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  • 99
    Millipore lentiviral mediated shrna infection lentivirus particles
    Lentiviral Mediated Shrna Infection Lentivirus Particles, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc lentivirus infection lentiviral vector
    Lentivirus Infection Lentiviral Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Genecopoeia lentivirus infection lentivirus
    Scanning EM analysis of the effect of anti‐CD11b, anti‐CD18 antibodies and Itgam silencing on the bone resorption levels in vitro . Mouse OCPs were cultured on bone slices and treated for 7 days with sRANKL (100 ng/ml). ( A ) Bone resorption pits of cells treated with neutralizing antibodies (10 μg/ml) or <t>lentivirus</t> (MOI = 10). ( B ) Relative bone resorption area per osteoclast from different groups, n = 3. # P
    Lentivirus Infection Lentivirus, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genechem lentivirus infection lentiviruses
    GFP-labeled <t>lentivirus</t> (LV)-hsa-miR-200c and its negative control (NC) infected A549, H1975 and H1299 The infection efficiency is still keeping in the range of 80%-90%.
    Lentivirus Infection Lentiviruses, supplied by Genechem, used in various techniques. Bioz Stars score: 94/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company lentivirus infection lentiviral vectors
    GFP-labeled <t>lentivirus</t> (LV)-hsa-miR-200c and its negative control (NC) infected A549, H1975 and H1299 The infection efficiency is still keeping in the range of 80%-90%.
    Lentivirus Infection Lentiviral Vectors, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    System Biosciences Inc lentivirus infection lentiviruses
    GFP-labeled <t>lentivirus</t> (LV)-hsa-miR-200c and its negative control (NC) infected A549, H1975 and H1299 The infection efficiency is still keeping in the range of 80%-90%.
    Lentivirus Infection Lentiviruses, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Horizon Discovery lentivirus infection lentivirus
    AMPK/p27 Kip1 Signaling Mediates Cell Survival in the MuSC Geriatric MuSCs were cultured for 48 hr treated with or without AICAR and/or overexpression of p27 Kip1 mutant-p27 Kip1(198A) . (A) Percentage of TUNEL-positive cells with respective treatment (N = 4 independent experiments). (B) Upper: representative blot of total and cleaved PARP; lower: quantification of protein expression normalized to untreated geriatric cells (N = 3 independent experiments). (C) Percentage of TUNEL-positive cells during Atg5 knockdown with concurrent overexpression of p27 Kip1 mutants p27 Kip1(198D) or p27 Kip1(198A) (N = 3 independent experiments). (D) Percentage of TUNEL-positive cells during treatment of compound C to young MuSCs with concurrent overexpression of p27 Kip1 mutant p27 Kip1(198D) (N = 3 independent experiments). (E) Geriatric MuSCs treated with or without AICAR and infected with respective p27 Kip1 mutant <t>lentivirus.</t> Cells were transplanted into injured young SCID muscle and quantified 4 or 28 days later. (F) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 4 days after transplantation. (G) Quantification of GFP(+) cells per muscle (N = 6 independent experiments). (H) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 28 days after transplantation. (I) Quantification of GFP(+) myofibers per muscle (N = 6 independent experiments). Scale bar, 100 μm. ∗ Signifies difference from young or untreated control cells (p
    Lentivirus Infection Lentivirus, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lentivirus infection lentiviral expression vectors carrying shrnas
    C/EBPβ and Stat3 maintain the mesenchymal phenotype of human glioma cells a , Immunofluorescence for fibronectin, Col5A1 and YKL40 in BTSC-3408 infected with <t>lentiviruses</t> expressing Stat3, C/EBPβ, or Stat3 plus C/EBPβ shRNA. b , Quantification of fibronectin; c , Col5A1; and d , YKL40 positive cells. n = 3 independent experiments; Bars indicate Mean±SD. e , qRT-PCR of mesenchymal genes in BTSC-20 infected with lentiviruses expressing Stat3, C/EBPβ, or Stat3 plus C/EBPβ shRNA. Gene expression was normalized to the expression of 18S ribosomal RNA. n = 3; Bars indicate Mean±SD. f , Microphotograps of invading SNB19 cells infected with <t>lentiviral</t> vectors expressing control or shStat3 plus shC/EBPβ. g , Quantification of SNB19 invading cells. Bars indicate Mean±SD; n = 6 (two independent experiments, each performed in triplicate). h , Invading BTSC-3408 cells infected with shCtr, shStat3, shC/EBPβ or shStat3 plus shC/EBPβ lentiviruses. i , Quantification of invading BTSC-3408 cells. Bars indicate Mean±SD; n = 6 (two independent experiments, each performed in triplicate). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
    Lentivirus Infection Lentiviral Expression Vectors Carrying Shrnas, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company lentivirus infection recombinant lentiviruses
    Knockdown of DEPTOR promotes osteogenic differentiation of hBMSCs in vitro. hBMSCs transfected with <t>lentivirus</t> expressing DEPTOR shRNAs (shDEPTOR #1, shDEPTOR #2) or scrambled control (shNC), and cultured in proliferation medium (PM) or osteogenic medium (OM). a ALP staining on day 7, ARS staining on day 14, and VK staining on day 21 after proliferative culture or osteogenic induction. b, c ALP activity on day 7 and AZR mineralization assay on day 14 after proliferative culture or osteogenic induction. d, e Relative mRNA levels of ALP and RUNX2 measured by qRT-PCR on day 7 of proliferative culture or osteogenic induction. f, g Relative mRNA levels of OSX and OCN measured by qRT-PCR on day 14 of proliferative culture or osteogenic induction. GAPDH used for normalization. h Left: western blot analysis of RUNX2 and OCN protein levels on day 14 after osteogenic induction. GAPDH used as internal control. Right: quantification of band intensities. Data presented as mean ± SD. * P
    Lentivirus Infection Recombinant Lentiviruses, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shanghai Genechem lentivirus infection
    PLK1 inhibition induces AML cell autophagy via mTOR dephosphorylation. (A) Western blotting of mTOR, AMPK and ULK1 expression levels and phosphorylation in NB4 cells treated with RO3280 or BI2536. There was significantly decreased mTOR phosphorylation and ULK1 phosphorylation at Ser317. (B) Western blotting of mTOR expression levels and phosphorylation in NB4, K562 and HL-60 cells transfected with PLK1 RNA interference <t>lentivirus</t> at 100 and 200 MOI. Relative expression of p-mTOR/mTOR was analyzed with ImageJ software according to the user guide. **p
    Lentivirus Infection, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 93/100, based on 178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc lentivirus infection
    Regulation of RB1 in spermatogonia. (A) Suppression of GS cell proliferation through depletion of Cdk4 or Cdk6 . GS cells were infected with the indicated <t>lentivirus</t> expressing shRNA and passaged 4 days after transfection. Cell recovery was determined 6 days after passage (n = 4; MOI = 10). (B) Colony counts. Cells were transplanted 2 days after transfection. Results of two experiments (n = 8; MOI = 10). (C) Macroscopic appearance of recipient testes that received transplantation of GS cells depleted of Cdk4 or Cdk6 . (D) Western blot analysis of RB1. GS cells were cultured without cytokines for 3 days and stimulated with FGF2 and GDNF. Cells were recovered 1 h after cytokine stimulation. Note the relative increase in phosphorylated RB1 (pRB1) caused by cytokine stimulation. The graph shows relative band intensity (n = 3). (E) Western blot analysis of RB1 following depletion of Cdk4 or Cdk6 . Cells were recovered 4 days after transfection (MOI = 10). The graph shows relative band intensity (n = 3). (F) Suppression of GS cell proliferation through depletion of Cdkn1b . GS cells were infected with the indicated lentivrus, and cells were recovered 4 days after transfection (n = 4; MOI = 4). (G) Western blot analysis of cell cycle-related proteins following depletion of Cdkn1b . Cells were recovered 4 days after transfection (MOI = 4). The graph shows relative band intensity (n = 3). Bar = 1 mm (C).
    Lentivirus Infection, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company lentivirus infection
    RIPK1 has a protective role in SHK-induced apoptosis. ( a ) Nec-1 (50 μM) contributes to apoptosis induced by SHK (10 μM) and these effects could be partly abolished by ZVAD-FMK (20 μM). ( b ) Western blots indicated the decrease in RIPK1 with treatment of SHK. ( c ) Cells transfected with pANP7-RIPK1 plasmids or control were incubated with SHK (10 μM) with or without ZVAD-FMK (20 μM) for 24 h. Western blots were used to determine the effects of over-expression. ( d ) Cells infected with RIPK1 shRNA <t>lentivirus</t> particles or control for 48 h were incubated with SHK (10 μM) with or without ZVAD-FMK (20 μM) for 24 h. Western blots were used to determine the effect of knockdown. Values are presented as the mean ± SD for at least three independent experiments. * P
    Lentivirus Infection, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 96/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Anhui Medical University lentivirus infection
    Knockdown or overexpression KIF20A influences GC cell proliferation. Notes: ( A ) Expression levels of KIF20A determined by qRT-PCR in immortalized human gastric mucosal epithelial cell line GES-1 and five GC cell lines (MGC-803, BGC-823, NCI-N87, SGC-7901, and AGS). ( B ) Expression levels of KIF20A in MGC-803 and AGS cell lines were examined by Western blot after <t>lentivirus</t> infection. ( C ) Cell viability assays for cells transfected with KIF20A knockdown and overexpression. ( D ) Colony formation assays for cells transfected with KIF20A knockdown and overexpression plasmids. Values are mean±SEM. *, **, ***, or **** corresponded to P
    Lentivirus Infection, supplied by Anhui Medical University, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher infection lentivirus
    TUTase depletion does not affect miRNA levels. ( a ) Western blot analysis of <t>Lentivirus-mediated</t> knockdown of Zcchc11 and Zcchc6 in Hela cells. ( b ) Mature miRNA northern blot of let-7g levels in the indicated transduced Hela cell lines. Valyl tRNA serves as a loading control. ( c ) Total small RNA from shGFP and shZcchc6/shZcchc11 (shTUT) Hela cells was purified and cloned before sequencing. Each point represents a single miRNA species, and axes are scaled to reads/million (RPM). R 2 > 0.99. ( d ) The relative number of reads for WT (i.e. non-modified) motif-containing miRNAs is unchanged after TUTase depletion.
    Infection Lentivirus, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter lentivirus infection
    STAT2 is required for USP18-mediated inhibition of Type I IFN signaling (a–f) MIP or MIP-USP18 expressing 2fTGH, U1A, U2A, U4A, U5A, and U6A cells were treated with IFNα (1000 U/ml) for 15 minutes. The cell lysates were immunoblotted with the indicated antibodies. (g) IB analysis of STAT1 phosphorylation in 2fTGH and U6A cells in the presence or absence of (1000 U/ml) IFNα. (h–i) Stat2 −/− MEFs were infected with MIP control (−) or MIP-Usp18 (+) retroviruses, either in the presence or absence of rescue with C-terminally FLAG-tagged Stat2 cDNA. Where indicated cells were treated with either mouse IFNβ (500 U/ml) for 15 minutes (h) or 30 minutes (i), before cell lysates were collected and analyzed by Western blotting with indicated antibodies. (j) Validation of Stat2 knockdown. Ba/F3 cells were infected with control or Stat2-targeting shRNA <t>lentivirus.</t> After 5 days puromycin selection, Stat2 expression was examined by Western blotting. (k) Usp18 −/− bone marrow cells were infected with pCX4-bsr control (−) or pCX4-bsr-Usp18 (+), either in the presence or absence of control (shCtrl) or Stat2-knockdown (shStat2–3) shRNA expression. Two days following double drug selection (puromycin and blasticidin), cells were either left untreated (−) or treated (+) with mouse IFNβ (500 U/ml) for 30 minutes. Cell lysates were collected and analyzed by Western blotting with indicated antibodies.
    Lentivirus Infection, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    System Biosciences Inc infection lentivirus
    STAT2 is required for USP18-mediated inhibition of Type I IFN signaling (a–f) MIP or MIP-USP18 expressing 2fTGH, U1A, U2A, U4A, U5A, and U6A cells were treated with IFNα (1000 U/ml) for 15 minutes. The cell lysates were immunoblotted with the indicated antibodies. (g) IB analysis of STAT1 phosphorylation in 2fTGH and U6A cells in the presence or absence of (1000 U/ml) IFNα. (h–i) Stat2 −/− MEFs were infected with MIP control (−) or MIP-Usp18 (+) retroviruses, either in the presence or absence of rescue with C-terminally FLAG-tagged Stat2 cDNA. Where indicated cells were treated with either mouse IFNβ (500 U/ml) for 15 minutes (h) or 30 minutes (i), before cell lysates were collected and analyzed by Western blotting with indicated antibodies. (j) Validation of Stat2 knockdown. Ba/F3 cells were infected with control or Stat2-targeting shRNA <t>lentivirus.</t> After 5 days puromycin selection, Stat2 expression was examined by Western blotting. (k) Usp18 −/− bone marrow cells were infected with pCX4-bsr control (−) or pCX4-bsr-Usp18 (+), either in the presence or absence of control (shCtrl) or Stat2-knockdown (shStat2–3) shRNA expression. Two days following double drug selection (puromycin and blasticidin), cells were either left untreated (−) or treated (+) with mouse IFNβ (500 U/ml) for 30 minutes. Cell lysates were collected and analyzed by Western blotting with indicated antibodies.
    Infection Lentivirus, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Novoprotein lentivirus infection
    STAT2 is required for USP18-mediated inhibition of Type I IFN signaling (a–f) MIP or MIP-USP18 expressing 2fTGH, U1A, U2A, U4A, U5A, and U6A cells were treated with IFNα (1000 U/ml) for 15 minutes. The cell lysates were immunoblotted with the indicated antibodies. (g) IB analysis of STAT1 phosphorylation in 2fTGH and U6A cells in the presence or absence of (1000 U/ml) IFNα. (h–i) Stat2 −/− MEFs were infected with MIP control (−) or MIP-Usp18 (+) retroviruses, either in the presence or absence of rescue with C-terminally FLAG-tagged Stat2 cDNA. Where indicated cells were treated with either mouse IFNβ (500 U/ml) for 15 minutes (h) or 30 minutes (i), before cell lysates were collected and analyzed by Western blotting with indicated antibodies. (j) Validation of Stat2 knockdown. Ba/F3 cells were infected with control or Stat2-targeting shRNA <t>lentivirus.</t> After 5 days puromycin selection, Stat2 expression was examined by Western blotting. (k) Usp18 −/− bone marrow cells were infected with pCX4-bsr control (−) or pCX4-bsr-Usp18 (+), either in the presence or absence of control (shCtrl) or Stat2-knockdown (shStat2–3) shRNA expression. Two days following double drug selection (puromycin and blasticidin), cells were either left untreated (−) or treated (+) with mouse IFNβ (500 U/ml) for 30 minutes. Cell lysates were collected and analyzed by Western blotting with indicated antibodies.
    Lentivirus Infection, supplied by Novoprotein, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    OriGene lentivirus infection
    EZH2 is a target of miR-101. ( A , B ) Left: Schematic representation of the miR-101 site in theEZH2 3′-UTR (up: position 59–65; down: position 114–121);Right: The 3′-UTR reporter assay was carried out in hBMSCs infected with miR-101lentivirus or miR-NC <t>lentivirus.</t> The WT or Mut reporter plasmids were transfected with Lipo-2000. Luciferase assays were performed 48 h after transfection. Firefly luciferase activity was standardized to a Renilla luciferase control. ( C ) EZH2 protein expression and ( D ) mRNA level in hBMSCs infected with miR-101lentivirus or transfected with anti-miR-101 were measured after 48 h. All of the data are expressed as the mean ± SD. *p
    Lentivirus Infection, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SABiosciences lentivirus infection
    EZH2 is a target of miR-101. ( A , B ) Left: Schematic representation of the miR-101 site in theEZH2 3′-UTR (up: position 59–65; down: position 114–121);Right: The 3′-UTR reporter assay was carried out in hBMSCs infected with miR-101lentivirus or miR-NC <t>lentivirus.</t> The WT or Mut reporter plasmids were transfected with Lipo-2000. Luciferase assays were performed 48 h after transfection. Firefly luciferase activity was standardized to a Renilla luciferase control. ( C ) EZH2 protein expression and ( D ) mRNA level in hBMSCs infected with miR-101lentivirus or transfected with anti-miR-101 were measured after 48 h. All of the data are expressed as the mean ± SD. *p
    Lentivirus Infection, supplied by SABiosciences, used in various techniques. Bioz Stars score: 87/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lentivirus infection
    EZH2 is a target of miR-101. ( A , B ) Left: Schematic representation of the miR-101 site in theEZH2 3′-UTR (up: position 59–65; down: position 114–121);Right: The 3′-UTR reporter assay was carried out in hBMSCs infected with miR-101lentivirus or miR-NC <t>lentivirus.</t> The WT or Mut reporter plasmids were transfected with Lipo-2000. Luciferase assays were performed 48 h after transfection. Firefly luciferase activity was standardized to a Renilla luciferase control. ( C ) EZH2 protein expression and ( D ) mRNA level in hBMSCs infected with miR-101lentivirus or transfected with anti-miR-101 were measured after 48 h. All of the data are expressed as the mean ± SD. *p
    Lentivirus Infection, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 348 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company infective lentivirus
    EZH2 is a target of miR-101. ( A , B ) Left: Schematic representation of the miR-101 site in theEZH2 3′-UTR (up: position 59–65; down: position 114–121);Right: The 3′-UTR reporter assay was carried out in hBMSCs infected with miR-101lentivirus or miR-NC <t>lentivirus.</t> The WT or Mut reporter plasmids were transfected with Lipo-2000. Luciferase assays were performed 48 h after transfection. Firefly luciferase activity was standardized to a Renilla luciferase control. ( C ) EZH2 protein expression and ( D ) mRNA level in hBMSCs infected with miR-101lentivirus or transfected with anti-miR-101 were measured after 48 h. All of the data are expressed as the mean ± SD. *p
    Infective Lentivirus, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cellecta infection lentivirus
    EZH2 is a target of miR-101. ( A , B ) Left: Schematic representation of the miR-101 site in theEZH2 3′-UTR (up: position 59–65; down: position 114–121);Right: The 3′-UTR reporter assay was carried out in hBMSCs infected with miR-101lentivirus or miR-NC <t>lentivirus.</t> The WT or Mut reporter plasmids were transfected with Lipo-2000. Luciferase assays were performed 48 h after transfection. Firefly luciferase activity was standardized to a Renilla luciferase control. ( C ) EZH2 protein expression and ( D ) mRNA level in hBMSCs infected with miR-101lentivirus or transfected with anti-miR-101 were measured after 48 h. All of the data are expressed as the mean ± SD. *p
    Infection Lentivirus, supplied by Cellecta, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa infections lentivirus
    MDM2 maintains retinoblastoma cell proliferation in a p53-independent manner A. p53 association with MDM2 but not MDM4 in RB176 and Y79 cells detected by p53 or control IgG immunoprecipitation followed by MDM2, MDM4, and p53 western. *, a non-specific band seen with SMP-14 antibody. B. Western analysis after 6 h of treatment (left) and cell growth response (right) of RB176 cells treated with 10 μM or 20 μM PFT-α starting 4 days after infection with <t>lentivirus</t> expressing shRNA against MDM2 (shMDM2-1) or a scrambled control (Scr). C. Western analysis at day 4 (left) and cell growth response (right) of RB176 cells after infection with lentivirus co-expressing shMDM2 and shp53 or shMDM2 and Scr. Signal intensity was normalized to α-tubulin and expression relative to Scr control is indicated below corresponding panels. D. Western analysis at day 4 (left) and cell growth response (right) of TP53 R175H/R175H CHLA- RB215 cells after infection with lentivirus expressing shMDM2-2 or a scrambled control (Scr). A similar result was obtained separately using shMDM2-1 (not shown).
    Infections Lentivirus, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lentiviral infection
    Knockdown of AP-3δ redistributes Vangl2 from the plasma membrane. HEK293 cells were infected with <t>lentiviral</t> shRNA against nontargeting shRNA (scrambled) (A–C, C′) or with shRNA against AP-3δ subunit (D–F, F′) for 72 h before puromycin selection was started. Cells were then transfected with GFP-Vangl2 for 48 h; this was followed by a quick staining with WGA-594 to mark cell membranes. Vangl2 was not detected on the membrane and was observed in punctuate structures throughout the cell in most cells depleted of AP-3δ. C′ and F′ are large views of the boxed regions in C and F, respectively. The cultures were confluent for both scrambled and AP-3 shRNA conditions. Cultures with less confluency (Supplemental Figure S4) showed similar results. (G) Cell lysates were collected and subjected to Western blot analysis of protein lysates using anti–AP-3δ SA4 antibody, revealing a reduction of > 95% in AP-3 levels by shRNA against AP-3. (F) The percent of Vangl2 redistribution in AP-3–depleted cells was quantified (H). The numbers of cells with Vangl2 on the membrane were significantly lower than that of controls. One hundred cells were counted per condition in each experimental set, and the experiments were repeated three times. Vangl2 membrane localization was quantified by measuring Pearson’s coefficient of Vangl2 and WGA in shRNA Scrambled and shRNA AP-3 cells (I). N = 3; 50 cells analyzed. Scale bars: 19 µm. p
    Lentiviral Infection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 602 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc lentiviral infections
    NOTCH1 signaling regulates Group 3 medulloblastoma metastasis. a – d shRNAs specific to NOTCH1 (shNotch1) were introduced through <t>lentiviral</t> infections into luciferase-expressing D425 cells. 2 μg mL −1 of Dox was used to induce the expression of shRNA (shNotch1 + Dox). The un-induced infected cells (shNotch1-Dox) were used as controls. Recovery of NOTCH1 expression was performed by overexpressing NICD1 in NOTCH1-silenced Group 3 medulloblastoma cells (shNotch1 + Dox + NICD1). Bioluminescence imaging ( a ), hematoxylin/eosin staining ( b ), and quantification of total flux ( c ) from primary tumors and spinal metastases in mice injected with infected cells. ** P
    Lentiviral Infections, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene lentiviral infection lentiviral supernatants
    NOTCH1 signaling regulates Group 3 medulloblastoma metastasis. a – d shRNAs specific to NOTCH1 (shNotch1) were introduced through <t>lentiviral</t> infections into luciferase-expressing D425 cells. 2 μg mL −1 of Dox was used to induce the expression of shRNA (shNotch1 + Dox). The un-induced infected cells (shNotch1-Dox) were used as controls. Recovery of NOTCH1 expression was performed by overexpressing NICD1 in NOTCH1-silenced Group 3 medulloblastoma cells (shNotch1 + Dox + NICD1). Bioluminescence imaging ( a ), hematoxylin/eosin staining ( b ), and quantification of total flux ( c ) from primary tumors and spinal metastases in mice injected with infected cells. ** P
    Lentiviral Infection Lentiviral Supernatants, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PromoCell lentivirus infection human hepatocytes
    Sox2 negatively regulate HAMP2 expression in primary hepatocytes. Primary hepatocytes were cultured and <t>lentivirus</t> expressing Myc-Sox2 (A) or shRNA targeting Sox2 (B and C) infected the cells. Cells were harvested at 72 h later for Western blotting assay and RT-qPCR assay to determine Myc-Sox2 expression, HAMP expression and Sox2 knockdown efficiency. The experiments were repeated for three times with similar results and data were shown as means ± SE. Statistical analyses were conducted using a one-way ANOVA with Tukey’s multiple comparison test to determine individual p-values (B-C) or an unpaired, two-tailed student t-test (A) . Significant differences are indicated by *p
    Lentivirus Infection Human Hepatocytes, supplied by PromoCell, used in various techniques. Bioz Stars score: 89/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selleck Chemicals lentivirus infection xav939
    Sox2 negatively regulate HAMP2 expression in primary hepatocytes. Primary hepatocytes were cultured and <t>lentivirus</t> expressing Myc-Sox2 (A) or shRNA targeting Sox2 (B and C) infected the cells. Cells were harvested at 72 h later for Western blotting assay and RT-qPCR assay to determine Myc-Sox2 expression, HAMP expression and Sox2 knockdown efficiency. The experiments were repeated for three times with similar results and data were shown as means ± SE. Statistical analyses were conducted using a one-way ANOVA with Tukey’s multiple comparison test to determine individual p-values (B-C) or an unpaired, two-tailed student t-test (A) . Significant differences are indicated by *p
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    Millipore lentiviral infection
    Sox2 negatively regulate HAMP2 expression in primary hepatocytes. Primary hepatocytes were cultured and <t>lentivirus</t> expressing Myc-Sox2 (A) or shRNA targeting Sox2 (B and C) infected the cells. Cells were harvested at 72 h later for Western blotting assay and RT-qPCR assay to determine Myc-Sox2 expression, HAMP expression and Sox2 knockdown efficiency. The experiments were repeated for three times with similar results and data were shown as means ± SE. Statistical analyses were conducted using a one-way ANOVA with Tukey’s multiple comparison test to determine individual p-values (B-C) or an unpaired, two-tailed student t-test (A) . Significant differences are indicated by *p
    Lentiviral Infection, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 855 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company lentiviral infection
    Sox2 negatively regulate HAMP2 expression in primary hepatocytes. Primary hepatocytes were cultured and <t>lentivirus</t> expressing Myc-Sox2 (A) or shRNA targeting Sox2 (B and C) infected the cells. Cells were harvested at 72 h later for Western blotting assay and RT-qPCR assay to determine Myc-Sox2 expression, HAMP expression and Sox2 knockdown efficiency. The experiments were repeated for three times with similar results and data were shown as means ± SE. Statistical analyses were conducted using a one-way ANOVA with Tukey’s multiple comparison test to determine individual p-values (B-C) or an unpaired, two-tailed student t-test (A) . Significant differences are indicated by *p
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    Genecopoeia lentiviral infection
    Sox2 negatively regulate HAMP2 expression in primary hepatocytes. Primary hepatocytes were cultured and <t>lentivirus</t> expressing Myc-Sox2 (A) or shRNA targeting Sox2 (B and C) infected the cells. Cells were harvested at 72 h later for Western blotting assay and RT-qPCR assay to determine Myc-Sox2 expression, HAMP expression and Sox2 knockdown efficiency. The experiments were repeated for three times with similar results and data were shown as means ± SE. Statistical analyses were conducted using a one-way ANOVA with Tukey’s multiple comparison test to determine individual p-values (B-C) or an unpaired, two-tailed student t-test (A) . Significant differences are indicated by *p
    Lentiviral Infection, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 92/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa lentivirus infection system
    Sox2 negatively regulate HAMP2 expression in primary hepatocytes. Primary hepatocytes were cultured and <t>lentivirus</t> expressing Myc-Sox2 (A) or shRNA targeting Sox2 (B and C) infected the cells. Cells were harvested at 72 h later for Western blotting assay and RT-qPCR assay to determine Myc-Sox2 expression, HAMP expression and Sox2 knockdown efficiency. The experiments were repeated for three times with similar results and data were shown as means ± SE. Statistical analyses were conducted using a one-way ANOVA with Tukey’s multiple comparison test to determine individual p-values (B-C) or an unpaired, two-tailed student t-test (A) . Significant differences are indicated by *p
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    Image Search Results


