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  • 99
    Thermo Fisher superscript iii kit
    Autophagosome formation and ATG 9a trafficking are impaired upon loss of retromer Parental HeLa cells and VPS35 KO cells were transduced with mCherry‐Parkin and GFP‐LC3b and incubated with CCCP for 2 and 4 h. Co‐localization of GFP‐LC3b and endogenous TOM20 (blue) was quantified over two independent experiments. Parental HeLa cells and <t>VPS29</t> and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG16L1 (green) and endogenous TOM20 (blue). HeLa cells were transfected with humanized Cas9 and a mix of <t>three</t> distinct gRNAs targeting the ATG9a gene. Note that the vesicular ATG9a antibody signal (green) completely disappears in the CRISPR‐treated cells, indicating a high degree of specificity. Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG9a (green) and endogenous TOM20 (blue). Data information: All scale bars = 10 μm, all error bars = SD, and * P
    Superscript Iii Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript one step rt pcr kit
    <t>TPC2</t> signaling in mouse ES cells. ( A ) Expressions of TPC2 mRNAs in D3 ES cells were determined by <t>RT-PCR.</t> ( B ) NAADP-AM induced a Ca 2+ increase in TPC2 overexpressing ES cells in a bell-shaped concentration response curve. ( C ) Inhibition of Ca 2+ response triggered by NAADP-AM (50 nM) by bafilomycin A1 (100 nM) and esterase (50 units/ml). Data quantifications of [Ca 2+ ] i peak induced by drug treatment in (B) and (C) are expressed as mean ± S.E., n = 30–40 cells. The * symbols indicate the results of t Test analysis, p
    Superscript One Step Rt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1554 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii platinum sybr green one step qrt pcr kit
    Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with RNase-A. Virus RNA degradation was evaluated by <t>qRT-PCR.</t> The data represent mean values ± standard deviations (SD) for <t>three</t> independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p
    Superscript Iii Platinum Sybr Green One Step Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii reverse transcription kit
    Effect of LPA treatment on pro-migratory, pro-invasive, and pro-angiogenic gene expression. PMM were seeded onto 24-well plates, serum-starved overnight, and treated with 0.1% BSA (control) or LPA (1 μM). At the indicated time points, <t>RNA</t> was isolated, reverse-transcribed, and analyzed by qPCR. Expression ratios were normalized to HPRT. Results of <t>three</t> separate experiments in triplicate are expressed as mean + SD (* p
    Superscript Iii Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 413 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii reverse transcriptase kit
    Effects of AAs, 20E, insulin and HR3 on YPP genes. (A) Relative expression of gene—AAEL006563, Carboxypeptidase, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (E) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids and Insulin (AA+INS), Insulin and 20E (INS+20E), amino acids plus 20E (AA+20E), and amino acids plus 20E and Insulin (AA+20E+INS). Injecting double stranded <t>RNA</t> for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of <t>three</t> biological replicates, with three technical replicates and are illustrated as average ± SD, * P
    Superscript Iii Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5021 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii rt kit
    A charge-rebalance approach via reducing acidic residues of HBc. (A) A cartoon illustrates the charge rebalance approach. (B) The dimeric structure of HBc (PDB ID:1QGT) with each monomer in green and orange ribbon modes. The side chains of four glutamic acids are located close to the capsid interior: E40 (cyan), E46 (yellow), E113 (white) and E117 (blue) 18 70 71 90 . (C) The schematic presentation of a 40-mer HBc in the context of a 240-mer icosahedral particle (T = 4) is illustrated using PyMol (DeLano Scientific LLC, Palo Alto, CA, USA). Upper panel : E40 (cyan), E46 (yellow), E113 (white) and E117 (blue). Lower panel : all four acidic residues are in black. (D) Parental HBc mutant ARD-I + II + <t>III</t> + IV exhibited a very short DNA phenotype by Southern blot, which can be partially rescued by E-to-A single or double mutations at E46 or E113. (E) The Southern result in ( D ) can be confirmed by Whole Genome PCR analysis using core particle-associated viral DNAs as a template. (F) Upper panel : Northern blot analysis of HBc parental mutant ARD-I + II + III + IV and its charge rebalanced derivatives. Rescued viral <t>RNAs</t> with significantly increased size (between two dotted lines) were observed in HBc mutant E46A + E113A (last lane). Lower panels : Total cytoplasmic HBV RNA and GAPDH RNA were included as controls. (G) The Northern results in ( F ) was confirmed by RT-PCR analysis using core particle-associated viral RNAs. (H) Size distributions of encapsidated viral DNAs in WT, mutant ARD-I + II + III + IV, and its double-rescued mutant. Each clone was characterized by sequencing as described in Fig. 2D . The horizontal line represents the medium size of encapsidated viral DNA species. ***p
    Superscript Iii Rt Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii transcriptase kit
    MTase-defective rVSV (mtdVSV)-based vaccine induces high levels of <t>ZIKV-specific</t> antibody in A129 mice. A129 mice were immunized intramuscularly with pCI, pCI-prM-E, or pCI-NS1 at a dose of 50 µg DNA per mouse, and were boosted with the same plasmid at the same dose two weeks later. For VSV-based vaccines, mice were administered intramuscularly using a single dose (1 × 10 5 PFU). The body weight for each mouse was evaluated at indicated time points ( a ). The average body weights of five mice were shown. At day 7, two out of five mice in rVSV-prM-E-NS1 group were dead and the other <t>three</t> terminated at day 10. After immunization, blood samples were collected at weeks 1 and 3. ZIKV E-specific antibody was measured by ELISA at weeks 1 ( b ) and 3 ( c ) post-immunization. ZIKV-specific neutralizing Ab was measured at weeks 1 ( d ) and 3 ( e ) post-immunization. ZIKV NS1-specific Ab was measured by ELISA at weeks 1 ( f ) and 3 ( g ) post-immunization. ELISA titers shown are GMT of 5 mice ± standard deviation. At the termination of this experiment, brains were collected and the presence of the VSV RNA was quantified by real-time RT-PCR using primers annealing to the VSV L gene ( h ). Antibody and viral load data are expressed as the GMT of five mice (black bars). Exact P value (by Student’s t -test) in each panel: b **** P = 1.36 × 10 −6 ; d **** P = 5.44 × 10 −5 ; g **** P = 6.70 × 10 −5 ; h **** P = 4.32 × 10 −6 , N.S., not significant
    Superscript Iii Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher express one step superscript qrt pcr kit universal
    The MCMV m152 protein specifically targets STING‐dependent signaling 293T cells were co‐transfected with expression plasmids for Cherry‐STING, the murine IFNβ‐luciferase reporter (IFNβ‐Luc), a Renilla luciferase normalization control (pRL‐TK), and the indicated expression plasmids or empty vector (ev). Cells were additionally co‐transfected with expression plasmids for cGAS‐GFP (stimulated) or IRES‐GFP (unstimulated). 20 hours post‐transfection, cells were lysed and a dual‐luciferase assay was performed. An expression plasmid for RIG‐I N (stimulated) or ev (unstimulated) was co‐transfected with IFNβ‐Luc, pRL‐TK and the indicated expression plasmids in 293T cells and analyzed as in (A). An expression plasmid for TBK1 (stimulated) or ev (unstimulated) was co‐transfected together with IFNβ‐Luc, pRL‐TK and the indicated expression plasmids and analyzed as in (A). 293T cells were co‐transfected with a plasmid expressing constitutively active IRF3 (IRF3‐5D; stimulated) or IRES‐GFP (unstimulated) together with IFNβ‐Luc, pRL‐TK and the indicated expression plasmids and analyzed as in (A). The ISG56‐luciferase reporter, pRL‐TK, and the indicated expression plasmids were co‐transfected in 293T cells. 24 hours post‐transfection, cells were stimulated with 0.1 ng/μl human IFNβ or mock stimulated and analyzed 16 h later as described in (A). iMEF gt/gt stably expressing Cherry‐STING and either ev or V5‐tagged m152 were stimulated with 5 μg/ml ISD (F), 10 μg/ml poly(I:C) (G), or mock stimulated with Lipofectamine. 4 hours post‐stimulation, RNA was extracted to determine IFNβ mRNA transcripts by <t>qRT–PCR.</t> iBMDM stably expressing ev or m152‐V5 were stimulated in duplicates with 10 μg/ml cGAMP (H), 5 μg/ml ISD (I), Newcastle disease virus (NDV) infection (J), or 1 μM CpG DNA (K). 6 (H) or 16 (I‐K) hours later, secreted IFNβ (H‐J) or TNFα (K) levels were determined by ELISA. Data information: (A‐G) Data are combined from three independent experiments. (H‐K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t ‐test (unpaired, two‐tailed), n.s. not significant, * P
    Express One Step Superscript Qrt Pcr Kit Universal, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Autophagosome formation and ATG 9a trafficking are impaired upon loss of retromer Parental HeLa cells and VPS35 KO cells were transduced with mCherry‐Parkin and GFP‐LC3b and incubated with CCCP for 2 and 4 h. Co‐localization of GFP‐LC3b and endogenous TOM20 (blue) was quantified over two independent experiments. Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG16L1 (green) and endogenous TOM20 (blue). HeLa cells were transfected with humanized Cas9 and a mix of three distinct gRNAs targeting the ATG9a gene. Note that the vesicular ATG9a antibody signal (green) completely disappears in the CRISPR‐treated cells, indicating a high degree of specificity. Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG9a (green) and endogenous TOM20 (blue). Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Autophagosome formation and ATG 9a trafficking are impaired upon loss of retromer Parental HeLa cells and VPS35 KO cells were transduced with mCherry‐Parkin and GFP‐LC3b and incubated with CCCP for 2 and 4 h. Co‐localization of GFP‐LC3b and endogenous TOM20 (blue) was quantified over two independent experiments. Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG16L1 (green) and endogenous TOM20 (blue). HeLa cells were transfected with humanized Cas9 and a mix of three distinct gRNAs targeting the ATG9a gene. Note that the vesicular ATG9a antibody signal (green) completely disappears in the CRISPR‐treated cells, indicating a high degree of specificity. Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG9a (green) and endogenous TOM20 (blue). Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: Gene Knockout, Transduction, Incubation, Staining, Transfection, CRISPR

    Mitophagy is defective upon loss of retromer mCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were treated with CCCP over 16 h, followed by PFA fixation and staining of endogenous TOM20 (green). Residual TOM20 after CCCP treatment was quantified over two independent experiments. The cells described for panel (A) were lysed after 16‐h CCCP treatment, and residual TOM20 was detected by Western blotting (left) and quantified over three independent experiments (right). SHSY‐5Y cells that had been transduced with a construct expressing tandem mCherry‐eGFP‐FIS1TM (mitochondria‐anchored mCherry‐GFP tandem) were transfected with control and VPS35‐specific siRNA and treated with the iron chelator deferiprone for 24 h. The cells were then assessed for red‐shifted dots representing mitophagy events by confocal microscopy. Efficiency of the siRNA treatment was confirmed by Western blotting against endogenous VPS35. Mitophagy events were counted in 20 images per condition, acquired in two independent experiments. Parental HeLa cells and VPS29 and VPS35 KO cells were transduced with mCherry–Parkin, and mitophagy was induced for the indicated time points with the proton uncoupler CCCP. The cells were then fixed in methanol and co‐stained for endogenous LC3b (green) and TOM20 (blue). (D′) Co‐localization between LC3b and TOM20 was analyzed over 12 images per condition acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Mitophagy is defective upon loss of retromer mCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were treated with CCCP over 16 h, followed by PFA fixation and staining of endogenous TOM20 (green). Residual TOM20 after CCCP treatment was quantified over two independent experiments. The cells described for panel (A) were lysed after 16‐h CCCP treatment, and residual TOM20 was detected by Western blotting (left) and quantified over three independent experiments (right). SHSY‐5Y cells that had been transduced with a construct expressing tandem mCherry‐eGFP‐FIS1TM (mitochondria‐anchored mCherry‐GFP tandem) were transfected with control and VPS35‐specific siRNA and treated with the iron chelator deferiprone for 24 h. The cells were then assessed for red‐shifted dots representing mitophagy events by confocal microscopy. Efficiency of the siRNA treatment was confirmed by Western blotting against endogenous VPS35. Mitophagy events were counted in 20 images per condition, acquired in two independent experiments. Parental HeLa cells and VPS29 and VPS35 KO cells were transduced with mCherry–Parkin, and mitophagy was induced for the indicated time points with the proton uncoupler CCCP. The cells were then fixed in methanol and co‐stained for endogenous LC3b (green) and TOM20 (blue). (D′) Co‐localization between LC3b and TOM20 was analyzed over 12 images per condition acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: Gene Knockout, Transduction, Construct, Staining, Western Blot, Expressing, Transfection, Confocal Microscopy

    Loss of retromer leads to a pronounced shift in RAB 7 distribution Parental HeLa cells, two clonal VPS35 knockout cell lines, and one clonal VPS29 KO cell line were fixed in PFA and co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Co‐localization was analyzed across three independent experiments. A clonal RAB7a knockout cell line was mixed 1:1 with parental HeLa cells and seeded onto coverslips. Following PFA fixation, the mixed cells were stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Note that the RAB7a signal completely disappears in the cells not expressing RAB7a. Parental HeLa cells and clonal VPS35 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red, upper panel) or for endogenous RAB7a and a mCherry‐tagged ER marker (red, lower panel), and co‐localization between RAB7a and the respective marker was analyzed across two independent experiments. To show that RAB7a localizes to the ER and mitochondria, endogenous TOM20 (blue) was co‐stained in the lower panel. Parental HeLa cells and clonal VPS35 and VPS29 KO cells were transduced with a lentivirus expressing GFP‐FIS1TM (eGFP with a C‐terminal mitochondrial targeting sequence and transmembrane domain of the mitochondrial protein FIS1) and disrupted through a fine needle in detergent‐free sucrose buffer followed by isolation of the mitochondria from postnuclear supernatants with GFP‐trap agarose beads. The amount of RAB7 precipitating with the mitochondria was quantified over four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Loss of retromer leads to a pronounced shift in RAB 7 distribution Parental HeLa cells, two clonal VPS35 knockout cell lines, and one clonal VPS29 KO cell line were fixed in PFA and co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Co‐localization was analyzed across three independent experiments. A clonal RAB7a knockout cell line was mixed 1:1 with parental HeLa cells and seeded onto coverslips. Following PFA fixation, the mixed cells were stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Note that the RAB7a signal completely disappears in the cells not expressing RAB7a. Parental HeLa cells and clonal VPS35 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red, upper panel) or for endogenous RAB7a and a mCherry‐tagged ER marker (red, lower panel), and co‐localization between RAB7a and the respective marker was analyzed across two independent experiments. To show that RAB7a localizes to the ER and mitochondria, endogenous TOM20 (blue) was co‐stained in the lower panel. Parental HeLa cells and clonal VPS35 and VPS29 KO cells were transduced with a lentivirus expressing GFP‐FIS1TM (eGFP with a C‐terminal mitochondrial targeting sequence and transmembrane domain of the mitochondrial protein FIS1) and disrupted through a fine needle in detergent‐free sucrose buffer followed by isolation of the mitochondria from postnuclear supernatants with GFP‐trap agarose beads. The amount of RAB7 precipitating with the mitochondria was quantified over four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: Knock-Out, Gene Knockout, Staining, Expressing, Marker, Transduction, Sequencing, Isolation

