Journal: Oncotarget

Article Title: GNA13 as a prognostic factor and mediator of gastric cancer progression

doi: 10.18632/oncotarget.6780

Figure Lengend Snippet: ( A ) Ectopic expression of GNA13 in AGS and HGC-27 cells analyzed by western blotting. ( B and C ) Ectopic expression of GNA13 promoted proliferation ability of AGS and HGC-27 cell as determined by MTT assays (B) and colony formation assays (C). ( D ) Knockdown of endogenous GNA13 in specific shRNA transduced stable SGC-7901 and BGC-823 cells. ( E and F ), knockdown of GNA13 inhibits cell growth as determined by MTT assays (E) and colony formation assays (F). * P < 0.05; ** P < 0.01.

Article Snippet: The sequences of two human short hairpin RNA (shRNA) sequences to repress GNA13 expression are listed as follows: GNA13 shRNA#1: 5′-GCCCAAGGAATGGTGGAAACA-3′; GNA13 shRNA#2: 5′-GGATAACTTGGATAAACTTGG-3′(Genecopoeia, Guangzhou, China).

Techniques: Expressing, Western Blot, Knockdown, shRNA

Journal: Oncotarget

Article Title: GNA13 as a prognostic factor and mediator of gastric cancer progression

doi: 10.18632/oncotarget.6780

Figure Lengend Snippet: ( A ) Upper: representative histograms depicting cell cycle profiles of indicated cells. Cells were stained with PI and analyzed by flow cytometry. Lower: proportion of cells in various phases of the cell cycle. ( B ) Real-time PCR analysis of p21Cip1, p27Kip1, Ki67, and cyclinD1 mRNA expression in GNA13-transduced cells (upper panel) or GNA13 shRNA–infected cells (lower panel). Expression levels were normalized to GAPDH. ( C ) Western blot analysis of p21Cip1, p27Kip1, cyclin D1, and Ki67 proteins in GNA13-transduced cells or GNA13 shRNA–infected cells. GAPDH was used as a loading control. * P < 0.05; ** P < 0.01.

Article Snippet: The sequences of two human short hairpin RNA (shRNA) sequences to repress GNA13 expression are listed as follows: GNA13 shRNA#1: 5′-GCCCAAGGAATGGTGGAAACA-3′; GNA13 shRNA#2: 5′-GGATAACTTGGATAAACTTGG-3′(Genecopoeia, Guangzhou, China).

Techniques: Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, shRNA, Infection, Western Blot, Control

Journal: Oncotarget

Article Title: GNA13 as a prognostic factor and mediator of gastric cancer progression

doi: 10.18632/oncotarget.6780

Figure Lengend Snippet: ( A and B ) Related FOXO1 reporter activity (A) and c-Myc reporter activity ( B ) in GNA13-transduced cells or GNA13 shRNA–infected cells. ( C ), Western blot analysis of p-AKT, total AKT, p-ERK, total ERK, c-Myc, p-GSK-3β, total GSK-3β, p-FOXO1, and total FOXO1 in GNA13-transduced cells or GNA13 shRNA–infected cells. ( D ) AGS/GNA13 and HGC-27/GNA13 cells were treated with the AKT inhibitor LY294002 (20 lM), the ERK kinase inhibitor U0126 (20 lM) or DMSO for 24 h, then harvested to examine the expression levels of the indicated proteins by Western blotting. ( E , F and G ) AGS/GNA13 and HGC-27/GNA13 proliferation and tumorigenicity were determined by MTT (E), colony formation assays (F) and anchorage-independent growth assay (G) after treatment with LY294002, U0126 or DMSO. * P < 0.05; ** P < 0.01.

Article Snippet: The sequences of two human short hairpin RNA (shRNA) sequences to repress GNA13 expression are listed as follows: GNA13 shRNA#1: 5′-GCCCAAGGAATGGTGGAAACA-3′; GNA13 shRNA#2: 5′-GGATAACTTGGATAAACTTGG-3′(Genecopoeia, Guangzhou, China).

