Journal: Molecular Metabolism

Article Title: Impaired histone deacetylases 5 and 6 expression mimics the effects of obesity and hypoxia on adipocyte function

doi: 10.1016/j.molmet.2016.09.011

Figure Lengend Snippet: Effect of silencing of Hdac5 and Hdac6 levels in 3T3-L1 adipocytes . A ) Efficiency of siRNAs on Hdac5 and Hdac6 content by Western Blotting. 3T3-L1 adipocytes were electroporated with 5 nmol of control small interfering RNA duplexes directed against GFP (siCtl) or with siRNAs against Hdac5 (siH5) or Hdac6 (siH6). Total proteins were subjected to Western blotting experiments for the quantification of Hdac5, Hdac6, and α-tubulin as a loading control. The figure shows the results of a representative experiment out of three. Effect of siH5 and siH6 on the B ) expression of Icer , C ) Atf3 , and D ) Glut4 mRNA. Gene expression was monitored by quantitative real-time PCR 72 h after transfection with either control small interfering RNA duplexes directed against GFP (siCtl), or siH5, or siH6. mRNA levels were normalized against the Rplp0 and were expressed as the fold changes over the controls. Data are the mean of ±SEM of at least 3 independent experiments (***P < 0.001). E ) Efficiency of siH5 and siH6 on the 2-deoxy-glucose (DOG) uptake. Measurement of the labeled DOG in 3T3-L1 adipocytes cells was done 96 h after electroporation with siRNAs by measuring [1- 3 H]2-DOG (100 μm, 0.5 μCi/ml) uptake from unstimulated cells ( open bar ) or stimulated cell with 10 nM insulin ( filled bar ). Data are the mean of ±SEM of 4 independent experiments (**P < 0.01; *P < 0.05).

Article Snippet: The siRNA duplexes, targeting the GFP (5′-CGCTGACCCTGAAGTTCAT-3′), the mouse Hdac5 (5′-GCAAGCATTCTACAACGAT-3′), and Hdac6 (5′-CCAGGACGATCTCCAAGAT-3′) were purchased from Microsynth (Balgach, Switzerland).

Techniques: Western Blot, Control, Small Interfering RNA, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Transfection, Labeling, Electroporation

Journal: Molecules and Cells

Article Title: Differentiated Embryo Chondrocyte 1 (DEC1) Represses PPAR?2 Gene through Interacting with CCAAT/Enhancer Binding Protein ? (C/EBP?)

doi: 10.1007/s10059-012-0002-9

Figure Lengend Snippet: Hypoxia-induced DEC1 expression in 3T3-L1 cells. (A) The schematic diagram describing the sequential activation process of adipogenic transcription factors in response to MDI treatment. (B) The 3T3-L1 preadipocytes were differentiated to adipocytes in a normoxic (20–21% oxygen) or hypoxic conditions (0.1–0.5 % oxygen) for the indicated days. The levels of mRNAs were analyzed by RT-PCR using primer sets for DEC1, PPARγ2 and C/EBPα as described previously (Choi et al., 2008). The 18S rRNA was used as a loading control. (C) Adipogenesis was induced by MDI treatment in HIF-1α knockdown 3T3-L1 (shHIF-1α) cells and control 3T3-L1 (shControl) cells for the indicated times. DEC1, HIF-1α, and C/EBPβ protein levels were detected by western analyses. Tubulin was detected to check for equal loadings.

Article Snippet: si RNAs specific for DEC1 and green fluorescent protein (GFP, for Control), were synthesized by Samchully Pharm.

Techniques: Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Western Blot

Journal: Molecules and Cells

Article Title: Differentiated Embryo Chondrocyte 1 (DEC1) Represses PPAR?2 Gene through Interacting with CCAAT/Enhancer Binding Protein ? (C/EBP?)