    RAB 7 localizes to mitochondria and the ER All images show formaldehyde‐fixed HeLa cells. HeLa cells stained for endogenous RAB7a (green) and endogenous LAMP2 (red). White arrows indicate sites of vesicular RAB7 that appears to be budding from a network of RAB7. Immunofluorescent co‐staining of endogenous RAB7a (green) and the mitochondria marker TOM20 (red). Co‐staining of endogenous RAB7 (green) and the trans ‐Golgi network marker TGN46 (red). Co‐staining of endogenous RAB7 (green) and the ER marker protein disulfide isomerase (PDI, red). Co‐staining of lentivirally expressed GFP‐RAB7a and endogenous TOM20 (red). Co‐staining of endogenous RAB7 (green) and TOM20 (red) in cells treated with Cas9 and a mixture of three gRNAs targeting the RAB7a locus. The Western blot of this cell population shows an almost complete loss of RAB7 in the treated cells compared to parental HeLa cells. GFP‐RAB7 was expressed in RAB7a KO HeLa cells and imaged in live cells using a spinning disk confocal microscope. Mitochondria were visualized with MitoTracker Red. Parental HeLa cells and clonal VPS29 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red), and co‐localization was analyzed across two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: RAB 7 localizes to mitochondria and the ER All images show formaldehyde‐fixed HeLa cells. HeLa cells stained for endogenous RAB7a (green) and endogenous LAMP2 (red). White arrows indicate sites of vesicular RAB7 that appears to be budding from a network of RAB7. Immunofluorescent co‐staining of endogenous RAB7a (green) and the mitochondria marker TOM20 (red). Co‐staining of endogenous RAB7 (green) and the trans ‐Golgi network marker TGN46 (red). Co‐staining of endogenous RAB7 (green) and the ER marker protein disulfide isomerase (PDI, red). Co‐staining of lentivirally expressed GFP‐RAB7a and endogenous TOM20 (red). Co‐staining of endogenous RAB7 (green) and TOM20 (red) in cells treated with Cas9 and a mixture of three gRNAs targeting the RAB7a locus. The Western blot of this cell population shows an almost complete loss of RAB7 in the treated cells compared to parental HeLa cells. GFP‐RAB7 was expressed in RAB7a KO HeLa cells and imaged in live cells using a spinning disk confocal microscope. Mitochondria were visualized with MitoTracker Red. Parental HeLa cells and clonal VPS29 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red), and co‐localization was analyzed across two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: Staining, Marker, Western Blot, Gene Knockout, Microscopy

    RAB 7 activity state controls its localization to the ER , mitochondria, and lysosomes Mitochondria were purified from detergent‐free postnuclear supernatants using magnetic beads coated with a TOM22‐specific antibody from parental HeLa cells and from clonal VPS35 KO cells. The purified mitochondria were then subjected to a marker analysis using the indicated organelle markers. Note that only mitochondria (TOM20) and RAB7 are enriched over the inputs, with lower levels of RAB7 precipitating with the mitochondria of VPS35 KO cells. The quantification of RAB7 was done across three independent mitochondria isolations. RAB7a KO cells transduced with the indicated GFP‐RAB7a variants were fixed and co‐stained for endogenous LAMP2. Note that the inactive RAB7 (T22N) does not localize to lysosomes, whereas the hyperactive variant (Q67L) fully localizes to lysosomes. RAB7a KO cells transduced with the indicated GFP‐RAB7a variants and a mCherry‐ER marker were fixed and co‐stained for endogenous TOM20. RAB7a KO cells were infected with the indicated GFP‐RAB7 variants, labeled with MitoTracker Red, and subjected to live cell imaging using a spinning disk confocal microscope. Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: RAB 7 activity state controls its localization to the ER , mitochondria, and lysosomes Mitochondria were purified from detergent‐free postnuclear supernatants using magnetic beads coated with a TOM22‐specific antibody from parental HeLa cells and from clonal VPS35 KO cells. The purified mitochondria were then subjected to a marker analysis using the indicated organelle markers. Note that only mitochondria (TOM20) and RAB7 are enriched over the inputs, with lower levels of RAB7 precipitating with the mitochondria of VPS35 KO cells. The quantification of RAB7 was done across three independent mitochondria isolations. RAB7a KO cells transduced with the indicated GFP‐RAB7a variants were fixed and co‐stained for endogenous LAMP2. Note that the inactive RAB7 (T22N) does not localize to lysosomes, whereas the hyperactive variant (Q67L) fully localizes to lysosomes. RAB7a KO cells transduced with the indicated GFP‐RAB7a variants and a mCherry‐ER marker were fixed and co‐stained for endogenous TOM20. RAB7a KO cells were infected with the indicated GFP‐RAB7 variants, labeled with MitoTracker Red, and subjected to live cell imaging using a spinning disk confocal microscope. Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: Activity Assay, Purification, Magnetic Beads, Gene Knockout, Marker, Transduction, Staining, Variant Assay, Infection, Labeling, Live Cell Imaging, Microscopy

    A CRISPR /Cas9 screen identifies TBC 1D5 as the retromer‐associated component that controls RAB 7 activity and localization HeLa cells were co‐transfected with CRISPR/Cas9 constructs targeting the indicated genes and a puromycin resistance marker. Five days after puromycin selection of transfected cells, all cell lines were stained for endogenous RAB7a and the lysosomal marker LAMP2, and co‐localization was quantified across 12 images acquired in two independent experiments. The cell lines described above for panel (A) were lysed, and RAB7a activity was assayed with the GST‐RILP activity assay. Western blotting was used to confirm the efficiency of the CRISPR/CAS9 targeting approach. RILP‐bound RAB7a was quantified over three independent experiments. Methanol‐fixed HeLa cells and VPS35 and TBC1D5 KO cells were co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Methanol‐fixed parental HeLa cells and TBC1D5 KO cells were stained for endogenous VPS35 (green) and LAMP2 (red). Methanol‐fixed parental HeLa and TBC1D5 KO cells were co‐stained for endogenous RAB7a (green) and endogenous VPS35 (red), and co‐localization was quantified over two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: A CRISPR /Cas9 screen identifies TBC 1D5 as the retromer‐associated component that controls RAB 7 activity and localization HeLa cells were co‐transfected with CRISPR/Cas9 constructs targeting the indicated genes and a puromycin resistance marker. Five days after puromycin selection of transfected cells, all cell lines were stained for endogenous RAB7a and the lysosomal marker LAMP2, and co‐localization was quantified across 12 images acquired in two independent experiments. The cell lines described above for panel (A) were lysed, and RAB7a activity was assayed with the GST‐RILP activity assay. Western blotting was used to confirm the efficiency of the CRISPR/CAS9 targeting approach. RILP‐bound RAB7a was quantified over three independent experiments. Methanol‐fixed HeLa cells and VPS35 and TBC1D5 KO cells were co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Methanol‐fixed parental HeLa cells and TBC1D5 KO cells were stained for endogenous VPS35 (green) and LAMP2 (red). Methanol‐fixed parental HeLa and TBC1D5 KO cells were co‐stained for endogenous RAB7a (green) and endogenous VPS35 (red), and co‐localization was quantified over two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: CRISPR, Activity Assay, Transfection, Construct, Marker, Selection, Staining, Western Blot, Gene Knockout

    Control of RAB 7 activity is not required for retromer‐based sorting of integral membrane proteins All images show formaldehyde‐fixed cells. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were co‐stained for endogenous GLUT1 (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were surface‐biotinylated, followed by streptavidin isolation and Western blot‐based quantification of biotinylated surface proteins. Surface GLUT1 was quantified over four independent experiments. RAB7a knockout cells and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs cells were co‐stained for endogenous GLUT1 (red) and endogenous LAMP2 (blue), and co‐localization was quantified over two independent experiments. Parental HeLa cells, RAB7a knockout cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were co‐stained for endogenous CI‐MPR (red) and endogenous TGN46 (blue). Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Control of RAB 7 activity is not required for retromer‐based sorting of integral membrane proteins All images show formaldehyde‐fixed cells. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were co‐stained for endogenous GLUT1 (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were surface‐biotinylated, followed by streptavidin isolation and Western blot‐based quantification of biotinylated surface proteins. Surface GLUT1 was quantified over four independent experiments. RAB7a knockout cells and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs cells were co‐stained for endogenous GLUT1 (red) and endogenous LAMP2 (blue), and co‐localization was quantified over two independent experiments. Parental HeLa cells, RAB7a knockout cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were co‐stained for endogenous CI‐MPR (red) and endogenous TGN46 (blue). Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: Activity Assay, Gene Knockout, Transduction, Construct, Staining, Isolation, Western Blot, Knock-Out

    Retromer controls RAB 7 activity levels and mobility/membrane turnover GDP‐locked (inactive) GFP‐RAB7‐T22N and GTP‐locked (constitutively active) GFP‐RAB7‐Q67L were lentivirally expressed in RAB7 KO cells and co‐stained with the mitochondrial marker TOM20 (red). Co‐localization was analyzed over two independent experiments with 10 images each. Lysates from parental HeLa cells and clonal VPS35 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lysates from parental HeLa cells and clonal VPS29 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lysates from parental HeLa cells and clonal VPS29 KO cells and VPS29 KO cells with lentivirally re‐expressed VPS29‐myc were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lentivirally expressed GFP‐RAB7 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of the endogenous RAB‐chaperone GDI2. Lentivirally expressed GFP‐GDI1 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of endogenous RAB14 and endogenous RAB7a. GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FRAP imaging in live cells. The recovery kinetics were obtained by averaging 15 FRAP recoveries acquired in two independent experiments. GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FLIP imaging in live cells. The depletion kinetics (in area A, as indicated) were obtained by averaging 18 FLIP depletions acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Retromer controls RAB 7 activity levels and mobility/membrane turnover GDP‐locked (inactive) GFP‐RAB7‐T22N and GTP‐locked (constitutively active) GFP‐RAB7‐Q67L were lentivirally expressed in RAB7 KO cells and co‐stained with the mitochondrial marker TOM20 (red). Co‐localization was analyzed over two independent experiments with 10 images each. Lysates from parental HeLa cells and clonal VPS35 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lysates from parental HeLa cells and clonal VPS29 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lysates from parental HeLa cells and clonal VPS29 KO cells and VPS29 KO cells with lentivirally re‐expressed VPS29‐myc were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lentivirally expressed GFP‐RAB7 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of the endogenous RAB‐chaperone GDI2. Lentivirally expressed GFP‐GDI1 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of endogenous RAB14 and endogenous RAB7a. GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FRAP imaging in live cells. The recovery kinetics were obtained by averaging 15 FRAP recoveries acquired in two independent experiments. GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FLIP imaging in live cells. The depletion kinetics (in area A, as indicated) were obtained by averaging 18 FLIP depletions acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: Activity Assay, Gene Knockout, Staining, Marker, Western Blot, Imaging

    TBC 1D5 and retromer cooperate in the control of RAB 7 activity and mobility GFP‐trap IPs of the indicated GFP‐tagged VPS29 (upper panel) or TBC1D5 (lower panel) constructs confirm that the VPS29‐L152E and the TBC1D5‐L142E mutant lose binding to each other. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Note that re‐expression of both VPS29 variants fully restores the level of endogenous VPS35. PFA‐fixed parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells and VPS29 KO cells as well as VPS29 KO cells transduced with the indicated VPS29 rescue constructs were transduced with GFP‐RAB7, and RAB7 mobility/turnover was analyzed by FRAP in living cells. The displayed recovery kinetics were obtained by averaging kinetics from fifteen FRAP recoveries per condition acquired in two independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Parental HeLa cells and clonal TBC1D5 KO cells and TBC1D5 KO cells transduced with the indicated GFP‐TBC1D5 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: TBC 1D5 and retromer cooperate in the control of RAB 7 activity and mobility GFP‐trap IPs of the indicated GFP‐tagged VPS29 (upper panel) or TBC1D5 (lower panel) constructs confirm that the VPS29‐L152E and the TBC1D5‐L142E mutant lose binding to each other. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Note that re‐expression of both VPS29 variants fully restores the level of endogenous VPS35. PFA‐fixed parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells and VPS29 KO cells as well as VPS29 KO cells transduced with the indicated VPS29 rescue constructs were transduced with GFP‐RAB7, and RAB7 mobility/turnover was analyzed by FRAP in living cells. The displayed recovery kinetics were obtained by averaging kinetics from fifteen FRAP recoveries per condition acquired in two independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Parental HeLa cells and clonal TBC1D5 KO cells and TBC1D5 KO cells transduced with the indicated GFP‐TBC1D5 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: Activity Assay, Construct, Mutagenesis, Binding Assay, Gene Knockout, Transduction, Expressing, Staining

    Loss of TBC 1D5 does not perturb retromer‐mediated sorting of GLUT 1 and CI ‐ MPR HeLa, SNX5/6 double KO, and TBC1D5 KO cells were fixed in PFA and stained for CI‐MPR (red) and the trans ‐Golgi network marker TGN46. Note that knockout of SNX5/6 results in dispersal of CI‐MPR from the TGN. HeLa, VPS29, and TBC1D5 KO cells were fixed in PFA and stained for GLUT1 (green) and LAMP2 (red). Co‐localization between GLUT1 and LAMP2 was quantified over ten images for each condition. HeLa cells were transfected with a pool of three distinct CRISPR/Cas9 plasmids targeting the RAB7a gene at three sites together with a puromycin resistance plasmid. Following puromycin selection and 5 days of incubation, the RAB7a KO cells were transduced with lentiviruses expressing the indicated GFP‐RAB7 proteins. A Western blot for RAB7 confirms high knockout efficiency and demonstrates that the GFP‐RAB7 variants are expressed at low endogenous levels. Parental HeLa cells and clonal VPS29 and VPS35 KO cells transduced with GFP‐LC3b were starved in EBSS for 4 h without (EBSS) or with addition of bafilomycin A1 to the EBSS (EBSS + Bafi) to assess autophagic flux. Autophagic flux was calculated from the increase in lipidated GFP‐LC3 in the EBSS + bafilomycin samples compared to the EBSS‐only sample. The quantification was done across four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Loss of TBC 1D5 does not perturb retromer‐mediated sorting of GLUT 1 and CI ‐ MPR HeLa, SNX5/6 double KO, and TBC1D5 KO cells were fixed in PFA and stained for CI‐MPR (red) and the trans ‐Golgi network marker TGN46. Note that knockout of SNX5/6 results in dispersal of CI‐MPR from the TGN. HeLa, VPS29, and TBC1D5 KO cells were fixed in PFA and stained for GLUT1 (green) and LAMP2 (red). Co‐localization between GLUT1 and LAMP2 was quantified over ten images for each condition. HeLa cells were transfected with a pool of three distinct CRISPR/Cas9 plasmids targeting the RAB7a gene at three sites together with a puromycin resistance plasmid. Following puromycin selection and 5 days of incubation, the RAB7a KO cells were transduced with lentiviruses expressing the indicated GFP‐RAB7 proteins. A Western blot for RAB7 confirms high knockout efficiency and demonstrates that the GFP‐RAB7 variants are expressed at low endogenous levels. Parental HeLa cells and clonal VPS29 and VPS35 KO cells transduced with GFP‐LC3b were starved in EBSS for 4 h without (EBSS) or with addition of bafilomycin A1 to the EBSS (EBSS + Bafi) to assess autophagic flux. Autophagic flux was calculated from the increase in lipidated GFP‐LC3 in the EBSS + bafilomycin samples compared to the EBSS‐only sample. The quantification was done across four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: RILP, TBC1D5, RAB7, and VPS29 cDNA was cloned from HeLa cell cDNA prepared with the Superscript III kit (Invitrogen) using Kapa HiFi DNA polymerase.

