B) transmission electron microscopy (TEM). (C) Nano-CUR formulation demonstrates sustained release of curcumin . Cumulative release of curcumin from PLGA NPs was determined by UV spectrophotometer at 450 nm over a period of 18 days. (D) Nano-CUR effectively inhibits the growth of cisplatin resistant ovarian cancer cells . A2780CP cells were treated with Nano-CUR (5-80 μM) or PLGA NPs without curcumin (NPs control) for 48 hrs. Cell proliferation was determined by MTS assay and normalized to control cells treated with vehicle (PBS). (E) A2780CP cells internalize PLGA-NPs . A2780CP cells were incubated with FITC-PLGA NPs for 6 hrs and analyzed by fluorescent microscopy. Original magnifications 400×. Inset image represents PLGA NPs no FITC. (F) Strategy used for antibody conjugation of PLGA-NP for targeted delivery of curcumin to ovarian cancer cells. (G) PLGA-NPs can be conjugated with anti-TAG-72 MAb (CC49) . PLGA-NPs were incubated with anti-TAG-72 MAb. Nano-immunoconjugates were run on 10% SDS-PAGE, transferred to the PVDF membrane and were probed with an anti-mouse secondary antibody as indicated.

Article Snippet: After 48 hrs cells were washed twice with PBS, lysed in SDS buffer (Santa Cruz Biotechnology, Santa Cruz, CA) and kept at 4°C for 30 min.

Techniques: In Vitro, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Spectrophotometry, MTS Assay, Incubation, Microscopy, Conjugation Assay, SDS Page

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Peptides Selected for Binding to a Virulent Strain of Haemophilus influenzae by Phage Display Are Bactericidal

doi: 10.1128/AAC.49.7.2972-2978.2005

Figure Lengend Snippet: Immunoblot analysis with selected phage-displayed peptides. OMPs isolated from NTHi strain R2866 were separated by SDS-PAGE and blotted onto PVDF membranes (Millipore). The membranes were probed separately with unselected f88-4/15-mer library and phage

Article Snippet: For Western immunoblot analyses, OMPs (5 μg/sample) from R2866 were boiled for 5 min in sodium dodecyl sulfate (SDS) reducing buffer prior to being separated at 150 V on Bio-Rad SDS-12% polyacrylamide gel electrophoresis (PAGE) minigels ( ).

Techniques: Isolation, SDS Page