doi: 10.1007/s10059-012-0002-9

Figure Lengend Snippet: Effects of DEC1 on the activity of C/EBPβ. (A) 3xC/EBP-Luc reporter plasmid and the indicated plasmids were transfected into 3T3-L1 cells with pCHO110. After 24 h, the transfected cells were further incubated under either normoxic or hypoxic conditions (0.1–0.5% oxygen) for 24 h before harvest. The luciferase activity of the cell lysate was measured and normalized based on β-galactosidase activity as described previously. (B) The PPARγ2-Luc reporter plasmids and the indicated plasmids were transfected into 3T3-L1 cells with the pRL-TK plasmids as described above. The luciferase activity was measured and normalized based on the Renilla luciferase activity. The values indicated averages and standard deviations of three independent experiments. (C) 3T3-L1 cells were transfected with siRNA against DEC1 as described. Before harvest, the transfected cells were exposed to hypoxia (0.1–0.5% oxygen) for 24 h. The levels of DEC1 mRNA were quantified by qRT-PCR. (D) The PPARγ2-Luc reporter plasmids were transfected with the indicated plasmid or siRNA into 3T3-L1 cells. The transfected cells were incubated in hypoxia (0.1–0.5% oxygen) for 24 h before harvesting, and luciferase assays were performed. Values represent the means and standard deviations of three experiments. P values for the indicated sets are calculated by Student’s t-test; *p < 0.05 and **p < 0.01, respectively. (E, F) NIH3T3 cells were transfected with the indicated plasmids. At 48 h after transfection, cell lysate was harvested and immunoprecipitated (IP) with either anti-flag antibody (E) or anti-myc antibody (F), then the resulting immunoprecipitant was analyzed by Western blot (WB) using the indicated antibodies. Input indicates the Western blot of 10% amount of the lysates which were used for IP.

Article Snippet: si RNAs specific for DEC1 and green fluorescent protein (GFP, for Control), were synthesized by Samchully Pharm.

Techniques: Activity Assay, Plasmid Preparation, Transfection, Incubation, Luciferase, Quantitative RT-PCR, Immunoprecipitation, Western Blot

Journal: Molecules and Cells

Article Title: Differentiated Embryo Chondrocyte 1 (DEC1) Represses PPAR?2 Gene through Interacting with CCAAT/Enhancer Binding Protein ? (C/EBP?)

doi: 10.1007/s10059-012-0002-9

Figure Lengend Snippet: Effects of DEC1 on the expression of PPARγ2 in NIH-3T3/Cβ cells. (A) The stably expressed flag-tagged C/EBPβ in NIH-3T3/Cβ cells was detected using an anti-flag antibody (left panel). NIH-3T3/LacZ or Cβ cells were differentiated to adipocytes for 6 or 8 days under either normoxic (20–21% oxygen) or hypoxic (0.1–0.5% oxygen) conditions. The expression of PPARγ2 mRNA was determined by qRT-PCR (middle panel). The lipid droplets were stained with Oil Red-O (right panel). (B, C) NIH-3T3/Cβ cells were transiently transfected with myc-tagged DEC1 then treated with MDI for 36 h. Western analyses were performed using the indicated antibodies. The 14-3-3γ was detected to check for equal loadings (B). mRNA levels of PPARγ2 of each sample was measured by quantitative RT-PCR analyses. Expression level of 18S rRNA was used for normalization (C). (D) Confocal microscopic images of PPARγ (red) or myc-tagged DEC1 (green) in NIH3T3/Cβ cells which were treated with MDI for 36 h. An arrow indicates a NIH3T3/Cβ cell which is transfected with myc-tagged DEC1.

Article Snippet: si RNAs specific for DEC1 and green fluorescent protein (GFP, for Control), were synthesized by Samchully Pharm.

Techniques: Expressing, Stable Transfection, Quantitative RT-PCR, Staining, Transfection, Western Blot

Journal: Molecules and Cells

Article Title: Differentiated Embryo Chondrocyte 1 (DEC1) Represses PPAR?2 Gene through Interacting with CCAAT/Enhancer Binding Protein ? (C/EBP?)