    Techniques: Gene Knockout, Staining, Marker, Knock-Out, Transfection, CRISPR, Plasmid Preparation, Selection, Incubation, Transduction, Expressing, Western Blot

    Expression of PhHD-Zip in petunia flowers in response to ethylene and 1-MCP treatments. “ Ethylene ” Flowers harvested at anthesis and treated continuously with ethylene (3 ppm), “ 1-MCP/Ethylene” Flowers harvested at anthesis and treated with 1-MCP (50 nL/L) for 4 hours before a continuous ethylene treatment. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).

    Journal: PLoS ONE

    Article Title: A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    doi: 10.1371/journal.pone.0088320

    Figure Lengend Snippet: Expression of PhHD-Zip in petunia flowers in response to ethylene and 1-MCP treatments. “ Ethylene ” Flowers harvested at anthesis and treated continuously with ethylene (3 ppm), “ 1-MCP/Ethylene” Flowers harvested at anthesis and treated with 1-MCP (50 nL/L) for 4 hours before a continuous ethylene treatment. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).

    Article Snippet: 2 µg total RNA was used to synthesis first-strand cDNA using the SuperScript III kit (Invitrogen, CA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation

    Expression of PhHD-Zip in different tissues of petunia. A. A representative gel image from semi-quantitative PCR of RNA isolated from different tissues. 26S RNA: the internal control. Samples were analyzed after 33 cycles for PhHD-Zip , and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip in different tissues (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Journal: PLoS ONE

    Article Title: A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    doi: 10.1371/journal.pone.0088320

    Figure Lengend Snippet: Expression of PhHD-Zip in different tissues of petunia. A. A representative gel image from semi-quantitative PCR of RNA isolated from different tissues. 26S RNA: the internal control. Samples were analyzed after 33 cycles for PhHD-Zip , and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip in different tissues (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Article Snippet: 2 µg total RNA was used to synthesis first-strand cDNA using the SuperScript III kit (Invitrogen, CA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation

    Expression of senescence-related genes in D7 petunia flowers. Abundance of transcripts of genes associated with senescence were determined at D7 in purple control flowers (WT), in white flowers of plants inoculated with the CHS /TRV reporter construct (VW), and in white flowers of plant inoculated with the PhHD-Zip / CHS /TRV silencing construct. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas. 26S RNA: the internal control. Samples were analyzed after 33 cycles for ACS, after 30 cycles for other genes, and after 24 cycles for 26S RNA , respectively. B. Relative expression levels of different genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Journal: PLoS ONE

    Article Title: A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    doi: 10.1371/journal.pone.0088320

    Figure Lengend Snippet: Expression of senescence-related genes in D7 petunia flowers. Abundance of transcripts of genes associated with senescence were determined at D7 in purple control flowers (WT), in white flowers of plants inoculated with the CHS /TRV reporter construct (VW), and in white flowers of plant inoculated with the PhHD-Zip / CHS /TRV silencing construct. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas. 26S RNA: the internal control. Samples were analyzed after 33 cycles for ACS, after 30 cycles for other genes, and after 24 cycles for 26S RNA , respectively. B. Relative expression levels of different genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Article Snippet: 2 µg total RNA was used to synthesis first-strand cDNA using the SuperScript III kit (Invitrogen, CA, USA).

    Techniques: Expressing, Construct, Real-time Polymerase Chain Reaction, Isolation

    Expression of PhHD-Zip in petunia flower under abiotic stress. Petunia flowers harvested at anthesis were placed in tubes with water, without water, with 50°C. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, and after 24 cycles for 26S RNA. B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates). C. Relative expression levels of PhHD-Zip determined using the same RNA samples, but using real-time quantitative PCR (error bars correspond to SE of the means of three biological replicates).

    Journal: PLoS ONE

    Article Title: A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    doi: 10.1371/journal.pone.0088320

    Figure Lengend Snippet: Expression of PhHD-Zip in petunia flower under abiotic stress. Petunia flowers harvested at anthesis were placed in tubes with water, without water, with 50°C. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, and after 24 cycles for 26S RNA. B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates). C. Relative expression levels of PhHD-Zip determined using the same RNA samples, but using real-time quantitative PCR (error bars correspond to SE of the means of three biological replicates).

    Article Snippet: 2 µg total RNA was used to synthesis first-strand cDNA using the SuperScript III kit (Invitrogen, CA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation

    Expression of PhHD-Zip in petunia corollas during flower senescence. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals after anthesis. D0: at anthesis; D2, D4, D7: 2, 4, and 7 days after anthesis, respectively. 26S RNA: the internal control. Samples were analyzed after 30 cycles of amplification for PhHD-Zip , and after 24 cycles of amplification for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).

    Journal: PLoS ONE

    Article Title: A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    doi: 10.1371/journal.pone.0088320

    Figure Lengend Snippet: Expression of PhHD-Zip in petunia corollas during flower senescence. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals after anthesis. D0: at anthesis; D2, D4, D7: 2, 4, and 7 days after anthesis, respectively. 26S RNA: the internal control. Samples were analyzed after 30 cycles of amplification for PhHD-Zip , and after 24 cycles of amplification for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).

    Article Snippet: 2 µg total RNA was used to synthesis first-strand cDNA using the SuperScript III kit (Invitrogen, CA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Amplification

    Effect of transgenic over-expression of PhHD-Zip on abundance of transcripts of PhHD-Zip and of ethylene biosynthesis genes in petunia corollas. WT: wild type flower; #3, #7: two transgenic lines of 35S:: PhHD-Zip . A. A representative gel image from semi-quantitative PCR of RNA isolated from harvested corollas. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, ACO1 and ACO4 ; after 33 cycles for ACS ; and after 24 cycles for 26S RNA . B. Relative expression level of PhHD-Zip and ethylene biosynthesis genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Journal: PLoS ONE

    Article Title: A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    doi: 10.1371/journal.pone.0088320

    Figure Lengend Snippet: Effect of transgenic over-expression of PhHD-Zip on abundance of transcripts of PhHD-Zip and of ethylene biosynthesis genes in petunia corollas. WT: wild type flower; #3, #7: two transgenic lines of 35S:: PhHD-Zip . A. A representative gel image from semi-quantitative PCR of RNA isolated from harvested corollas. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, ACO1 and ACO4 ; after 33 cycles for ACS ; and after 24 cycles for 26S RNA . B. Relative expression level of PhHD-Zip and ethylene biosynthesis genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Article Snippet: 2 µg total RNA was used to synthesis first-strand cDNA using the SuperScript III kit (Invitrogen, CA, USA).

    Techniques: Transgenic Assay, Over Expression, Real-time Polymerase Chain Reaction, Isolation, Expressing

    The SNX-BAR proteins are needed to prevent lysosomal degradation of the activated IGF1R. (A) HeLa cells, clonal SNX1/2 and SNX5/6 double-KO cells, and two VPS35-KO cell lines were serum starved and treated with 10 nM IGF-1 for the indicated periods. The level of endogenous IGF1R and the INSR were detected by Western blotting, and the IGF1R degradation kinetics were quantified over four independent experiments. (B) HeLa cells, clonal SNX1/2, and SNX5/6 double-KO cells as well as VPS35-KO cell lines were transduced with a lentiviruses expressing human IGF1R-myc, serum starved, and then treated with 10 nM IGF-1 for 1 h. The IGF1R and endogenous LAMP1 were stained by immunofluorescence, and the colocalization was quantified across 16 images acquired in two independent experiments. (C) HeLa cells and SNX1/2 and SNX5/6 double-KO cell lines were serum starved and treated with 5 µg/ml insulin for the indicated periods, and the level of the endogenous INSR was quantified by Western blotting over three independent experiments. (D) SNX5/6 double-KO cells were transduced with lentiviruses expressing GFP-SNX5 or GFP-SNX6 and with GFP only as a control. The cells were serum starved and treated with 10 nM IGF-1 for the indicated periods, followed by Western blot–based detection of the levels of the endogenous IGF1R. The level of the endogenous IGF1R in starved cells (top bar graph) as well as the degradation kinetics in IGF-1–treated cells were quantified over four independent experiments. (E) HeLa cells and clonal SNX5/6 double-KO cells were serum starved and treated with 10 nM IGF-1 for the indicated periods. One set of the SNX5/6 KO cells was treated with the lysosomal inhibitor bafilomycin-A1. The degradation kinetics were quantified over four independent experiments. Bars, 10 µm. Error bars indicate SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport

    doi: 10.1083/jcb.201702137

    Figure Lengend Snippet: The SNX-BAR proteins are needed to prevent lysosomal degradation of the activated IGF1R. (A) HeLa cells, clonal SNX1/2 and SNX5/6 double-KO cells, and two VPS35-KO cell lines were serum starved and treated with 10 nM IGF-1 for the indicated periods. The level of endogenous IGF1R and the INSR were detected by Western blotting, and the IGF1R degradation kinetics were quantified over four independent experiments. (B) HeLa cells, clonal SNX1/2, and SNX5/6 double-KO cells as well as VPS35-KO cell lines were transduced with a lentiviruses expressing human IGF1R-myc, serum starved, and then treated with 10 nM IGF-1 for 1 h. The IGF1R and endogenous LAMP1 were stained by immunofluorescence, and the colocalization was quantified across 16 images acquired in two independent experiments. (C) HeLa cells and SNX1/2 and SNX5/6 double-KO cell lines were serum starved and treated with 5 µg/ml insulin for the indicated periods, and the level of the endogenous INSR was quantified by Western blotting over three independent experiments. (D) SNX5/6 double-KO cells were transduced with lentiviruses expressing GFP-SNX5 or GFP-SNX6 and with GFP only as a control. The cells were serum starved and treated with 10 nM IGF-1 for the indicated periods, followed by Western blot–based detection of the levels of the endogenous IGF1R. The level of the endogenous IGF1R in starved cells (top bar graph) as well as the degradation kinetics in IGF-1–treated cells were quantified over four independent experiments. (E) HeLa cells and clonal SNX5/6 double-KO cells were serum starved and treated with 10 nM IGF-1 for the indicated periods. One set of the SNX5/6 KO cells was treated with the lysosomal inhibitor bafilomycin-A1. The degradation kinetics were quantified over four independent experiments. Bars, 10 µm. Error bars indicate SD. *, P

    Article Snippet: The SNX-BAR cDNA as well as the IGF1R and IGF2R cDNA was cloned from HeLa cell cDNA prepared with the Superscript-III kit (Invitrogen) using KAPA HiFi DNA polymerase (Kapa Biosystems).

    Techniques: Gene Knockout, Western Blot, Transduction, Expressing, Staining, Immunofluorescence

    The CI-MPR accumulates in VPS35-positive sorting endosomes but does not localize to the VPS35-decorated subdomain. (A) Immunofluorescent staining of endogenous CI-MPR (red) and endogenous EEA1 (green) in HeLa cells and clonal VPS35, SNX1/2, and SNX5/6 double-KO cell lines. The colocalization was quantified over three independent experiments. (B) Immunofluorescent staining of endogenous CI-MPR (red) and endogenous VPS35 (green) in HeLa cells and clonal SNX1/2 and SNX5/6 double-KO cell lines. The colocalization was quantified over three independent experiments. (C) Costaining of indicated endogenous proteins in WT HeLa cells and analysis of colocalization by conventional confocal microscopy and STED superresolution microscopy. The colocalization between CI-MPR and VPS35 and CI-MPR and SNX1 was quantified across 12 images acquired in two independent experiments with conventional confocal microscopy. Bars: (main images) 10 µm; (insets) 3 µm. Error bars indicate SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport

    doi: 10.1083/jcb.201702137

    Figure Lengend Snippet: The CI-MPR accumulates in VPS35-positive sorting endosomes but does not localize to the VPS35-decorated subdomain. (A) Immunofluorescent staining of endogenous CI-MPR (red) and endogenous EEA1 (green) in HeLa cells and clonal VPS35, SNX1/2, and SNX5/6 double-KO cell lines. The colocalization was quantified over three independent experiments. (B) Immunofluorescent staining of endogenous CI-MPR (red) and endogenous VPS35 (green) in HeLa cells and clonal SNX1/2 and SNX5/6 double-KO cell lines. The colocalization was quantified over three independent experiments. (C) Costaining of indicated endogenous proteins in WT HeLa cells and analysis of colocalization by conventional confocal microscopy and STED superresolution microscopy. The colocalization between CI-MPR and VPS35 and CI-MPR and SNX1 was quantified across 12 images acquired in two independent experiments with conventional confocal microscopy. Bars: (main images) 10 µm; (insets) 3 µm. Error bars indicate SD. *, P

    Article Snippet: The SNX-BAR cDNA as well as the IGF1R and IGF2R cDNA was cloned from HeLa cell cDNA prepared with the Superscript-III kit (Invitrogen) using KAPA HiFi DNA polymerase (Kapa Biosystems).

    Techniques: Staining, Gene Knockout, Confocal Microscopy, Microscopy

    KO of the SNX-BAR proteins causes a complete loss of retrograde sorting of the CI-MPR in HeLa cells. (A) Immunofluorescent analysis of the endogenous CI-MPR (red) and endogenous TGN46 (green) in clonal VPS35 and SNX1/2 and SNX5/6 double-KO cells. The colocalization was quantified over three independent experiments. (B) Uptake assay with an antibody against the extracellular domain of the endogenous CI-MPR over 30 and 60 min of uptake at 37°C. Delivery of the antibody/receptor duplex to the TGN was analyzed through costaining of the internalized antibody (red) with endogenous TGN46 (green). The colocalization was quantified over three independent experiments. (C) GFP-tagged SNX5 and GFP-tagged SNX6 were lentivirally expressed in SNX5/6 double-KO cells lines, and localization of endogenous CI-MPR (red) and endogenous TGN46 (blue) was quantified over three independent experiments. Bars, 10 µm. Error bars indicate SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport

    doi: 10.1083/jcb.201702137

    Figure Lengend Snippet: KO of the SNX-BAR proteins causes a complete loss of retrograde sorting of the CI-MPR in HeLa cells. (A) Immunofluorescent analysis of the endogenous CI-MPR (red) and endogenous TGN46 (green) in clonal VPS35 and SNX1/2 and SNX5/6 double-KO cells. The colocalization was quantified over three independent experiments. (B) Uptake assay with an antibody against the extracellular domain of the endogenous CI-MPR over 30 and 60 min of uptake at 37°C. Delivery of the antibody/receptor duplex to the TGN was analyzed through costaining of the internalized antibody (red) with endogenous TGN46 (green). The colocalization was quantified over three independent experiments. (C) GFP-tagged SNX5 and GFP-tagged SNX6 were lentivirally expressed in SNX5/6 double-KO cells lines, and localization of endogenous CI-MPR (red) and endogenous TGN46 (blue) was quantified over three independent experiments. Bars, 10 µm. Error bars indicate SD. *, P

    Article Snippet: The SNX-BAR cDNA as well as the IGF1R and IGF2R cDNA was cloned from HeLa cell cDNA prepared with the Superscript-III kit (Invitrogen) using KAPA HiFi DNA polymerase (Kapa Biosystems).

