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    MISSION shRNA Product Offerings
    Description:
    Sigma Aldrich is a global supplier of leading RNA interference RNAi tools The MISSION TRC shRNA libraries are a comprehensive collection of 150 000 pre cloned shRNA constructs targeting 15 000 human and 15 000 mouse genes These lentiviral based constructs allow flexible delivery of long or short term silencing in a broad range of mammalian cell types including primary and non dividing cells The MISSION TRC shRNA libraries provide solutions for pathway elucidation target identification and are ideal for high content screens The libraries are offered with a variety of options and formats Entire libraries gene family sets target sets or individual clones are available as bacterial glycerol stocks purified DNA and lentiviral transduction particles To find more information including protocols references and frequently asked questions visit the RNAi website
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    Millipore shrna sirna knockdown experiments shrnas
    MISSION shRNA Product Offerings
    Sigma Aldrich is a global supplier of leading RNA interference RNAi tools The MISSION TRC shRNA libraries are a comprehensive collection of 150 000 pre cloned shRNA constructs targeting 15 000 human and 15 000 mouse genes These lentiviral based constructs allow flexible delivery of long or short term silencing in a broad range of mammalian cell types including primary and non dividing cells The MISSION TRC shRNA libraries provide solutions for pathway elucidation target identification and are ideal for high content screens The libraries are offered with a variety of options and formats Entire libraries gene family sets target sets or individual clones are available as bacterial glycerol stocks purified DNA and lentiviral transduction particles To find more information including protocols references and frequently asked questions visit the RNAi website
    https://www.bioz.com/result/shrna sirna knockdown experiments shrnas/product/Millipore
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    shrna sirna knockdown experiments shrnas - by Bioz Stars, 2020-09
    99/100 stars

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    1) Product Images from "Notch inhibition allows oncogene independent generation of iPS cells"

    Article Title: Notch inhibition allows oncogene independent generation of iPS cells

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1552

    NOTCH inhibition suppresses p21 without reducing p53 activity a , qPCR analysis of p53-dependent genes in human neonatal keratinocytes 3 days after transduction of GFP or OCT4 and SOX2 . b , Western blot of p53 levels in human neonatal keratinocytes with DMSO or 10 μM DAPT treatment for 3 days. Full blot shown in Supplementary Figure 7g . c , qPCR analysis of p53-dependent genes after 10 μM DAPT or 2 μM DBZ treatment for 3 days in OCT4, SOX2 -transduced human keratinocytes. d , The efficiency of NANOG+/TRA-1-81+ iPSC generation in OCT4, SOX2, KLF4 , and CMYC -transduced human neonatal keratinocytes transduced with p53DD or GFP with or without exposure to UV irradiation. e , γH2AX immunostaining in human neonatal keratinocytes 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC and treatment with DAPT, p53DD, or p53 shRNA. Scale bars = 50 μm. f , The percentage of TUNEL-positive cells in human neonatal keratinocyte reprogramming cultures with active or inactive p53 (p53DD expression) 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC . g , The number of insertions or deletions (indels) per iPSC line derived under normal, DAPT, or p53DD conditions, as determined by array CGH. For all experiments, error bars represent the standard deviation between two biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test. * denotes significance p-value
    Figure Legend Snippet: NOTCH inhibition suppresses p21 without reducing p53 activity a , qPCR analysis of p53-dependent genes in human neonatal keratinocytes 3 days after transduction of GFP or OCT4 and SOX2 . b , Western blot of p53 levels in human neonatal keratinocytes with DMSO or 10 μM DAPT treatment for 3 days. Full blot shown in Supplementary Figure 7g . c , qPCR analysis of p53-dependent genes after 10 μM DAPT or 2 μM DBZ treatment for 3 days in OCT4, SOX2 -transduced human keratinocytes. d , The efficiency of NANOG+/TRA-1-81+ iPSC generation in OCT4, SOX2, KLF4 , and CMYC -transduced human neonatal keratinocytes transduced with p53DD or GFP with or without exposure to UV irradiation. e , γH2AX immunostaining in human neonatal keratinocytes 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC and treatment with DAPT, p53DD, or p53 shRNA. Scale bars = 50 μm. f , The percentage of TUNEL-positive cells in human neonatal keratinocyte reprogramming cultures with active or inactive p53 (p53DD expression) 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC . g , The number of insertions or deletions (indels) per iPSC line derived under normal, DAPT, or p53DD conditions, as determined by array CGH. For all experiments, error bars represent the standard deviation between two biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test. * denotes significance p-value

    Techniques Used: Inhibition, Activity Assay, Real-time Polymerase Chain Reaction, Transduction, Western Blot, Irradiation, Immunostaining, shRNA, TUNEL Assay, Expressing, Derivative Assay, Standard Deviation, Two Tailed Test

    Notch inhibition promotes keratinocyte reprogramming by suppressing p21 a , Schematic of the DAPT treatment time course on human neonatal keratinocytes. b , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, SOX2, KLF4 , and CMYC and treated with intervals of 10 μM DAPT or c , 2 μM DBZ. d , Western blot for p21 in human neonatal keratinocytes transduced with OCT4 and SOX2 and treated with DMSO or 10 μM DAPT. Full blot shown in Supplementary Figure 7c . e , Western blot for INVOLUCRIN in human neonatal keratinocytes treated with DMSO, 10 μM DAPT, or 1.2 mM calcium chloride for 6 days. Calcium was used as a positive control to induce keratinocyte differentiation. Full blot shown in Supplementary Figure 7d . f , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, KLF4, SOX2 , and CMYC and a scrambled shRNA or a p21 shRNA at day 0 of reprogramming. DAPT was added at 10 μM. g , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4 and SOX2 and a scrambled shRNA control or a p21 shRNA at day 0 of reprogramming. DAPT was added at 2.5 μM. h , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT, SOX2, KLF4 , and CMYC and GFP or p21 and treated with DMSO or 10 μM DAPT from days 1-18 post-transduction. For all experiments, error bars represent the standard deviation between two-three biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test.
    Figure Legend Snippet: Notch inhibition promotes keratinocyte reprogramming by suppressing p21 a , Schematic of the DAPT treatment time course on human neonatal keratinocytes. b , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, SOX2, KLF4 , and CMYC and treated with intervals of 10 μM DAPT or c , 2 μM DBZ. d , Western blot for p21 in human neonatal keratinocytes transduced with OCT4 and SOX2 and treated with DMSO or 10 μM DAPT. Full blot shown in Supplementary Figure 7c . e , Western blot for INVOLUCRIN in human neonatal keratinocytes treated with DMSO, 10 μM DAPT, or 1.2 mM calcium chloride for 6 days. Calcium was used as a positive control to induce keratinocyte differentiation. Full blot shown in Supplementary Figure 7d . f , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, KLF4, SOX2 , and CMYC and a scrambled shRNA or a p21 shRNA at day 0 of reprogramming. DAPT was added at 10 μM. g , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4 and SOX2 and a scrambled shRNA control or a p21 shRNA at day 0 of reprogramming. DAPT was added at 2.5 μM. h , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT, SOX2, KLF4 , and CMYC and GFP or p21 and treated with DMSO or 10 μM DAPT from days 1-18 post-transduction. For all experiments, error bars represent the standard deviation between two-three biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test.

    Techniques Used: Inhibition, Transduction, Western Blot, Positive Control, shRNA, Standard Deviation, Two Tailed Test

    Related Articles

    Clone Assay:

    Article Title: Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells
    Article Snippet: .. shRNA knockdown Four shRNA clones targeting human UGT1 and the pLKO.1 control plasmid were purchased through TRC consortium from Sigma. .. To generate lentivirus, 3 μg of shRNA clone, 3 μg of pCMV8.2ΔR, and 1.5 μg VSV-G expression plasmid were transfected into 293T cells in 6-cm plates by lipofectamine 2000 (Invitrogen).

    Article Title: Lentiviral shRNA knockdown of ADAMTS-5 and -9 restores matrix deposition in 3D chondrocyte culture
    Article Snippet: .. Cloned shRNA-lentiviral construct target sets for the two aggrecanases were from the MISSION™ TRC-Hs 1.0 (Human) library, designed by the RNAi consortium and available via Sigma-Aldrich (Poole, UK) ( ). .. Unique construct designations used were TRCN0000050493 to 50497 (ADAMTS-5) and TRCN0000046633 to 46637 (ADAMTS-9).