    Scanning EM analysis of the effect of anti‐CD11b, anti‐CD18 antibodies and Itgam silencing on the bone resorption levels in vitro . Mouse OCPs were cultured on bone slices and treated for 7 days with sRANKL (100 ng/ml). ( A ) Bone resorption pits of cells treated with neutralizing antibodies (10 μg/ml) or lentivirus (MOI = 10). ( B ) Relative bone resorption area per osteoclast from different groups, n = 3. # P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: CD11b promotes the differentiation of osteoclasts induced by RANKL through the spleen tyrosine kinase signalling pathway

    doi: 10.1111/jcmm.13254

    Figure Lengend Snippet: Scanning EM analysis of the effect of anti‐CD11b, anti‐CD18 antibodies and Itgam silencing on the bone resorption levels in vitro . Mouse OCPs were cultured on bone slices and treated for 7 days with sRANKL (100 ng/ml). ( A ) Bone resorption pits of cells treated with neutralizing antibodies (10 μg/ml) or lentivirus (MOI = 10). ( B ) Relative bone resorption area per osteoclast from different groups, n = 3. # P

    Article Snippet: Lentivirus infection Lentivirus (GeneCopoeia Inc., Rockville, USA) carrying Itgam shRNA (shCD11b‐1, shCD11b‐2) and empty lentivirus (Mock) were added to the medium (multiplicity of infection index, MOI = 10) after OCPs were seeded in the plate.