    Techniques: Gene Knockout

    TALEN-mediated deletion of VPS35 in U2OS and knockdown of VPS35 in HeLa cells does not cause retrograde sorting defects of CI-MPR. (A) Western blot analysis of clonal U2OS osteosarcoma cells with a TALEN-mediated disruption of exon 4 of the VPS35 gene. The graph displays the level of endogenous CI-MPR/GAPDH quantified over three independent experiments. (B) Immunofluorescent staining of endogenous CI-MPR and endogenous TGN46 in human U2OS, with colocalization quantified over three independent experiments. (C) CI-MPR degradation experiment in U2OS cells treated with the ribosome inhibitor cycloheximide for the indicated periods. The abundance of CI-MPR was adjusted by the GAPDH signal and quantified over three independent experiments. (D) Immunofluorescent staining of endogenous GLUT1 (red) and endogenous LAMP1 (green) in HeLa cells treated with scrambled and VPS35-specific siRNA. The colocalization between LAMP1 and GLUT1 was quantified across 12 images acquired in two independent experiments. The knockdown (KD) efficiency was verified by Western blotting. (E) Immunofluorescent staining of endogenous CI-MPR (red) with the lysosomal marker LAMP1 (blue) and the TGN marker TGN46 (green). The colocalization of CI-MPR with the two markers was quantified over three independent experiments. Bars, 10 µm. Error bars indicate SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport

    doi: 10.1083/jcb.201702137

    Figure Lengend Snippet: TALEN-mediated deletion of VPS35 in U2OS and knockdown of VPS35 in HeLa cells does not cause retrograde sorting defects of CI-MPR. (A) Western blot analysis of clonal U2OS osteosarcoma cells with a TALEN-mediated disruption of exon 4 of the VPS35 gene. The graph displays the level of endogenous CI-MPR/GAPDH quantified over three independent experiments. (B) Immunofluorescent staining of endogenous CI-MPR and endogenous TGN46 in human U2OS, with colocalization quantified over three independent experiments. (C) CI-MPR degradation experiment in U2OS cells treated with the ribosome inhibitor cycloheximide for the indicated periods. The abundance of CI-MPR was adjusted by the GAPDH signal and quantified over three independent experiments. (D) Immunofluorescent staining of endogenous GLUT1 (red) and endogenous LAMP1 (green) in HeLa cells treated with scrambled and VPS35-specific siRNA. The colocalization between LAMP1 and GLUT1 was quantified across 12 images acquired in two independent experiments. The knockdown (KD) efficiency was verified by Western blotting. (E) Immunofluorescent staining of endogenous CI-MPR (red) with the lysosomal marker LAMP1 (blue) and the TGN marker TGN46 (green). The colocalization of CI-MPR with the two markers was quantified over three independent experiments. Bars, 10 µm. Error bars indicate SD. *, P

    Article Snippet: The SNX-BAR cDNA as well as the IGF1R and IGF2R cDNA was cloned from HeLa cell cDNA prepared with the Superscript-III kit (Invitrogen) using KAPA HiFi DNA polymerase (Kapa Biosystems).

    Techniques: Western Blot, Staining, Marker

    Genomic deletion of VPS35 does not cause retrograde sorting defects of CI-MPR. (A) Western blot of lysates from clonal HeLa cell lines with a disruption of the VPS35 gene in exon 5 or exon 8 (left). Immunofluorescent staining of endogenous GLUT1 (red) and endogenous LAMP1 (green) in HeLa cells and in two distinct VPS35-KO cell lines (right). (B) Staining of endogenous CI-MPR (red) and TGN46 (green) in parental HeLa cells and two VPS35-KO cell lines. The colocalization (coloc) between the TGN marker TGN46 and CI-MPR was quantified over three independent experiments. (C) Uptake assay with an antibody against the extracellular domain of CI-MPR in HeLa and VPS35-KO cell lines. Delivery of the CI-MPR–antibody complex (red) to the TGN46 (green)-labeled TGN was quantified after 30 and 60 min of uptake at 37°C over three independent experiments. (D) CI-MPR degradation assays in parental HeLa and VPS35-KO cells treated with the ribosomal inhibitor cycloheximide for indicated time points (left). Graph shows the degradation kinetics averaged over four independent experiments. Immunofluorescence of endogenous CI-MPR (red) and endogenous LAMP1 (green) in HeLa and VPS35-KO cell lines quantified over three independent experiments (right). Bars, 10 µm. Error bars indicate SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport

    doi: 10.1083/jcb.201702137

    Figure Lengend Snippet: Genomic deletion of VPS35 does not cause retrograde sorting defects of CI-MPR. (A) Western blot of lysates from clonal HeLa cell lines with a disruption of the VPS35 gene in exon 5 or exon 8 (left). Immunofluorescent staining of endogenous GLUT1 (red) and endogenous LAMP1 (green) in HeLa cells and in two distinct VPS35-KO cell lines (right). (B) Staining of endogenous CI-MPR (red) and TGN46 (green) in parental HeLa cells and two VPS35-KO cell lines. The colocalization (coloc) between the TGN marker TGN46 and CI-MPR was quantified over three independent experiments. (C) Uptake assay with an antibody against the extracellular domain of CI-MPR in HeLa and VPS35-KO cell lines. Delivery of the CI-MPR–antibody complex (red) to the TGN46 (green)-labeled TGN was quantified after 30 and 60 min of uptake at 37°C over three independent experiments. (D) CI-MPR degradation assays in parental HeLa and VPS35-KO cells treated with the ribosomal inhibitor cycloheximide for indicated time points (left). Graph shows the degradation kinetics averaged over four independent experiments. Immunofluorescence of endogenous CI-MPR (red) and endogenous LAMP1 (green) in HeLa and VPS35-KO cell lines quantified over three independent experiments (right). Bars, 10 µm. Error bars indicate SD. *, P

    Article Snippet: The SNX-BAR cDNA as well as the IGF1R and IGF2R cDNA was cloned from HeLa cell cDNA prepared with the Superscript-III kit (Invitrogen) using KAPA HiFi DNA polymerase (Kapa Biosystems).

    Techniques: Western Blot, Staining, Gene Knockout, Marker, Labeling, Immunofluorescence

    Genomic deletion of the SNX-BAR binding site abrogates retrograde transport of endogenous as well as reexpressed CI-MPR. (A) CRISPR-Cas9 was used to introduce a 6-aa deletion of the WLM SNX-BAR binding motif into the endogenous CI-MPR locus of a HeLa cell line. The immunofluorescence shows the colocalization between endogenous CI-MPR and the TGN marker TGN46. (B) 3D reconstruction of the CI-MPR and TGN46 staining in a HeLa cell and in a cell carrying the SNX-BAR–binding site deletion. The colocalization between CI-MPR and TGN46 was quantified over three independent experiments. (C) Reexpression of WT and WLM-AAA CI-MPR in a CI-MPR–KO HeLa cell line. The transfected cells were stained for CI-MPR and endogenous TGN46, and colocalization was quantified over three independent experiments. Bars, 10 µm. Error bars indicate SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport

    doi: 10.1083/jcb.201702137

    Figure Lengend Snippet: Genomic deletion of the SNX-BAR binding site abrogates retrograde transport of endogenous as well as reexpressed CI-MPR. (A) CRISPR-Cas9 was used to introduce a 6-aa deletion of the WLM SNX-BAR binding motif into the endogenous CI-MPR locus of a HeLa cell line. The immunofluorescence shows the colocalization between endogenous CI-MPR and the TGN marker TGN46. (B) 3D reconstruction of the CI-MPR and TGN46 staining in a HeLa cell and in a cell carrying the SNX-BAR–binding site deletion. The colocalization between CI-MPR and TGN46 was quantified over three independent experiments. (C) Reexpression of WT and WLM-AAA CI-MPR in a CI-MPR–KO HeLa cell line. The transfected cells were stained for CI-MPR and endogenous TGN46, and colocalization was quantified over three independent experiments. Bars, 10 µm. Error bars indicate SD. *, P

    Article Snippet: The SNX-BAR cDNA as well as the IGF1R and IGF2R cDNA was cloned from HeLa cell cDNA prepared with the Superscript-III kit (Invitrogen) using KAPA HiFi DNA polymerase (Kapa Biosystems).

    Techniques: Binding Assay, CRISPR, Introduce, Immunofluorescence, Marker, Staining, Gene Knockout, Transfection

    The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: Inhibition, Real-time Polymerase Chain Reaction

    Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: Activity Assay, Mutagenesis, Immunoprecipitation, shRNA, Selection, Southern Blot, Real-time Polymerase Chain Reaction

    Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: Expressing, Plasmid Preparation, Transfection, Cotransfection, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: CRISPR, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Modification

    PKP2 loss triggers an increase in TGF-β1 expression and transcriptional signaling. (A) RNA isolated from control (CT) and PKP2 KD CMs were analyzed for mRNA levels of the TGF-β genes. Tgfb1 (but not Tgfb2 or Tgfb3 ) mRNA levels were significantly increased on KD of PKP2. (B). Levels of TGF-β1 were analyzed in the cell culture supernatants of control and PKP2 KD cells 72 h after KD by performing a TGF-β1 ELISA. These data indicate a significant increase in secreted TGF-β1 on loss of PKP2. (C) RNA isolated from wild-type and PKP2 +/− mouse heart tissue were analyzed for mRNA levels of Tgfb1 . Haploinsufficiency of PKP2 results in a significant increase in Tgfb1 mRNA in vivo. (D–F) SMAD3, STAT3, and NF-κB transcriptional activity was followed via luciferase reporter arrays (see Materials and methods). After control and PKP2 KD in 96-well plates, CMs were infected with luciferase reporter constructs for SMAD3, STAT3, or NF-κB and luciferase expression followed in the same cells by noninvasive imaging for a period of 6 d. KD of PKP2 caused a significant increase in transcriptional activity of SMAD3, STAT3, and NF-κB. For all graphs, fold change values from three independent samples are represented with error bars indicating SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: Plakophilin-2 loss promotes TGF-β1/p38 MAPK-dependent fibrotic gene expression in cardiomyocytes

    doi: 10.1083/jcb.201507018

    Figure Lengend Snippet: PKP2 loss triggers an increase in TGF-β1 expression and transcriptional signaling. (A) RNA isolated from control (CT) and PKP2 KD CMs were analyzed for mRNA levels of the TGF-β genes. Tgfb1 (but not Tgfb2 or Tgfb3 ) mRNA levels were significantly increased on KD of PKP2. (B). Levels of TGF-β1 were analyzed in the cell culture supernatants of control and PKP2 KD cells 72 h after KD by performing a TGF-β1 ELISA. These data indicate a significant increase in secreted TGF-β1 on loss of PKP2. (C) RNA isolated from wild-type and PKP2 +/− mouse heart tissue were analyzed for mRNA levels of Tgfb1 . Haploinsufficiency of PKP2 results in a significant increase in Tgfb1 mRNA in vivo. (D–F) SMAD3, STAT3, and NF-κB transcriptional activity was followed via luciferase reporter arrays (see Materials and methods). After control and PKP2 KD in 96-well plates, CMs were infected with luciferase reporter constructs for SMAD3, STAT3, or NF-κB and luciferase expression followed in the same cells by noninvasive imaging for a period of 6 d. KD of PKP2 caused a significant increase in transcriptional activity of SMAD3, STAT3, and NF-κB. For all graphs, fold change values from three independent samples are represented with error bars indicating SD. *, P

    Article Snippet: Total RNA concentrations were equalized between samples, and cDNA was prepared using the Superscript III First Strand kit (Invitrogen). qPCR was performed using SYBR Green PCR master mix (Applied Biosystems) and gene-specific primers in a StepOnePlus instrument (Applied Biosystems).

    Techniques: Expressing, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, In Vivo, Activity Assay, Luciferase, Infection, Construct, Imaging

    Activation of TGF-β1/p38 MAPK signaling in cardiac cells is regulated by DP expression. (A–C). Control (CT), PKP2 KD, and PKP2 KD CMs treated with either the TAK1 inhibitor (5Z)-7-oxozeaenol or p38 MAPK inhibitor SB203580 were analyzed for total protein levels of DP (A), solubility of DP using Triton X-100 fractionation assays (B), and junctional localization of DP (C). Neither TAK1 nor p38 MAPK inhibition was able to rescue the loss of DP protein expression, increase in DP solubility, or loss of junctional localization of DP, all of which are seen on loss of PKP2 KD in CMs. Bar, 20 µm. (D) Control and DP KD cells were analyzed for levels of phospho–p38 MAPK and Tgfb1 mRNA, both of which were up-regulated on loss of DP expression. (E) Tissue samples from wild-type (WT) and DP epidermal knockout (DPeKO) mice were processed for protein and RNA analysis. Loss of DP expression resulted in activation of phospho–p38 MAPK and an increase in Tgfb1 mRNA in the epidermis. For all graphs, fold change values from three or more independent samples are represented with error bars indicating SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: Plakophilin-2 loss promotes TGF-β1/p38 MAPK-dependent fibrotic gene expression in cardiomyocytes

    doi: 10.1083/jcb.201507018

    Figure Lengend Snippet: Activation of TGF-β1/p38 MAPK signaling in cardiac cells is regulated by DP expression. (A–C). Control (CT), PKP2 KD, and PKP2 KD CMs treated with either the TAK1 inhibitor (5Z)-7-oxozeaenol or p38 MAPK inhibitor SB203580 were analyzed for total protein levels of DP (A), solubility of DP using Triton X-100 fractionation assays (B), and junctional localization of DP (C). Neither TAK1 nor p38 MAPK inhibition was able to rescue the loss of DP protein expression, increase in DP solubility, or loss of junctional localization of DP, all of which are seen on loss of PKP2 KD in CMs. Bar, 20 µm. (D) Control and DP KD cells were analyzed for levels of phospho–p38 MAPK and Tgfb1 mRNA, both of which were up-regulated on loss of DP expression. (E) Tissue samples from wild-type (WT) and DP epidermal knockout (DPeKO) mice were processed for protein and RNA analysis. Loss of DP expression resulted in activation of phospho–p38 MAPK and an increase in Tgfb1 mRNA in the epidermis. For all graphs, fold change values from three or more independent samples are represented with error bars indicating SD. *, P

    Article Snippet: Total RNA concentrations were equalized between samples, and cDNA was prepared using the Superscript III First Strand kit (Invitrogen). qPCR was performed using SYBR Green PCR master mix (Applied Biosystems) and gene-specific primers in a StepOnePlus instrument (Applied Biosystems).

