Journal: Cancers

Article Title: Soluble Guanylate Cyclase β1 Subunit Represses Human Glioblastoma Growth

doi: 10.3390/cancers15051567

Figure Lengend Snippet: The effects of sGCβ1 overexpression or silencing on glioblastoma growth. ( a ) Serial analysis of gene expression (SAGA; GEO database GSE15309, n = 327). The results of analysis of the transcript levels in malignant and normal human tissues under various conditions indicated a statistically significant reduction in the levels of sGCβ1 transcript in human glioma specimens compared with that in normal brain tissue. ( b ) The proliferation of U87 cells transfected with a control vector or the vectors for overexpression of sGCβ1 or sGCβ1 Cys105 . Untransfected U87 cells were used as a control ( n = 18 per group; the data were obtained by the MTT assay). ( c ) Colony formation assay of glioblastoma cells. U87 cells were transfected with a vector control or the vectors for overexpression of sGCβ1 or sGCβ1 Cys105 ( n = 6 wells per group). Average colony size and the numbers of the colonies larger than 1 mm 2 are shown. ( d , e ) In vivo antitumor activity of sGCβ1 in athymic mice after intracerebral xenotransplantation of human glioblastoma cells transfected with a vector control or with the vector for overexpression of sGCβ1 Cys105 . Untransfected U87 cells were used as a control. The survival rate ( d ) and average survival time ( e ) are shown ( n = 6 for each group). Log-rank test with Bonferroni correction was used to compare the survival curves in panel ( d ): control vs. vector p = 0.133 (not significant); control vs. sGCβ1 Cys105 p = 0.005; and vector vs. sGCβ1 Cys105 p = 0.009. ( f ) Proliferation assay of human neuroblastoma BE2 cells transfected with sGCβ1 shRNA or scrambled control on days 1, 3, 5, and 7 after plating. The data are the mean ± S.E.M.; ** p < 0.01 (vs. empty vector or control determined by one-way ANOVA with Bonferroni correction for panel ( e ).

Article Snippet: For sGCβ1 knockdown, BE2 cells were transfected with nonsilencing control shRNA or sGCβ1 shRNA (Origene).

Techniques: Over Expression, Gene Expression, Transfection, Control, Plasmid Preparation, MTT Assay, Colony Assay, In Vivo, Activity Assay, Proliferation Assay, shRNA

Journal: Cancers

Article Title: Soluble Guanylate Cyclase β1 Subunit Represses Human Glioblastoma Growth

doi: 10.3390/cancers15051567

Figure Lengend Snippet: Assay of cGMP levels and the effects of sGC activators or inhibitors on sGCβ1-overexpressing cells. ( a ) cGMP levels were not significantly changed in U87 and pCDNA3.1-, sGCβ1 Cys105 -, and sGCβ1-transfected cells. ( b ) Proliferation assay of the cells with overexpression of sGCβ1 Cys105 and sGCβ1 treated with ODQ (10 µM), Bay41-2272 (1 µM), or YC-1 (10 µM). Cell numbers were normalized to the numbers of untreated cells. There were no significant differences between the groups ( p > 0.05).

Article Snippet: For sGCβ1 knockdown, BE2 cells were transfected with nonsilencing control shRNA or sGCβ1 shRNA (Origene).

Techniques: Transfection, Proliferation Assay, Over Expression

Journal: Cancers

Article Title: Soluble Guanylate Cyclase β1 Subunit Represses Human Glioblastoma Growth

doi: 10.3390/cancers15051567

Figure Lengend Snippet: Cell cycle and proliferation/survival analysis of sGCβ1-overexpressing cells. ( a ) Cell cycle analysis of sGCβ1-overexpressing cells stained with propidium iodide by flow cytometry. G0/G1, S, and G2/M phases are indicated. ( b ) DNA and RNA content analysis of sGCβ1-overexpressing cells stained with 7AAD and Pyronin Y by flow cytometry. S/G2/M, G1, and G0 phase are indicated. The data were analyzed by using FlowJo software. The data are the mean ± S.E.M. *, p < 0.05; **, p < 0.01 (vs. empty vector or control).

Article Snippet: For sGCβ1 knockdown, BE2 cells were transfected with nonsilencing control shRNA or sGCβ1 shRNA (Origene).

Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Software, Plasmid Preparation, Control

Journal: Cancers

Article Title: Soluble Guanylate Cyclase β1 Subunit Represses Human Glioblastoma Growth

doi: 10.3390/cancers15051567

Figure Lengend Snippet: Subcellular localization of sGCβ1 and sGCα1 in human glioblastoma cells. ( a ) Immunoblotting analysis of U87 cells, U87 cells transfected with pcDNA3.1D control vector or the vectors for overexpression of sGCβ1 Cys105 , sGCα1 plus sGCβ1 Cys105 , and sGCβ1. An anti-sGCβ1 antibody was used to detect the distribution of sGCβ1 in the whole cell extract (left panel), cytosol (middle panel), and nucleus (right panel). Detection by an antilamin A/C antibody was used as a loading control. ( b ) Immunoblotting analysis of untransfected human glioblastoma U87 cells, U87 cells transfected with pcDNA3.1D control vector alone, and U87 cells with sGCα1 overexpression. An anti-sGCα1 antibody was used to detect the α1 subunit in the cytosol and nucleus. The lack of cross-contamination between the nucleus and cytoplasm was confirmed using the nuclear marker lamin A/C and cytoplasmic marker α-tubulin. ( c ) Immunostaining of sGCβ1 in control U87 cells (upper panel), an sGCα1 + β1 Cys105 –overexpressing stable clone (middle panel), and an sGCβ1 Cys105 –overexpressing stable clone (bottom panel). Blue represents the nuclei stained by DAPI, and green represents FITC staining of sGCβ1. The images were acquired by confocal microscopy at 600× magnification. Scale bars: 20 μm.

Article Snippet: For sGCβ1 knockdown, BE2 cells were transfected with nonsilencing control shRNA or sGCβ1 shRNA (Origene).

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Over Expression, Marker, Immunostaining, Stable Transfection, Staining, Confocal Microscopy

Journal: Cancers

Article Title: Soluble Guanylate Cyclase β1 Subunit Represses Human Glioblastoma Growth

doi: 10.3390/cancers15051567

Figure Lengend Snippet: sGCβ1 interferes with p53 transcription. qRT-PCR (( a ); n = 6) and Western blot (( b , c ); n = 3) of p53 expression promoted by sGCβ1. A significant increase in the levels of p53 was detected after sGCβ1 overexpression (( b , c ); n = 3). An anti-β-actin antibody was used as a loading control for whole cell extract. Original Western blot figure can be found in , and a representative staining of actin is shown in b,e,h). Antilamin A/C and anti-α-tubulin antibodies were used as the controls for the nuclear and cytoplasmic fractions, respectively. The data were normalized to the level of β-actin in the whole cell extract. ( d ) ChIP analysis of sGCβ1 binding to the TP53 promoter. An anti-sGCβ1 antibody was used to immunoprecipitate sGCβ1 from a stable clone of sGCβ1-overexpressing U87 cells, and qRT-PCR was performed to amplify the DNA regions involved in sGCβ1 binding (1 kb downstream of the TSS) and a nonbinding region (1 kb upstream of the TSS). An IgG was used as a control. ( e ) Schematic representation of the cloned TP53 promoter constructs. The 50 bp deletion mutation sites are shown. Dual luciferase assay was performed by using the TP53 promoter constructs cloned into the pGL3 vector and the corresponding deletion mutants of these constructs without sGCβ1-binding sites. sGCβ1 overexpression plasmid was cotransfected into U87 cells with these plasmids. pcDNA3.1 was used as a control. The experiments were repeated 3 times, and the p -values were obtained by Welch- and Boferroni-corrected one-way ANOVA. The data are the mean ± S.E.M. **, p < 0.01.

Article Snippet: For sGCβ1 knockdown, BE2 cells were transfected with nonsilencing control shRNA or sGCβ1 shRNA (Origene).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Control, Staining, Binding Assay, Stable Transfection, Clone Assay, Construct, Mutagenesis, Luciferase, Plasmid Preparation

Journal: Cancers

Article Title: Soluble Guanylate Cyclase β1 Subunit Represses Human Glioblastoma Growth

doi: 10.3390/cancers15051567

Figure Lengend Snippet: Effects of sGCβ1 overexpression on signaling in glioblastoma multiforme. qRT-PCR analysis of p21 ( a ), CDK4, CDK6 ( d ), and integrin α6 ( g ) expression in sGCβ1-overexpressing cells. Western blot analysis of p21 ( b , c ), Original Western blot figure can be found in , CDK4, CDK6 ( e , f ), Original Western blot figure can be found in , and integrin α6 ( h , i ) levels in sGCβ1-overexpressing cells in the whole cell extract, Original Western blot figure can be found in . The data were normalized to the level of β-actin. pcDNA3.1D-transfected cells were used as a control. The data are the mean ± S.E.M. *, p < 0.05; **, p < 0.01 (vs. empty vector or control).