Techniques: Activity Assay, shRNA, Infection, Western Blot, Expressing, Growth Assay

Journal: BMC Cell Biology

Article Title: Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation

doi: 10.1186/1471-2121-11-44

Figure Lengend Snippet: ITGA5 Controls IGF2/IGFBP2 Expression in hMSCs . Adult hMSCs were transduced with shITGA5, shITGB1 or a non relevant shRNA (shNR), total RNA was used for quantitative RT-PCR analysis of IGF2 ( A ) and IGFBP2 ( B ). Adult hMSCs were transduced with a lentiviral vector encoding ITGA5, or treated with the agonist peptide CRRETAWAC (100 μg/ml) (CRRETAWAC) or with a non relevant control peptide (GRGESP; 100 μg/ml), or with a conformation-dependent anti-α5 monoclonal antibody (SNAKA51; 10 μg/ml), and IGF2 and IGFBP2 mRNA expression was determined by quantitative RT-PCR analysis ( C-D ). Results are expressed as mean ± SD of treated over control ratio after normalization to 18 S expression. *: significant difference with untreated cells ( P <0.05).

Article Snippet: Non relevant shRNA (scrambled sequence that does not lead to specific degradation of any known cellular mRNA), ITGB1 shRNA and human FAK shRNA lentiviral particles (mixtures of viral particles containing 3 target-specific constructs that encode shRNA designed to knock down gene expression) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Transduction, shRNA, Quantitative RT-PCR, Plasmid Preparation, Control

Journal: BMC Cell Biology

Article Title: Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation

doi: 10.1186/1471-2121-11-44

Figure Lengend Snippet: Inhibition of ITGA5-Induced Signalling Abrogates IGF2/IGFBP2 Expression in hMSCs . Adult hMSCs transduced with a lentiviral vector encoding ITGA5 or treated with the agonist peptide (CRRETAWAC; 100 μg/ml), and control cells were transiently transfected with a specific shRNA targeting FAK. Total RNA was collected and used for quantitative RT-PCR analysis of IGF2 ( A ) and IGFBP2 ( B ). Adult hMSCs were treated with the MEK inhibitor U0126 (10 μM), or the PI3K inhibitor wortmannin (50 nM) for 24 hours and IGF2 and IGFBP2 mRNA levels were determined by quantitative RT-PCR analysis ( C-D ). Results are expressed as mean ± SD of treated over control ratio after normalization to 18 S expression. *: significant difference with untreated cells ( P <0.05).

Article Snippet: Non relevant shRNA (scrambled sequence that does not lead to specific degradation of any known cellular mRNA), ITGB1 shRNA and human FAK shRNA lentiviral particles (mixtures of viral particles containing 3 target-specific constructs that encode shRNA designed to knock down gene expression) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Inhibition, Expressing, Transduction, Plasmid Preparation, Control, Transfection, shRNA, Quantitative RT-PCR

Journal: Cell metabolism

Article Title: Targeting Peripheral CB 1 Receptors Reduces Ethanol Intake via a Gut-Brain Axis

doi: 10.1016/j.cmet.2019.04.012

Figure Lengend Snippet: (A) Time course and rate of increase in oleate-driven respiratory activity in MGN3–1 cells. Cells were treated with 10 or 100 nM JD5037, 2.5 M of FCCP (positive control) or vehicle in the presence of the fluorescent extracellular O2 consumption reagent. Oxidative respiration was recorded every 90 sec and the slope of the initial linear increase was calculated. Points and bars are means ± SEM from n = 11–15 experiments, as indicated. *P < 0.05 compared to vehicle. (B) Verification of Cnr1 knockdown by rtPCR in cells used in panel C. Cellular uptake of the constructs was verified by fluorescent microscopy (20x magnification) and the degree of knockdown was determined by rt-PCR, *P < 0.05, n = 4. (C) Oleate-driven oxidative activity in MGN3–1 cells with shRNA-mediated knockdown of Cnr1 expression and their mock-transfected controls, n = 3–8 experiments, as indicated, *P < 0.05.

Article Snippet: Scramble shRNA with GFP Adenovirus (Ad-scramble-shRNA) , Vector Biolabs , 1122.