    Transfection:

    Article Title: TGF-? Signaling Is Often Attenuated during Hepatotumorigenesis, but Is Retained for the Malignancy of Hepatocellular Carcinoma Cells
    Article Snippet: .. Transfection and Determination of Knockdown of TGFBRII and Smad4 The TβRII shRNA, Smad4 shRNA and control shRNA in lentiviral vector pLK0.1-puro (Sigma, St Louis, MO, USA) were provided by Dr. John A. Copland. .. The sequence of shRNA is: TGFBRII: 5′-CCG GCC TGA CTT GTT GCT AGT CAT ACT CGA GTA TGA CTA GCA ACA AGT CAG GTT TTT G-3′ as described previously ; Smad4∶5′-CCG GCG AGT TGT ATC ACC TGG AAT TCT CGA GAA TTC CAG GTG ATA CAA CTC GTT TTT G-3′.

    shRNA:

    Article Title: ZEB1‐mediated melanoma cell plasticity enhances resistance to MAPK inhibitors
    Article Snippet: .. IPTG‐inducible shRNA control and shRNA‐ZEB1 (TRCN0000369266 and TRCN0000369267) in pLKO_IPTG_3xLacO were purchased from Sigma‐Aldrich (St‐Louis, MO, USA). .. For activation of shRNA expression, IPTG (Sigma) was added to the culture medium (100–200 μM) every two days.

    Article Title: TGF-? Signaling Is Often Attenuated during Hepatotumorigenesis, but Is Retained for the Malignancy of Hepatocellular Carcinoma Cells
    Article Snippet: .. Transfection and Determination of Knockdown of TGFBRII and Smad4 The TβRII shRNA, Smad4 shRNA and control shRNA in lentiviral vector pLK0.1-puro (Sigma, St Louis, MO, USA) were provided by Dr. John A. Copland. .. The sequence of shRNA is: TGFBRII: 5′-CCG GCC TGA CTT GTT GCT AGT CAT ACT CGA GTA TGA CTA GCA ACA AGT CAG GTT TTT G-3′ as described previously ; Smad4∶5′-CCG GCG AGT TGT ATC ACC TGG AAT TCT CGA GAA TTC CAG GTG ATA CAA CTC GTT TTT G-3′.

    Article Title: VSL#3 Probiotic Stimulates T-Cell Protein Tyrosine Phosphatase-Mediated Recovery of IFN-γ Induced Intestinal Epithelial Barrier Defects
    Article Snippet: .. The shRNA targeting human PTPN2 and scrambled control shRNA were purchased from Sigma-Aldrich (St. Louis, MO). ..

    Article Title: Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells
    Article Snippet: .. shRNA knockdown Four shRNA clones targeting human UGT1 and the pLKO.1 control plasmid were purchased through TRC consortium from Sigma. .. To generate lentivirus, 3 μg of shRNA clone, 3 μg of pCMV8.2ΔR, and 1.5 μg VSV-G expression plasmid were transfected into 293T cells in 6-cm plates by lipofectamine 2000 (Invitrogen).

    Article Title: Inflammation in the hippocampus affects IGF1 receptor signaling and contributes to neurological sequelae in rheumatoid arthritis
    Article Snippet: .. Lentiviral particles, MISSION TRCN0000023490 and TRCN0000023493 (Sigma-Aldrich), encoding shRNA targeting IGF1R (shIGF1R), or nontargeting shRNA controls, shNT (SHC002H; Sigma-Aldrich), were injected intraperitoneally (106 to 107 particles in 100 µL per mouse) on days 19, 26, and 33. .. The optimal dose and delivery frequency was set in cultures of PMA-stimulated splenocytes and in a pilot in vivo experiment ( ).

    Article Title: Lentiviral shRNA knockdown of ADAMTS-5 and -9 restores matrix deposition in 3D chondrocyte culture
    Article Snippet: .. Cloned shRNA-lentiviral construct target sets for the two aggrecanases were from the MISSION™ TRC-Hs 1.0 (Human) library, designed by the RNAi consortium and available via Sigma-Aldrich (Poole, UK) ( ). .. Unique construct designations used were TRCN0000050493 to 50497 (ADAMTS-5) and TRCN0000046633 to 46637 (ADAMTS-9).

    Article Title: Inducibly decreased MITF levels do not affect proliferation and phenotype switching but reduce differentiation of melanoma cells
    Article Snippet: .. Cells bearing the inducible shRNA against MITF or control cells were treated for 6 days with the indicated concentrations of DOX, replated onto 12‐well plates, and viability was determined next day by the MTT viability kit (Sigma‐Aldrich) according to the manufacturer's instructions. ..

    Article Title: Otud7b facilitates T cell activation and inflammatory responses by regulating Zap70 ubiquitination
    Article Snippet: .. Otud7b shRNA and a control luciferase shRNA in pLKO.1 lentiviral vector were obtained from Sigma-Aldrich; Sts1 shRNAs and a nonsilencing control shRNA in pGIPZ lentiviral vector were purchased from Thermo Fisher Scientific. .. The expression vectors encoding HA-ubiquitin was cloned into pcDNA vector as previously described , and Myc-tagged human Zap70 in pSXSR vector (pSXSR-Myc-hZap70) was obtained from L.E.

    MTT Assay:

    Article Title: Inducibly decreased MITF levels do not affect proliferation and phenotype switching but reduce differentiation of melanoma cells
    Article Snippet: .. Cells bearing the inducible shRNA against MITF or control cells were treated for 6 days with the indicated concentrations of DOX, replated onto 12‐well plates, and viability was determined next day by the MTT viability kit (Sigma‐Aldrich) according to the manufacturer's instructions. ..

    Construct:

    Article Title: Lentiviral shRNA knockdown of ADAMTS-5 and -9 restores matrix deposition in 3D chondrocyte culture
    Article Snippet: .. Cloned shRNA-lentiviral construct target sets for the two aggrecanases were from the MISSION™ TRC-Hs 1.0 (Human) library, designed by the RNAi consortium and available via Sigma-Aldrich (Poole, UK) ( ). .. Unique construct designations used were TRCN0000050493 to 50497 (ADAMTS-5) and TRCN0000046633 to 46637 (ADAMTS-9).

    Luciferase:

    Article Title: Otud7b facilitates T cell activation and inflammatory responses by regulating Zap70 ubiquitination
    Article Snippet: .. Otud7b shRNA and a control luciferase shRNA in pLKO.1 lentiviral vector were obtained from Sigma-Aldrich; Sts1 shRNAs and a nonsilencing control shRNA in pGIPZ lentiviral vector were purchased from Thermo Fisher Scientific. .. The expression vectors encoding HA-ubiquitin was cloned into pcDNA vector as previously described , and Myc-tagged human Zap70 in pSXSR vector (pSXSR-Myc-hZap70) was obtained from L.E.

    Injection:

    Article Title: Inflammation in the hippocampus affects IGF1 receptor signaling and contributes to neurological sequelae in rheumatoid arthritis
    Article Snippet: .. Lentiviral particles, MISSION TRCN0000023490 and TRCN0000023493 (Sigma-Aldrich), encoding shRNA targeting IGF1R (shIGF1R), or nontargeting shRNA controls, shNT (SHC002H; Sigma-Aldrich), were injected intraperitoneally (106 to 107 particles in 100 µL per mouse) on days 19, 26, and 33. .. The optimal dose and delivery frequency was set in cultures of PMA-stimulated splenocytes and in a pilot in vivo experiment ( ).

    Plasmid Preparation:

    Article Title: TGF-? Signaling Is Often Attenuated during Hepatotumorigenesis, but Is Retained for the Malignancy of Hepatocellular Carcinoma Cells
    Article Snippet: .. Transfection and Determination of Knockdown of TGFBRII and Smad4 The TβRII shRNA, Smad4 shRNA and control shRNA in lentiviral vector pLK0.1-puro (Sigma, St Louis, MO, USA) were provided by Dr. John A. Copland. .. The sequence of shRNA is: TGFBRII: 5′-CCG GCC TGA CTT GTT GCT AGT CAT ACT CGA GTA TGA CTA GCA ACA AGT CAG GTT TTT G-3′ as described previously ; Smad4∶5′-CCG GCG AGT TGT ATC ACC TGG AAT TCT CGA GAA TTC CAG GTG ATA CAA CTC GTT TTT G-3′.

    Article Title: Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells
    Article Snippet: .. shRNA knockdown Four shRNA clones targeting human UGT1 and the pLKO.1 control plasmid were purchased through TRC consortium from Sigma. .. To generate lentivirus, 3 μg of shRNA clone, 3 μg of pCMV8.2ΔR, and 1.5 μg VSV-G expression plasmid were transfected into 293T cells in 6-cm plates by lipofectamine 2000 (Invitrogen).