Article Snippet: For sGCβ1 knockdown, BE2 cells were transfected with nonsilencing control shRNA or sGCβ1 shRNA (Origene).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Control, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Defective Flux of Thrombospondin-4 through the Secretory Pathway Impairs Cardiomyocyte Membrane Stability and Causes Cardiomyopathy

doi: 10.1128/MCB.00114-18

Figure Lengend Snippet: Impaired sarcolemmal glycoprotein trafficking in hearts overexpressing secretion-defective Thbs4. (A) Western blotting for membrane glycoproteins purified from hearts of transgenic mice overexpressing wild-type Thbs4 (DTG-Thbs4) or the secretion-defective mutant (DTG-mCa2+). Cadherin was used as a loading control. (B) Representative experiment of immunocytochemistry for β-sarcoglycan and the Golgi marker GM130 in cardiomyocytes isolated from transgenic hearts of the indicated genotype. Scale bar = 50 μm. Similar results were obtained in 2 additional experiments.

Article Snippet: Briefly, paraformaldehyde-fixed paraffin-embedded cardiac sections were incubated in blocking solution (PBS, 5% goat serum, 1% bovine serum albumin [BSA], 1% glycine, 0.2% Triton X-100) for 1 h and then with anti-Thbs4 primary antibody (Santa Cruz) diluted 1:500 in blocking solution overnight, followed by incubation with Alexa Fluor-labeled secondary antibody (Invitrogen; 1:1,000) for 1 h. For immunocytochemistry, adult mouse cardiomyocytes were isolated from mouse hearts by Langendorff perfusion, fixed in 4% paraformaldehyde, and immunostained in suspension as described previously ( 51 ) using antibodies against β-sarcoglycan (Novus Biologicals) and GM130 (BD Biosciences) coupled to Alexa Fluor-labeled secondary antibodies (Invitrogen).

Techniques: Western Blot, Membrane, Purification, Transgenic Assay, Mutagenesis, Control, Immunocytochemistry, Marker, Isolation

Journal: Cancer medicine

Article Title: MiRNA-545 negatively regulates the oncogenic activity of EMS1 in gastric cancer.

doi: 10.1002/cam4.1520

Figure Lengend Snippet: Figure 2. The expression of EMS1 in GC cells was controlled by miR-545. (A) Expression profile of miR-545 in GC cells, as assayed by qRT-PCR. (B) Scatter diagram showing the differential expression of miR-545 in CTs and para-CTs, as assayed by qRT-PCR. (C~E) Histograms showing the expression level of miR-545 and EMS1 in BGC-823 and SGC-7901 cells after transfection with miR-545 oligonucleotides, as assayed by qRT-PCR and Western blotting. (F) Proliferation of GC cells after transfection with miR-545 oligonucleotides, as assayed by MTT.

Article Snippet: MaterialsandMethods Cellculture Human normal gastric mucosal cells GES- 1 and GC cells BGC- 823, MGC- 803, HGC- 027, and SGC- 7901 were purchased from BOSTER (Wuhan, Hubei, China) or the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Transfection, Western Blot

Journal: Cancer medicine

Article Title: MiRNA-545 negatively regulates the oncogenic activity of EMS1 in gastric cancer.

doi: 10.1002/cam4.1520

Figure Lengend Snippet: Figure 3. EMT in GC cell lines BGC-823 and SGC-7901 was regulated by miR-545. (A and B) Alteration of migratory behavior of GC cells after transfection with miR-545 oligonucleotides, as assayed by wound healing. (C and D) Migration ability of GC cells after transfection with miR-545 oligonucleotides, as evaluated by Transwell assays. (E) Ability of GC cells to adhere to the matrix, as examined by adhesion assays. (F) The expression levels of EMT-associated markers, as assayed by Western blotting.