    Techniques: In Vitro, Cell Culture

    GFP-labeled lentivirus (LV)-hsa-miR-200c and its negative control (NC) infected A549, H1975 and H1299 The infection efficiency is still keeping in the range of 80%-90%.

    Journal: Oncotarget

    Article Title: MiR-200c overexpression is associated with better efficacy of EGFR-TKIs in non-small cell lung cancer patients with EGFR wild-type

    doi:

    Figure Lengend Snippet: GFP-labeled lentivirus (LV)-hsa-miR-200c and its negative control (NC) infected A549, H1975 and H1299 The infection efficiency is still keeping in the range of 80%-90%.

    Article Snippet: Lentivirus Infection Lentivirus (8×108 TU/mL) packaging of green fluorescent protein (GFP) LV-hsa-mir-200c and negative control (NC) were constructed in Genechem (Shanghai, China).

    Techniques: Labeling, Negative Control, Infection

    SHCBP1 participates in TGF-β1-induced EMT. a Western blot of SHCBP1, vimentin and E-cadherin in the indicated cells in response to treatment with 10 ng/mL TGF-β1 for 0, 24, and 48 h. b Twenty-four hours post-transfection of LV-Control or LV-shSHCBP1 lentivirus, the cells were treated with TGF-β1 (2 ng/ml) for an additional 48 h. The expressions of SHCBP1, E-cadherin and vimentin were detected by Western blot. c Representative images and data of a transwell invasion assay for HS-SY-II and SW982 cells. TGF-β1 stimulation significantly increased the invasiveness of both tumor cell lines compared with that in the absence of TGF-β1 (LV-Control) or LV-shSHCBP1 lentivirus cells. Each bar represents the mean ± SD, ** P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: SHCBP1 promotes synovial sarcoma cell metastasis via targeting TGF-β1/Smad signaling pathway and is associated with poor prognosis

    doi: 10.1186/s13046-017-0616-z

    Figure Lengend Snippet: SHCBP1 participates in TGF-β1-induced EMT. a Western blot of SHCBP1, vimentin and E-cadherin in the indicated cells in response to treatment with 10 ng/mL TGF-β1 for 0, 24, and 48 h. b Twenty-four hours post-transfection of LV-Control or LV-shSHCBP1 lentivirus, the cells were treated with TGF-β1 (2 ng/ml) for an additional 48 h. The expressions of SHCBP1, E-cadherin and vimentin were detected by Western blot. c Representative images and data of a transwell invasion assay for HS-SY-II and SW982 cells. TGF-β1 stimulation significantly increased the invasiveness of both tumor cell lines compared with that in the absence of TGF-β1 (LV-Control) or LV-shSHCBP1 lentivirus cells. Each bar represents the mean ± SD, ** P

    Article Snippet: Vector construction and lentivirus infection The lentiviral vector containing SHCBP1 DNA sequence (LV-SHCBP1), lentiviral vector containing SHCBP1 siRNA sequence (LV-shSHCBP1), and non-effective scrambled shRNA (LV-Control) were constructed by Genechem (Shanghai, China).

    Techniques: Western Blot, Transfection, Transwell Invasion Assay

    Construct and verify the lentiviral interference model of lncRNA-OBFC2A (A) AHH-1 cells were transfected by lentivirus vectors with lncRNA-OBFC2A or empty lentiviral vectors for 72 hours. (B) The expression of lncRNA-OBFC2A in AHH-1 cells was detected by qRT-PCR. *p

    Journal: Oncotarget

    Article Title: Benzene and its metabolite decreases cell proliferation via LncRNA-OBFC2A-mediated anti-proliferation effect involving NOTCH1 and KLF15

    doi: 10.18632/oncotarget.16588

    Figure Lengend Snippet: Construct and verify the lentiviral interference model of lncRNA-OBFC2A (A) AHH-1 cells were transfected by lentivirus vectors with lncRNA-OBFC2A or empty lentiviral vectors for 72 hours. (B) The expression of lncRNA-OBFC2A in AHH-1 cells was detected by qRT-PCR. *p

    Article Snippet: Lentivirus infection Lentivirus vectors were constructed in Genechem (Shanghai, People's Republic of China).

    Techniques: Construct, Transfection, Expressing, Quantitative RT-PCR

    Effect of CD13 expression on drug resistance. PLC/PRF/5 cells were infected with lentivirus. After puromycin screening, PLC/PRF/5 cells with stable CD13 overexpression or knockdown were obtained (A) . A representative immunoblot from three independent experiments giving similar results is shown for each western blot experiment. Densitometry for western blot was performed using AlphaEaseFC-v4.0.0 program. 1 × 10 3 PLC/PRF/5 cells, vector control, stable CD13 overexpression or knockdown PLC/PRF/5 cells were seeded in 6-well plates. Approximately 1 week later, cells were dyed with 0.1% crystal violet, and then photographs were taken (B) . The inhibition rate of different cytotoxic agents on overexpressed or knocked down cells were determined (C) . Data represent mean ± SD ( n = 3). ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: CD13 Inhibition Enhances Cytotoxic Effect of Chemotherapy Agents

    doi: 10.3389/fphar.2018.01042

    Figure Lengend Snippet: Effect of CD13 expression on drug resistance. PLC/PRF/5 cells were infected with lentivirus. After puromycin screening, PLC/PRF/5 cells with stable CD13 overexpression or knockdown were obtained (A) . A representative immunoblot from three independent experiments giving similar results is shown for each western blot experiment. Densitometry for western blot was performed using AlphaEaseFC-v4.0.0 program. 1 × 10 3 PLC/PRF/5 cells, vector control, stable CD13 overexpression or knockdown PLC/PRF/5 cells were seeded in 6-well plates. Approximately 1 week later, cells were dyed with 0.1% crystal violet, and then photographs were taken (B) . The inhibition rate of different cytotoxic agents on overexpressed or knocked down cells were determined (C) . Data represent mean ± SD ( n = 3). ∗ P

    Article Snippet: Lentivirus Infection Lentivirus particles was supplied by GeneChem.

    Techniques: Expressing, Planar Chromatography, Infection, Over Expression, Western Blot, Plasmid Preparation, Inhibition

    Downregulation of DDIT4 activates the mitogen-activated protein kinase (MAPK) and p53 pathways in GC cells. a The total and phosphorylated levels of AKT, mTOR and 4EBP1 in DDIT4 -silenced and DDIT4 -overexpressing cells were examined by western blot. b The total and phosphorylated levels of MEK1, P42/44-MAPK, BCL-1, BAD, p53, and p21 Cip1 in DDIT4 -silenced and DDIT4 -overexpressing cells were examined by western blot. c , d SGC7901 and BGC823 cells were infected shDDIT4 lentivirus and then were treated with inhibitors specific to p53 (A15201) or MAPK (PD98059). c The protein expression levels of phosphorylated and total ERK, BCL-2, p53 and p21 Cip1 were analyzed by western blot. d Cell proliferation was analyzed by CCK8 assay

    Journal: Cancer Communications

    Article Title: DDIT4 promotes gastric cancer proliferation and tumorigenesis through the p53 and MAPK pathways

    doi: 10.1186/s40880-018-0315-y

    Figure Lengend Snippet: Downregulation of DDIT4 activates the mitogen-activated protein kinase (MAPK) and p53 pathways in GC cells. a The total and phosphorylated levels of AKT, mTOR and 4EBP1 in DDIT4 -silenced and DDIT4 -overexpressing cells were examined by western blot. b The total and phosphorylated levels of MEK1, P42/44-MAPK, BCL-1, BAD, p53, and p21 Cip1 in DDIT4 -silenced and DDIT4 -overexpressing cells were examined by western blot. c , d SGC7901 and BGC823 cells were infected shDDIT4 lentivirus and then were treated with inhibitors specific to p53 (A15201) or MAPK (PD98059). c The protein expression levels of phosphorylated and total ERK, BCL-2, p53 and p21 Cip1 were analyzed by western blot. d Cell proliferation was analyzed by CCK8 assay

    Article Snippet: Lentivirus infection DDIT4 -overexpression or sh-DDIT4 lentivirus infection was conducted by GeneChem (Shanghai, China).

    Techniques: Western Blot, Infection, Expressing, CCK-8 Assay

    AMPK/p27 Kip1 Signaling Mediates Cell Survival in the MuSC Geriatric MuSCs were cultured for 48 hr treated with or without AICAR and/or overexpression of p27 Kip1 mutant-p27 Kip1(198A) . (A) Percentage of TUNEL-positive cells with respective treatment (N = 4 independent experiments). (B) Upper: representative blot of total and cleaved PARP; lower: quantification of protein expression normalized to untreated geriatric cells (N = 3 independent experiments). (C) Percentage of TUNEL-positive cells during Atg5 knockdown with concurrent overexpression of p27 Kip1 mutants p27 Kip1(198D) or p27 Kip1(198A) (N = 3 independent experiments). (D) Percentage of TUNEL-positive cells during treatment of compound C to young MuSCs with concurrent overexpression of p27 Kip1 mutant p27 Kip1(198D) (N = 3 independent experiments). (E) Geriatric MuSCs treated with or without AICAR and infected with respective p27 Kip1 mutant lentivirus. Cells were transplanted into injured young SCID muscle and quantified 4 or 28 days later. (F) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 4 days after transplantation. (G) Quantification of GFP(+) cells per muscle (N = 6 independent experiments). (H) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 28 days after transplantation. (I) Quantification of GFP(+) myofibers per muscle (N = 6 independent experiments). Scale bar, 100 μm. ∗ Signifies difference from young or untreated control cells (p

    Journal: Stem Cell Reports

    Article Title: The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells

    doi: 10.1016/j.stemcr.2018.06.014

    Figure Lengend Snippet: AMPK/p27 Kip1 Signaling Mediates Cell Survival in the MuSC Geriatric MuSCs were cultured for 48 hr treated with or without AICAR and/or overexpression of p27 Kip1 mutant-p27 Kip1(198A) . (A) Percentage of TUNEL-positive cells with respective treatment (N = 4 independent experiments). (B) Upper: representative blot of total and cleaved PARP; lower: quantification of protein expression normalized to untreated geriatric cells (N = 3 independent experiments). (C) Percentage of TUNEL-positive cells during Atg5 knockdown with concurrent overexpression of p27 Kip1 mutants p27 Kip1(198D) or p27 Kip1(198A) (N = 3 independent experiments). (D) Percentage of TUNEL-positive cells during treatment of compound C to young MuSCs with concurrent overexpression of p27 Kip1 mutant p27 Kip1(198D) (N = 3 independent experiments). (E) Geriatric MuSCs treated with or without AICAR and infected with respective p27 Kip1 mutant lentivirus. Cells were transplanted into injured young SCID muscle and quantified 4 or 28 days later. (F) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 4 days after transplantation. (G) Quantification of GFP(+) cells per muscle (N = 6 independent experiments). (H) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 28 days after transplantation. (I) Quantification of GFP(+) myofibers per muscle (N = 6 independent experiments). Scale bar, 100 μm. ∗ Signifies difference from young or untreated control cells (p

    Article Snippet: Lentivirus Infection Lentivirus for GFP, sh-Atg5 and scrambled sh were purchased from Dharmacon (GE Lifesciences) with titer 108 transduction units (TU)/mL.