Techniques: Activity Assay, Positive Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Construct, Microscopy, shRNA, Expressing, Transfection

Journal: Journal of Bone and Mineral Research

Article Title: Effects of 25-Hydroxyvitamin D 3 on Proliferation and Osteoblast Differentiation of Human Marrow Stromal Cells Require CYP27B1/1α-Hydroxylase

doi: 10.1002/jbmr.298

Figure Lengend Snippet: Primer Sets Used for RT-PCR

Article Snippet: Transient transfection of siRNA into hMSCs hi-1α was performed by electroporation with the Human MSC Nucleofector Kit (Lonza/Amaxa Biosystems, Walkersville, MD, USA) with either CYP27B1 siRNA, nonsilencing control siRNA (a nonhomologous, scrambled sequence equivalent; Santa Cruz Biotechnology, Inc.), or PBS according to the manufacturer's instructions and as described previously. ( ) In brief, hMSCs hi-1α were harvested by trypsinization and resuspended at 10 6 cells in 100 μL of Nucleofector Solution (Lonza/Amaxa Biosystems) with 10 or 100 pmol of CYP27B1 siRNA.

Techniques: Sequencing

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: BRD4 knockdown attenuates the proliferation and promotes the apoptosis of U251 cells. (A) A Cell counting kit-8 assay was performed to detect the proliferation of U251 cells transduced with BRD4-shRNA or Scr-shRNA. Each experiment was performed in triplicate. (B) An EdU assay was performed to determine cell proliferation rates following BRD4 knockdown. Nuclei were counterstained blue with DAPI. Red indicates the cells undergoing proliferation. Scale bar, 20 µ m. (C) Quantification of the EdU assay results demonstrated that U251 cell proliferation was reduced following BRD4 knockdown. (D) Representative plots and quantification of flow cytometry analysis of apoptosis following BRD4 knockdown in U251 cells. Data are presented as the mean ± standard deviation of three independent experiments. ** P<0.01. BRD4, bromodomain containing 4; sh, short hairpin; Scr, scrambled control; OD, optical density.

Article Snippet: Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as the control. siRNA targeting KRAS proto-oncogene GTPase (KRAS) and the negative control siRNA (cat. nos. sc-35731 and sc-37007, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, Cell Counting, Transduction, shRNA, EdU Assay, Flow Cytometry, Standard Deviation, Control

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: Ten key genes identified by global signal transduction network analysis.

Article Snippet: Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as the control. siRNA targeting KRAS proto-oncogene GTPase (KRAS) and the negative control siRNA (cat. nos. sc-35731 and sc-37007, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Transduction

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: Experimental validation of microarray results. (A) Reverse transcription-quantitative polymerase chain reaction results for the mRNA expression levels of the ten key genes identified by global signal transduction network analysis. (B) Western blotting validation of the protein expression changes of key genes in the BRD4-shRNA and the Scr-shRNA groups. GAPDH was used as an internal control. (C) Representative photographs and quantification of KRAS immunostaining in normal brain tissue and glioma tissues of grades II, III and IV. Scale bar, 20 µ m. (D) Western blot analysis of KRAS levels in HA and U251 cells. (E) KRAS silencing following siRNA transfection in U251 cells was confirmed by western blotting (at 72 h post-transfection). (F) A cell counting kit-8 assay was performed to detect the proliferation rates of siKRAS and or siCon-transfected U251 cells. Each experiment was performed in triplicate. (G) The apoptosis rates of siKRAS and siCon-transfected U251 cells were determined by TUNEL staining (red). Nuclei were counterstained with DAPI (blue). Scar bar, 50 µ m. Experimental data are presented as the mean ± standard deviation of at least three experiments. * P<0.05 and ** P<0.01. BRD4, bromodomain containing 4; sh, short hairpin; Scr, scrambled control; KRAS, KRAS proto-oncogene GTPase; HA, human astrocytes; si, small interfering; Con, control; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; OD, optical density.