Article Snippet: MaterialsandMethods Cellculture Human normal gastric mucosal cells GES- 1 and GC cells BGC- 823, MGC- 803, HGC- 027, and SGC- 7901 were purchased from BOSTER (Wuhan, Hubei, China) or the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Techniques: Transfection, Migration, Expressing, Western Blot

Journal: Movement Disorders

Article Title: Myoclonus dystonia and muscular dystrophy: ɛ‐sarcoglycan is part of the dystrophin‐associated protein complex in brain

doi: 10.1002/mds.26738

Figure Lengend Snippet: Detection of sarcoglycan transcripts and protein in brain. PCR products were amplified from single‐stranded DNA prepared from RNA extracted from adult rat skeletal muscle and brain (A). PCR primers for amplifying rat sarcoglycan cDNA were described previously. Sizes of the expected PCR product are shown in base pairs. Note that α‐sarcoglycan and γ‐sarcoglycan transcripts were not detected in brain. Sarcoglycan levels in the BIO14.6 hamster brain (B). Membrane fractions prepared from wild‐type controls and δ‐sarcoglycan‐deficient BIO14.6 brain were analyzed by Western blotting using antibodies against the sarcoglycans (ANA‐ɛ and ANA‐ζ), β‐dystroglycan and Dp71. The absence of δ‐sarcoglycan in the BIO14.6 hamster is associated with a reduced level of β‐sarcoglycan but not ɛ‐ and ζ‐sarcoglycan. Note that the 2 proteins detected with the anti‐ɛ‐sarcoglycan antibodies correspond to the brain‐specific (upper) and ubiquitous ɛ‐sarcoglycan isoforms. The levels of Dp71 and β‐dystroglycan are similarly unaltered in the BIO14.6 hamster compared with the wild‐type control.

Article Snippet: Myc‐tagged ɛ‐sarcoglycan isoforms, FLAG‐tagged human β‐sarcoglycan (wild‐type and T182A, c.544A>G mutant , HA‐tagged δ‐sarcoglycan and myc‐tagged ζ‐sarcoglycan were produced by polymerase chain reaction (PCR) and cloned into either pCI‐neo (Promega) or pCMV‐myc (Clontech).

Techniques: Amplification, Membrane, Western Blot, Control

Journal: Movement Disorders

Article Title: Myoclonus dystonia and muscular dystrophy: ɛ‐sarcoglycan is part of the dystrophin‐associated protein complex in brain

doi: 10.1002/mds.26738

Figure Lengend Snippet: IAP of ɛ‐sarcoglycan containing protein complexes from rat tissue. Esg‐4990‐protein A agarose beads were used to purify ɛ‐sarcoglycan and associated proteins from rat brain, heart, and lung. Proteins eluted from esg‐4990‐protein A agarose beads (IAP) or protein A agarose beads (control) were resolved by SDS‐PAGE using a 4%‐12% bis‐Tris gradient gel and visualized with colloidal Coomassie blue G250 dye (A). Bands (A1‐A5) were excised and processed for mass spectrometry. The identity of the major protein in each gel plug is indicated. Note that the strongly stained bands in each IAP correspond to the IgG heavy and light chains and partially obscure ɛ‐sarcoglycan at 50 kDa. Western blots of protein extracts used for IAP derived from brain (B) and heart (C). Each panel shows the original RIPA‐extract (lysate), the precleared extract (pre‐IAP), the IAP, the flow‐through following IAP (IAP unbound), and the flow‐through from the protein A agarose control. Positive controls (brain extract, ɛ‐sarcoglycan; HEK cells expressing β‐sarcoglycan, β‐sarcoglycan) for each antibody are also shown. As expected, ɛ‐sarcoglycan is highly enriched in the IAP and depleted from the flow‐through in both tissues. Similarly, β‐sarcoglycan is present in the IAP, although in the brain the cognate protein could not be detected in the original RIPA‐extract probably because of its relatively low abundance.

Article Snippet: Myc‐tagged ɛ‐sarcoglycan isoforms, FLAG‐tagged human β‐sarcoglycan (wild‐type and T182A, c.544A>G mutant , HA‐tagged δ‐sarcoglycan and myc‐tagged ζ‐sarcoglycan were produced by polymerase chain reaction (PCR) and cloned into either pCI‐neo (Promega) or pCMV‐myc (Clontech).