    Techniques: Cell Culture, Over Expression, Mutagenesis, TUNEL Assay, Expressing, Infection, Transplantation Assay

    Aged MuSCs Are Highly Susceptible to Apoptosis during Conditions of Autophagy Impairment (A) Percentage of TUNEL-positive cells in young and geriatric groups infected with scramble or Atg5 sh lentivirus (N = 3 independent experiments). (B) A 72-hr time course of EdU labeling in young and geriatric MuSCs with or without an sh to Atg5 (N = 3 independent experiments). (C) Percentage of TUNEL-positive cells in young and geriatric cells infected with Atg5 sh with or without caspase inhibitor Q-VD-OPh (N = 4 independent experiments). (D) Upper: representative images of young and geriatric MuSCs infected with GFP scramble or shAtg5 lentivirus for 48 hr in culture. Lower: quantification of relative cell size normalized to young control cells (N = 3 independent experiments). Scale bar, 5 μm. Actin, green; nuclei, blue. ∗ Difference among age-matched treatment groups; # different from treatment-matched young cells; † difference between age-matched groups at 48 and 72 hr (p

    Journal: Stem Cell Reports

    Article Title: The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells

    doi: 10.1016/j.stemcr.2018.06.014

    Figure Lengend Snippet: Aged MuSCs Are Highly Susceptible to Apoptosis during Conditions of Autophagy Impairment (A) Percentage of TUNEL-positive cells in young and geriatric groups infected with scramble or Atg5 sh lentivirus (N = 3 independent experiments). (B) A 72-hr time course of EdU labeling in young and geriatric MuSCs with or without an sh to Atg5 (N = 3 independent experiments). (C) Percentage of TUNEL-positive cells in young and geriatric cells infected with Atg5 sh with or without caspase inhibitor Q-VD-OPh (N = 4 independent experiments). (D) Upper: representative images of young and geriatric MuSCs infected with GFP scramble or shAtg5 lentivirus for 48 hr in culture. Lower: quantification of relative cell size normalized to young control cells (N = 3 independent experiments). Scale bar, 5 μm. Actin, green; nuclei, blue. ∗ Difference among age-matched treatment groups; # different from treatment-matched young cells; † difference between age-matched groups at 48 and 72 hr (p

    Article Snippet: Lentivirus Infection Lentivirus for GFP, sh-Atg5 and scrambled sh were purchased from Dharmacon (GE Lifesciences) with titer 108 transduction units (TU)/mL.

    Techniques: TUNEL Assay, Infection, Labeling

    Restored AMPK Activation Rescues Inherent Apoptotic Susceptibility in Geriatric MuSCs (A) Phosphorylated and total protein expression of young MuSC AMPK and p27 Kip1 throughout 48 hr in culture. (B) Phosphorylated and total protein expression of AMPK and p27 Kip1 at 48 hr in culture across different ages. (C) TUNEL-positive cells in young, geriatric and geriatric MuSCs treated with AICAR (N = 3 independent experiments). (D). Left: representative blot of total and cleaved PARP; right: quantification of cleaved PARP protein expression normalized to young cells (N = 3 independent experiments). (E) Young and geriatric MuSCs infected with respective AMPK lentivirus, transplanted into injured SCID muscle and quantified 4 or 28 days later. (F and G) Upper: young GFP-labeled MuSCs overexpressing GFP control or a dominant-negative (DN) AMPK 4 days; lower: 28 days after transplantation (N = 5 independent experiments) (F). Upper: geriatric GFP-labeled MuSCs overexpressing GFP control or constitutively active (CA) AMPK 4 days; lower: 28 days after transplantation. Quantification includes counts of GFP(+) cells (4d) or myofibers (28d) per muscle (N = 5 independent experiments) (G). ∗ Signifies difference from young cells; # signifies difference from GFP control transplanted cells (p

    Journal: Stem Cell Reports

    Article Title: The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells

    doi: 10.1016/j.stemcr.2018.06.014

    Figure Lengend Snippet: Restored AMPK Activation Rescues Inherent Apoptotic Susceptibility in Geriatric MuSCs (A) Phosphorylated and total protein expression of young MuSC AMPK and p27 Kip1 throughout 48 hr in culture. (B) Phosphorylated and total protein expression of AMPK and p27 Kip1 at 48 hr in culture across different ages. (C) TUNEL-positive cells in young, geriatric and geriatric MuSCs treated with AICAR (N = 3 independent experiments). (D). Left: representative blot of total and cleaved PARP; right: quantification of cleaved PARP protein expression normalized to young cells (N = 3 independent experiments). (E) Young and geriatric MuSCs infected with respective AMPK lentivirus, transplanted into injured SCID muscle and quantified 4 or 28 days later. (F and G) Upper: young GFP-labeled MuSCs overexpressing GFP control or a dominant-negative (DN) AMPK 4 days; lower: 28 days after transplantation (N = 5 independent experiments) (F). Upper: geriatric GFP-labeled MuSCs overexpressing GFP control or constitutively active (CA) AMPK 4 days; lower: 28 days after transplantation. Quantification includes counts of GFP(+) cells (4d) or myofibers (28d) per muscle (N = 5 independent experiments) (G). ∗ Signifies difference from young cells; # signifies difference from GFP control transplanted cells (p

    Article Snippet: Lentivirus Infection Lentivirus for GFP, sh-Atg5 and scrambled sh were purchased from Dharmacon (GE Lifesciences) with titer 108 transduction units (TU)/mL.

    Techniques: Activation Assay, Expressing, TUNEL Assay, Infection, Labeling, Dominant Negative Mutation, Transplantation Assay

    C/EBPβ and Stat3 maintain the mesenchymal phenotype of human glioma cells a , Immunofluorescence for fibronectin, Col5A1 and YKL40 in BTSC-3408 infected with lentiviruses expressing Stat3, C/EBPβ, or Stat3 plus C/EBPβ shRNA. b , Quantification of fibronectin; c , Col5A1; and d , YKL40 positive cells. n = 3 independent experiments; Bars indicate Mean±SD. e , qRT-PCR of mesenchymal genes in BTSC-20 infected with lentiviruses expressing Stat3, C/EBPβ, or Stat3 plus C/EBPβ shRNA. Gene expression was normalized to the expression of 18S ribosomal RNA. n = 3; Bars indicate Mean±SD. f , Microphotograps of invading SNB19 cells infected with lentiviral vectors expressing control or shStat3 plus shC/EBPβ. g , Quantification of SNB19 invading cells. Bars indicate Mean±SD; n = 6 (two independent experiments, each performed in triplicate). h , Invading BTSC-3408 cells infected with shCtr, shStat3, shC/EBPβ or shStat3 plus shC/EBPβ lentiviruses. i , Quantification of invading BTSC-3408 cells. Bars indicate Mean±SD; n = 6 (two independent experiments, each performed in triplicate). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Journal: Nature

    Article Title: The transcriptional network for mesenchymal transformation of brain tumors

    doi: 10.1038/nature08712

    Figure Lengend Snippet: C/EBPβ and Stat3 maintain the mesenchymal phenotype of human glioma cells a , Immunofluorescence for fibronectin, Col5A1 and YKL40 in BTSC-3408 infected with lentiviruses expressing Stat3, C/EBPβ, or Stat3 plus C/EBPβ shRNA. b , Quantification of fibronectin; c , Col5A1; and d , YKL40 positive cells. n = 3 independent experiments; Bars indicate Mean±SD. e , qRT-PCR of mesenchymal genes in BTSC-20 infected with lentiviruses expressing Stat3, C/EBPβ, or Stat3 plus C/EBPβ shRNA. Gene expression was normalized to the expression of 18S ribosomal RNA. n = 3; Bars indicate Mean±SD. f , Microphotograps of invading SNB19 cells infected with lentiviral vectors expressing control or shStat3 plus shC/EBPβ. g , Quantification of SNB19 invading cells. Bars indicate Mean±SD; n = 6 (two independent experiments, each performed in triplicate). h , Invading BTSC-3408 cells infected with shCtr, shStat3, shC/EBPβ or shStat3 plus shC/EBPβ lentiviruses. i , Quantification of invading BTSC-3408 cells. Bars indicate Mean±SD; n = 6 (two independent experiments, each performed in triplicate). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Article Snippet: Lentivirus infection Lentiviral expression vectors carrying shRNAs were purchased from Sigma.

    Techniques: Immunofluorescence, Infection, Expressing, shRNA, Quantitative RT-PCR

    Knockdown of DEPTOR promotes osteogenic differentiation of hBMSCs in vitro. hBMSCs transfected with lentivirus expressing DEPTOR shRNAs (shDEPTOR #1, shDEPTOR #2) or scrambled control (shNC), and cultured in proliferation medium (PM) or osteogenic medium (OM). a ALP staining on day 7, ARS staining on day 14, and VK staining on day 21 after proliferative culture or osteogenic induction. b, c ALP activity on day 7 and AZR mineralization assay on day 14 after proliferative culture or osteogenic induction. d, e Relative mRNA levels of ALP and RUNX2 measured by qRT-PCR on day 7 of proliferative culture or osteogenic induction. f, g Relative mRNA levels of OSX and OCN measured by qRT-PCR on day 14 of proliferative culture or osteogenic induction. GAPDH used for normalization. h Left: western blot analysis of RUNX2 and OCN protein levels on day 14 after osteogenic induction. GAPDH used as internal control. Right: quantification of band intensities. Data presented as mean ± SD. * P

    Journal: Stem Cell Research & Therapy

    Article Title: DEPTOR regulates osteogenic differentiation via inhibiting MEG3-mediated activation of BMP4 signaling and is involved in osteoporosis

    doi: 10.1186/s13287-018-0935-9

    Figure Lengend Snippet: Knockdown of DEPTOR promotes osteogenic differentiation of hBMSCs in vitro. hBMSCs transfected with lentivirus expressing DEPTOR shRNAs (shDEPTOR #1, shDEPTOR #2) or scrambled control (shNC), and cultured in proliferation medium (PM) or osteogenic medium (OM). a ALP staining on day 7, ARS staining on day 14, and VK staining on day 21 after proliferative culture or osteogenic induction. b, c ALP activity on day 7 and AZR mineralization assay on day 14 after proliferative culture or osteogenic induction. d, e Relative mRNA levels of ALP and RUNX2 measured by qRT-PCR on day 7 of proliferative culture or osteogenic induction. f, g Relative mRNA levels of OSX and OCN measured by qRT-PCR on day 14 of proliferative culture or osteogenic induction. GAPDH used for normalization. h Left: western blot analysis of RUNX2 and OCN protein levels on day 14 after osteogenic induction. GAPDH used as internal control. Right: quantification of band intensities. Data presented as mean ± SD. * P

    Article Snippet: Lentivirus infection Recombinant lentiviruses expressing short hairpin RNAs targeting DEPTOR (shDEPTOR #1, shDEPTOR #2) and the scrambled control (shNC) were purchased from GenePharma (Shanghai, China); the sequences are presented in Additional file : Table S1. hBMSCs were exposed with the viral supernatant at a multiplicity of infection of 100 together with polybrene (5 μg·ml− 1 ) for 24 h. Puromycin at 1 μg·ml− 1 was then added to the culture medium for at least 3 days to establish stable cell lines.

    Techniques: In Vitro, Transfection, Expressing, Cell Culture, ALP Assay, Staining, Activity Assay, Mineralization Assay, Quantitative RT-PCR, Western Blot

    PLK1 inhibition induces AML cell autophagy via mTOR dephosphorylation. (A) Western blotting of mTOR, AMPK and ULK1 expression levels and phosphorylation in NB4 cells treated with RO3280 or BI2536. There was significantly decreased mTOR phosphorylation and ULK1 phosphorylation at Ser317. (B) Western blotting of mTOR expression levels and phosphorylation in NB4, K562 and HL-60 cells transfected with PLK1 RNA interference lentivirus at 100 and 200 MOI. Relative expression of p-mTOR/mTOR was analyzed with ImageJ software according to the user guide. **p

    Journal: Oncology Reports

    Article Title: Inhibiting PLK1 induces autophagy of acute myeloid leukemia cells via mammalian target of rapamycin pathway dephosphorylation

    doi: 10.3892/or.2017.5417

    Figure Lengend Snippet: PLK1 inhibition induces AML cell autophagy via mTOR dephosphorylation. (A) Western blotting of mTOR, AMPK and ULK1 expression levels and phosphorylation in NB4 cells treated with RO3280 or BI2536. There was significantly decreased mTOR phosphorylation and ULK1 phosphorylation at Ser317. (B) Western blotting of mTOR expression levels and phosphorylation in NB4, K562 and HL-60 cells transfected with PLK1 RNA interference lentivirus at 100 and 200 MOI. Relative expression of p-mTOR/mTOR was analyzed with ImageJ software according to the user guide. **p

    Article Snippet: Lentivirus infection was according to the manufacturer (Shanghai Genechem Co., Ltd.) at a final concentration of 100–200 MOI.

    Techniques: Inhibition, De-Phosphorylation Assay, Western Blot, Expressing, Transfection, Software

    PLK1 inhibition by RNA interference induces more LC3-II in AML cells. (A) NB4 cells were treated with 500 nM RO3280 or BI2536 for 4 to 12 h. There was more LC3-II in NB4 cells treated with 500 nM RO3280 or BI2536. (B) Western blotting of PLK1 and LC3-I/LC3-II in NB4, K562 and HL-60 cells transfected with RNA interference lentivirus. (C) NB4, K562 and HL-60 cells transfected with RNA interference lentivirus observed under fluorescence confocal microscopy for analysis of autophagy activity. White arrows indicate autophagosomes. Autophagy activity in NB4, K562 and HL-60 cells. **p

    Journal: Oncology Reports

    Article Title: Inhibiting PLK1 induces autophagy of acute myeloid leukemia cells via mammalian target of rapamycin pathway dephosphorylation

    doi: 10.3892/or.2017.5417

    Figure Lengend Snippet: PLK1 inhibition by RNA interference induces more LC3-II in AML cells. (A) NB4 cells were treated with 500 nM RO3280 or BI2536 for 4 to 12 h. There was more LC3-II in NB4 cells treated with 500 nM RO3280 or BI2536. (B) Western blotting of PLK1 and LC3-I/LC3-II in NB4, K562 and HL-60 cells transfected with RNA interference lentivirus. (C) NB4, K562 and HL-60 cells transfected with RNA interference lentivirus observed under fluorescence confocal microscopy for analysis of autophagy activity. White arrows indicate autophagosomes. Autophagy activity in NB4, K562 and HL-60 cells. **p

    Article Snippet: Lentivirus infection was according to the manufacturer (Shanghai Genechem Co., Ltd.) at a final concentration of 100–200 MOI.