    Article Title: Otud7b facilitates T cell activation and inflammatory responses by regulating Zap70 ubiquitination
    Article Snippet: .. Otud7b shRNA and a control luciferase shRNA in pLKO.1 lentiviral vector were obtained from Sigma-Aldrich; Sts1 shRNAs and a nonsilencing control shRNA in pGIPZ lentiviral vector were purchased from Thermo Fisher Scientific. .. The expression vectors encoding HA-ubiquitin was cloned into pcDNA vector as previously described , and Myc-tagged human Zap70 in pSXSR vector (pSXSR-Myc-hZap70) was obtained from L.E.

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    Millipore anti smurf2 antibody
    <t>Smurf2</t> −/− mice have increased bone resorption. ( a – i ) Histomorphometric analysis of proximal tibia from 5-week-old WT and Smurf2 −/− male mice for bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Ths), number of osteoclasts per bone perimeter (N.Oc/B.Pm), osteoclast surface per bone surface (Oc.S/BS), bone formation rate per bone volume (BFR/BV), number of osteoblasts per bone perimeter (N.Ob/B.Pm) and osteoblast surface per bone surface (Ob.S/BS). Scale bar, 500 μm. Data represent mean±s.d. ( n =6 for each genotype, ** P
    Anti Smurf2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti smurf2 antibody/product/Millipore
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    Millipore anti bmp4 antibody
    <t>BMP4</t> regulation of ERα depends on levels of estrogens. (A) Western blotting analysis of ERα expression level in adipose tissue from male BMP4 knockout and control mice on HFD at age of 6 months. Relative grey intensity of the band was quantified using Image J software. n = 6. (B) Multipotent C3H10T1/2 cells stably expressing ERα-Flag were induced to differentiation and treated with 20 ng/ml BMP4 in presence of estradiol at different concentration for 12 h, and the protein level of ERα was examined with anti-Flag antibody by Western blot. Relative grey intensity of the band from 4 individual experiments was quantified using Image J software.
    Anti Bmp4 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti ev71 antibody
    <t>EV71</t> viruses bind to galectin-1. (A) Galectin-1 serum levels are increased in EV71-infected patients. Seven clinical severe EV71-infected patient sera, five non-severe EV71-infected patient sera and twelve non-infected control sera were collected to detect galectin-1 by ELISA. *p
    Anti Ev71 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Smurf2 −/− mice have increased bone resorption. ( a – i ) Histomorphometric analysis of proximal tibia from 5-week-old WT and Smurf2 −/− male mice for bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Ths), number of osteoclasts per bone perimeter (N.Oc/B.Pm), osteoclast surface per bone surface (Oc.S/BS), bone formation rate per bone volume (BFR/BV), number of osteoblasts per bone perimeter (N.Ob/B.Pm) and osteoblast surface per bone surface (Ob.S/BS). Scale bar, 500 μm. Data represent mean±s.d. ( n =6 for each genotype, ** P

    Journal: Nature Communications

    Article Title: SMURF2 regulates bone homeostasis by disrupting SMAD3 interaction with vitamin D receptor in osteoblasts

    doi: 10.1038/ncomms14570

    Figure Lengend Snippet: Smurf2 −/− mice have increased bone resorption. ( a – i ) Histomorphometric analysis of proximal tibia from 5-week-old WT and Smurf2 −/− male mice for bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Ths), number of osteoclasts per bone perimeter (N.Oc/B.Pm), osteoclast surface per bone surface (Oc.S/BS), bone formation rate per bone volume (BFR/BV), number of osteoblasts per bone perimeter (N.Ob/B.Pm) and osteoblast surface per bone surface (Ob.S/BS). Scale bar, 500 μm. Data represent mean±s.d. ( n =6 for each genotype, ** P

    Article Snippet: Anti-SMURF2 antibody (ABS59; 1:500) was obtained from Millipore.

    Techniques: Mouse Assay

    Smurf2 −/− mice have a reduced bone mass phenotype. ( a ) Alcian blue/alizarin red staining of the whole skeleton from 1-week-old WT and Smurf2 −/− male littermates. ( b ) Alcian blue/alizarin red staining of forelimb and hindlimb from 1-week-old WT and Smurf2 −/− male littermates. ( c ) Haematoxylin and eosin staining of distal femur of 12-week-old male WT and Smurf2 −/− mice. Scale bar, 500 μm. ( d – h ) μ-QCT analysis of distal femurs from 12-week-old male WT and Smurf2 −/− mice for bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and cortical thickness (C.Th). Scale bar, 500 μm. ( i – m ) μ-QCT analysis of the fifth lumbar vertebra from 12-week-old male WT and Smurf2 −/− mice for BV/TV, Tb.N, Tb.Th and C.Th. Scale bar, 500 μm. Data represent mean±s.d. ( n =6 for each genotype, ** P

    Journal: Nature Communications

    Article Title: SMURF2 regulates bone homeostasis by disrupting SMAD3 interaction with vitamin D receptor in osteoblasts

    doi: 10.1038/ncomms14570

    Figure Lengend Snippet: Smurf2 −/− mice have a reduced bone mass phenotype. ( a ) Alcian blue/alizarin red staining of the whole skeleton from 1-week-old WT and Smurf2 −/− male littermates. ( b ) Alcian blue/alizarin red staining of forelimb and hindlimb from 1-week-old WT and Smurf2 −/− male littermates. ( c ) Haematoxylin and eosin staining of distal femur of 12-week-old male WT and Smurf2 −/− mice. Scale bar, 500 μm. ( d – h ) μ-QCT analysis of distal femurs from 12-week-old male WT and Smurf2 −/− mice for bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and cortical thickness (C.Th). Scale bar, 500 μm. ( i – m ) μ-QCT analysis of the fifth lumbar vertebra from 12-week-old male WT and Smurf2 −/− mice for BV/TV, Tb.N, Tb.Th and C.Th. Scale bar, 500 μm. Data represent mean±s.d. ( n =6 for each genotype, ** P

    Article Snippet: Anti-SMURF2 antibody (ABS59; 1:500) was obtained from Millipore.

    Techniques: Mouse Assay, Staining

    SMAD3 and VDR function together to induce RANKL expression. ( a ) Expression of RANKL, SMAD3 and TUBULIN in WT and Smurf2 −/− neonatal calvarial osteoblasts infected with shRNA lentivirus targeting Smad3 or Egfp shRNA. ( b ) Expression of RANKL, SMAD3, p-SMAD3 and TUBULIN in WT and Smurf2 −/− neonatal calvarial osteoblasts with or without 10 μM SB525334. ( c ) Co-immunoprecipitation of HA-VDR and Flag SMAD3 in 293T cells with dimethylsulphoxide (DMSO) or 10 nM 1,25D treatment for 24 h. ( d ) Co-immunoprecipitation of endogenous VDR and SMAD3 in calvarial osteoblasts. ( e , f ) 293T cells were transfected with the HA-VDR or Flag SMAD3 construct, and 48 h post transfection, total cell lysates (TCLs) were recovered to perform GST pull-down analysis with the indicated GST-fusion proteins. ( g , h ) mRNA and protein level of RANKL in C3H10 T1/2 cells infected with SMAD3- and VDR-expressing lentivirus. ( i ) C3H10 T1/2 cells were transiently co-transfected with a RANKL 2-kb promoter luciferase reporter and pRL-Renilla plasmids together with empty vector or Smad3 or VDR. The cells were collected for dual-luciferase reporter assay after transfection for 48 h.

    Journal: Nature Communications

    Article Title: SMURF2 regulates bone homeostasis by disrupting SMAD3 interaction with vitamin D receptor in osteoblasts

    doi: 10.1038/ncomms14570

    Figure Lengend Snippet: SMAD3 and VDR function together to induce RANKL expression. ( a ) Expression of RANKL, SMAD3 and TUBULIN in WT and Smurf2 −/− neonatal calvarial osteoblasts infected with shRNA lentivirus targeting Smad3 or Egfp shRNA. ( b ) Expression of RANKL, SMAD3, p-SMAD3 and TUBULIN in WT and Smurf2 −/− neonatal calvarial osteoblasts with or without 10 μM SB525334. ( c ) Co-immunoprecipitation of HA-VDR and Flag SMAD3 in 293T cells with dimethylsulphoxide (DMSO) or 10 nM 1,25D treatment for 24 h. ( d ) Co-immunoprecipitation of endogenous VDR and SMAD3 in calvarial osteoblasts. ( e , f ) 293T cells were transfected with the HA-VDR or Flag SMAD3 construct, and 48 h post transfection, total cell lysates (TCLs) were recovered to perform GST pull-down analysis with the indicated GST-fusion proteins. ( g , h ) mRNA and protein level of RANKL in C3H10 T1/2 cells infected with SMAD3- and VDR-expressing lentivirus. ( i ) C3H10 T1/2 cells were transiently co-transfected with a RANKL 2-kb promoter luciferase reporter and pRL-Renilla plasmids together with empty vector or Smad3 or VDR. The cells were collected for dual-luciferase reporter assay after transfection for 48 h.