Techniques: Control, SDS Page, Mass Spectrometry, Staining, Western Blot, Derivative Assay, Expressing

Journal: Movement Disorders

Article Title: Myoclonus dystonia and muscular dystrophy: ɛ‐sarcoglycan is part of the dystrophin‐associated protein complex in brain

doi: 10.1002/mds.26738

Figure Lengend Snippet: Brain sarcoglycan complex associates with DGC components. Sarcoglycan complexes were immunoaffinity‐purified from mouse brain and lung tissue solubilized in either digitonin‐(DIG) or RIPA (RIPA)‐containing buffers using the pan anti‐ɛ‐sarcoglycan antibody. The biotinylated anti‐ɛ‐sarcoglycan antibody detects ɛ‐sarcoglycan‐1 and ‐2 in brain and ɛ‐sarcoglycan‐1 in lung. β‐Dystroglycan and dystrophin Dp71 only copurify with ɛ‐sarcoglycan in digitonin‐containing buffer. Note that ɛ‐sarcoglycan is more effectively solubilized in RIPA buffer compared with digitonin. Similar results were also obtained using antibodies that recognize only ɛ‐sarcoglycan‐2 (data not shown).

Article Snippet: Myc‐tagged ɛ‐sarcoglycan isoforms, FLAG‐tagged human β‐sarcoglycan (wild‐type and T182A, c.544A>G mutant , HA‐tagged δ‐sarcoglycan and myc‐tagged ζ‐sarcoglycan were produced by polymerase chain reaction (PCR) and cloned into either pCI‐neo (Promega) or pCMV‐myc (Clontech).

Techniques: Purification

Journal: Movement Disorders

Article Title: Myoclonus dystonia and muscular dystrophy: ɛ‐sarcoglycan is part of the dystrophin‐associated protein complex in brain

doi: 10.1002/mds.26738

Figure Lengend Snippet: Membrane trafficking of the brain sarcoglycan complex. HEK293T cells were transfected with different combinations of epitope‐tagged sarcoglycans as indicated. After surface biotinylation, esg‐4990 beads were used to immunoprecipitate ɛ‐sarcoglycan and associated proteins. Biotinylated surface proteins that coimmunoprecipitate with ɛ‐sarcoglycan were identified using streptavidin‐Alexa Fluor 680 (A). For comparative purposes, data are presented in groups of 3 (lanes 1‐12) corresponding to the 4 major brain ɛ‐sarcoglycan isoforms. Each group follows the same scheme; ɛ‐sarcoglycan isoform alone, ɛ‐sarcoglycan isoform in a tetramer with wild‐type β‐sarcoglycan and ɛ‐sarcoglycan isoform in a tetramer with mutant β‐sarcoglycan. Lanes 13 and 14 show that trimers that form in the absence of transfected ɛ‐sarcoglycan with either wild‐type or mutant β‐sarcoglycan do not traffic to the cell surface. Although ɛ‐sarcoglycan isoforms traffic to the cell surface in the absence of the other sarcoglycans (lanes 1, 4, 7, and 10), the highest levels of sarcoglycans at the membrane were observed in cells coexpressing ɛ‐, ζ‐, and δ‐sarcoglycan with wild‐type β‐sarcoglycan (lanes 2, 5, 8, and 11). By contrast, levels of δ‐sarcoglycan and, to a lesser extent, ɛ‐ and ζ‐sarcoglycan were drastically reduced in cells expressing the LGMD2E‐associated β‐sarcoglycan T182A mutant (lanes 3, 6, 9, and 12). Complex formation was demonstrated by coimmunoprecipitation for all four ɛ‐sarcoglycan isoforms (B). Robust coimmunoprecipitation of each sarcoglycan is observed for all heterotetramers that contain wild‐type β‐sarcoglycan (lanes 2, 5, 8, and 11). By contrast, βδ‐sarcoglycan core formation was severely disrupted in cells expressing mutant β‐sarcoglycan compared with wild type; however, the association of ɛ‐ and ζ‐sarcoglycan was apparently unaffected by the mutant (lanes 3, 6, 9, and 12). Whole‐cell lysates are shown for comparative purposes, whereas α‐tubulin was used as a loading control (C).

Article Snippet: Myc‐tagged ɛ‐sarcoglycan isoforms, FLAG‐tagged human β‐sarcoglycan (wild‐type and T182A, c.544A>G mutant , HA‐tagged δ‐sarcoglycan and myc‐tagged ζ‐sarcoglycan were produced by polymerase chain reaction (PCR) and cloned into either pCI‐neo (Promega) or pCMV‐myc (Clontech).

Techniques: Membrane, Transfection, Mutagenesis, Expressing, Control