    Techniques: Inhibition, Western Blot, Transfection, Fluorescence, Confocal Microscopy, Activity Assay

    Regulation of RB1 in spermatogonia. (A) Suppression of GS cell proliferation through depletion of Cdk4 or Cdk6 . GS cells were infected with the indicated lentivirus expressing shRNA and passaged 4 days after transfection. Cell recovery was determined 6 days after passage (n = 4; MOI = 10). (B) Colony counts. Cells were transplanted 2 days after transfection. Results of two experiments (n = 8; MOI = 10). (C) Macroscopic appearance of recipient testes that received transplantation of GS cells depleted of Cdk4 or Cdk6 . (D) Western blot analysis of RB1. GS cells were cultured without cytokines for 3 days and stimulated with FGF2 and GDNF. Cells were recovered 1 h after cytokine stimulation. Note the relative increase in phosphorylated RB1 (pRB1) caused by cytokine stimulation. The graph shows relative band intensity (n = 3). (E) Western blot analysis of RB1 following depletion of Cdk4 or Cdk6 . Cells were recovered 4 days after transfection (MOI = 10). The graph shows relative band intensity (n = 3). (F) Suppression of GS cell proliferation through depletion of Cdkn1b . GS cells were infected with the indicated lentivrus, and cells were recovered 4 days after transfection (n = 4; MOI = 4). (G) Western blot analysis of cell cycle-related proteins following depletion of Cdkn1b . Cells were recovered 4 days after transfection (MOI = 4). The graph shows relative band intensity (n = 3). Bar = 1 mm (C).

    Journal: The Journal of Reproduction and Development

    Article Title: The CDKN1B-RB1-E2F1 pathway protects mouse spermatogonial stem cells from genomic damage

    doi: 10.1262/jrd.2015-027

    Figure Lengend Snippet: Regulation of RB1 in spermatogonia. (A) Suppression of GS cell proliferation through depletion of Cdk4 or Cdk6 . GS cells were infected with the indicated lentivirus expressing shRNA and passaged 4 days after transfection. Cell recovery was determined 6 days after passage (n = 4; MOI = 10). (B) Colony counts. Cells were transplanted 2 days after transfection. Results of two experiments (n = 8; MOI = 10). (C) Macroscopic appearance of recipient testes that received transplantation of GS cells depleted of Cdk4 or Cdk6 . (D) Western blot analysis of RB1. GS cells were cultured without cytokines for 3 days and stimulated with FGF2 and GDNF. Cells were recovered 1 h after cytokine stimulation. Note the relative increase in phosphorylated RB1 (pRB1) caused by cytokine stimulation. The graph shows relative band intensity (n = 3). (E) Western blot analysis of RB1 following depletion of Cdk4 or Cdk6 . Cells were recovered 4 days after transfection (MOI = 10). The graph shows relative band intensity (n = 3). (F) Suppression of GS cell proliferation through depletion of Cdkn1b . GS cells were infected with the indicated lentivrus, and cells were recovered 4 days after transfection (n = 4; MOI = 4). (G) Western blot analysis of cell cycle-related proteins following depletion of Cdkn1b . Cells were recovered 4 days after transfection (MOI = 4). The graph shows relative band intensity (n = 3). Bar = 1 mm (C).

    Article Snippet: Lentivirus infection For gene OE experiments, PSM-human Rb1 (Addgene, Cambridge, MA, USA), human papillomavirus E7 (Addgene), mouse Bbc3 (transOMIC technologies, Huntsville, AL, USA), mouse Cdkn1a (Addgene) and mouse Cdkn2a (p16 ; Addgene) were cloned into the CSII-EF-IRES2-Venus vector.

    Techniques: Infection, Expressing, shRNA, Transfection, Transplantation Assay, Western Blot, Cell Culture

    Induction of DNA DSBs by E2F1 activation. (A) Suppression of GS cell proliferation following transfection of E2f1 . GS cells were infected with the indicated lentivirus and passaged 4 days after transfection. Cell recovery was determined 6 days after passage (n = 3; MOI = 10). (B) Colony counts. Results of two experiments (n = 8). (C) Macroscopic appearance of recipient testis. (D) Quantification of apoptotic cells following E2f1 or E7 OE using TUNEL staining. At least 536 cells were counted 10 days after AxCANCre transfection. Results of four experiments (MOI = 10). (E) Karyotype analysis and metaphase spread of GS cells. Cells were analyzed 2 weeks after AxCANCre transfection (n = 88 for control; n = 75 for KO). The number in the metaphase spread indicates the chromosome number. (F–H) Quantification of γH2AX- (F), RAD51- (G) and TRP53BP1 (H)-positive cells of Rb1 KO GS cells. Cells were recovered 2 weeks after AxCANCre transfection (n =3). At least 338 (F), 401 (G) and 149 (H) cells were counted. Representative pictures are shown in Supplementary Fig. 2 (online only). (I) Comet assay. Data are expressed as the percent damage compared with the control sample. The arrowhead indicates a cell with DNA damage. (J) Immunocytochemistry of GS cells transfected with E2f1 or E7 for γH2AX. Cells were recovered 10 days after transfection (n = 3). At least 304 cells were counted. (K) Suppression of γH2AX staining following depletion of E2f1 . Cells were co-transfected with AxCANCre and a lentivirus expressing shRNA against E2f1 and recovered 6 days after transfection (n = 3; MOI = 4). At least 252 cells were counted. Bar = 1 mm (C), 50 μm (E, I).

    Journal: The Journal of Reproduction and Development

    Article Title: The CDKN1B-RB1-E2F1 pathway protects mouse spermatogonial stem cells from genomic damage

    doi: 10.1262/jrd.2015-027

    Figure Lengend Snippet: Induction of DNA DSBs by E2F1 activation. (A) Suppression of GS cell proliferation following transfection of E2f1 . GS cells were infected with the indicated lentivirus and passaged 4 days after transfection. Cell recovery was determined 6 days after passage (n = 3; MOI = 10). (B) Colony counts. Results of two experiments (n = 8). (C) Macroscopic appearance of recipient testis. (D) Quantification of apoptotic cells following E2f1 or E7 OE using TUNEL staining. At least 536 cells were counted 10 days after AxCANCre transfection. Results of four experiments (MOI = 10). (E) Karyotype analysis and metaphase spread of GS cells. Cells were analyzed 2 weeks after AxCANCre transfection (n = 88 for control; n = 75 for KO). The number in the metaphase spread indicates the chromosome number. (F–H) Quantification of γH2AX- (F), RAD51- (G) and TRP53BP1 (H)-positive cells of Rb1 KO GS cells. Cells were recovered 2 weeks after AxCANCre transfection (n =3). At least 338 (F), 401 (G) and 149 (H) cells were counted. Representative pictures are shown in Supplementary Fig. 2 (online only). (I) Comet assay. Data are expressed as the percent damage compared with the control sample. The arrowhead indicates a cell with DNA damage. (J) Immunocytochemistry of GS cells transfected with E2f1 or E7 for γH2AX. Cells were recovered 10 days after transfection (n = 3). At least 304 cells were counted. (K) Suppression of γH2AX staining following depletion of E2f1 . Cells were co-transfected with AxCANCre and a lentivirus expressing shRNA against E2f1 and recovered 6 days after transfection (n = 3; MOI = 4). At least 252 cells were counted. Bar = 1 mm (C), 50 μm (E, I).

    Article Snippet: Lentivirus infection For gene OE experiments, PSM-human Rb1 (Addgene, Cambridge, MA, USA), human papillomavirus E7 (Addgene), mouse Bbc3 (transOMIC technologies, Huntsville, AL, USA), mouse Cdkn1a (Addgene) and mouse Cdkn2a (p16 ; Addgene) were cloned into the CSII-EF-IRES2-Venus vector.

    Techniques: Activation Assay, Transfection, Infection, TUNEL Assay, Staining, Single Cell Gel Electrophoresis, Immunocytochemistry, Expressing, shRNA

    Increased Bbc3 expression causes apoptosis of GS cells upon Rb1 deletion. (A) Western blot analysis of TRP53. Cells were recovered 3 days after AxCANCre transfection. The graph shows relative band intensity (n = 3). Note the relative increase in phosphorylated TRP53 (pTRP53) in Rb1 KO GS cells. (B) Increased recovery of Rb1 KO GS cells following depletion of Trp53 . Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Trp53 and recovered 1 week after infection (n = 3; MOI = 4). (C) Quantification of apoptotic cells using TUNEL staining following depletion of Trp53 (n = 3). Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Trp53 (MOI = 4) and recovered 1 week after infection. At least 545 cells were counted in three experiments. (D) Increased cell recovery of E7 -transfected Trp53 KO GS cells (n = 3; MOI = 10). Cells were transfected with a lentivirus expressing E7 and passaged 4 days after transfection. Cells were recovered 6 days after passage. (E) Real-time PCR analysis of apoptosis-related genes following Rb1 gene deletion. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Trp53 , and recovered 3 days after infection (n = 3; MOI = 4). (F) Reduced GS cell recovery upon Bbc3 OE. GS cells were transfected with a lentivirus expressing Bbc3 . Cells were recovered 4 days after transfection (n = 3; MOI = 10). (G) Quantification of apoptotic cells after Bbc3 OE using TUNEL staining (n = 3; MOI = 10). At least 239 cells were counted 4 days after transfection. (H) Increased recovery of Rb1 KO GS cells following depletion of Bbc3 . Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Bbc3 and recovered 1 week after infection (n = 6; MOI = 10). (I) Quantification of apoptosis of Rb1 KO cells using TUNEL staining after Bbc3 depletion. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Bbc3 and recovered 1 week after infection (n = 3; MOI = 10). At least 270 cells were counted.

    Journal: The Journal of Reproduction and Development

    Article Title: The CDKN1B-RB1-E2F1 pathway protects mouse spermatogonial stem cells from genomic damage

    doi: 10.1262/jrd.2015-027

    Figure Lengend Snippet: Increased Bbc3 expression causes apoptosis of GS cells upon Rb1 deletion. (A) Western blot analysis of TRP53. Cells were recovered 3 days after AxCANCre transfection. The graph shows relative band intensity (n = 3). Note the relative increase in phosphorylated TRP53 (pTRP53) in Rb1 KO GS cells. (B) Increased recovery of Rb1 KO GS cells following depletion of Trp53 . Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Trp53 and recovered 1 week after infection (n = 3; MOI = 4). (C) Quantification of apoptotic cells using TUNEL staining following depletion of Trp53 (n = 3). Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Trp53 (MOI = 4) and recovered 1 week after infection. At least 545 cells were counted in three experiments. (D) Increased cell recovery of E7 -transfected Trp53 KO GS cells (n = 3; MOI = 10). Cells were transfected with a lentivirus expressing E7 and passaged 4 days after transfection. Cells were recovered 6 days after passage. (E) Real-time PCR analysis of apoptosis-related genes following Rb1 gene deletion. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Trp53 , and recovered 3 days after infection (n = 3; MOI = 4). (F) Reduced GS cell recovery upon Bbc3 OE. GS cells were transfected with a lentivirus expressing Bbc3 . Cells were recovered 4 days after transfection (n = 3; MOI = 10). (G) Quantification of apoptotic cells after Bbc3 OE using TUNEL staining (n = 3; MOI = 10). At least 239 cells were counted 4 days after transfection. (H) Increased recovery of Rb1 KO GS cells following depletion of Bbc3 . Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Bbc3 and recovered 1 week after infection (n = 6; MOI = 10). (I) Quantification of apoptosis of Rb1 KO cells using TUNEL staining after Bbc3 depletion. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Bbc3 and recovered 1 week after infection (n = 3; MOI = 10). At least 270 cells were counted.

    Article Snippet: Lentivirus infection For gene OE experiments, PSM-human Rb1 (Addgene, Cambridge, MA, USA), human papillomavirus E7 (Addgene), mouse Bbc3 (transOMIC technologies, Huntsville, AL, USA), mouse Cdkn1a (Addgene) and mouse Cdkn2a (p16 ; Addgene) were cloned into the CSII-EF-IRES2-Venus vector.

    Techniques: Expressing, Western Blot, Transfection, shRNA, Infection, TUNEL Assay, Staining, Real-time Polymerase Chain Reaction

    Functional analysis of Rb1 in self-renewal division. (A) Suppression of GS cell proliferation following transfection of constitutively active RB1 (PSM-RB1). GS cells were infected with the indicated lentivirus and passaged 4 days after transfection. Cell recovery was determined 6 days after passage (n = 3; MOI = 10). OE was confirmed by RT-PCR 2 days after transfection. (B) Suppression of GS cell proliferation following transfection of E7 . GS cells were infected with the indicated lentivirus and passaged 4 days after transfection. Cell recovery was determined 6 days after passage (n = 3; MOI = 10). OE was confirmed by RT-PCR 2 days after transfection. (C) Conditional mutant mice used in the experiment. Exon 19 of the Rb1 gene was deleted using Cre -mediated recombination. The indicated probe was used for Southern blot analysis. (D) Colony counts. Results of three experiments (n = 12). (E) Macroscopic appearance of recipient testis. (F) Testicular weight (n = 8). (G) Tubules with spermatogenesis, defined as the presence of multiple layers of germ cells in the entire circumference of the tubules. At least 169 tubules were counted (n = 7). (H) Histological appearance of recipient testis. Bar = 1 mm (E), 20 μm (H).