doi: 10.1007/s10059-012-0002-9

Figure Lengend Snippet: Proteins bound to the PPARγ2 promoter. NIH3T3/Cβ cells were transiently transfected with myc-tagged DEC1 and then treated with MDI for 36 h. By using nuclear extracts from the transfected cells, ChIP assays were performed with the indicated antibodies and a primer set for PPARγ2 promoter region (−474 bp to −185 bp). (A) ChIP assays using either anti-C/EBPβ (top panel) or anti-Flag antibodies (bottom panel). (B) ChIP assay using anti-Myc antibody. (C) EMSA was performed as described using in vitro transcribed and translated C/EBPβ and DEC1 (Park and Park, 2010). Oligonucleotides containing the C/EBP binding element (5′-TGC AGA TTG CGC AAT CTG CA-3′) were radiolabeled with [α-32P]-dATP. (D) NIH3T3 cells were transfected with myc-tagged DEC1 and flag-tagged HDAC1. The cell lysates were harvested and immunoprecipitated (IP) with anti-myc antibody then the resulting immunocomplex was analyzed by Western blot (WB) using indicated antibodies. Input indicates the western blot of 10% amount of the lysates which were used for IP (left panel). NIH3T3/Cβ cells were transfected with myc-tagged DEC1 and treated with MDI. At 36 h after treated MDI, cell lysate was harvested and immunoprecipitated (IP) with anti-myc antibody. The resulting immunocomplex was analyzed by Western blot (WB) using indicated antibodies. Input indicates the Western blot of 10% amount of the lysates which were used for IP (right panel). (E) ChIP assay using anti-HDAC1 antibody or anti-acetylated K9/K14 residues of histone3 (H3K9K14Ac). (F) Hypothetical mechanism by which hypoxia-induced DEC1 represses PPARγ2 gene.

Article Snippet: si RNAs specific for DEC1 and green fluorescent protein (GFP, for Control), were synthesized by Samchully Pharm.

Techniques: Transfection, In Vitro, Binding Assay, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Inhibitor of Apoptosis-Stimulating Protein of p53 (iASPP) Is Required for Neuronal Survival after Axonal Injury

doi: 10.1371/journal.pone.0094175

Figure Lengend Snippet: A significant reduction of iASPP in the GCL was observed by immunohistochemistry of axotomized retinas at 24 hrs after intravitreal delivery of siRNA against iASPP (si-iASPP) compared to intact retinas, while control siRNA against GFP (siGFP) had no effect (A–C). RBPMS labeling confirmed that siRNA-mediated knockdown of iASPP occurred in RGCs (D–F). Scale bars: (A–C) = 50 μm; (D–F) = 15 μm. Western blot analysis confirmed that intravitreal delivery of si-iASPP led to marked reduction of retinal iASPP protein at 24 hrs after delivery, while siGFP had no effect (G, H; Student's T-test, *** = p<0.001). Quantitative analysis of RGC survival at one week after axotomy following intraocular injection of si-iASPP (black), or control siGFP (grey) (n = 4/group, ANOVA, * = p<0.05). The density of RGCs in intact, uninjured Sprague-Dawley rat retinas with si-iASPP intravitreal injection (hatched bar) or without (open bar) are shown as reference. Data are expressed as the mean ± S.E.M. ONL: Outer Nuclear Layer; OPL: Outer Plexiform Layer; INL: Inner Nuclear Layer; IPL: Inner Plexiform Layer; GCL: Ganglion Cell Layer.

Article Snippet: The control siRNA against GFP was kindly provided by Dr. Elena Feinstein (Quark Pharmaceuticals Inc.) and has been described elsewhere .

Techniques: Immunohistochemistry, Control, Labeling, Knockdown, Western Blot, Injection

Journal: Frontiers in Immunology

Article Title: Pathogen-Specific Binding Soluble Down Syndrome Cell Adhesion Molecule (Dscam) Regulates Phagocytosis via Membrane-Bound Dscam in Crab

doi: 10.3389/fimmu.2018.00801

Figure Lengend Snippet: Soluble Es Dscam promotes phagocytosis via membrane-bound Es Dscam in hemocytes. (A) Schematic diagram showing the RNAi region recognized by primers in the conserved transmembrane region of ds Es Dscam. (B–D) Knockdown of Es Dscam expression. Crab hemnocyte were transfected with Es Dscam dsRNA (5 nM) and equal amounts of GFP dsRNA served as a control. RNA samples were collected at 24 h post-transfection to check the silencing efficiency by RT-PCR ( left panel ) and qRT-PCR ( right panel ) with β-actin as a reference. And the protein samples also were collected and detected by Western blotting with β-actin as a reference. RT-PCR data are representative of two independent repeats. At least three crab were used for each sample, and qRT-PCR were performed three times. The results are expressed as the mean ± SD, data were analyzed by Student’s t -test. ** p < 0.01. ( Right panel ) Immunocytochemical analysis of Es Dscam dsRNA transfected hemocyte showing decreased Es Dscam protein expression compared with dsGFP-treated control cells. Scale bar = 15 µm. Data are representative of three independent repeats. (E,F) Membrane-bound Es Dscam regulate soluble Es Dscam promoted phagocytosis in hemocyte. Rate of hemocyte phagocytosis of (E) Staphylococcus aureus and (F) Vibrio parahemolyticus following Es Dscam knockdown. Phagocytic rates of hemocytes were calculated according to the formula presented in the Materials and Methods section. Three independent repeats were performed, and results are expressed as the mean ± SD. Data were analyzed by one-way ANOVA and the letters (a, b, c) presented significant differences ( p < 0.05).