    Article Snippet: Anti-SMURF2 antibody (ABS59; 1:500) was obtained from Millipore.

    Techniques: Expressing, Infection, shRNA, Immunoprecipitation, Transfection, Construct, Luciferase, Plasmid Preparation, Reporter Assay

    Smurf2 −/− osteoblasts express high levels of RANKL driving osteoclastogenesis. ( a – f ) Osteoclastogenesis of bone marrow cells from 4-week-old mice in vitro . ( a ) TRAP staining of osteoclasts in culture. Scale bar, 0.5 cm. ( b ) Statistical analysis of TRAP-positive cell numbers in the osteoclast differentiation culture. ( c ) Culture supernatants were assayed for TRAP activity via colorimetric readout (A405). ( d – f ) Gene expression analysis of osteoclast cultures was examined by quantitative reverse transcriptase–PCR. ( g – m ) Osteoclastogenesis of osteoblasts/bone marrow cells by OB-OC co-culture in vitro using neonatal calvarial osteoblasts. ( g ) TRAP staining of osteoclasts in co-culture, Scale bar, 0.5 mm. ( h ) Statistical analysis of TRAP-positive cell numbers in the co-culture shown in g . ( i ) Co-culture supernatants were assayed for TRAP activity via colorimetric readout (A405). Red line, osteoblast cells were from Smurf2 −/− mice; black line, osteoblast cells were from WT mice. ( j – m ) Gene expression analysis of osteoblasts/bone marrow cells co-cultures was examined by quantitative reverse transcriptase–PCR. ( n ) Gene expression of major osteoclast differentiation regulators in neonatal calvarial osteoblasts. ( o ) Serum RANKL was determined from 5-week-old male WT and Smurf2 −/− mice. Data represent mean±s.d. ( n =10 for each genotype, *** P

    Journal: Nature Communications

    Article Title: SMURF2 regulates bone homeostasis by disrupting SMAD3 interaction with vitamin D receptor in osteoblasts

    doi: 10.1038/ncomms14570

    Figure Lengend Snippet: Smurf2 −/− osteoblasts express high levels of RANKL driving osteoclastogenesis. ( a – f ) Osteoclastogenesis of bone marrow cells from 4-week-old mice in vitro . ( a ) TRAP staining of osteoclasts in culture. Scale bar, 0.5 cm. ( b ) Statistical analysis of TRAP-positive cell numbers in the osteoclast differentiation culture. ( c ) Culture supernatants were assayed for TRAP activity via colorimetric readout (A405). ( d – f ) Gene expression analysis of osteoclast cultures was examined by quantitative reverse transcriptase–PCR. ( g – m ) Osteoclastogenesis of osteoblasts/bone marrow cells by OB-OC co-culture in vitro using neonatal calvarial osteoblasts. ( g ) TRAP staining of osteoclasts in co-culture, Scale bar, 0.5 mm. ( h ) Statistical analysis of TRAP-positive cell numbers in the co-culture shown in g . ( i ) Co-culture supernatants were assayed for TRAP activity via colorimetric readout (A405). Red line, osteoblast cells were from Smurf2 −/− mice; black line, osteoblast cells were from WT mice. ( j – m ) Gene expression analysis of osteoblasts/bone marrow cells co-cultures was examined by quantitative reverse transcriptase–PCR. ( n ) Gene expression of major osteoclast differentiation regulators in neonatal calvarial osteoblasts. ( o ) Serum RANKL was determined from 5-week-old male WT and Smurf2 −/− mice. Data represent mean±s.d. ( n =10 for each genotype, *** P

    Article Snippet: Anti-SMURF2 antibody (ABS59; 1:500) was obtained from Millipore.

    Techniques: Mouse Assay, In Vitro, Staining, Activity Assay, Expressing, Polymerase Chain Reaction, Co-Culture Assay

    SMURF2-mediated SMAD3 monoubiquitination impedes formation of the SMAD3–VDR complex. ( a ) Ubiquitinated and non-ubiquitinated HA-SMAD3 prepared from transfected 293T cells were performed GST pull-down experiments with the indicated GST-fusion proteins. ( b ) Immunoblot analysis of total cell lysates (TCLs) derived from 293T cells transfected with HA-VDR and the indicated Flag SMAD3 constructs. ( c ) Ubiquitinated and non-ubiquitinated HA-SMAD3 1–231 truncations prepared from transfected 293T cells were performed GST pull-down experiments with the indicated GST-fusion proteins. ( d ) Ubiquitination analysis of SMAD3 and N-terminal ubiquitin acceptor site lysine mutants in 293T cells. ( e , f ) mRNA and protein level of RANKL in C3H10 T1/2 cell line infected with lentivirus-expressing SMAD3, SMAD3-K3R and VDR. ( g ) Co-immunoprecipitation experiments were conducted in 293T cells transfected with Flag SMAD3/SMAD3-K3R and HA-VDR. ( h ) Co-immunoprecipitation of Flag SMAD3/SMAD3-K3R with endogenous VDR in calvarial osteoblast cells infected with lentivirus-expressing SMAD3/SMAD3-K3R mutant protein. ( i ) Immunoblot for RANKL, SMURF2, VDR and TUBULIN in response to 10 nM 1,25D treatment for 24 h in WT and Smurf2 −/− calvarial osteoblasts. ( j , k ) WT and Smurf2 −/− calvarial osteoblasts were subjected to immunoprecipitation with IgG, SMAD3 or VDR antibody. Quantitative PCR analysis of DNA precipitates to amplify VDRE site in the mouse Rankl promoter. Three sets of independent experiments were performed to generate each data and the error bars represent mean±s.d. ( n =3 for each genotype, * P

    Journal: Nature Communications

    Article Title: SMURF2 regulates bone homeostasis by disrupting SMAD3 interaction with vitamin D receptor in osteoblasts

    doi: 10.1038/ncomms14570

    Figure Lengend Snippet: SMURF2-mediated SMAD3 monoubiquitination impedes formation of the SMAD3–VDR complex. ( a ) Ubiquitinated and non-ubiquitinated HA-SMAD3 prepared from transfected 293T cells were performed GST pull-down experiments with the indicated GST-fusion proteins. ( b ) Immunoblot analysis of total cell lysates (TCLs) derived from 293T cells transfected with HA-VDR and the indicated Flag SMAD3 constructs. ( c ) Ubiquitinated and non-ubiquitinated HA-SMAD3 1–231 truncations prepared from transfected 293T cells were performed GST pull-down experiments with the indicated GST-fusion proteins. ( d ) Ubiquitination analysis of SMAD3 and N-terminal ubiquitin acceptor site lysine mutants in 293T cells. ( e , f ) mRNA and protein level of RANKL in C3H10 T1/2 cell line infected with lentivirus-expressing SMAD3, SMAD3-K3R and VDR. ( g ) Co-immunoprecipitation experiments were conducted in 293T cells transfected with Flag SMAD3/SMAD3-K3R and HA-VDR. ( h ) Co-immunoprecipitation of Flag SMAD3/SMAD3-K3R with endogenous VDR in calvarial osteoblast cells infected with lentivirus-expressing SMAD3/SMAD3-K3R mutant protein. ( i ) Immunoblot for RANKL, SMURF2, VDR and TUBULIN in response to 10 nM 1,25D treatment for 24 h in WT and Smurf2 −/− calvarial osteoblasts. ( j , k ) WT and Smurf2 −/− calvarial osteoblasts were subjected to immunoprecipitation with IgG, SMAD3 or VDR antibody. Quantitative PCR analysis of DNA precipitates to amplify VDRE site in the mouse Rankl promoter. Three sets of independent experiments were performed to generate each data and the error bars represent mean±s.d. ( n =3 for each genotype, * P

    Article Snippet: Anti-SMURF2 antibody (ABS59; 1:500) was obtained from Millipore.