Article Snippet: Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as the control. siRNA targeting KRAS proto-oncogene GTPase (KRAS) and the negative control siRNA (cat. nos. sc-35731 and sc-37007, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Biomarker Discovery, Microarray, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transduction, Western Blot, shRNA, Control, Immunostaining, Transfection, Cell Counting, TUNEL Assay, Staining, Standard Deviation

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Affinity purification of SirT7-binding factors identified the histone H2A variant mH2A1 by MS analysis (table S2). HA, hemagglutinin. ( B ) Schematic representation of the three mH2A isoforms, of which, only mH2A1.1 binds to ADP-ribose . ( C ) Endogenous SirT7 specifically immunoprecipitates mH2A1.1 in HEK293F cells. IgG, immunoglobulin G. ( D and E ) mH2A1.1 recognizes and binds ADP-ribosylated SirT7. Immunoprecipitation (IP) of bacterially expressed rSirT7 WT or N189A preincubated ± NAD + and added to nuclear extracts of HEK293F SirT7 KO cells (CRISPR-Cas9–mediated KO of SIRT7) expressing mH2A1.1 WT or G224E, a mutant deficient in ADP-ribose binding. Inputs (I) and elutions (E) are shown. A similar experiment with H187Y is shown in fig. S3A. ( F ) Top: Interaction between endogenous SirT7 and mH2A1.1 under high-stringency conditions upon different types of stress in HEK293 cells. C, untreated; IR, 7-gray ionizing irradiation; H 2 O 2 , oxidative stress. Western blot of the input and elution of immunoprecipitation experiments with anti-SirT7 antibody under these conditions. Bottom: Summary of the ADP-ribosylation events detected by MS in SirT7-FLAG expressed in the same cells and purified under the same stress conditions. Further details are shown in table in fig. S3B and in table S1. ( G ) Superose 6 gel filtration chromatography of nuclear endogenous proteins from nuclear extracts of HEK293F cells under normal cell growth (NT) or upon GS. Fraction numbers and approximate molecular weights (MW) are indicated. Western blot of SirT7 and mH2A1.1 are shown. ( H ) High-stringency immunoprecipitation of endogenous mH2A1.1 by WT or N189A SirT7 in HEK293F cells treated under normal conditions or under GS. Inputs (I) and elutions (E) are shown.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Affinity Purification, Binding Assay, Variant Assay, Immunoprecipitation, CRISPR, Expressing, Mutagenesis, Irradiation, Western Blot, Purification, Filtration, Chromatography

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Levels of three mH2A isoforms in WCE of NIH3T3 cells transfected with scramble shRNA or mH2A1 shRNA and cultured under normal or GS conditions. ( B ) Levels of endogenous SirT7 and histone H3 in chromatin and nucleoplasm fractions purified from Wt and Sirt7 −/− MEFs cultured under normal or GS conditions during the indicated times. ( C ) Venn diagrams showing the intersection of SIRT7-associated genes with mH2A1-enriched genes in Wt cells under NT (top) or GS (bottom). SirT7-associated and mH2A1-enriched genes were derived from GREAT analysis (fig. S3B and Materials and Methods). ( D ) Average enrichment of mH2A1 at all genes in Wt (left) or SirT7 −/− MEF (right) cells under NT (black) or GS (red) conditions. Data are expressed as the log 2 ratio of reads per kilobase of transcript per million mapped reads–normalized ChIP/input signals. ( E ) Distribution of sites occupied by SirT7 upon GS around the TSS by GREAT analysis. The values for each bin from the TSS are shown above each bar (TSS: −5 kb, 5 to 50 kb, 50 to 500 kb, and >500 kb). ( F ) KEGG cell signaling pathways for SirT7-associated genes mapped by GREAT analysis under GS in MEF cells. The signaling pathways were ranked by their combined score provided by Enrichr analysis. cGMP-PKG, guanosine 3′,5′-monophosphate–protein kinase G; cAMP, cyclic adenosine 3′,5′-monophosphate; TCA, tricarboxylic acid. ( G ) SirT7 ChIP-qPCR (quantitative polymerase chain reaction) analysis of SirT7 binding sites associated with mH2A1 at distal regions upon shRNA-mediated down-regulation of mH2A1 under normal and GS conditions in NIH3T3 cells. The amplified regions (red) and their distance to each gene are indicated in the upper part of each graph. Each SirT7 ChIP was normalized with respect to its own input. SEM from n = 4. Two-tailed t test (* P < 0.05 and *** P < 0.005).