Article Snippet: Briefly, siRNA against exon 4.24- and exon 6.20-containing Es Dscam isoforms and GFP (as control) were synthesized by GenePharma (Shanghai, China); the primer sequences used are listed in Table .

Techniques: Membrane, Knockdown, Expressing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: Pathogen-Specific Binding Soluble Down Syndrome Cell Adhesion Molecule (Dscam) Regulates Phagocytosis via Membrane-Bound Dscam in Crab

doi: 10.3389/fimmu.2018.00801

Figure Lengend Snippet: Interaction of membrane-bound and soluble Es Dscam is essential for promotion of phagocytosis. (A) Colocalization of Es -mDscam and Es -sDscam in hemocyte surface. Each group that including r Es Dscam 4.6,6.9 , Staphylococcus aureus coated r Es Dscam 4.24,6.19 , and Vibrio parahemolyticus coated r Es Dscam 4.12,6.20 was used to stimulate cultured crab hemocyte. Hemocytes were collected 1 h later and subjected to immunocytochemistry detected by the antibodies of Es Dscam (membrane-bound Es Dscam) and His-tag (soluble Es Dscam). Scale bar = 15 µm. Data are representative of two independent repeats. (B) Schematic diagram showing the RNAi region recognized by siExon 4.24 and siExon 6.20 primers in the epitope I region. (C,D) Knockdown of exon 4.24 and exon 6.20 expression. Crab hemocyte were transfected with 5 nM exon 4.24 siRNA ( left panel ) or exon 6.20 siRNA ( right panel ), with equal amounts of GFP siRNA served as a control. RNA samples were collected at 24 h post-transfection to check the silencing efficiency by RT-PCR with β-actin as a reference. RT-PCR data are representative of two independent repeats. At least three crab were used for each sample, and qRT-PCR were performed three times. The results are expressed as the mean ± SD, data were analyzed by Student’s t -test. ** p < 0.01, * p < 0.05. (E,F) Specific membrane-bound Es Dscam regulate soluble Es Dscam promoted phagocytosis in hemocyte. Rate of hemocyte phagocytosis of (E) Staphylococcus aureus and (F) V. parahemolyticus following knockdown of exon 4.24 and exon 6.20, respectively. Phagocytic rates of hemocytes were calculated according to the formula presented in Section “ .” At least 15 fields were included and data represent the mean ± SD of three independent experiments. The results were analyzed statistically using one-way ANOVA and the letters (a, b, c) presented significant differences ( p < 0.05). (G) Interaction between membrane-bound and soluble Es Dscam in vitro . The bait protein r Es Dscam 4.24,6.19 -His was first separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and renatured by adding a series of different concentration of guanidine-HCl. The undetected proteins were added to recognize the bait protein on the membrane before the attached proteins were detected by Western blotting using the specific antibodies. Data are representative of two independent repeats. (H) Interaction between membrane-bound and soluble Es Dscam in vivo . Lysates from S2 cells transiently transfected with pAc5.1-r Es Dscam 4.24,6.19 - His, together with pAc5.1-r Es Dscam 4.12,6.20 - V5 or pAc5.1-r Es Dscam 4.24,6.19 - V5, were subjected to immunoprecipitation with anti-His Ab or anti-V5 Ab, followed by Western blot analysis with anti-His Ab and anti-V5 Ab. The input controls were also analyzed by Western blot. Data are representative of two independent repeats.

Article Snippet: Briefly, siRNA against exon 4.24- and exon 6.20-containing Es Dscam isoforms and GFP (as control) were synthesized by GenePharma (Shanghai, China); the primer sequences used are listed in Table .

Techniques: Membrane, Cell Culture, Immunocytochemistry, Knockdown, Expressing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, In Vitro, SDS Page, Concentration Assay, Western Blot, In Vivo, Immunoprecipitation