    Techniques: Transfection, Derivative Assay, Construct, Infection, Expressing, Immunoprecipitation, Mutagenesis, Real-time Polymerase Chain Reaction

    Osteoblast differentiation increased in Smurf2 knockdown and knockout cells. ( a , b ) Analysis of ALP and Smurf2 expression in human mesenchymal stem cells (hMSCs) infected with lentivirus-expressing GFP control or Smurf2 shRNAs. ( c ) Von Kossa staining representative images for combined ALP (blue) and Von Kossa (brown) staining of hMSCs infected with lentivirus-expressing control or Smurf2 shRNAs cultured in osteoblast differentiation medium for 21 days. Scale bar, 0.1 cm. ( d ) Bone nodule mineralization of hMSCs determined by Von Kossa staining. The bar graph displays the quantification of Von Kossa-positive mineralized nodules. ( e , f ) Analysis of ALP expression and Von Kossa staining of primary osteoblasts cultured in osteoblast differentiation medium for 7 and 21 days. Scale bar, 0.1 cm. ( g ) Quantitative reverse transcriptase–PCR analysis of osteoblast genes in primary osteoblasts from neonatal 5-day-old mice cultured in osteoblast differentiation medium for 7 days. Data represent mean±s.d. ( n =10 for each genotype, * P

    Journal: Nature Communications

    Article Title: SMURF2 regulates bone homeostasis by disrupting SMAD3 interaction with vitamin D receptor in osteoblasts

    doi: 10.1038/ncomms14570

    Figure Lengend Snippet: Osteoblast differentiation increased in Smurf2 knockdown and knockout cells. ( a , b ) Analysis of ALP and Smurf2 expression in human mesenchymal stem cells (hMSCs) infected with lentivirus-expressing GFP control or Smurf2 shRNAs. ( c ) Von Kossa staining representative images for combined ALP (blue) and Von Kossa (brown) staining of hMSCs infected with lentivirus-expressing control or Smurf2 shRNAs cultured in osteoblast differentiation medium for 21 days. Scale bar, 0.1 cm. ( d ) Bone nodule mineralization of hMSCs determined by Von Kossa staining. The bar graph displays the quantification of Von Kossa-positive mineralized nodules. ( e , f ) Analysis of ALP expression and Von Kossa staining of primary osteoblasts cultured in osteoblast differentiation medium for 7 and 21 days. Scale bar, 0.1 cm. ( g ) Quantitative reverse transcriptase–PCR analysis of osteoblast genes in primary osteoblasts from neonatal 5-day-old mice cultured in osteoblast differentiation medium for 7 days. Data represent mean±s.d. ( n =10 for each genotype, * P

    Article Snippet: Anti-SMURF2 antibody (ABS59; 1:500) was obtained from Millipore.

    Techniques: Knock-Out, ALP Assay, Expressing, Infection, Staining, Cell Culture, Polymerase Chain Reaction, Mouse Assay

    Deletion of Smurf2 in mesenchymal cells leads to increased bone resorption. ( a – e ) μ-QCT analysis of proximal femur from 5-week-old Smurf2 fl/fl and Smurf2 prx1 male mice for bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and cortical thickness (C.Th). Scale bar, 500 μm. ( f ) Histological sections of tibias from 5-week-old Smurf2 fl/fl and Smurf2 prx1 male mice stained for tartrate-resistant acid phosphatase (TRAP). Scale bar, 100 μm. ( g ) Serum CTX levels in 5-week-old Smurf2 fl/fl and Smurf2 prx1 male mice. Value represent means±s.d. ( n =6 for each genotype, ** P

    Journal: Nature Communications

    Article Title: SMURF2 regulates bone homeostasis by disrupting SMAD3 interaction with vitamin D receptor in osteoblasts

    doi: 10.1038/ncomms14570

    Figure Lengend Snippet: Deletion of Smurf2 in mesenchymal cells leads to increased bone resorption. ( a – e ) μ-QCT analysis of proximal femur from 5-week-old Smurf2 fl/fl and Smurf2 prx1 male mice for bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and cortical thickness (C.Th). Scale bar, 500 μm. ( f ) Histological sections of tibias from 5-week-old Smurf2 fl/fl and Smurf2 prx1 male mice stained for tartrate-resistant acid phosphatase (TRAP). Scale bar, 100 μm. ( g ) Serum CTX levels in 5-week-old Smurf2 fl/fl and Smurf2 prx1 male mice. Value represent means±s.d. ( n =6 for each genotype, ** P

    Article Snippet: Anti-SMURF2 antibody (ABS59; 1:500) was obtained from Millipore.

    Techniques: Mouse Assay, Staining

    BMP4 regulation of ERα depends on levels of estrogens. (A) Western blotting analysis of ERα expression level in adipose tissue from male BMP4 knockout and control mice on HFD at age of 6 months. Relative grey intensity of the band was quantified using Image J software. n = 6. (B) Multipotent C3H10T1/2 cells stably expressing ERα-Flag were induced to differentiation and treated with 20 ng/ml BMP4 in presence of estradiol at different concentration for 12 h, and the protein level of ERα was examined with anti-Flag antibody by Western blot. Relative grey intensity of the band from 4 individual experiments was quantified using Image J software.

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: BMP4 regulation of ERα depends on levels of estrogens. (A) Western blotting analysis of ERα expression level in adipose tissue from male BMP4 knockout and control mice on HFD at age of 6 months. Relative grey intensity of the band was quantified using Image J software. n = 6. (B) Multipotent C3H10T1/2 cells stably expressing ERα-Flag were induced to differentiation and treated with 20 ng/ml BMP4 in presence of estradiol at different concentration for 12 h, and the protein level of ERα was examined with anti-Flag antibody by Western blot. Relative grey intensity of the band from 4 individual experiments was quantified using Image J software.

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Western Blot, Expressing, Knock-Out, Mouse Assay, Software, Stable Transfection, Concentration Assay

    Estradiol reduced obesity and insulin resistance of ovariectomized female BMP4-knockout mice. WT and KO female mice at age of 2 months were ovariectomized and maintained for 8 weeks to deplete ovarian steroid hormone, followed by ip administration of estradiol for 6 weeks. (A) Western blotting analysis of BMP4 expression level in WAT. Relative grey intensity of the band was quantitated using Image J software. n = 4. (B) Body weight of BMP4 knockout and control mice after ovariectomized. n = 4. (C) Change of body weight of ovariectomized female mice during the period of estradiol administration. n = 4. (D) Glucose concentrations during an intraperitoneal glucose tolerance test (n = 8) and quantification of AUG (area under curve) from ovariectomized BMP4 knockout and control mice. n = 6. (E) Glucose concentrations during an intraperitoneal insulin tolerance test (n = 7) and quantification of AUG from ovariectomized BMP4 knockout and control mice. n = 6. (F) Glucose concentrations during an intraperitoneal glucose tolerance test (n = 8) and quantification of AUG (area under curve) from ovariectomized BMP4 knockout and control mice treated with estradiol. n = 6. (G) Glucose concentrations during an intraperitoneal insulin tolerance test (n = 7) and quantification of AUG from ovariectomized BMP4 knockout and control mice treated with estradiol. n = 6.

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: Estradiol reduced obesity and insulin resistance of ovariectomized female BMP4-knockout mice. WT and KO female mice at age of 2 months were ovariectomized and maintained for 8 weeks to deplete ovarian steroid hormone, followed by ip administration of estradiol for 6 weeks. (A) Western blotting analysis of BMP4 expression level in WAT. Relative grey intensity of the band was quantitated using Image J software. n = 4. (B) Body weight of BMP4 knockout and control mice after ovariectomized. n = 4. (C) Change of body weight of ovariectomized female mice during the period of estradiol administration. n = 4. (D) Glucose concentrations during an intraperitoneal glucose tolerance test (n = 8) and quantification of AUG (area under curve) from ovariectomized BMP4 knockout and control mice. n = 6. (E) Glucose concentrations during an intraperitoneal insulin tolerance test (n = 7) and quantification of AUG from ovariectomized BMP4 knockout and control mice. n = 6. (F) Glucose concentrations during an intraperitoneal glucose tolerance test (n = 8) and quantification of AUG (area under curve) from ovariectomized BMP4 knockout and control mice treated with estradiol. n = 6. (G) Glucose concentrations during an intraperitoneal insulin tolerance test (n = 7) and quantification of AUG from ovariectomized BMP4 knockout and control mice treated with estradiol. n = 6.

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Knock-Out, Mouse Assay, Western Blot, Expressing, Software

    The correlation between BMP4 level and body mass index (BMI) dependents on activation of estrogen/ERα. (A) Real-time PCR determined the BMP4 mRNA level in subcutaneous WAT of pre and post-menopausal women. (B C) Linear regression analysis between BMI and BMP4 mRNA level in subcutaneous adipose tissue of premenopausal and postmenopausal women. (D) Schematic Model: Counterbalance between BMP4 and estrogen/ERα signaling in adipose tissue development and energy metabolism.

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: The correlation between BMP4 level and body mass index (BMI) dependents on activation of estrogen/ERα. (A) Real-time PCR determined the BMP4 mRNA level in subcutaneous WAT of pre and post-menopausal women. (B C) Linear regression analysis between BMI and BMP4 mRNA level in subcutaneous adipose tissue of premenopausal and postmenopausal women. (D) Schematic Model: Counterbalance between BMP4 and estrogen/ERα signaling in adipose tissue development and energy metabolism.