    Techniques: Activation Assay, Expressing, Solubility, Fractionation, Inhibition, Knock-Out, Mouse Assay

    Loss of PKP2 induces inflammatory and fibrotic gene expression. (A and B) Freshly isolated neonatal CMs were infected with adenovirus containing either control (CT) or PKP2 KD constructs, and samples were analyzed 72 h postinfection. RNA isolated from control and PKP2 KD CMs were analyzed for mRNA levels of different genes by qPCR. KD of PKP2 in neonatal CMs resulted in a significant increase in mRNA levels of proinflammatory markers interleukin-1α ( Il1a ) and Ccl12 (A) and ECM genes fibronectin ( Fn1 ) and collagen ( Col2A1 and Col3a1 ) (B). Expression of Elastin ( Eln ) was reduced on loss of PKP2. (C) KD of PKP2 results in an increase in FN protein levels, as indicated by quantification of FN band intensity (normalized to GAPDH). (D) Control and PKP2 KD CMs were stained with an anti-FN antibody. KD of PKP2 results in an increase in FN expression as indicated by the increase in staining intensity of FN by immunofluorescence. Bar, 20 µm. (E) Analysis of RNA from wild-type (WT) and PKP2 +/− mouse hearts demonstrated a significant increase in expression of fibrotic genes such as Fn1 and Col3a1 on loss of PKP2 expression in vivo. All images and blots shown are representative of three independent experiments. For all graphs, fold change values from three or more independent samples are represented with error bars indicating SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: Plakophilin-2 loss promotes TGF-β1/p38 MAPK-dependent fibrotic gene expression in cardiomyocytes

    doi: 10.1083/jcb.201507018

    Figure Lengend Snippet: Loss of PKP2 induces inflammatory and fibrotic gene expression. (A and B) Freshly isolated neonatal CMs were infected with adenovirus containing either control (CT) or PKP2 KD constructs, and samples were analyzed 72 h postinfection. RNA isolated from control and PKP2 KD CMs were analyzed for mRNA levels of different genes by qPCR. KD of PKP2 in neonatal CMs resulted in a significant increase in mRNA levels of proinflammatory markers interleukin-1α ( Il1a ) and Ccl12 (A) and ECM genes fibronectin ( Fn1 ) and collagen ( Col2A1 and Col3a1 ) (B). Expression of Elastin ( Eln ) was reduced on loss of PKP2. (C) KD of PKP2 results in an increase in FN protein levels, as indicated by quantification of FN band intensity (normalized to GAPDH). (D) Control and PKP2 KD CMs were stained with an anti-FN antibody. KD of PKP2 results in an increase in FN expression as indicated by the increase in staining intensity of FN by immunofluorescence. Bar, 20 µm. (E) Analysis of RNA from wild-type (WT) and PKP2 +/− mouse hearts demonstrated a significant increase in expression of fibrotic genes such as Fn1 and Col3a1 on loss of PKP2 expression in vivo. All images and blots shown are representative of three independent experiments. For all graphs, fold change values from three or more independent samples are represented with error bars indicating SD. *, P

    Article Snippet: Total RNA concentrations were equalized between samples, and cDNA was prepared using the Superscript III First Strand kit (Invitrogen). qPCR was performed using SYBR Green PCR master mix (Applied Biosystems) and gene-specific primers in a StepOnePlus instrument (Applied Biosystems).

    Techniques: Expressing, Isolation, Infection, Construct, Real-time Polymerase Chain Reaction, Staining, Immunofluorescence, In Vivo

    PKP2 KD in neonatal CMs disrupts area composita formation via loss of DP localization and stability. Freshly isolated neonatal CMs were infected with adenovirus containing either control or PKP2 KD constructs, and samples were analyzed 72 h postinfection. (A) Control (CT) and PKP2 KD CMs grown on coverslips were stained for cell–cell junction components, including PKP2, PG, DP, β-catenin, p120-catenin, and Cnx43. PKP2 KD specifically results in a major loss of DP and Cnx43 from junctions, but not other junctional markers. Bar, 20 µm. (B) Protein levels of cell–cell junction proteins are not perturbed on loss of PKP2, except DP, whose expression is reduced by 60–70%. (C) Re-expression of human V5-tagged PKP2 in rat PKP2 KD CMs rescues DP protein levels. A pool of two antibodies was used to analyze PKP2 levels in this experiment (described in Materials and methods). (D) PKP2 KD results in an increase in DP solubility, as indicated by a decrease in DP levels in the Triton X-100 (Tx)–insoluble fraction and a concomitant increase in the Triton X-100–soluble fraction. Solubility of the intermediate filament protein Desmin is also increased, but solubility of other junctional markers such as β-catenin is not affected. Vinculin is included as a loading control. (E) RNA extracted from control and PKP2 KD cells was analyzed for Dsp mRNA levels by qPCR. Transcription of DP is not decreased on loss of PKP2 KD in CMs. (F) Control and PKP2 KD cells were treated with cycloheximide (CHX) for the indicated times, and samples are blotted for DP and tubulin. Degradation of DP is increased in PKP2 KD cells compared with control, as indicated by quantification of DP band intensity (normalized to tubulin) at the various time points. All images and blots shown are representative of three independent experiments. (G) Control, PKP2 KD, and PKP2 KD CMs treated with either a proteasomal inhibitor (MG132) or a lysosomal inhibitor (chloroquine) were blotted for DP and tubulin (loading control). DP expression in PKP2 KD cells was restored by proteasomal inhibition, but not lysosomal inhibition.

    Journal: The Journal of Cell Biology

    Article Title: Plakophilin-2 loss promotes TGF-β1/p38 MAPK-dependent fibrotic gene expression in cardiomyocytes

    doi: 10.1083/jcb.201507018

    Figure Lengend Snippet: PKP2 KD in neonatal CMs disrupts area composita formation via loss of DP localization and stability. Freshly isolated neonatal CMs were infected with adenovirus containing either control or PKP2 KD constructs, and samples were analyzed 72 h postinfection. (A) Control (CT) and PKP2 KD CMs grown on coverslips were stained for cell–cell junction components, including PKP2, PG, DP, β-catenin, p120-catenin, and Cnx43. PKP2 KD specifically results in a major loss of DP and Cnx43 from junctions, but not other junctional markers. Bar, 20 µm. (B) Protein levels of cell–cell junction proteins are not perturbed on loss of PKP2, except DP, whose expression is reduced by 60–70%. (C) Re-expression of human V5-tagged PKP2 in rat PKP2 KD CMs rescues DP protein levels. A pool of two antibodies was used to analyze PKP2 levels in this experiment (described in Materials and methods). (D) PKP2 KD results in an increase in DP solubility, as indicated by a decrease in DP levels in the Triton X-100 (Tx)–insoluble fraction and a concomitant increase in the Triton X-100–soluble fraction. Solubility of the intermediate filament protein Desmin is also increased, but solubility of other junctional markers such as β-catenin is not affected. Vinculin is included as a loading control. (E) RNA extracted from control and PKP2 KD cells was analyzed for Dsp mRNA levels by qPCR. Transcription of DP is not decreased on loss of PKP2 KD in CMs. (F) Control and PKP2 KD cells were treated with cycloheximide (CHX) for the indicated times, and samples are blotted for DP and tubulin. Degradation of DP is increased in PKP2 KD cells compared with control, as indicated by quantification of DP band intensity (normalized to tubulin) at the various time points. All images and blots shown are representative of three independent experiments. (G) Control, PKP2 KD, and PKP2 KD CMs treated with either a proteasomal inhibitor (MG132) or a lysosomal inhibitor (chloroquine) were blotted for DP and tubulin (loading control). DP expression in PKP2 KD cells was restored by proteasomal inhibition, but not lysosomal inhibition.

    Article Snippet: Total RNA concentrations were equalized between samples, and cDNA was prepared using the Superscript III First Strand kit (Invitrogen). qPCR was performed using SYBR Green PCR master mix (Applied Biosystems) and gene-specific primers in a StepOnePlus instrument (Applied Biosystems).

    Techniques: Isolation, Infection, Construct, Staining, Expressing, Solubility, Real-time Polymerase Chain Reaction, Inhibition

    TPC2 signaling in mouse ES cells. ( A ) Expressions of TPC2 mRNAs in D3 ES cells were determined by RT-PCR. ( B ) NAADP-AM induced a Ca 2+ increase in TPC2 overexpressing ES cells in a bell-shaped concentration response curve. ( C ) Inhibition of Ca 2+ response triggered by NAADP-AM (50 nM) by bafilomycin A1 (100 nM) and esterase (50 units/ml). Data quantifications of [Ca 2+ ] i peak induced by drug treatment in (B) and (C) are expressed as mean ± S.E., n = 30–40 cells. The * symbols indicate the results of t Test analysis, p

    Journal: PLoS ONE

    Article Title: Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

    doi: 10.1371/journal.pone.0066077

    Figure Lengend Snippet: TPC2 signaling in mouse ES cells. ( A ) Expressions of TPC2 mRNAs in D3 ES cells were determined by RT-PCR. ( B ) NAADP-AM induced a Ca 2+ increase in TPC2 overexpressing ES cells in a bell-shaped concentration response curve. ( C ) Inhibition of Ca 2+ response triggered by NAADP-AM (50 nM) by bafilomycin A1 (100 nM) and esterase (50 units/ml). Data quantifications of [Ca 2+ ] i peak induced by drug treatment in (B) and (C) are expressed as mean ± S.E., n = 30–40 cells. The * symbols indicate the results of t Test analysis, p

    Article Snippet: RT-PCR of TPC2 using SuperScript® One-Step RT-PCR kit (Life Technologies) was performed with Takara PCR Thermal Cycler Dice (Takara).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Inhibition

    TPC2 knockdown inhibited neuronal differentiation from mouse ES cells derived neural progenitors. (A) and (B) RNA was harvested at indicated time points during neural differentiation in control and TPC2 knockdown ES cells and analyzed for expression of Mash1 (A) and Nurr1 (B) by qRT-PCR. (C) Immunofluorescent analysis of Tuj1 expression (TujI, Green; DAPI, blue) in control and TPC2 knockdown ES cells harvested at indicated time points during differentiation (Scale bar = 200 µm). Data quantification was presented as Tuj1-positive cells/DAPI-stained cells ± S.E., n = 5 (40–50 cells per experiment). The * symbols indicate the results of t Test analysis, p

    Journal: PLoS ONE

    Article Title: Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

    doi: 10.1371/journal.pone.0066077

    Figure Lengend Snippet: TPC2 knockdown inhibited neuronal differentiation from mouse ES cells derived neural progenitors. (A) and (B) RNA was harvested at indicated time points during neural differentiation in control and TPC2 knockdown ES cells and analyzed for expression of Mash1 (A) and Nurr1 (B) by qRT-PCR. (C) Immunofluorescent analysis of Tuj1 expression (TujI, Green; DAPI, blue) in control and TPC2 knockdown ES cells harvested at indicated time points during differentiation (Scale bar = 200 µm). Data quantification was presented as Tuj1-positive cells/DAPI-stained cells ± S.E., n = 5 (40–50 cells per experiment). The * symbols indicate the results of t Test analysis, p

    Article Snippet: RT-PCR of TPC2 using SuperScript® One-Step RT-PCR kit (Life Technologies) was performed with Takara PCR Thermal Cycler Dice (Takara).

    Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Staining

    TPC2 knockdown did not affect glia differentiation of mouse ES cells. (A) and (B) RNA was harvested at indicated time points during neural differentiation and analyzed for expression of GFAP (A) and Olig2 (B) in both control and TPC2 knockdown cells by qRT-PCR. (C) Cell lysates were harvested at indicated time points during neural differentiation in both control and TPC2 knockdown cells, and analyzed for expression of GFAP and olig1 by western blot analyses.

    Journal: PLoS ONE

    Article Title: Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

    doi: 10.1371/journal.pone.0066077

    Figure Lengend Snippet: TPC2 knockdown did not affect glia differentiation of mouse ES cells. (A) and (B) RNA was harvested at indicated time points during neural differentiation and analyzed for expression of GFAP (A) and Olig2 (B) in both control and TPC2 knockdown cells by qRT-PCR. (C) Cell lysates were harvested at indicated time points during neural differentiation in both control and TPC2 knockdown cells, and analyzed for expression of GFAP and olig1 by western blot analyses.

    Article Snippet: RT-PCR of TPC2 using SuperScript® One-Step RT-PCR kit (Life Technologies) was performed with Takara PCR Thermal Cycler Dice (Takara).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    TPC2 knockdown facilitated neural lineage entry of mouse ES cells while overexpression of TPC2 inhibited it. (A) and (B) RNA was harvested at the indicated time points during neural differentiation of scramble shRNA infected, TPC2 knockdown, and TPC2 overexpressing D3 ES cells and analyzed for expression of Sox1 (A) and Nestin (B) by quantitative real-time RT-PCR. (C) Immunostaining analyses (Nestin, Green; DAPI, blue) of scramble shRNA infected, TPC2 knockdown, and TPC2 overexpressing D3 cells harvested at the indicated time point during differentiation. Scale bar = 200 µm. (D) Quantification of Nestin positive cells in (C). Data are presented as % of Nestin positive cells ± S.E., n = 5 (40–50 cells per experiment). (E) Quantification of FACS analyses of control, TPC2 knockdown, and TPC2 overexpressing 46C Sox1-GFP ES cells harvested at the indicated time point during neural differentiation. Data are expressed as mean ± S.E. from three independent experiments. The * symbols indicate the results of t Test analysis, p

    Journal: PLoS ONE

    Article Title: Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

    doi: 10.1371/journal.pone.0066077

    Figure Lengend Snippet: TPC2 knockdown facilitated neural lineage entry of mouse ES cells while overexpression of TPC2 inhibited it. (A) and (B) RNA was harvested at the indicated time points during neural differentiation of scramble shRNA infected, TPC2 knockdown, and TPC2 overexpressing D3 ES cells and analyzed for expression of Sox1 (A) and Nestin (B) by quantitative real-time RT-PCR. (C) Immunostaining analyses (Nestin, Green; DAPI, blue) of scramble shRNA infected, TPC2 knockdown, and TPC2 overexpressing D3 cells harvested at the indicated time point during differentiation. Scale bar = 200 µm. (D) Quantification of Nestin positive cells in (C). Data are presented as % of Nestin positive cells ± S.E., n = 5 (40–50 cells per experiment). (E) Quantification of FACS analyses of control, TPC2 knockdown, and TPC2 overexpressing 46C Sox1-GFP ES cells harvested at the indicated time point during neural differentiation. Data are expressed as mean ± S.E. from three independent experiments. The * symbols indicate the results of t Test analysis, p

    Article Snippet: RT-PCR of TPC2 using SuperScript® One-Step RT-PCR kit (Life Technologies) was performed with Takara PCR Thermal Cycler Dice (Takara).