    Journal: The Journal of Reproduction and Development

    Article Title: The CDKN1B-RB1-E2F1 pathway protects mouse spermatogonial stem cells from genomic damage

    doi: 10.1262/jrd.2015-027

    Figure Lengend Snippet: Functional analysis of Rb1 in self-renewal division. (A) Suppression of GS cell proliferation following transfection of constitutively active RB1 (PSM-RB1). GS cells were infected with the indicated lentivirus and passaged 4 days after transfection. Cell recovery was determined 6 days after passage (n = 3; MOI = 10). OE was confirmed by RT-PCR 2 days after transfection. (B) Suppression of GS cell proliferation following transfection of E7 . GS cells were infected with the indicated lentivirus and passaged 4 days after transfection. Cell recovery was determined 6 days after passage (n = 3; MOI = 10). OE was confirmed by RT-PCR 2 days after transfection. (C) Conditional mutant mice used in the experiment. Exon 19 of the Rb1 gene was deleted using Cre -mediated recombination. The indicated probe was used for Southern blot analysis. (D) Colony counts. Results of three experiments (n = 12). (E) Macroscopic appearance of recipient testis. (F) Testicular weight (n = 8). (G) Tubules with spermatogenesis, defined as the presence of multiple layers of germ cells in the entire circumference of the tubules. At least 169 tubules were counted (n = 7). (H) Histological appearance of recipient testis. Bar = 1 mm (E), 20 μm (H).

    Article Snippet: Lentivirus infection For gene OE experiments, PSM-human Rb1 (Addgene, Cambridge, MA, USA), human papillomavirus E7 (Addgene), mouse Bbc3 (transOMIC technologies, Huntsville, AL, USA), mouse Cdkn1a (Addgene) and mouse Cdkn2a (p16 ; Addgene) were cloned into the CSII-EF-IRES2-Venus vector.

    Techniques: Functional Assay, Transfection, Infection, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Mouse Assay, Southern Blot

    Suppression of the cell cycle by Cdkn1a induction following induction of Rb1 deficiency. (A) Real-time PCR analysis of cell cycle-related genes following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection (n = 3). (B) Western blot analysis of CDKI proteins following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection. The graph shows relative band intensity (n = 3). (C) Cell cycle analysis by Hoechst 33342 following Cdkn1a or Cdkn2a OE (MOI = 10). (D) Increased cell recovery of Rb1 KO GS cells following depletion of Cdkn1a . Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Cdkn1a and recovered 1 week after infection (n = 3; MOI = 4). (E) Increased cell recovery of Rb1 KO GS cells following double depletion of Bbc3 and Cdkn1a . Cells were transfected with AxCANCre and a lentivirus expressing shRNA against the indicated genes and recovered 1 week after infection (n = 8; MOI = 4).

    Journal: The Journal of Reproduction and Development

    Article Title: The CDKN1B-RB1-E2F1 pathway protects mouse spermatogonial stem cells from genomic damage

    doi: 10.1262/jrd.2015-027

    Figure Lengend Snippet: Suppression of the cell cycle by Cdkn1a induction following induction of Rb1 deficiency. (A) Real-time PCR analysis of cell cycle-related genes following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection (n = 3). (B) Western blot analysis of CDKI proteins following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection. The graph shows relative band intensity (n = 3). (C) Cell cycle analysis by Hoechst 33342 following Cdkn1a or Cdkn2a OE (MOI = 10). (D) Increased cell recovery of Rb1 KO GS cells following depletion of Cdkn1a . Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Cdkn1a and recovered 1 week after infection (n = 3; MOI = 4). (E) Increased cell recovery of Rb1 KO GS cells following double depletion of Bbc3 and Cdkn1a . Cells were transfected with AxCANCre and a lentivirus expressing shRNA against the indicated genes and recovered 1 week after infection (n = 8; MOI = 4).

    Article Snippet: Lentivirus infection For gene OE experiments, PSM-human Rb1 (Addgene, Cambridge, MA, USA), human papillomavirus E7 (Addgene), mouse Bbc3 (transOMIC technologies, Huntsville, AL, USA), mouse Cdkn1a (Addgene) and mouse Cdkn2a (p16 ; Addgene) were cloned into the CSII-EF-IRES2-Venus vector.

    Techniques: Real-time Polymerase Chain Reaction, Transfection, Western Blot, Cell Cycle Assay, Expressing, shRNA, Infection

    RIPK1 has a protective role in SHK-induced apoptosis. ( a ) Nec-1 (50 μM) contributes to apoptosis induced by SHK (10 μM) and these effects could be partly abolished by ZVAD-FMK (20 μM). ( b ) Western blots indicated the decrease in RIPK1 with treatment of SHK. ( c ) Cells transfected with pANP7-RIPK1 plasmids or control were incubated with SHK (10 μM) with or without ZVAD-FMK (20 μM) for 24 h. Western blots were used to determine the effects of over-expression. ( d ) Cells infected with RIPK1 shRNA lentivirus particles or control for 48 h were incubated with SHK (10 μM) with or without ZVAD-FMK (20 μM) for 24 h. Western blots were used to determine the effect of knockdown. Values are presented as the mean ± SD for at least three independent experiments. * P

    Journal: Scientific Reports

    Article Title: Shikonin induces mitochondria-mediated apoptosis and enhances chemotherapeutic sensitivity of gastric cancer through reactive oxygen species

    doi: 10.1038/srep38267

    Figure Lengend Snippet: RIPK1 has a protective role in SHK-induced apoptosis. ( a ) Nec-1 (50 μM) contributes to apoptosis induced by SHK (10 μM) and these effects could be partly abolished by ZVAD-FMK (20 μM). ( b ) Western blots indicated the decrease in RIPK1 with treatment of SHK. ( c ) Cells transfected with pANP7-RIPK1 plasmids or control were incubated with SHK (10 μM) with or without ZVAD-FMK (20 μM) for 24 h. Western blots were used to determine the effects of over-expression. ( d ) Cells infected with RIPK1 shRNA lentivirus particles or control for 48 h were incubated with SHK (10 μM) with or without ZVAD-FMK (20 μM) for 24 h. Western blots were used to determine the effect of knockdown. Values are presented as the mean ± SD for at least three independent experiments. * P

    Article Snippet: RNA interference, plasmid transfection and lentivirus infection The siRNA against Endo G (target sequence of 5′-CCAUGGACGACACGUUCUA-3′), AIF (target sequence of 5′-GCAGUGGCAAGUUACUUAU-3′) and control siRNA (target sequence of 5′-UUCUCCGAACGUGUCACGU-3′) were synthesized by GenePharma Technologies (Shanghai, China).

    Techniques: Western Blot, Transfection, Incubation, Over Expression, Infection, shRNA

    MiR-10b promotes osteogenic differentiation and inhibits adipogenic differentiation of hADSCs. (A) The miR-10b-expressing lentivirus increased the expression of mature miR-10b in hADSCs, analyzed by stem-loop qRT-PCR. (B and C) ALP staining was performed on day 4 and ALP activity was detected during osteogenic differentiation. (D) Alizarin red staining was performed to indicate mineral deposition on day 12. (E and F) Lenti-10b increased the mRNA and protein expression levels of osteogenic-specific markers on day 6 of osteogenic differentiation. (G) Oil red O staining was performed to detect the lipid droplets formation on day 10 of adipogenic differentiation. (H) The dye of oil red O-positive cells was extracted by isopropanol, and the OD value was quantified at 510 nm wavelength. (I and J) Lenti-10b decreased the mRNA and protein expression levels of adipogenic-specific markers. The data, normalized to U6 or GAPDH, are averages of 3 independent experiments (mean ± SD). * P

    Journal: Aging and Disease

    Article Title: MiRNA-10b Reciprocally Stimulates Osteogenesis and Inhibits Adipogenesis Partly through the TGF-β/SMAD2 Signaling Pathway

    doi: 10.14336/AD.2018.0214

    Figure Lengend Snippet: MiR-10b promotes osteogenic differentiation and inhibits adipogenic differentiation of hADSCs. (A) The miR-10b-expressing lentivirus increased the expression of mature miR-10b in hADSCs, analyzed by stem-loop qRT-PCR. (B and C) ALP staining was performed on day 4 and ALP activity was detected during osteogenic differentiation. (D) Alizarin red staining was performed to indicate mineral deposition on day 12. (E and F) Lenti-10b increased the mRNA and protein expression levels of osteogenic-specific markers on day 6 of osteogenic differentiation. (G) Oil red O staining was performed to detect the lipid droplets formation on day 10 of adipogenic differentiation. (H) The dye of oil red O-positive cells was extracted by isopropanol, and the OD value was quantified at 510 nm wavelength. (I and J) Lenti-10b decreased the mRNA and protein expression levels of adipogenic-specific markers. The data, normalized to U6 or GAPDH, are averages of 3 independent experiments (mean ± SD). * P

    Article Snippet: Lentiviral vector preparation and infection Lentivirus production was carried out according to protocols from GenePharma ( www.genepharma.com/ ).

    Techniques: Expressing, Quantitative RT-PCR, ALP Assay, Staining, Activity Assay

    Knockdown or overexpression KIF20A influences GC cell proliferation. Notes: ( A ) Expression levels of KIF20A determined by qRT-PCR in immortalized human gastric mucosal epithelial cell line GES-1 and five GC cell lines (MGC-803, BGC-823, NCI-N87, SGC-7901, and AGS). ( B ) Expression levels of KIF20A in MGC-803 and AGS cell lines were examined by Western blot after lentivirus infection. ( C ) Cell viability assays for cells transfected with KIF20A knockdown and overexpression. ( D ) Colony formation assays for cells transfected with KIF20A knockdown and overexpression plasmids. Values are mean±SEM. *, **, ***, or **** corresponded to P

    Journal: Cancer Management and Research

    Article Title: Upregulation of KIF20A correlates with poor prognosis in gastric cancer

    doi: 10.2147/CMAR.S176147

    Figure Lengend Snippet: Knockdown or overexpression KIF20A influences GC cell proliferation. Notes: ( A ) Expression levels of KIF20A determined by qRT-PCR in immortalized human gastric mucosal epithelial cell line GES-1 and five GC cell lines (MGC-803, BGC-823, NCI-N87, SGC-7901, and AGS). ( B ) Expression levels of KIF20A in MGC-803 and AGS cell lines were examined by Western blot after lentivirus infection. ( C ) Cell viability assays for cells transfected with KIF20A knockdown and overexpression. ( D ) Colony formation assays for cells transfected with KIF20A knockdown and overexpression plasmids. Values are mean±SEM. *, **, ***, or **** corresponded to P

    Article Snippet: Cell culture and lentivirus infection This study was approved by the Ethics Committee of Anhui Medical University.

    Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Infection, Transfection

    TUTase depletion does not affect miRNA levels. ( a ) Western blot analysis of Lentivirus-mediated knockdown of Zcchc11 and Zcchc6 in Hela cells. ( b ) Mature miRNA northern blot of let-7g levels in the indicated transduced Hela cell lines. Valyl tRNA serves as a loading control. ( c ) Total small RNA from shGFP and shZcchc6/shZcchc11 (shTUT) Hela cells was purified and cloned before sequencing. Each point represents a single miRNA species, and axes are scaled to reads/million (RPM). R 2 > 0.99. ( d ) The relative number of reads for WT (i.e. non-modified) motif-containing miRNAs is unchanged after TUTase depletion.

    Journal: Nucleic Acids Research

    Article Title: Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4)

    doi: 10.1093/nar/gku805

    Figure Lengend Snippet: TUTase depletion does not affect miRNA levels. ( a ) Western blot analysis of Lentivirus-mediated knockdown of Zcchc11 and Zcchc6 in Hela cells. ( b ) Mature miRNA northern blot of let-7g levels in the indicated transduced Hela cell lines. Valyl tRNA serves as a loading control. ( c ) Total small RNA from shGFP and shZcchc6/shZcchc11 (shTUT) Hela cells was purified and cloned before sequencing. Each point represents a single miRNA species, and axes are scaled to reads/million (RPM). R 2 > 0.99. ( d ) The relative number of reads for WT (i.e. non-modified) motif-containing miRNAs is unchanged after TUTase depletion.

    Article Snippet: Lentivirus production and infection Lentivirus was prepared according to the manufacturer's protocol (Invitrogen #K4975-00) and supernatant was filtered through a 0.45 μm filter before being stored at −80°C or used immediately.

    Techniques: Western Blot, Northern Blot, Purification, Clone Assay, Sequencing, Modification

    STAT2 is required for USP18-mediated inhibition of Type I IFN signaling (a–f) MIP or MIP-USP18 expressing 2fTGH, U1A, U2A, U4A, U5A, and U6A cells were treated with IFNα (1000 U/ml) for 15 minutes. The cell lysates were immunoblotted with the indicated antibodies. (g) IB analysis of STAT1 phosphorylation in 2fTGH and U6A cells in the presence or absence of (1000 U/ml) IFNα. (h–i) Stat2 −/− MEFs were infected with MIP control (−) or MIP-Usp18 (+) retroviruses, either in the presence or absence of rescue with C-terminally FLAG-tagged Stat2 cDNA. Where indicated cells were treated with either mouse IFNβ (500 U/ml) for 15 minutes (h) or 30 minutes (i), before cell lysates were collected and analyzed by Western blotting with indicated antibodies. (j) Validation of Stat2 knockdown. Ba/F3 cells were infected with control or Stat2-targeting shRNA lentivirus. After 5 days puromycin selection, Stat2 expression was examined by Western blotting. (k) Usp18 −/− bone marrow cells were infected with pCX4-bsr control (−) or pCX4-bsr-Usp18 (+), either in the presence or absence of control (shCtrl) or Stat2-knockdown (shStat2–3) shRNA expression. Two days following double drug selection (puromycin and blasticidin), cells were either left untreated (−) or treated (+) with mouse IFNβ (500 U/ml) for 30 minutes. Cell lysates were collected and analyzed by Western blotting with indicated antibodies.