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction

    Aged BMP4-knockout female mice are prone to developing obesity and insulin resistance. (A) Western blotting analysis of BMP4 expression level in gonadal white adipose tissue from BMP4 knockout and control mice at different ages. Relative grey intensity of the band was quantitated using Image J software. n = 6. (B) Fat index of inguinal WAT and gonadal WAT in aged BMP4 knockout and control mice. n = 6–7. (C) H E staining of inguinal WAT and gonadal WAT from BMP4 knockout and control mice. Scale bar: 20 μm. (D) Glucose concentrations during an intraperitoneal glucose tolerance test and quantification of AUG (area under curve) from aged BMP4 knockout and control mice. n = 7. (E) Glucose concentrations during an intraperitoneal insulin tolerance test and quantification of AUG from aged BMP4 knockout and control mice. n = 6. Data are collected from mice on NC at age different ages (Young: 6 months; Aged 22 months), and expressed as means ± SEM, *p

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: Aged BMP4-knockout female mice are prone to developing obesity and insulin resistance. (A) Western blotting analysis of BMP4 expression level in gonadal white adipose tissue from BMP4 knockout and control mice at different ages. Relative grey intensity of the band was quantitated using Image J software. n = 6. (B) Fat index of inguinal WAT and gonadal WAT in aged BMP4 knockout and control mice. n = 6–7. (C) H E staining of inguinal WAT and gonadal WAT from BMP4 knockout and control mice. Scale bar: 20 μm. (D) Glucose concentrations during an intraperitoneal glucose tolerance test and quantification of AUG (area under curve) from aged BMP4 knockout and control mice. n = 7. (E) Glucose concentrations during an intraperitoneal insulin tolerance test and quantification of AUG from aged BMP4 knockout and control mice. n = 6. Data are collected from mice on NC at age different ages (Young: 6 months; Aged 22 months), and expressed as means ± SEM, *p

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Knock-Out, Mouse Assay, Western Blot, Expressing, Software, Staining

    Disruption of BMP4 expression prevent female mice from fatty liver and inflammation in adipose tissue. (A) Hematoxylin and eosin staining of liver from BMP4 knockout and control mice on HFD. Scale bar: 25 μm. (B) RT-qPCR analysis of adipose tissue for expression of inflammatory factors in visceral WATfromBMP4 knockout and control male mice. n = 4. (C) RT-qPCR analysis of adipose tissue for expression of inflammatory factors in visceral WAT fromBMP4 knockout and control male mice. n = 4.

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: Disruption of BMP4 expression prevent female mice from fatty liver and inflammation in adipose tissue. (A) Hematoxylin and eosin staining of liver from BMP4 knockout and control mice on HFD. Scale bar: 25 μm. (B) RT-qPCR analysis of adipose tissue for expression of inflammatory factors in visceral WATfromBMP4 knockout and control male mice. n = 4. (C) RT-qPCR analysis of adipose tissue for expression of inflammatory factors in visceral WAT fromBMP4 knockout and control male mice. n = 4.

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Expressing, Mouse Assay, Staining, Knock-Out, Quantitative RT-PCR

    Adiposity and glucose metabolism of BMP4 knockout mice. (A) Growth curve of BMP4 knockout and wild type control mice on HFD from 8 to 26 weeks. n = 6. (B) Representative pictures of mice and fat pads of BMP4 knockout (left panel) and control mice (right panel). (C, D) Fat index (percentage of fat pad weight to the whole body weight) of inguinal WAT and gonadal WAT in BMP4 knockout and control male (C) and female (D) mice. n = 8. (E) H E staining of inguinal WAT and gonadal WAT from BMP4 knockout and control mice. Scale bar: 25 μm. (F) Quantification of adipocyte diameter of inguinal WAT, gonadal WAT and BAT from BMP4 knockout and control mice. (Data were collected using Image J software from H E staining section of 3 individual mice, 5 fields per mouse, 10–15 cells per field in each group). (G) Glucose concentrations during an intraperitoneal glucose tolerance test (I) (n = 8) and quantification of AUG (area under curve) from BMP4 knockout and control mice. n = 6–9. (H) Glucose concentrations during an intraperitoneal insulin tolerance test (n = 7) and quantification of AUG from BMP4 knockout and control mice. n = 6–10. BMP4 +/+: wild type control; BMP4 −/−: BMP4 knockout. Data are collected from mice on HFD at age of 6 months except indicated, and expressed as means ± SEM, *p

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: Adiposity and glucose metabolism of BMP4 knockout mice. (A) Growth curve of BMP4 knockout and wild type control mice on HFD from 8 to 26 weeks. n = 6. (B) Representative pictures of mice and fat pads of BMP4 knockout (left panel) and control mice (right panel). (C, D) Fat index (percentage of fat pad weight to the whole body weight) of inguinal WAT and gonadal WAT in BMP4 knockout and control male (C) and female (D) mice. n = 8. (E) H E staining of inguinal WAT and gonadal WAT from BMP4 knockout and control mice. Scale bar: 25 μm. (F) Quantification of adipocyte diameter of inguinal WAT, gonadal WAT and BAT from BMP4 knockout and control mice. (Data were collected using Image J software from H E staining section of 3 individual mice, 5 fields per mouse, 10–15 cells per field in each group). (G) Glucose concentrations during an intraperitoneal glucose tolerance test (I) (n = 8) and quantification of AUG (area under curve) from BMP4 knockout and control mice. n = 6–9. (H) Glucose concentrations during an intraperitoneal insulin tolerance test (n = 7) and quantification of AUG from BMP4 knockout and control mice. n = 6–10. BMP4 +/+: wild type control; BMP4 −/−: BMP4 knockout. Data are collected from mice on HFD at age of 6 months except indicated, and expressed as means ± SEM, *p

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Knock-Out, Mouse Assay, Staining, Software

    BMP4 knockout Increased protein stability of ERα in adipocytes. (A) Preadipocytes isolated from adipose tissue of female BMP4 knockout and control mice were stimulated to adipogenesis. Differentiated adipocytes were maintained for more 12 h in presence of 100 nM estradiol, and RT-qPCR analysis of BMP4 expression level. (B) Western blotting analysis of ERα expression level in differentiated adipocyte indicated in A. (C) Differentiated multipotent C3H10T1/2 cells stably expressing FLAG tagged ERα were treated with BMP4 at different concentration in presence of 100 nM estradiol for 12 h, and the level of ERα was examined with anti-Flag antibody by Western blot. (D) Multipotent C3H10T1/2 cells stably expressing ERα-FLAG were induced to differentiation and at day 4 and cells were infected with adenovirus expressing BMP4 shRNA and control Lac Z sh, the of ERα was examined with anti-Flag antibody by Western blot 48 h after infection. (E) Differentiated adipocytes from C3H10T1/2 cells stably expressing ERα-FLAG treated with or without of BMP4 were incubated with cycloheximide (20 μM) for the indicated times and ERα was examined by Western blot. (F) Differentiated C3H10T1/2 cells pretreated with cycloheximide (20 μM) and incubated with combination of 20 ng/ml BMP4 and 25 μM MG132 for 8 h. ERα was examined by Western blot. For Western blot, relative grey intensity of the band from three individual experiments was quantitated using Image J software.

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: BMP4 knockout Increased protein stability of ERα in adipocytes. (A) Preadipocytes isolated from adipose tissue of female BMP4 knockout and control mice were stimulated to adipogenesis. Differentiated adipocytes were maintained for more 12 h in presence of 100 nM estradiol, and RT-qPCR analysis of BMP4 expression level. (B) Western blotting analysis of ERα expression level in differentiated adipocyte indicated in A. (C) Differentiated multipotent C3H10T1/2 cells stably expressing FLAG tagged ERα were treated with BMP4 at different concentration in presence of 100 nM estradiol for 12 h, and the level of ERα was examined with anti-Flag antibody by Western blot. (D) Multipotent C3H10T1/2 cells stably expressing ERα-FLAG were induced to differentiation and at day 4 and cells were infected with adenovirus expressing BMP4 shRNA and control Lac Z sh, the of ERα was examined with anti-Flag antibody by Western blot 48 h after infection. (E) Differentiated adipocytes from C3H10T1/2 cells stably expressing ERα-FLAG treated with or without of BMP4 were incubated with cycloheximide (20 μM) for the indicated times and ERα was examined by Western blot. (F) Differentiated C3H10T1/2 cells pretreated with cycloheximide (20 μM) and incubated with combination of 20 ng/ml BMP4 and 25 μM MG132 for 8 h. ERα was examined by Western blot. For Western blot, relative grey intensity of the band from three individual experiments was quantitated using Image J software.