    Techniques: Over Expression, shRNA, Infection, Expressing, Quantitative RT-PCR, Immunostaining, FACS

    Expression pattern of TPC2 during in vitro neural differentiation of mouse ES cells. (A) Schematic of in vitro neural differentiation of mouse ES cells initiated by monolayer adherent culture. (B–E) Induction of neural differentiation of mouse ES cells by the monolayer adherent culture is indicated by Sox1GFP (B), Nestin (C), Tuj1 (D), and GFAP (E) positive cells. Scale bar = 200 µm. (F–H) Expressions of Sox1 (F), Nurr1 (G), and S100beta (H) in the neural differentiation of mouse ES cells were determined by quantitative real-time RT-PCR. (I) and (J) Expression of TPC2 in the neural differentiation of D3 ES cells was determined by quantitative real-time RT-PCR (I) and western blot analyses (J). The * symbols indicate the results of t Test analysis, p

    Journal: PLoS ONE

    Article Title: Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

    doi: 10.1371/journal.pone.0066077

    Figure Lengend Snippet: Expression pattern of TPC2 during in vitro neural differentiation of mouse ES cells. (A) Schematic of in vitro neural differentiation of mouse ES cells initiated by monolayer adherent culture. (B–E) Induction of neural differentiation of mouse ES cells by the monolayer adherent culture is indicated by Sox1GFP (B), Nestin (C), Tuj1 (D), and GFAP (E) positive cells. Scale bar = 200 µm. (F–H) Expressions of Sox1 (F), Nurr1 (G), and S100beta (H) in the neural differentiation of mouse ES cells were determined by quantitative real-time RT-PCR. (I) and (J) Expression of TPC2 in the neural differentiation of D3 ES cells was determined by quantitative real-time RT-PCR (I) and western blot analyses (J). The * symbols indicate the results of t Test analysis, p

    Article Snippet: RT-PCR of TPC2 using SuperScript® One-Step RT-PCR kit (Life Technologies) was performed with Takara PCR Thermal Cycler Dice (Takara).

    Techniques: Expressing, In Vitro, Quantitative RT-PCR, Western Blot

    TPC2 overexpression or knockdown did not affect the self-renewal and pluripotency of mouse ES cells. (A) TPC2 knockdown by two distinctive shRNAs in D3 ES cells was verified by qRT-PCR analysis. (B) Bright field, lysotracker red staining, and GFP cell imaging of the D3 TPC2GFP cell line (Scale bar = 50 µm). (C) NAADP-AM (20 nM) induced a Ca 2+ increase in mouse ES cells that was blocked by TPC2 knockdown but enhanced by TPC2 overexpression. Data quantifications of [Ca 2+ ] i peak induced by drug treatment are expressed as mean ± S.E., n = 30–40 cells. (D) WST assay of control, TPC2 knockdown, and TPC2 overexpressing D3 ES cells. (E) FACS analyses of DNA contents (PI staining) in control, TPC2 knockdown, and TPC2 overexpressing D3 ES cells. (F) Alkaline phosphatase (AP) staining of control, TPC2 knockdown, and TPC2 overexpressing D3 ES cells. Quantification of AP staining were presented as AP positive colonies/total colonies ± S.E., n = 5 (40–50 cells per experiment). (G) Immunostaining analyses of pluripotent markers, Oct4 and SSEA-1, in control, TPC2 knockdown, and TPC2 overexpressing D3 ES cells (Oct4 and SSEA-1, Green; Dapi, Blue). Scale bar = 200 µm.

    Journal: PLoS ONE

    Article Title: Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

    doi: 10.1371/journal.pone.0066077

    Figure Lengend Snippet: TPC2 overexpression or knockdown did not affect the self-renewal and pluripotency of mouse ES cells. (A) TPC2 knockdown by two distinctive shRNAs in D3 ES cells was verified by qRT-PCR analysis. (B) Bright field, lysotracker red staining, and GFP cell imaging of the D3 TPC2GFP cell line (Scale bar = 50 µm). (C) NAADP-AM (20 nM) induced a Ca 2+ increase in mouse ES cells that was blocked by TPC2 knockdown but enhanced by TPC2 overexpression. Data quantifications of [Ca 2+ ] i peak induced by drug treatment are expressed as mean ± S.E., n = 30–40 cells. (D) WST assay of control, TPC2 knockdown, and TPC2 overexpressing D3 ES cells. (E) FACS analyses of DNA contents (PI staining) in control, TPC2 knockdown, and TPC2 overexpressing D3 ES cells. (F) Alkaline phosphatase (AP) staining of control, TPC2 knockdown, and TPC2 overexpressing D3 ES cells. Quantification of AP staining were presented as AP positive colonies/total colonies ± S.E., n = 5 (40–50 cells per experiment). (G) Immunostaining analyses of pluripotent markers, Oct4 and SSEA-1, in control, TPC2 knockdown, and TPC2 overexpressing D3 ES cells (Oct4 and SSEA-1, Green; Dapi, Blue). Scale bar = 200 µm.

    Article Snippet: RT-PCR of TPC2 using SuperScript® One-Step RT-PCR kit (Life Technologies) was performed with Takara PCR Thermal Cycler Dice (Takara).

    Techniques: Over Expression, Quantitative RT-PCR, Staining, Imaging, WST Assay, FACS, Immunostaining

    Curcumin induces Bex genes in N2a neuroblastoma cells in a dose-dependent manner. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated either with 10, 25 or 50 μM of curcumin or with equal amount of DMSO (controls) in serum free media for 2 hours. Total RNA was isolated by Trizol reagent and treated with DNase I to remove any DNA contamination. Reverse transcription on 5 μg of DNA-free RNA was performed and Bex cDNAs were amplified either for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis shows a dose-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S9 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex4 ( d ) and Bex6 ( e ) mRNA amplicons was performed using Image lab software and values obtained were normalized with respective GAPDH band intensity, and were plotted as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Curcumin induces Bex genes in N2a neuroblastoma cells in a dose-dependent manner. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated either with 10, 25 or 50 μM of curcumin or with equal amount of DMSO (controls) in serum free media for 2 hours. Total RNA was isolated by Trizol reagent and treated with DNase I to remove any DNA contamination. Reverse transcription on 5 μg of DNA-free RNA was performed and Bex cDNAs were amplified either for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis shows a dose-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S9 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex4 ( d ) and Bex6 ( e ) mRNA amplicons was performed using Image lab software and values obtained were normalized with respective GAPDH band intensity, and were plotted as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Superscript III RT PCR kit (Invitrogen) was used to synthesize cDNA from 5 μg of DNA free RNA.

    Techniques: Cell Culture, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Software, Two Tailed Test

    Curcumin is a specific inducer of all the Bex genes. ( a ) Approximately 80% confluent N2a cells were serum starved for 2 hours and treated with either 2.5, 5, 10 μM of MPTQ or 25 μM of curcumin in serum free DMEM for 4 hours. Equivalent amount of DMSO treated N2a cells were considered as controls. Total RNA was isolated, DNase I treated and expression of Bex mRNAs was studied by RT-PCR analysis. GAPDH was used as loading control. The original gel images are shown in Supplementary Fig. S11 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products was performed and normalized with corresponding GAPDH band intensity. Values are displayed as histograms of mean ± standard deviation from three independent experiments, which indicates induction of all endogenous Bex genes are observed in curcumin but not in MPTQ treated N2a cells. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Curcumin is a specific inducer of all the Bex genes. ( a ) Approximately 80% confluent N2a cells were serum starved for 2 hours and treated with either 2.5, 5, 10 μM of MPTQ or 25 μM of curcumin in serum free DMEM for 4 hours. Equivalent amount of DMSO treated N2a cells were considered as controls. Total RNA was isolated, DNase I treated and expression of Bex mRNAs was studied by RT-PCR analysis. GAPDH was used as loading control. The original gel images are shown in Supplementary Fig. S11 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products was performed and normalized with corresponding GAPDH band intensity. Values are displayed as histograms of mean ± standard deviation from three independent experiments, which indicates induction of all endogenous Bex genes are observed in curcumin but not in MPTQ treated N2a cells. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Superscript III RT PCR kit (Invitrogen) was used to synthesize cDNA from 5 μg of DNA free RNA.

    Techniques: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation, Two Tailed Test

    Optimization of curcumin treatment duration for the induction of Bex genes in N2a cells. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated with 25 μM of curcumin for 2, 4, 8 or 24 hours. Control cells were treated with equivalent amount of DMSO for 2 hours. Total RNA was isolated at indicated time points using Trizol reagent and treated with DNase I for 15 minutes at room temperature. RT-PCR analysis of DNA-free Bex mRNA was performed for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis clearly demonstrates a time-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S10 . Intensity of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products were obtained and normalized with corresponding GAPDH band intensity, and displayed as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Optimization of curcumin treatment duration for the induction of Bex genes in N2a cells. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated with 25 μM of curcumin for 2, 4, 8 or 24 hours. Control cells were treated with equivalent amount of DMSO for 2 hours. Total RNA was isolated at indicated time points using Trizol reagent and treated with DNase I for 15 minutes at room temperature. RT-PCR analysis of DNA-free Bex mRNA was performed for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis clearly demonstrates a time-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S10 . Intensity of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products were obtained and normalized with corresponding GAPDH band intensity, and displayed as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Superscript III RT PCR kit (Invitrogen) was used to synthesize cDNA from 5 μg of DNA free RNA.

    Techniques: Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Two Tailed Test

    Molecular mechanisms involved in the induction of Bex genes and its association with curcumin-mediated N2a neuroblastoma cells apoptosis. ( a ) Approximately 80% confluent N2a cells were treated with wortmannin (4 nM), SP600125 (300 nM) or pifithrin-α (25 μM) for 30 minutes after two hours of serum starvation. Cells were then treated with either curcumin (25 μM) alone or along with these inhibitors. DMSO treated cells were used as controls. Total RNA was isolated at 4 hours of curcumin treatment. DNA-free RNA was used for RT-PCR analysis as described earlier, which demonstrates wortmannin, SP600125 and pifithrin-α abrogate curcumin-mediated induction of Bex genes. The original gel images are shown in Supplementary Fig. S12 . ( b ) Intensities of Bex1, Bex2, Bex3, Bex4 and Bex6 PCR products were measured and normalized values were displayed as histograms of mean ± standard error from three independent experiments. ( c ) LIVE/DEAD analysis was performed as described earlier except pifithrin-α was treated 30 minutes before curcumin treatment. Multiple images were captured and displayed with equal image settings. ( d ) Percentage of dead cells in each group was calculated from three independent experiments and plotted as histograms. ( e ) Cell lysates were prepared after 24 hours either from DMSO or curcumin (25 μM) alone or along with pifithrin-α (25 μM) treated N2a cells. Western blotting was performed using antibodies against caspase-9, caspase-3 or PARP1. GAPDH immunoblotting was used as loading and transfer control. The original immunoblot images are shown in Supplementary Fig. S13 . ( f ) N2a cells were transfected with Bex1, 2, 4 or 6 siRNA duplexes either individually or in combination for 72 hours followed by curcumin (25 μM) treatment for 24 hours. Scrambled siRNA transfected N2a cells were used as negative controls. Untransfected N2a cells treated with 25 μM of curcumin or DMSO were used as positive and negative controls respectively. LIVE/DEAD assay was performed at 24 hours of curcumin treatment and fluorescent images from nine random fields were captured at 30X magnification indicating inhibition of Bex siRNAs against curcumin-mediated cell death. ( g ) The number of dead cells (red) were counted manually and plotted as histograms. P-values displayed as numbers or * were calculated using two tailed unpaired Student’s t-test. P-value ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Molecular mechanisms involved in the induction of Bex genes and its association with curcumin-mediated N2a neuroblastoma cells apoptosis. ( a ) Approximately 80% confluent N2a cells were treated with wortmannin (4 nM), SP600125 (300 nM) or pifithrin-α (25 μM) for 30 minutes after two hours of serum starvation. Cells were then treated with either curcumin (25 μM) alone or along with these inhibitors. DMSO treated cells were used as controls. Total RNA was isolated at 4 hours of curcumin treatment. DNA-free RNA was used for RT-PCR analysis as described earlier, which demonstrates wortmannin, SP600125 and pifithrin-α abrogate curcumin-mediated induction of Bex genes. The original gel images are shown in Supplementary Fig. S12 . ( b ) Intensities of Bex1, Bex2, Bex3, Bex4 and Bex6 PCR products were measured and normalized values were displayed as histograms of mean ± standard error from three independent experiments. ( c ) LIVE/DEAD analysis was performed as described earlier except pifithrin-α was treated 30 minutes before curcumin treatment. Multiple images were captured and displayed with equal image settings. ( d ) Percentage of dead cells in each group was calculated from three independent experiments and plotted as histograms. ( e ) Cell lysates were prepared after 24 hours either from DMSO or curcumin (25 μM) alone or along with pifithrin-α (25 μM) treated N2a cells. Western blotting was performed using antibodies against caspase-9, caspase-3 or PARP1. GAPDH immunoblotting was used as loading and transfer control. The original immunoblot images are shown in Supplementary Fig. S13 . ( f ) N2a cells were transfected with Bex1, 2, 4 or 6 siRNA duplexes either individually or in combination for 72 hours followed by curcumin (25 μM) treatment for 24 hours. Scrambled siRNA transfected N2a cells were used as negative controls. Untransfected N2a cells treated with 25 μM of curcumin or DMSO were used as positive and negative controls respectively. LIVE/DEAD assay was performed at 24 hours of curcumin treatment and fluorescent images from nine random fields were captured at 30X magnification indicating inhibition of Bex siRNAs against curcumin-mediated cell death. ( g ) The number of dead cells (red) were counted manually and plotted as histograms. P-values displayed as numbers or * were calculated using two tailed unpaired Student’s t-test. P-value ≤ 0.05 is considered statistically significant.

    Article Snippet: Superscript III RT PCR kit (Invitrogen) was used to synthesize cDNA from 5 μg of DNA free RNA.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot, Transfection, Live Dead Assay, Inhibition, Two Tailed Test