    Journal: Nature structural & molecular biology

    Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

    doi: 10.1038/nsmb.3378

    Figure Lengend Snippet: STAT2 is required for USP18-mediated inhibition of Type I IFN signaling (a–f) MIP or MIP-USP18 expressing 2fTGH, U1A, U2A, U4A, U5A, and U6A cells were treated with IFNα (1000 U/ml) for 15 minutes. The cell lysates were immunoblotted with the indicated antibodies. (g) IB analysis of STAT1 phosphorylation in 2fTGH and U6A cells in the presence or absence of (1000 U/ml) IFNα. (h–i) Stat2 −/− MEFs were infected with MIP control (−) or MIP-Usp18 (+) retroviruses, either in the presence or absence of rescue with C-terminally FLAG-tagged Stat2 cDNA. Where indicated cells were treated with either mouse IFNβ (500 U/ml) for 15 minutes (h) or 30 minutes (i), before cell lysates were collected and analyzed by Western blotting with indicated antibodies. (j) Validation of Stat2 knockdown. Ba/F3 cells were infected with control or Stat2-targeting shRNA lentivirus. After 5 days puromycin selection, Stat2 expression was examined by Western blotting. (k) Usp18 −/− bone marrow cells were infected with pCX4-bsr control (−) or pCX4-bsr-Usp18 (+), either in the presence or absence of control (shCtrl) or Stat2-knockdown (shStat2–3) shRNA expression. Two days following double drug selection (puromycin and blasticidin), cells were either left untreated (−) or treated (+) with mouse IFNβ (500 U/ml) for 30 minutes. Cell lysates were collected and analyzed by Western blotting with indicated antibodies.

    Article Snippet: For the retrovirus or lentivirus infection, spin infection (2000g, 3hours, 30°C; Allegra X12R (Beckman Coulter)) in the presence of polybrene (8ug/ml) was performed.

    Techniques: Inhibition, Expressing, Infection, Western Blot, shRNA, Selection

    EZH2 is a target of miR-101. ( A , B ) Left: Schematic representation of the miR-101 site in theEZH2 3′-UTR (up: position 59–65; down: position 114–121);Right: The 3′-UTR reporter assay was carried out in hBMSCs infected with miR-101lentivirus or miR-NC lentivirus. The WT or Mut reporter plasmids were transfected with Lipo-2000. Luciferase assays were performed 48 h after transfection. Firefly luciferase activity was standardized to a Renilla luciferase control. ( C ) EZH2 protein expression and ( D ) mRNA level in hBMSCs infected with miR-101lentivirus or transfected with anti-miR-101 were measured after 48 h. All of the data are expressed as the mean ± SD. *p

    Journal: Scientific Reports

    Article Title: MiR-101 Targets the EZH2/Wnt/β-Catenin the Pathway to Promote the Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    doi: 10.1038/srep36988

    Figure Lengend Snippet: EZH2 is a target of miR-101. ( A , B ) Left: Schematic representation of the miR-101 site in theEZH2 3′-UTR (up: position 59–65; down: position 114–121);Right: The 3′-UTR reporter assay was carried out in hBMSCs infected with miR-101lentivirus or miR-NC lentivirus. The WT or Mut reporter plasmids were transfected with Lipo-2000. Luciferase assays were performed 48 h after transfection. Firefly luciferase activity was standardized to a Renilla luciferase control. ( C ) EZH2 protein expression and ( D ) mRNA level in hBMSCs infected with miR-101lentivirus or transfected with anti-miR-101 were measured after 48 h. All of the data are expressed as the mean ± SD. *p

    Article Snippet: Lentivirus infection and oligonucleotide transfection The miR-101, anti-miR-101 and EZH2 siRNA were purchased from Origene (Beijing, China).

    Techniques: Reporter Assay, Infection, Transfection, Luciferase, Activity Assay, Expressing

    miR-101 promotes the osteogenic differentiation of hBMSCs. ( A ) miR-101 mRNA expression in hBMSCs infected by miR101-lentivirus or transfected with anti-miR101 was analyzed by qRT-PCR with U6 as a control before osteogenic differentiation. ( B ) The osteoblastic marker genes RUNX2, ALP, OPN and OCN were analyzed by Western Blot at day 15 of osteogenic differentiation with β-actin as a control. ( C ) ALP activity was measured at day 15. ( D ) Alizarin Red staining was measured at day 15 and quantification is shown at right. All of the data are expressed as the mean ± SD. *p

    Journal: Scientific Reports

    Article Title: MiR-101 Targets the EZH2/Wnt/β-Catenin the Pathway to Promote the Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    doi: 10.1038/srep36988

    Figure Lengend Snippet: miR-101 promotes the osteogenic differentiation of hBMSCs. ( A ) miR-101 mRNA expression in hBMSCs infected by miR101-lentivirus or transfected with anti-miR101 was analyzed by qRT-PCR with U6 as a control before osteogenic differentiation. ( B ) The osteoblastic marker genes RUNX2, ALP, OPN and OCN were analyzed by Western Blot at day 15 of osteogenic differentiation with β-actin as a control. ( C ) ALP activity was measured at day 15. ( D ) Alizarin Red staining was measured at day 15 and quantification is shown at right. All of the data are expressed as the mean ± SD. *p

    Article Snippet: Lentivirus infection and oligonucleotide transfection The miR-101, anti-miR-101 and EZH2 siRNA were purchased from Origene (Beijing, China).

    Techniques: Expressing, Infection, Transfection, Quantitative RT-PCR, Marker, ALP Assay, Western Blot, Activity Assay, Staining

    MDM2 maintains retinoblastoma cell proliferation in a p53-independent manner A. p53 association with MDM2 but not MDM4 in RB176 and Y79 cells detected by p53 or control IgG immunoprecipitation followed by MDM2, MDM4, and p53 western. *, a non-specific band seen with SMP-14 antibody. B. Western analysis after 6 h of treatment (left) and cell growth response (right) of RB176 cells treated with 10 μM or 20 μM PFT-α starting 4 days after infection with lentivirus expressing shRNA against MDM2 (shMDM2-1) or a scrambled control (Scr). C. Western analysis at day 4 (left) and cell growth response (right) of RB176 cells after infection with lentivirus co-expressing shMDM2 and shp53 or shMDM2 and Scr. Signal intensity was normalized to α-tubulin and expression relative to Scr control is indicated below corresponding panels. D. Western analysis at day 4 (left) and cell growth response (right) of TP53 R175H/R175H CHLA- RB215 cells after infection with lentivirus expressing shMDM2-2 or a scrambled control (Scr). A similar result was obtained separately using shMDM2-1 (not shown).

    Journal: Oncogene

    Article Title: MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation

    doi: 10.1038/onc.2016.350

    Figure Lengend Snippet: MDM2 maintains retinoblastoma cell proliferation in a p53-independent manner A. p53 association with MDM2 but not MDM4 in RB176 and Y79 cells detected by p53 or control IgG immunoprecipitation followed by MDM2, MDM4, and p53 western. *, a non-specific band seen with SMP-14 antibody. B. Western analysis after 6 h of treatment (left) and cell growth response (right) of RB176 cells treated with 10 μM or 20 μM PFT-α starting 4 days after infection with lentivirus expressing shRNA against MDM2 (shMDM2-1) or a scrambled control (Scr). C. Western analysis at day 4 (left) and cell growth response (right) of RB176 cells after infection with lentivirus co-expressing shMDM2 and shp53 or shMDM2 and Scr. Signal intensity was normalized to α-tubulin and expression relative to Scr control is indicated below corresponding panels. D. Western analysis at day 4 (left) and cell growth response (right) of TP53 R175H/R175H CHLA- RB215 cells after infection with lentivirus expressing shMDM2-2 or a scrambled control (Scr). A similar result was obtained separately using shMDM2-1 (not shown).

    Article Snippet: Lentivirus production and infections Lentivirus was produced in 15cm dishes by reverse transfection of Lenti-X cells (Clontech) using 17.5 μg vector, 8.75 μg pVSV-G, 17.5 μg pCMV-dR8.91 and 219 μl 0.6 μg/μl polyethylenimine (PEI) (Polysciences).

    Techniques: Immunoprecipitation, Western Blot, Infection, Expressing, shRNA

    MDM2 regulates MYCN translation A. Diagram of protein translation assay. Nascent proteins were labeled with the azide-modified alanine analog- azidohomoalanine (AHA), immunoprecipitated, and incorporated AHA reacted with a fluorescent TAMRA labeled alkyne. TAMRA labeled proteins were separated by SDS-PAGE and detected by immunoblot using anti-TAMRA antibody (in Fig. 5b) or by UV fluorescence (in Supplementary Fig. S4 ). B. Protein translation assay performed on AHA-labeled RB176 cells 4 days after infection with lentivirus expressing either of two shRNAs against MDM2 or a scrambled control (Scr). AHA-labeled cell lysates either were sequentially immunoprecipitated with MYCN and then α-tubulin antibodies followed by anti-TAMRA immunoblot ( top ) or were used for direct Western analysis of MDM2, MYCN, and α-tubulin ( bottom ). C. Quantitation of total MYCN protein (the average and s.d. of two western analyses of the same lysate) and newly translated MYCN protein, both normalized to α-tubulin. D. qRT-PCR analysis of MYCN RNA from the same AHA-labeled cells as used for protein analyses (average and s.d. of triplicate technical replicates).

    Journal: Oncogene

    Article Title: MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation

    doi: 10.1038/onc.2016.350

    Figure Lengend Snippet: MDM2 regulates MYCN translation A. Diagram of protein translation assay. Nascent proteins were labeled with the azide-modified alanine analog- azidohomoalanine (AHA), immunoprecipitated, and incorporated AHA reacted with a fluorescent TAMRA labeled alkyne. TAMRA labeled proteins were separated by SDS-PAGE and detected by immunoblot using anti-TAMRA antibody (in Fig. 5b) or by UV fluorescence (in Supplementary Fig. S4 ). B. Protein translation assay performed on AHA-labeled RB176 cells 4 days after infection with lentivirus expressing either of two shRNAs against MDM2 or a scrambled control (Scr). AHA-labeled cell lysates either were sequentially immunoprecipitated with MYCN and then α-tubulin antibodies followed by anti-TAMRA immunoblot ( top ) or were used for direct Western analysis of MDM2, MYCN, and α-tubulin ( bottom ). C. Quantitation of total MYCN protein (the average and s.d. of two western analyses of the same lysate) and newly translated MYCN protein, both normalized to α-tubulin. D. qRT-PCR analysis of MYCN RNA from the same AHA-labeled cells as used for protein analyses (average and s.d. of triplicate technical replicates).

    Article Snippet: Lentivirus production and infections Lentivirus was produced in 15cm dishes by reverse transfection of Lenti-X cells (Clontech) using 17.5 μg vector, 8.75 μg pVSV-G, 17.5 μg pCMV-dR8.91 and 219 μl 0.6 μg/μl polyethylenimine (PEI) (Polysciences).

    Techniques: Labeling, Modification, Immunoprecipitation, SDS Page, Fluorescence, Infection, Expressing, Western Blot, Quantitation Assay, Quantitative RT-PCR

    MDM2 but not MDM4 maintains retinoblastoma cell proliferation A. Western analysis of MDM2 (with SMP14), MDM4, p53, MYCN, and α-tubulin expression in post-fertilization week 19 human fetal retina and in four retinoblastoma (RB) and two neuroblastoma (NB) cell lines B. Western analysis at day 4 (left) and cell growth response (right) of RB176 cells after infection with lentivirus expressing shRNAs against MDM2 (shMDM2-1 and shMDM2-2) or against MDM4 (shMDM4-1), or expressing a scrambled shRNA control (Scr). Values and error bars denote mean and standard deviation (s.d.) of triplicate assays. C. Tumors formed up to 3.5 months after xenograft of RB176-luc cells transduced with shScr, shMDM2-1, or shMDM4-1 and engrafted into the sub-retinal space of athymic ( nude ) mice. P values are from two-tailed Fisher’s exact test. D. R epresentative tumor growth tracked over 1.5 months by bioluminescent imaging, ( left ) and MDM4 and HuNu expression in the tumors examined by Western blot sequentially probed with anti-HuNu ( bottom ) followed without stripping with anti-MDM2 ( top ).