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Knock-Out, Isolation, Mouse Assay, Quantitative RT-PCR, Expressing, Western Blot, Stable Transfection, Concentration Assay, Infection, shRNA, Incubation, Software

    Enhanced ERα signaling in WAT of BMP4 knockout female mice. (A) Serum 17β-estradiol level showed by Elisa in BMP4 knockout and control mice. n = 4. (B) RT-qPCR analysis of WAT for expression of ERα from female BMP4 knockout and control mice on HFD at age of 6 months. n = 5. (C) Representative Western blotting analysis of ERα expression level in adipose tissue from BMP4 knockout and control female mice on HFD at age of 6 months. Relative grey intensity of the band was quantitated using Image J software. n = 6–7. (D) RT-qPCR analysis of WAT for expression of ERα target genes GPX3, LXRα and LXRβ from 6 months old mice on HFD. n = 6. (E) Daily food intake of control and knockout female mice maintained on a HFD, 8 mice per group, weigh each week for 5 weeks. (F) Whole-body oxygen consumption rate (VO2) of control and knockout female mice of 6 months during a 12-hour dark/12-hour light cycle measured in metabolic cage, n = 8. (G) RT-qPCR analysis of WAT for expression of genes of β oxidation from female BMP4 knockout and control mice on HFD at age of 6 months, n = 8. Data are expressed as means ± SEM, *p

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: Enhanced ERα signaling in WAT of BMP4 knockout female mice. (A) Serum 17β-estradiol level showed by Elisa in BMP4 knockout and control mice. n = 4. (B) RT-qPCR analysis of WAT for expression of ERα from female BMP4 knockout and control mice on HFD at age of 6 months. n = 5. (C) Representative Western blotting analysis of ERα expression level in adipose tissue from BMP4 knockout and control female mice on HFD at age of 6 months. Relative grey intensity of the band was quantitated using Image J software. n = 6–7. (D) RT-qPCR analysis of WAT for expression of ERα target genes GPX3, LXRα and LXRβ from 6 months old mice on HFD. n = 6. (E) Daily food intake of control and knockout female mice maintained on a HFD, 8 mice per group, weigh each week for 5 weeks. (F) Whole-body oxygen consumption rate (VO2) of control and knockout female mice of 6 months during a 12-hour dark/12-hour light cycle measured in metabolic cage, n = 8. (G) RT-qPCR analysis of WAT for expression of genes of β oxidation from female BMP4 knockout and control mice on HFD at age of 6 months, n = 8. Data are expressed as means ± SEM, *p

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Knock-Out, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot, Software

    Decreased level of estrogens up regulates BMP4 expression in adipose tissue. (A) Western blotting analysis of ERα expression level in inguinal and gonadal white adipose tissue from young (6 months old) and (22 months old) old female mice. (B) Relative grey intensity of the band showed in A quantitated using Image J software. n = 3–4. (C) Western blotting analysis of BMP4 expression level in inguinal and gonadal white adipose tissue from female mice at indicated ages. Relative grey intensity of the band was quantitated using Image J software. n = 4. (D) Serum 17β-estradiol level showed by Elisa in female mice at indicated ages. n = 4–8. (E) RT-qPCR analysis for expression of BMP4 in differentiated adipocytes from primary isolated preadipocytes treated with estradiol (100 ng/ml). (data from three independent experiments). (F) ERα expression level showed by RT-qPCR in adipocytes with ERα SiRNA transfection (data from three independent experiments). (G) Relative expression level of BMP4 showed by RT-qPCR in differentiated adipocytes (data from three independent experiments). BMP4 +/+: wild type control; BMP4 −/−: BMP4 knockout.

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: Decreased level of estrogens up regulates BMP4 expression in adipose tissue. (A) Western blotting analysis of ERα expression level in inguinal and gonadal white adipose tissue from young (6 months old) and (22 months old) old female mice. (B) Relative grey intensity of the band showed in A quantitated using Image J software. n = 3–4. (C) Western blotting analysis of BMP4 expression level in inguinal and gonadal white adipose tissue from female mice at indicated ages. Relative grey intensity of the band was quantitated using Image J software. n = 4. (D) Serum 17β-estradiol level showed by Elisa in female mice at indicated ages. n = 4–8. (E) RT-qPCR analysis for expression of BMP4 in differentiated adipocytes from primary isolated preadipocytes treated with estradiol (100 ng/ml). (data from three independent experiments). (F) ERα expression level showed by RT-qPCR in adipocytes with ERα SiRNA transfection (data from three independent experiments). (G) Relative expression level of BMP4 showed by RT-qPCR in differentiated adipocytes (data from three independent experiments). BMP4 +/+: wild type control; BMP4 −/−: BMP4 knockout.

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Expressing, Western Blot, Mouse Assay, Software, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation, Transfection, Knock-Out

    Phenotype of BMP4 knockout (BMP4 −/−) and control (BMP4 +/+) mice of different gender. (A) Growth curve of BMP4 knockout (BMP4 −/−) and control (BMP4 +/+) mice from 8 to 24 weeks. n = 8. (B) Comparison of BMP4 knockout and control mice of different gender. (C) Comparison of adipose tissue from BMP4 knockout and control mice of different gender. (D) Hematoxylin and eosin staining of liver from BMP4 knockout and control mice on HFD. Scale bar: 25 μm. (E) Fat index (percentage of fat pad weight to the whole body weight) of BAT in BMP4 knockout and control male (C) and female (D) mice. n = 8. Data were collected from mice on normal chow diet and expressed as means ± SEM, *p

    Journal: EBioMedicine

    Article Title: BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

    doi: 10.1016/j.ebiom.2016.07.034

    Figure Lengend Snippet: Phenotype of BMP4 knockout (BMP4 −/−) and control (BMP4 +/+) mice of different gender. (A) Growth curve of BMP4 knockout (BMP4 −/−) and control (BMP4 +/+) mice from 8 to 24 weeks. n = 8. (B) Comparison of BMP4 knockout and control mice of different gender. (C) Comparison of adipose tissue from BMP4 knockout and control mice of different gender. (D) Hematoxylin and eosin staining of liver from BMP4 knockout and control mice on HFD. Scale bar: 25 μm. (E) Fat index (percentage of fat pad weight to the whole body weight) of BAT in BMP4 knockout and control male (C) and female (D) mice. n = 8. Data were collected from mice on normal chow diet and expressed as means ± SEM, *p

    Article Snippet: The anti-BMP4 antibody was from Millipore (catalog number: MAB 1094; Dilution: 1:2000), anti-ERα was from Santa Cruz Biotechnology (catalog number: SC-787; Dilution: 1:500), and anti-HSP90 was also from Santa Cruz Biotechnology (catalog number: SC-7947; Dilution: 1:1000).

    Techniques: Knock-Out, Mouse Assay, Staining

    EV71 viruses bind to galectin-1. (A) Galectin-1 serum levels are increased in EV71-infected patients. Seven clinical severe EV71-infected patient sera, five non-severe EV71-infected patient sera and twelve non-infected control sera were collected to detect galectin-1 by ELISA. *p

    Journal: PLoS ONE

    Article Title: Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

    doi: 10.1371/journal.pone.0116278

    Figure Lengend Snippet: EV71 viruses bind to galectin-1. (A) Galectin-1 serum levels are increased in EV71-infected patients. Seven clinical severe EV71-infected patient sera, five non-severe EV71-infected patient sera and twelve non-infected control sera were collected to detect galectin-1 by ELISA. *p

    Article Snippet: The bound virus was detected by anti-EV71 antibody and HRP-conjugated goat anti-mouse IgG antibody. (TIF) Click here for additional data file.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay

    Galectin-1 is associated with released EV71 virions. (A) Detection of galectin-1/EV71 virion complexes by ELISA. EV71 viruses in 10-fold serial dilutions from 10 6 to 10 4 PFU were added to anti-EV71 antibody-coated (left panel) or anti-galectin-1 antibody-coated (right panel) 96-well plates to detect galectin-1 or EV71, respectively. *p

    Journal: PLoS ONE

    Article Title: Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

    doi: 10.1371/journal.pone.0116278

    Figure Lengend Snippet: Galectin-1 is associated with released EV71 virions. (A) Detection of galectin-1/EV71 virion complexes by ELISA. EV71 viruses in 10-fold serial dilutions from 10 6 to 10 4 PFU were added to anti-EV71 antibody-coated (left panel) or anti-galectin-1 antibody-coated (right panel) 96-well plates to detect galectin-1 or EV71, respectively. *p

    Article Snippet: The bound virus was detected by anti-EV71 antibody and HRP-conjugated goat anti-mouse IgG antibody. (TIF) Click here for additional data file.