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Transfection, shRNA, Cell Culture, Purification, Derivative Assay, Protein-Protein interactions, ChIP-qPCR, Real-time Polymerase Chain Reaction, Binding Assay, Amplification, Two Tailed Test

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Heat map showing RNA expression changes relative to NT (as log 2 magnitude of difference between GS and NT) conditions in Wt and SirT7-deficient MEFs. ( B ) Pipeline applied to RNA-seq data to filter genes associated with SirT7/mH2A1 in Wt and Sirt7 −/− MEF cells treated under GS or NT. The analysis was restricted to genes that (i) were associated with SirT7 via GREAT and were mH2A1-enriched, (ii) showed a log 2 fold change (FC) of expression between WT-GS and WT-NT >0.6, and (iii) showed a difference between WT and KO log 2 FC (GS versus NT) of >0.3 (table S3). ( C ) mH2A1 ChIP-seq signals across ctgf , a gene differentially enriched in mH2A1 upon GS in Wt MEFs compared with NT. ( D ) Top: Real-time qPCR (RT-qPCR) analysis of genes regulated by SirT7 upon GS and NT. The expression of SirT7 in SirT7 −/− MEFs was rescued by retroviral-mediated gene transfer of SirT7 WT, H187Y(HY), N189A(NA), and empty vector (−). SEM from n = 4. Two-tailed t tests (* P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001). Bottom: Relative mH2A1 enrichment (GS versus NT) by ChIP-qPCR analysis at specific regions around the TSS of the indicated genes [ gbp6 , −6 kb; necab1 , −3 kb; lair1 , −27.5 kb; ctgf , +600 base pairs (bp); adra2a , −5 kb; and nrip3 , +7 kb]. SEM from n = 3. One-way analysis of variance (ANOVA) (* P <0.05, ** P < 0.01, and *** P < 0.005). a.u., arbitrary units. ( E ) Chromatin state transitions induced by GS in Wt and SirT7-deficient cells. The colors of the arrows indicate the frequency (%) of the transition as stated in the color scale (right). Bottom right: State map illustrating the specific combination of mH2A1 and/or H3K27me3 in the four chromatin states defined in the analysis. U1, without H3K27me3 or mH2A1; U2, mH2A1; U3, H3K27me3; U4, enriched by H3K27me3 and mH2A1.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: RNA Expression, RNA Sequencing, Expressing, ChIP-sequencing, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Two Tailed Test, ChIP-qPCR

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Model validation studies in WT and Sirt7 −/− mice fed AL or calorie restricted (CR, 30%) for 8 weeks. ( B ) RT-qPCR analysis of the indicated genes in liver samples from Wt and Sirt7 −/− mice fed AL or CR. Three animals were analyzed for each condition. Each quantification was generated from three replicates. Probabilities are those associated with one-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.005). ( C ) Levels of SirT7 in liver samples from Wt and Sirt7 −/− mice AL and CR after subcellular fractionation. WCE and chromatin fractions are shown. ( D ) SirT7 immunoprecipitation of mH2A1 in the same liver samples. Inputs (I) and elutions (E) are shown. ( E ) Autophagy activity in the Wt and Sirt7 −/− livers under AL or CR monitored by levels of formation of LCIII-2. Left: A representative Western blot of n = 5 replicates used in the quantification shown. Right: Quantification of the relative accumulation of LCIII-2 compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) analyzed with a two-tailed t test (* P < 0.05). GAPDH was used as a loading control, as we did not detect a significant alteration of the levels of the protein in our conditions (data not shown). ( F ) Similar analysis ( n = 5) of Beclin-1 as in (E). ( G ) Model proposed for the dual SirT7/mH2A regulatory axis in GS. On the basis of our data, we speculate that this axis is also involved in CR and aging. Nutrient stress induces SirT7 auto-mADPRT, which leads to mH2A-dependent recruitment of SirT7 to distal regulatory regions and subsequent mH2A enrichment around the associated genes. This axis plays a key role in CR in vivo and possibly in aging by modulating key signaling pathways.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Generated, Fractionation, Immunoprecipitation, Activity Assay, Western Blot, Two Tailed Test, Control, In Vivo, Protein-Protein interactions