    Journal: Oncogene

    Article Title: MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation

    doi: 10.1038/onc.2016.350

    Figure Lengend Snippet: MDM2 but not MDM4 maintains retinoblastoma cell proliferation A. Western analysis of MDM2 (with SMP14), MDM4, p53, MYCN, and α-tubulin expression in post-fertilization week 19 human fetal retina and in four retinoblastoma (RB) and two neuroblastoma (NB) cell lines B. Western analysis at day 4 (left) and cell growth response (right) of RB176 cells after infection with lentivirus expressing shRNAs against MDM2 (shMDM2-1 and shMDM2-2) or against MDM4 (shMDM4-1), or expressing a scrambled shRNA control (Scr). Values and error bars denote mean and standard deviation (s.d.) of triplicate assays. C. Tumors formed up to 3.5 months after xenograft of RB176-luc cells transduced with shScr, shMDM2-1, or shMDM4-1 and engrafted into the sub-retinal space of athymic ( nude ) mice. P values are from two-tailed Fisher’s exact test. D. R epresentative tumor growth tracked over 1.5 months by bioluminescent imaging, ( left ) and MDM4 and HuNu expression in the tumors examined by Western blot sequentially probed with anti-HuNu ( bottom ) followed without stripping with anti-MDM2 ( top ).

    Article Snippet: Lentivirus production and infections Lentivirus was produced in 15cm dishes by reverse transfection of Lenti-X cells (Clontech) using 17.5 μg vector, 8.75 μg pVSV-G, 17.5 μg pCMV-dR8.91 and 219 μl 0.6 μg/μl polyethylenimine (PEI) (Polysciences).

    Techniques: Western Blot, Expressing, Infection, shRNA, Standard Deviation, Transduction, Mouse Assay, Two Tailed Test, Imaging, Stripping Membranes

    MDM2 maintains retinoblastoma cell proliferation in part by promoting MYCN expression A. Western analysis at day 4 (left) and cell growth response (right) of RB176 cells after infection with lentivirus expressing shRNA against MYCN or a scrambled control (Scr). B. Tumors formed by RB176-luc cells transduced with shScr but not by those transduced with shMYCN in mouse subretinal xenografts (right, P-value from two-tailed Fisher’s exact test). Western analysis of the cells used for xenograft (left). C. Western analysis at day 4 (left) and cell growth response (right) of CHLA-RB215 cells after infection with lentivirus expressing shRNA against MYCN or Scr. D. Western analysis at days 4 and 25 (left and middle) and cell growth response (right) of RB176 cells after co-infection with lentivirus expressing shRNA against MDM2 or Scr and with lentivirus expressing MYCN under control of the EF1α promoter (BN-MYCN) or the empty vector (BN). E. Western analysis at day 4 (left) and cell growth response (right) of RB176 cells after co-infection with lentivirus expressing shMDM2 or Scr and with lentivirus expressing shp27 or Scr.

    Journal: Oncogene

    Article Title: MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation

    doi: 10.1038/onc.2016.350

    Figure Lengend Snippet: MDM2 maintains retinoblastoma cell proliferation in part by promoting MYCN expression A. Western analysis at day 4 (left) and cell growth response (right) of RB176 cells after infection with lentivirus expressing shRNA against MYCN or a scrambled control (Scr). B. Tumors formed by RB176-luc cells transduced with shScr but not by those transduced with shMYCN in mouse subretinal xenografts (right, P-value from two-tailed Fisher’s exact test). Western analysis of the cells used for xenograft (left). C. Western analysis at day 4 (left) and cell growth response (right) of CHLA-RB215 cells after infection with lentivirus expressing shRNA against MYCN or Scr. D. Western analysis at days 4 and 25 (left and middle) and cell growth response (right) of RB176 cells after co-infection with lentivirus expressing shRNA against MDM2 or Scr and with lentivirus expressing MYCN under control of the EF1α promoter (BN-MYCN) or the empty vector (BN). E. Western analysis at day 4 (left) and cell growth response (right) of RB176 cells after co-infection with lentivirus expressing shMDM2 or Scr and with lentivirus expressing shp27 or Scr.

    Article Snippet: Lentivirus production and infections Lentivirus was produced in 15cm dishes by reverse transfection of Lenti-X cells (Clontech) using 17.5 μg vector, 8.75 μg pVSV-G, 17.5 μg pCMV-dR8.91 and 219 μl 0.6 μg/μl polyethylenimine (PEI) (Polysciences).

    Techniques: Expressing, Western Blot, Infection, shRNA, Transduction, Two Tailed Test, Plasmid Preparation

    MDM2 regulates MYCN protein expression without altering its stability A. Western analysis of RB176 cells 3 or 4 days after infection with lentivirus expressing shRNA against MDM2 or MDM4 or with a scrambled control (Scr). Signal intensity of MYCN was normalized to α-tubulin. Numbers below the MYCN panel indicate relative MYCN expression upon MDM2 knockdown compared to Scr control. B. qRT-PCR analysis of the same samples plotted after normalization to GAPDH . Values and error bars denote mean and s.d. of duplicate assays. C. Western analysis of RB176 cells treated with 30 μM CHX 4 days after infection with lentivirus expressing shRNA against MDM2 and Scr ( left ). Boxed MYCN panels show either the same exposure (for Scr samples) or a longer exposure (for shMDM2 samples) of the western analysis in the above panel. The level of MYCN at each time point was quantified using the longer and more equivalent shMDM2 exposure, normalized to α-tubulin, and the Log 2 values plotted ( right ), and MYCN half life determined. Data is representative of three independent experiments.

    Journal: Oncogene

    Article Title: MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation

    doi: 10.1038/onc.2016.350

    Figure Lengend Snippet: MDM2 regulates MYCN protein expression without altering its stability A. Western analysis of RB176 cells 3 or 4 days after infection with lentivirus expressing shRNA against MDM2 or MDM4 or with a scrambled control (Scr). Signal intensity of MYCN was normalized to α-tubulin. Numbers below the MYCN panel indicate relative MYCN expression upon MDM2 knockdown compared to Scr control. B. qRT-PCR analysis of the same samples plotted after normalization to GAPDH . Values and error bars denote mean and s.d. of duplicate assays. C. Western analysis of RB176 cells treated with 30 μM CHX 4 days after infection with lentivirus expressing shRNA against MDM2 and Scr ( left ). Boxed MYCN panels show either the same exposure (for Scr samples) or a longer exposure (for shMDM2 samples) of the western analysis in the above panel. The level of MYCN at each time point was quantified using the longer and more equivalent shMDM2 exposure, normalized to α-tubulin, and the Log 2 values plotted ( right ), and MYCN half life determined. Data is representative of three independent experiments.

    Article Snippet: Lentivirus production and infections Lentivirus was produced in 15cm dishes by reverse transfection of Lenti-X cells (Clontech) using 17.5 μg vector, 8.75 μg pVSV-G, 17.5 μg pCMV-dR8.91 and 219 μl 0.6 μg/μl polyethylenimine (PEI) (Polysciences).

    Techniques: Expressing, Western Blot, Infection, shRNA, Quantitative RT-PCR

    Knockdown of AP-3δ redistributes Vangl2 from the plasma membrane. HEK293 cells were infected with lentiviral shRNA against nontargeting shRNA (scrambled) (A–C, C′) or with shRNA against AP-3δ subunit (D–F, F′) for 72 h before puromycin selection was started. Cells were then transfected with GFP-Vangl2 for 48 h; this was followed by a quick staining with WGA-594 to mark cell membranes. Vangl2 was not detected on the membrane and was observed in punctuate structures throughout the cell in most cells depleted of AP-3δ. C′ and F′ are large views of the boxed regions in C and F, respectively. The cultures were confluent for both scrambled and AP-3 shRNA conditions. Cultures with less confluency (Supplemental Figure S4) showed similar results. (G) Cell lysates were collected and subjected to Western blot analysis of protein lysates using anti–AP-3δ SA4 antibody, revealing a reduction of > 95% in AP-3 levels by shRNA against AP-3. (F) The percent of Vangl2 redistribution in AP-3–depleted cells was quantified (H). The numbers of cells with Vangl2 on the membrane were significantly lower than that of controls. One hundred cells were counted per condition in each experimental set, and the experiments were repeated three times. Vangl2 membrane localization was quantified by measuring Pearson’s coefficient of Vangl2 and WGA in shRNA Scrambled and shRNA AP-3 cells (I). N = 3; 50 cells analyzed. Scale bars: 19 µm. p

    Journal: Molecular Biology of the Cell

    Article Title: Adaptor protein-3 complex is required for Vangl2 trafficking and planar cell polarity of the inner ear

    doi: 10.1091/mbc.E16-08-0592

    Figure Lengend Snippet: Knockdown of AP-3δ redistributes Vangl2 from the plasma membrane. HEK293 cells were infected with lentiviral shRNA against nontargeting shRNA (scrambled) (A–C, C′) or with shRNA against AP-3δ subunit (D–F, F′) for 72 h before puromycin selection was started. Cells were then transfected with GFP-Vangl2 for 48 h; this was followed by a quick staining with WGA-594 to mark cell membranes. Vangl2 was not detected on the membrane and was observed in punctuate structures throughout the cell in most cells depleted of AP-3δ. C′ and F′ are large views of the boxed regions in C and F, respectively. The cultures were confluent for both scrambled and AP-3 shRNA conditions. Cultures with less confluency (Supplemental Figure S4) showed similar results. (G) Cell lysates were collected and subjected to Western blot analysis of protein lysates using anti–AP-3δ SA4 antibody, revealing a reduction of > 95% in AP-3 levels by shRNA against AP-3. (F) The percent of Vangl2 redistribution in AP-3–depleted cells was quantified (H). The numbers of cells with Vangl2 on the membrane were significantly lower than that of controls. One hundred cells were counted per condition in each experimental set, and the experiments were repeated three times. Vangl2 membrane localization was quantified by measuring Pearson’s coefficient of Vangl2 and WGA in shRNA Scrambled and shRNA AP-3 cells (I). N = 3; 50 cells analyzed. Scale bars: 19 µm. p

    Article Snippet: The oligonucleotides for AP-3δ (RHS4533-NM_003938) shRNA in a pLKO.1 vector for lentiviral infection were obtained from Open Biosystems (Huntsville, AL) and used to silence expression of human AP-3.

    Techniques: Infection, shRNA, Selection, Transfection, Staining, Whole Genome Amplification, Western Blot

    NOTCH1 signaling regulates Group 3 medulloblastoma metastasis. a – d shRNAs specific to NOTCH1 (shNotch1) were introduced through lentiviral infections into luciferase-expressing D425 cells. 2 μg mL −1 of Dox was used to induce the expression of shRNA (shNotch1 + Dox). The un-induced infected cells (shNotch1-Dox) were used as controls. Recovery of NOTCH1 expression was performed by overexpressing NICD1 in NOTCH1-silenced Group 3 medulloblastoma cells (shNotch1 + Dox + NICD1). Bioluminescence imaging ( a ), hematoxylin/eosin staining ( b ), and quantification of total flux ( c ) from primary tumors and spinal metastases in mice injected with infected cells. ** P

    Journal: Nature Communications

    Article Title: Notch1 regulates the initiation of metastasis and self-renewal of Group 3 medulloblastoma

    doi: 10.1038/s41467-018-06564-9

    Figure Lengend Snippet: NOTCH1 signaling regulates Group 3 medulloblastoma metastasis. a – d shRNAs specific to NOTCH1 (shNotch1) were introduced through lentiviral infections into luciferase-expressing D425 cells. 2 μg mL −1 of Dox was used to induce the expression of shRNA (shNotch1 + Dox). The un-induced infected cells (shNotch1-Dox) were used as controls. Recovery of NOTCH1 expression was performed by overexpressing NICD1 in NOTCH1-silenced Group 3 medulloblastoma cells (shNotch1 + Dox + NICD1). Bioluminescence imaging ( a ), hematoxylin/eosin staining ( b ), and quantification of total flux ( c ) from primary tumors and spinal metastases in mice injected with infected cells. ** P

    Article Snippet: Lentiviral infections The lentivirus pWPT-NICD1 was generated by subcloning the activated Notch1 intracellular domain (EF.hICN1.CMV.GFP Addgene plasmid 17623) into the pWPT GCCACC backbone .

    Techniques: Luciferase, Expressing, shRNA, Infection, Imaging, Staining, Mouse Assay, Injection

    Sox2 negatively regulate HAMP2 expression in primary hepatocytes. Primary hepatocytes were cultured and lentivirus expressing Myc-Sox2 (A) or shRNA targeting Sox2 (B and C) infected the cells. Cells were harvested at 72 h later for Western blotting assay and RT-qPCR assay to determine Myc-Sox2 expression, HAMP expression and Sox2 knockdown efficiency. The experiments were repeated for three times with similar results and data were shown as means ± SE. Statistical analyses were conducted using a one-way ANOVA with Tukey’s multiple comparison test to determine individual p-values (B-C) or an unpaired, two-tailed student t-test (A) . Significant differences are indicated by *p

    Journal: Biological Research

    Article Title: Sox2 function as a negative regulator to control HAMP expression

    doi: 10.1186/s40659-015-0013-z

    Figure Lengend Snippet: Sox2 negatively regulate HAMP2 expression in primary hepatocytes. Primary hepatocytes were cultured and lentivirus expressing Myc-Sox2 (A) or shRNA targeting Sox2 (B and C) infected the cells. Cells were harvested at 72 h later for Western blotting assay and RT-qPCR assay to determine Myc-Sox2 expression, HAMP expression and Sox2 knockdown efficiency. The experiments were repeated for three times with similar results and data were shown as means ± SE. Statistical analyses were conducted using a one-way ANOVA with Tukey’s multiple comparison test to determine individual p-values (B-C) or an unpaired, two-tailed student t-test (A) . Significant differences are indicated by *p

    Article Snippet: Primary human hepatocytes culture, and lentivirus infection Human hepatocytes, purchased from PromoCell, were maintained in Hepatocyte Growth Medium (PromoCell, HD, GRE).

    Techniques: Expressing, Cell Culture, shRNA, Infection, Western Blot, Quantitative RT-PCR, Two Tailed Test