    Techniques: Enzyme-linked Immunosorbent Assay

    Galectin-1-/- EV71 shows less cellular binding, infectivity, mice neurological syndromes, and mortality. (A) Galectin-1-/- EV71 viruses reduce their cell binding activity. SK-N-SH cells were incubated with WT, galectin-1 -/- EV71 or galectin-1 -/- EV71 viruses with recombinant galectin-1 (25 ng/ml) at 4°C (MOI = 100) for 3 h. The surface-bound EV71 was detected by anti-EV71 antibody and hence analyzed by flow cytometry. *p

    Journal: PLoS ONE

    Article Title: Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

    doi: 10.1371/journal.pone.0116278

    Figure Lengend Snippet: Galectin-1-/- EV71 shows less cellular binding, infectivity, mice neurological syndromes, and mortality. (A) Galectin-1-/- EV71 viruses reduce their cell binding activity. SK-N-SH cells were incubated with WT, galectin-1 -/- EV71 or galectin-1 -/- EV71 viruses with recombinant galectin-1 (25 ng/ml) at 4°C (MOI = 100) for 3 h. The surface-bound EV71 was detected by anti-EV71 antibody and hence analyzed by flow cytometry. *p

    Article Snippet: The bound virus was detected by anti-EV71 antibody and HRP-conjugated goat anti-mouse IgG antibody. (TIF) Click here for additional data file.

    Techniques: Binding Assay, Infection, Mouse Assay, Activity Assay, Incubation, Recombinant, Flow Cytometry, Cytometry

    EV71 viruses interact with cellular galectin-1 in EV71-infected cells. (A) Co-localization of galectin-1 and EV71 in EV71-infected cells. SK-N-SH or RD cells were infected with EV71 viruses with MOI = 1 for 8 h and then stained with Hoechst 33258, anti-EV71, and anti-galectin-1 antibodies. The distributions of these proteins were analyzed by confocal microscopy. The co-localization of EV71 and galectin-1 is indicated by arrows. (B) Co-immunoprecipitation of galectin-1 and EV71virus from EV71-infected cells. SK-N-SH and RD cells were infected with EV71 virus with MOI = 1 for 8 h. Cells were lysed and then precipitated by anti-galectin-1 antibody. The precipitated proteins were analyzed by Western blotting. EV71 viruses input as positive control. (C) Galectin-1 interacts with VP1 and VP3 of EV71. 293T cells were co-transfected with pEGFP-galectin-1 and pFLAG-MSCV (VP1, VP2 or VP3) for 24 hours. The cells were fixed and stained with anti-Flag antibody. The colocalization of three VP proteins and galectin-1 was detected by confocal microscopy. The arrows indicate colocalization of the VP protein and galectin-1. (D) Co-immunoprecipitation of galectin-1 and EV71 VP proteins. Galecin-1-GFP, VP1-FLAG, VP2-FLAG and VP3-FLAG plasmids were introduced to 293T cells. Cells were lysed and then precipitated by anti-FLAG antibody. The precipitated proteins were detected by Western blotting. Results are representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

    doi: 10.1371/journal.pone.0116278

    Figure Lengend Snippet: EV71 viruses interact with cellular galectin-1 in EV71-infected cells. (A) Co-localization of galectin-1 and EV71 in EV71-infected cells. SK-N-SH or RD cells were infected with EV71 viruses with MOI = 1 for 8 h and then stained with Hoechst 33258, anti-EV71, and anti-galectin-1 antibodies. The distributions of these proteins were analyzed by confocal microscopy. The co-localization of EV71 and galectin-1 is indicated by arrows. (B) Co-immunoprecipitation of galectin-1 and EV71virus from EV71-infected cells. SK-N-SH and RD cells were infected with EV71 virus with MOI = 1 for 8 h. Cells were lysed and then precipitated by anti-galectin-1 antibody. The precipitated proteins were analyzed by Western blotting. EV71 viruses input as positive control. (C) Galectin-1 interacts with VP1 and VP3 of EV71. 293T cells were co-transfected with pEGFP-galectin-1 and pFLAG-MSCV (VP1, VP2 or VP3) for 24 hours. The cells were fixed and stained with anti-Flag antibody. The colocalization of three VP proteins and galectin-1 was detected by confocal microscopy. The arrows indicate colocalization of the VP protein and galectin-1. (D) Co-immunoprecipitation of galectin-1 and EV71 VP proteins. Galecin-1-GFP, VP1-FLAG, VP2-FLAG and VP3-FLAG plasmids were introduced to 293T cells. Cells were lysed and then precipitated by anti-FLAG antibody. The precipitated proteins were detected by Western blotting. Results are representative of two independent experiments.

    Article Snippet: The bound virus was detected by anti-EV71 antibody and HRP-conjugated goat anti-mouse IgG antibody. (TIF) Click here for additional data file.

    Techniques: Infection, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Positive Control, Transfection

    EV71 viruses propagated from galectin-1 silenced cells do not carry galectin-1. (A) Galectin-1 is silenced by lentiviral vector-based shRNA. SK-N-SH cells were infected with lentiviruses encoding a galectin-1 shRNA for 48 h. The silencing of galectin-1 in SK-N-SH cells was determined by Western blotting. (B) Co-immunoprecipitation of WT EV71 or galetin-1 -/- EV71 virus with galectin-1. WT or galectin-1 -/- EV71 viruses were immunoprecipitated by anti-EV71 antibody. The expressions of galectin-1 and EV71 were analyzed by Western blotting. (C) Detection of galectin-1 on WT or galectinl-1 -/- EV71 viruses by ELISA. EV71 in 10 fold serial dilutions from 10 6 to 10 4 PFU was added to anti-EV71 antibody-coated (left panel) or anti-galectin-1 antibody-coated (right panel) 96-well plate to detect galectin-1 or EV71, respectively. *p

    Journal: PLoS ONE

    Article Title: Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

    doi: 10.1371/journal.pone.0116278

    Figure Lengend Snippet: EV71 viruses propagated from galectin-1 silenced cells do not carry galectin-1. (A) Galectin-1 is silenced by lentiviral vector-based shRNA. SK-N-SH cells were infected with lentiviruses encoding a galectin-1 shRNA for 48 h. The silencing of galectin-1 in SK-N-SH cells was determined by Western blotting. (B) Co-immunoprecipitation of WT EV71 or galetin-1 -/- EV71 virus with galectin-1. WT or galectin-1 -/- EV71 viruses were immunoprecipitated by anti-EV71 antibody. The expressions of galectin-1 and EV71 were analyzed by Western blotting. (C) Detection of galectin-1 on WT or galectinl-1 -/- EV71 viruses by ELISA. EV71 in 10 fold serial dilutions from 10 6 to 10 4 PFU was added to anti-EV71 antibody-coated (left panel) or anti-galectin-1 antibody-coated (right panel) 96-well plate to detect galectin-1 or EV71, respectively. *p

    Article Snippet: The bound virus was detected by anti-EV71 antibody and HRP-conjugated goat anti-mouse IgG antibody. (TIF) Click here for additional data file.

    Techniques: Plasmid Preparation, shRNA, Infection, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

    Galectin-1 facilitates EV71 viruses against thermal and environmental stress. WT EV71, galectin-1 -/- EV71 or galectin-1 -/- EV71 viruses with recombinant galectin-1 (25 ng/ml) were heated to 39°C for 4 h, and then infected SK-N-SH cells at 37°C (MOI = 0.5) for another 12 h. The infection rates of EV71 viruses were detected and analyzed by flow cytometry. (B) WT EV71, galectin-1 -/- EV71 or galectin-1 -/- EV71 viruses with recombinant galectin-1 (25 ng/ml) were stored at 4°C for 30 days, and then infected SK-N-SH cells at 37°C (MOI = 0.5) for 12 h. The relative infection rate was compared to freshly isolated EV71 virus of each group. *p

    Journal: PLoS ONE

    Article Title: Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

    doi: 10.1371/journal.pone.0116278

    Figure Lengend Snippet: Galectin-1 facilitates EV71 viruses against thermal and environmental stress. WT EV71, galectin-1 -/- EV71 or galectin-1 -/- EV71 viruses with recombinant galectin-1 (25 ng/ml) were heated to 39°C for 4 h, and then infected SK-N-SH cells at 37°C (MOI = 0.5) for another 12 h. The infection rates of EV71 viruses were detected and analyzed by flow cytometry. (B) WT EV71, galectin-1 -/- EV71 or galectin-1 -/- EV71 viruses with recombinant galectin-1 (25 ng/ml) were stored at 4°C for 30 days, and then infected SK-N-SH cells at 37°C (MOI = 0.5) for 12 h. The relative infection rate was compared to freshly isolated EV71 virus of each group. *p

    Article Snippet: The bound virus was detected by anti-EV71 antibody and HRP-conjugated goat anti-mouse IgG antibody. (TIF) Click here for additional data file.

    Techniques: Recombinant, Infection, Flow Cytometry, Cytometry, Isolation