    Hyperphosphorylation of p53 at ser15 and its association with curcumin-mediated Bex gene induction in N2a cells. ( a ) Western blot analysis of p53 phosphorylation at ser15 in curcumin (25 μM)-treated N2a cells at indicated time points using anti-phospho-p53-ser15, anti-p53 or anti-GAPDH antibodies. The original immunoblot images are shown in Supplementary Fig. S14 . ( b ) Fold change of p53 and phospho-p53-ser15 level after normalizing with GAPDH level was calculated and plotted as histograms of mean ± standard deviation from three independent experiments. Immunofluorocytochemical analysis of phospho-p53-ser15 ( c ) and p53 ( d ) level at 30 minutes of curcumin treated or untreated N2a cells. ( e ) Percent phospho-p53-ser15 and p53 positive cells were calculated in both the groups and plotted as histograms. ( f ) Interaction of activated p53 to the promoters of Bex genes by chromatin immunoprecipitation (ChIP) assay. N2a cells were treated with curcumin (25 μM) for 30 minutes followed by ChIP using anti-phospho-p53-ser15 antibody. PCR amplification and densitometric analysis (see material and methods) of p53 binding elements of Bex1 ( f ), Bex2 ( g ), Bex3 ( h ), Bex4 ( i ) and Bex6 ( j ) genes to phospho-p53-ser15 show at least one p53 binding element of each Bex gene interact more with phospho-p53-ser15 in curcumin treated N2a cells than corresponding controls. ( k ) Immunofluorocytochemical analysis demonstrates wortmannin, SP600125 and pifithrin-α reduces p53-ser15 phosphorylation at 30 minutes of curcumin treated N2a cells. ( l ) Average nuclear phospho-p53-ser15 intensities with or without inhibitors were measured and plotted as histograms from three independent experiments. The original gels images used for f-j are shown in Supplementary Fig. S15 . ( m ) Effect of wortmannin (4 nM), SP600125 (300 nM) and pifithrin-α (25 μM) on phospho-p53-ser15 DNA binding activity to Bex genes at 30 minutes of curcumin (25 μM) treated N2a cells. Chromatin immunoprecipitation was performed as described above. PCR amplification was performed corresponding to p53 binding element/s of Bex1, Bex2, Bex3, Bex4 and Bex6 gene promoters. Input DNA PCR was also used as loading control. The original gel images are shown in Supplementary Fig. S16 . ( n ) Band intensities of ChIP PCR products were normalized with corresponding input DNA PCR products and fold change was calculated. Values were plotted as histograms. P-values displayed were calculated using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Hyperphosphorylation of p53 at ser15 and its association with curcumin-mediated Bex gene induction in N2a cells. ( a ) Western blot analysis of p53 phosphorylation at ser15 in curcumin (25 μM)-treated N2a cells at indicated time points using anti-phospho-p53-ser15, anti-p53 or anti-GAPDH antibodies. The original immunoblot images are shown in Supplementary Fig. S14 . ( b ) Fold change of p53 and phospho-p53-ser15 level after normalizing with GAPDH level was calculated and plotted as histograms of mean ± standard deviation from three independent experiments. Immunofluorocytochemical analysis of phospho-p53-ser15 ( c ) and p53 ( d ) level at 30 minutes of curcumin treated or untreated N2a cells. ( e ) Percent phospho-p53-ser15 and p53 positive cells were calculated in both the groups and plotted as histograms. ( f ) Interaction of activated p53 to the promoters of Bex genes by chromatin immunoprecipitation (ChIP) assay. N2a cells were treated with curcumin (25 μM) for 30 minutes followed by ChIP using anti-phospho-p53-ser15 antibody. PCR amplification and densitometric analysis (see material and methods) of p53 binding elements of Bex1 ( f ), Bex2 ( g ), Bex3 ( h ), Bex4 ( i ) and Bex6 ( j ) genes to phospho-p53-ser15 show at least one p53 binding element of each Bex gene interact more with phospho-p53-ser15 in curcumin treated N2a cells than corresponding controls. ( k ) Immunofluorocytochemical analysis demonstrates wortmannin, SP600125 and pifithrin-α reduces p53-ser15 phosphorylation at 30 minutes of curcumin treated N2a cells. ( l ) Average nuclear phospho-p53-ser15 intensities with or without inhibitors were measured and plotted as histograms from three independent experiments. The original gels images used for f-j are shown in Supplementary Fig. S15 . ( m ) Effect of wortmannin (4 nM), SP600125 (300 nM) and pifithrin-α (25 μM) on phospho-p53-ser15 DNA binding activity to Bex genes at 30 minutes of curcumin (25 μM) treated N2a cells. Chromatin immunoprecipitation was performed as described above. PCR amplification was performed corresponding to p53 binding element/s of Bex1, Bex2, Bex3, Bex4 and Bex6 gene promoters. Input DNA PCR was also used as loading control. The original gel images are shown in Supplementary Fig. S16 . ( n ) Band intensities of ChIP PCR products were normalized with corresponding input DNA PCR products and fold change was calculated. Values were plotted as histograms. P-values displayed were calculated using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Superscript III RT PCR kit (Invitrogen) was used to synthesize cDNA from 5 μg of DNA free RNA.

    Techniques: Western Blot, Standard Deviation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Binding Assay, Activity Assay, Two Tailed Test

    Characterization of strain with PfNCR1 apt in PMII-GFP background. ( A ) PCR with gDNA preparation from clonal parasites showing integration of K/D construct and disruption of wild-type locus. Expected size for pfncr1 locus is 2.3 kbp, for aptamer-modified locus is 13.1kbp. ( B ) PfNCR1 expression in PMII-GFP background is regulated by aTc. Image is western blot 22 hr post washout. Top blot was probed with α-HA antibody, bottom blot (loading control) was probed with α-PM-V antibody. ( C ) Western blot showing premature leakage of cytoplasmic HAD1 protein after aTc washout (22 hr) following saponin isolation of parasites in the PfNCR1 K/D + PMII GFP clone. Blot was probed with α-HAD1 and α-PM-V antibodies. ( D ) Western blot on osmotically released (5% sorbitol, 5 min, room temperature) PMII-GFP DVs that were treated with increasing saponin concentrations following incubation with MMV009108 (1 μM, 2 hr). This blot was probed with α-GFP antibody. Expected sizes: PfNCR1-HA = 171 kDa, PM-V = 69 kDa, HAD1 = 33 kDa, pro-PMII-GFP=79 kDa, free GFP = 27 kDa.

    Journal: eLife

    Article Title: Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis

    doi: 10.7554/eLife.40529

    Figure Lengend Snippet: Characterization of strain with PfNCR1 apt in PMII-GFP background. ( A ) PCR with gDNA preparation from clonal parasites showing integration of K/D construct and disruption of wild-type locus. Expected size for pfncr1 locus is 2.3 kbp, for aptamer-modified locus is 13.1kbp. ( B ) PfNCR1 expression in PMII-GFP background is regulated by aTc. Image is western blot 22 hr post washout. Top blot was probed with α-HA antibody, bottom blot (loading control) was probed with α-PM-V antibody. ( C ) Western blot showing premature leakage of cytoplasmic HAD1 protein after aTc washout (22 hr) following saponin isolation of parasites in the PfNCR1 K/D + PMII GFP clone. Blot was probed with α-HAD1 and α-PM-V antibodies. ( D ) Western blot on osmotically released (5% sorbitol, 5 min, room temperature) PMII-GFP DVs that were treated with increasing saponin concentrations following incubation with MMV009108 (1 μM, 2 hr). This blot was probed with α-GFP antibody. Expected sizes: PfNCR1-HA = 171 kDa, PM-V = 69 kDa, HAD1 = 33 kDa, pro-PMII-GFP=79 kDa, free GFP = 27 kDa.

    Article Snippet: For complementation, RNA was prepared from 3D7 parasites using TRIzol (ThermoFisher), pfncr1 RNA was amplified using a SuperScript RT-PCR kit (Invitrogen) with primers Comp1 and Comp2, cloned into the XhoI/AvrII sites of the pTEOE random integration vector with the PiggyBac transposase as described ( ; ).

    Techniques: Polymerase Chain Reaction, Construct, Modification, Expressing, Western Blot, Isolation, Incubation

    Preparation of PfNCR1-GFP11. PCR of gDNA from two independent transfection each of PfNCR1-GFP11 into cytoplasmic or vacuolar expressed GFP1-10 parasites. The primers Int1 and Plasmid1 were used for the amplification. The size ladder on the left is in kbase pairs. Cyto-GFP11 means PfNCR1-GFP11 is expressed in the background of cytoplasmic GFP1-10, SP-GFP11 means PfNCR1-GFP11 is expressed in the background of vacuolar GFP1-10. Expected size: 1.46 kbp.

    Journal: eLife

    Article Title: Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis

    doi: 10.7554/eLife.40529

    Figure Lengend Snippet: Preparation of PfNCR1-GFP11. PCR of gDNA from two independent transfection each of PfNCR1-GFP11 into cytoplasmic or vacuolar expressed GFP1-10 parasites. The primers Int1 and Plasmid1 were used for the amplification. The size ladder on the left is in kbase pairs. Cyto-GFP11 means PfNCR1-GFP11 is expressed in the background of cytoplasmic GFP1-10, SP-GFP11 means PfNCR1-GFP11 is expressed in the background of vacuolar GFP1-10. Expected size: 1.46 kbp.

    Article Snippet: For complementation, RNA was prepared from 3D7 parasites using TRIzol (ThermoFisher), pfncr1 RNA was amplified using a SuperScript RT-PCR kit (Invitrogen) with primers Comp1 and Comp2, cloned into the XhoI/AvrII sites of the pTEOE random integration vector with the PiggyBac transposase as described ( ; ).

    Techniques: Polymerase Chain Reaction, Transfection, Amplification

    Editing of the PfNCR1 locus to generate aptamer-regulated strains and generation of the PfNCR1 complementation. ( A ) Schematic of the CRISPR/Cas9 modification of the pfncr1 locus. Aptamers = 10X aptamer sequence, BS = blasticidin deaminase resistance cassette, 2A = T2A viral skip peptide, DOZI-TetR = tet repressor protein fused to DOZI effector. ( B ) PCR product from gDNA of pfncr1 locus in PfNCR1 apt parasite clones. Primers Int1 (anneals 5’ of the integration site) and Int2 (reverse, anneals to 3’ of the integration site) were used. Expected size for pfncr1 locus is 2.3 kbp, for aptamer-modified locus it is 13.1kbp. ( C ) Sequence of PfNCR1 apt clone two illustrating the Cas9 shield mutations, the AatII sites and the HA-sequence. ( D ) Western blot of PfNCR1 apt complemented parasites. Expression from the genomic locus is regulated by aTc (left), while there is no change in the level of expression of the additional copies of PfNCR1-GFP (right). Expected size for HA-tagged PfNCR1 apt is 171 kDa (arrow on left), expected size for second copy PfNCR1-GFP is 197 kDa (arrow on right).

    Journal: eLife

    Article Title: Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis

    doi: 10.7554/eLife.40529

    Figure Lengend Snippet: Editing of the PfNCR1 locus to generate aptamer-regulated strains and generation of the PfNCR1 complementation. ( A ) Schematic of the CRISPR/Cas9 modification of the pfncr1 locus. Aptamers = 10X aptamer sequence, BS = blasticidin deaminase resistance cassette, 2A = T2A viral skip peptide, DOZI-TetR = tet repressor protein fused to DOZI effector. ( B ) PCR product from gDNA of pfncr1 locus in PfNCR1 apt parasite clones. Primers Int1 (anneals 5’ of the integration site) and Int2 (reverse, anneals to 3’ of the integration site) were used. Expected size for pfncr1 locus is 2.3 kbp, for aptamer-modified locus it is 13.1kbp. ( C ) Sequence of PfNCR1 apt clone two illustrating the Cas9 shield mutations, the AatII sites and the HA-sequence. ( D ) Western blot of PfNCR1 apt complemented parasites. Expression from the genomic locus is regulated by aTc (left), while there is no change in the level of expression of the additional copies of PfNCR1-GFP (right). Expected size for HA-tagged PfNCR1 apt is 171 kDa (arrow on left), expected size for second copy PfNCR1-GFP is 197 kDa (arrow on right).

    Article Snippet: For complementation, RNA was prepared from 3D7 parasites using TRIzol (ThermoFisher), pfncr1 RNA was amplified using a SuperScript RT-PCR kit (Invitrogen) with primers Comp1 and Comp2, cloned into the XhoI/AvrII sites of the pTEOE random integration vector with the PiggyBac transposase as described ( ; ).

    Techniques: CRISPR, Modification, Sequencing, Polymerase Chain Reaction, Clone Assay, Western Blot, Expressing

    Characterization of 1108 allelic exchange clones. ( A ) Scheme of Southern blot for clones with the A1108T mutation. A C-terminal fragment of PfNCR1 (bp 2305–4893) was amplified from gDNA and cloned into the pPM2GT vector with restriction sites XhoI and AvrII. Site-directed mutagenesis was used to introduce the mutation expressing A1108T (nucleotide G3322A) as well as a mutation at bp 3294 to introduce a synonymous BspHI site. The location of the probe (bp 2624–3298) is shown in red. For integrants, and normally a second truncated and promoterless copy is introduced into the genome. Primers used for PCR analysis of clones are shown in blue. Primers Int4, Int5, Int6 anneal to the transfected plasmid, wild-type parasites sequences and allelic exchange clones. Int3 is outside the plasmid sequence, while GFP1/2 are not present in the parent. ( B ) Southern blot of plasmid DNA, 3D7 parental DNA, two clones in which homologous recombination occurred such that the A1108T mutation is expressed (Mutant 1 and 2), as well as two clones in which homologous recombination occurred downstream from the mutation (Wild-type 1 and 2). A size ladder (in kb) is shown on the left. The expected sizes from the BspHI/BamHI double digest are: 1.3 kbp (plasmid), 4.0 kbp (genomic locus), 4.5 kbp (integration of vector, 3’ GFP), 1.1 kbp (integration of vector and expression of A1108T mutation, 3’ GFP). The expected promoterless second copy in the mutation integrant parasites used in Figure 1 is not present. It is possible that this second copy has looped out via homologous crossover after the BspHI site in the integrated concatamerized plasmid. Clones with or without this second copy displayed similar resistance (see D, below). ( C ) PCR diagnostic tests on A1108 allelic exchange clones using primers indicated in A). PCRs show integration at endogenous locus and introduction of mutation in A1108T mutant clones (as indicated by BspHI restriction). Expected sizes of PCR products are as follows: Int3 +Int4=2747; Int3 +GFP2=2860, Int5 +Int6=521. * indicates non-specific product, # denotes primers. In clones expressing A1108T digest of Int3 +Int4 product results in two fragments of 1145 bp and 1602 bp, and digest of Int3 +GFP2 product produces 1145 bp and 1750 bp fragments. ( D ) Parasitemia of allelic replacement clones after 72 hr incubation with 1.2 µM MMV009108. In some clones, the second (promoter-less) copy has looped out. Clones expressing A1108T with or without second copy were resistant to MMV009108. Wild-type refers to A1108A expressing clones, M-2 nd copy refers to A1108T clones with looped out promoter-less copy, while M+2 nd copy have the promoter-less copy. This experiment was done once with biological duplicates.

    Journal: eLife

    Article Title: Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis

    doi: 10.7554/eLife.40529

    Figure Lengend Snippet: Characterization of 1108 allelic exchange clones. ( A ) Scheme of Southern blot for clones with the A1108T mutation. A C-terminal fragment of PfNCR1 (bp 2305–4893) was amplified from gDNA and cloned into the pPM2GT vector with restriction sites XhoI and AvrII. Site-directed mutagenesis was used to introduce the mutation expressing A1108T (nucleotide G3322A) as well as a mutation at bp 3294 to introduce a synonymous BspHI site. The location of the probe (bp 2624–3298) is shown in red. For integrants, and normally a second truncated and promoterless copy is introduced into the genome. Primers used for PCR analysis of clones are shown in blue. Primers Int4, Int5, Int6 anneal to the transfected plasmid, wild-type parasites sequences and allelic exchange clones. Int3 is outside the plasmid sequence, while GFP1/2 are not present in the parent. ( B ) Southern blot of plasmid DNA, 3D7 parental DNA, two clones in which homologous recombination occurred such that the A1108T mutation is expressed (Mutant 1 and 2), as well as two clones in which homologous recombination occurred downstream from the mutation (Wild-type 1 and 2). A size ladder (in kb) is shown on the left. The expected sizes from the BspHI/BamHI double digest are: 1.3 kbp (plasmid), 4.0 kbp (genomic locus), 4.5 kbp (integration of vector, 3’ GFP), 1.1 kbp (integration of vector and expression of A1108T mutation, 3’ GFP). The expected promoterless second copy in the mutation integrant parasites used in Figure 1 is not present. It is possible that this second copy has looped out via homologous crossover after the BspHI site in the integrated concatamerized plasmid. Clones with or without this second copy displayed similar resistance (see D, below). ( C ) PCR diagnostic tests on A1108 allelic exchange clones using primers indicated in A). PCRs show integration at endogenous locus and introduction of mutation in A1108T mutant clones (as indicated by BspHI restriction). Expected sizes of PCR products are as follows: Int3 +Int4=2747; Int3 +GFP2=2860, Int5 +Int6=521. * indicates non-specific product, # denotes primers. In clones expressing A1108T digest of Int3 +Int4 product results in two fragments of 1145 bp and 1602 bp, and digest of Int3 +GFP2 product produces 1145 bp and 1750 bp fragments. ( D ) Parasitemia of allelic replacement clones after 72 hr incubation with 1.2 µM MMV009108. In some clones, the second (promoter-less) copy has looped out. Clones expressing A1108T with or without second copy were resistant to MMV009108. Wild-type refers to A1108A expressing clones, M-2 nd copy refers to A1108T clones with looped out promoter-less copy, while M+2 nd copy have the promoter-less copy. This experiment was done once with biological duplicates.

    Article Snippet: For complementation, RNA was prepared from 3D7 parasites using TRIzol (ThermoFisher), pfncr1 RNA was amplified using a SuperScript RT-PCR kit (Invitrogen) with primers Comp1 and Comp2, cloned into the XhoI/AvrII sites of the pTEOE random integration vector with the PiggyBac transposase as described ( ; ).

    Techniques: Clone Assay, Southern Blot, Mutagenesis, Amplification, Plasmid Preparation, Introduce, Expressing, Polymerase Chain Reaction, Transfection, Sequencing, Homologous Recombination, Diagnostic Assay, Incubation

    Characterization of A1208E and F1436I clones. PCR diagnostic tests using primers indicated in Figure 1—figure supplement 1A ). ( A ) PCRs show integration at endogenous locus and introduction of mutation at 1208 (as indicated by EcoRI restriction). Expected sizes of PCR products are as follows: Int3 +Int4=2747; Int3 +GFP2=2860, Int5 +Int6=521. Digest of Int3 +Int4 product results in two fragments of 1291 bp and 1456 bp. Digest of Int3 +GFP2 product results in a doublet (1404 bp and 1456 bp). ( B ) PCRs show integration at endogenous locus and introduction of mutation at 1436 (as indicated by mutated HincII restriction). Expected sizes of PCR products are as follows: Int3 + Int4=2747; Int3 + GFP2=2860, Int5 +Int6=521. Digest of Int3 + 4 PCR from parent gDNA with HincII decreases the 2747 bp product by 317 bp (small fragment is not visible on gel). The HincII site is mutated in the mutant clone. * indicates non-specific product. ( C ) Sequencing traces of gDNA purified from allelic exchange parasites showing the introduction of the desired mutations (highlighted by red stars). The primers Int3 and GFP1 were used to amplify the sequence of PfNCR1.

    Journal: eLife

    Article Title: Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis

    doi: 10.7554/eLife.40529

    Figure Lengend Snippet: Characterization of A1208E and F1436I clones. PCR diagnostic tests using primers indicated in Figure 1—figure supplement 1A ). ( A ) PCRs show integration at endogenous locus and introduction of mutation at 1208 (as indicated by EcoRI restriction). Expected sizes of PCR products are as follows: Int3 +Int4=2747; Int3 +GFP2=2860, Int5 +Int6=521. Digest of Int3 +Int4 product results in two fragments of 1291 bp and 1456 bp. Digest of Int3 +GFP2 product results in a doublet (1404 bp and 1456 bp). ( B ) PCRs show integration at endogenous locus and introduction of mutation at 1436 (as indicated by mutated HincII restriction). Expected sizes of PCR products are as follows: Int3 + Int4=2747; Int3 + GFP2=2860, Int5 +Int6=521. Digest of Int3 + 4 PCR from parent gDNA with HincII decreases the 2747 bp product by 317 bp (small fragment is not visible on gel). The HincII site is mutated in the mutant clone. * indicates non-specific product. ( C ) Sequencing traces of gDNA purified from allelic exchange parasites showing the introduction of the desired mutations (highlighted by red stars). The primers Int3 and GFP1 were used to amplify the sequence of PfNCR1.

    Article Snippet: For complementation, RNA was prepared from 3D7 parasites using TRIzol (ThermoFisher), pfncr1 RNA was amplified using a SuperScript RT-PCR kit (Invitrogen) with primers Comp1 and Comp2, cloned into the XhoI/AvrII sites of the pTEOE random integration vector with the PiggyBac transposase as described ( ; ).

    Techniques: Clone Assay, Polymerase Chain Reaction, Diagnostic Assay, Mutagenesis, Sequencing, Purification

    Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p

    Journal: PLoS ONE

    Article Title: Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    doi: 10.1371/journal.pone.0112351

    Figure Lengend Snippet: Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p

    Article Snippet: Viral RNA detection was carried out by real-time RT-PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, USA), as previously described .

    Techniques: Quantitative RT-PCR

    Virucidal assay. DENV-2 was treated with different concentrations of PLA 2 -CB (a) and crotoxin (b) and then used to infect Vero cells for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (* p

    Journal: PLoS ONE

    Article Title: Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    doi: 10.1371/journal.pone.0112351

    Figure Lengend Snippet: Virucidal assay. DENV-2 was treated with different concentrations of PLA 2 -CB (a) and crotoxin (b) and then used to infect Vero cells for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (* p

    Article Snippet: Viral RNA detection was carried out by real-time RT-PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, USA), as previously described .

    Techniques: Proximity Ligation Assay, Cell Culture, Quantitative RT-PCR

    Analysis of the exposure of DENV-2 genomic RNA. DENV-2 was first treated with PLA 2 -CB, crotoxin (8 ng/µL each) or PBS at 37°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p

    Journal: PLoS ONE

    Article Title: Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    doi: 10.1371/journal.pone.0112351

    Figure Lengend Snippet: Analysis of the exposure of DENV-2 genomic RNA. DENV-2 was first treated with PLA 2 -CB, crotoxin (8 ng/µL each) or PBS at 37°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p

    Article Snippet: Viral RNA detection was carried out by real-time RT-PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, USA), as previously described .

    Techniques: Proximity Ligation Assay, Quantitative RT-PCR

    Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with PLA 2 -CB, crotoxin (8 ng/µL each) and PBS at 37°C and 28°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p

    Journal: PLoS ONE

    Article Title: Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    doi: 10.1371/journal.pone.0112351

    Figure Lengend Snippet: Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with PLA 2 -CB, crotoxin (8 ng/µL each) and PBS at 37°C and 28°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p

    Article Snippet: Viral RNA detection was carried out by real-time RT-PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, USA), as previously described .

    Techniques: Proximity Ligation Assay, Quantitative RT-PCR

    Pre-treatment assay. Vero cells were treated with different concentrations of PLA 2 -CB (a) and crotoxin (b) and then infected with DENV-2 for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated cells (*p

    Journal: PLoS ONE

    Article Title: Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    doi: 10.1371/journal.pone.0112351

    Figure Lengend Snippet: Pre-treatment assay. Vero cells were treated with different concentrations of PLA 2 -CB (a) and crotoxin (b) and then infected with DENV-2 for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated cells (*p

    Article Snippet: Viral RNA detection was carried out by real-time RT-PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, USA), as previously described .

    Techniques: Proximity Ligation Assay, Infection, Cell Culture, Quantitative RT-PCR

    Lin28a regulates miR-9 levels differentiating P19 cells ( a ) Western blot analysis of protein extracts from mock-depleted P19 cells (Lane 1) and Lin28a-depleted P19 cells (Lane 2). Lanes 3 through 7 show serial dilutions of total protein extracts from mock-depleted P19 cells, providing an estimation of the linearity of the Western blot assay and the limit of detection. ( b ) Real-time qRT-PCR analysis of the mature miR-101, miR-122, miR-9 and let-7a levels on day 4 (d4) of RA-induced neuronal differentiation. The results from the mock-depleted cells are shown as white bars; the results from Lin28a-depleted cells are shown as black bars. The values were normalized to the miR-16 level. The fold change was plotted relative to values derived from mock-depleted cells, which were set to 1. Mean values and standard deviations (SD) of three independent biological replicates are shown. Statistical significance was calculated using t -test (**) −P ≤ 0.01, (***) −P ≤ 0.001.

    Journal: Nature communications

    Article Title: Lin28a regulates neuronal differentiation and controls miR-9 production

    doi: 10.1038/ncomms4687

    Figure Lengend Snippet: Lin28a regulates miR-9 levels differentiating P19 cells ( a ) Western blot analysis of protein extracts from mock-depleted P19 cells (Lane 1) and Lin28a-depleted P19 cells (Lane 2). Lanes 3 through 7 show serial dilutions of total protein extracts from mock-depleted P19 cells, providing an estimation of the linearity of the Western blot assay and the limit of detection. ( b ) Real-time qRT-PCR analysis of the mature miR-101, miR-122, miR-9 and let-7a levels on day 4 (d4) of RA-induced neuronal differentiation. The results from the mock-depleted cells are shown as white bars; the results from Lin28a-depleted cells are shown as black bars. The values were normalized to the miR-16 level. The fold change was plotted relative to values derived from mock-depleted cells, which were set to 1. Mean values and standard deviations (SD) of three independent biological replicates are shown. Statistical significance was calculated using t -test (**) −P ≤ 0.01, (***) −P ≤ 0.001.

    Article Snippet: Real time qRT–PCR was performed using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Life Technologies) following the manufacturer’s instructions on a Roche 480 LightCycler.

    Techniques: Western Blot, Quantitative RT-PCR, Derivative Assay

    Lin28a induction in differentiated P19 cells results in reduction of miR-9 levels. ( a ) Western blot analysis of protein extracts from control P19 cells (Lane 1 – d0 and Lane 2 – d9) and d9 of three colonies of P19 Tet-On 3G Lin28a cells. Lanes 3, 5 and 7 represent results from the corresponding cell lines without Dox. Lanes 4, 6 and 8 show results from the corresponding cell lines induced with Dox (100ng/ml). ( b ) Real-time qRT-PCR analysis of mature miR-9, let-7a and miR-101 levels of Dox-induced cells after RA-mediated neuronal differentiation. The results from P19 Tet-On 3G Lin28a #2, which failed to induce Lin28a, is shown as white bars; the results from P19 Tet-On 3G Lin28a #3, which induced Lin28a, are shown as black bars. The values were normalized to miR-16 levels. The fold change was plotted relative to values derived from -Dox cells, which were set to 100. Mean values and standard deviations (SD) of three independent biological replicates are shown. Statistical significance was calculated using t -test (*) −P ≤ 0.05. ( c ) Real-time qRT-PCR analysis of the primary miR-9-1, miR-9-2 and let-7a-1 of Dox-induced cells after RA-mediated neuronal differentiation. The results from P19 Tet-On 3G Lin28a #2, which failed to induce Lin28a, is shown as white bars; the results from P19 Tet-On 3G Lin28a #3, which induced Lin28a, are shown as black bars. The values were normalized to cyclophilin A mRNA levels. Mean values and standard deviations (SD) of three independent experiments are shown (*) −P ≤ 0.05, (**) −P ≤ 0.01.

    Journal: Nature communications

    Article Title: Lin28a regulates neuronal differentiation and controls miR-9 production

    doi: 10.1038/ncomms4687

    Figure Lengend Snippet: Lin28a induction in differentiated P19 cells results in reduction of miR-9 levels. ( a ) Western blot analysis of protein extracts from control P19 cells (Lane 1 – d0 and Lane 2 – d9) and d9 of three colonies of P19 Tet-On 3G Lin28a cells. Lanes 3, 5 and 7 represent results from the corresponding cell lines without Dox. Lanes 4, 6 and 8 show results from the corresponding cell lines induced with Dox (100ng/ml). ( b ) Real-time qRT-PCR analysis of mature miR-9, let-7a and miR-101 levels of Dox-induced cells after RA-mediated neuronal differentiation. The results from P19 Tet-On 3G Lin28a #2, which failed to induce Lin28a, is shown as white bars; the results from P19 Tet-On 3G Lin28a #3, which induced Lin28a, are shown as black bars. The values were normalized to miR-16 levels. The fold change was plotted relative to values derived from -Dox cells, which were set to 100. Mean values and standard deviations (SD) of three independent biological replicates are shown. Statistical significance was calculated using t -test (*) −P ≤ 0.05. ( c ) Real-time qRT-PCR analysis of the primary miR-9-1, miR-9-2 and let-7a-1 of Dox-induced cells after RA-mediated neuronal differentiation. The results from P19 Tet-On 3G Lin28a #2, which failed to induce Lin28a, is shown as white bars; the results from P19 Tet-On 3G Lin28a #3, which induced Lin28a, are shown as black bars. The values were normalized to cyclophilin A mRNA levels. Mean values and standard deviations (SD) of three independent experiments are shown (*) −P ≤ 0.05, (**) −P ≤ 0.01.

    Article Snippet: Real time qRT–PCR was performed using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Life Technologies) following the manufacturer’s instructions on a Roche 480 LightCycler.

    Techniques: Western Blot, Quantitative RT-PCR, Derivative Assay

    Constitutive expression of untagged but not GFP-tagged Lin28a affects miR-9 biogenesis during P19 cell differentiation ( a ) Western blot analysis of Lin28a levels in P19 stable cell lines expressing untagged human Lin28a. Lanes 1 and 2 include d0 and d9 P19 cells with an integrated FRT site, respectively. Lanes 3 and 4 show the P19 FRT/Lin28a clone, which does not express Lin28a at day 9. Lanes 5 and 6 include P19 FRT/Lin28a clones with stable Lin28a expression in differentiated cells. Tubulin served as a loading control. ( b ) Western blot analysis of the Lin28a levels in P19 stable cell lines expressing GFP-tagged human Lin28a. Lanes 1 and 2 are d0 and d9 P19 cells with integrated GFP, respectively. Lanes 3 and 4 are P19 Lin28a-GFP cell lines with stable expression of GFP-tagged human Lin28a in undifferentiated and differentiated cells, respectively. Tubulin served as a loading control. ( c ) Bright-field microscopy images showing representative images of P19 embryonic bodies at day 4 of differentiation. ( d ) Quantification of P19 embryonic bodies sizes at day 4 of differentiation represented as box plots. Statistical significance was calculated using the Mann-Whitney U -test (**) - P ≤ 0.01, (***) - P ≤ 0.001. ( e ) Real-time qRT-PCR analysis of mature miR-9, let-7a and miR-101 on day 9 of P19 FRT (white bars), P19 GFP (light grey bars), P19 GFP-Lin28a (black bars), P19 FRT/Lin28a 3# (horizontal lines bars) and P19 FRT/Lin28a 4# (dark grey bars). The values were normalized to miR-16 levels. The fold change was plotted relative to values derived from the corresponding control cell lines P19 FRT and P19 GFP, which were set to 1. Mean values and standard deviations (SD) of three independent biological replicates are shown. Statistical significance was calculated using t -test (***) −P ≤ 0.001, (****) - P ≤ 0.0001.

    Journal: Nature communications

    Article Title: Lin28a regulates neuronal differentiation and controls miR-9 production

    doi: 10.1038/ncomms4687

    Figure Lengend Snippet: Constitutive expression of untagged but not GFP-tagged Lin28a affects miR-9 biogenesis during P19 cell differentiation ( a ) Western blot analysis of Lin28a levels in P19 stable cell lines expressing untagged human Lin28a. Lanes 1 and 2 include d0 and d9 P19 cells with an integrated FRT site, respectively. Lanes 3 and 4 show the P19 FRT/Lin28a clone, which does not express Lin28a at day 9. Lanes 5 and 6 include P19 FRT/Lin28a clones with stable Lin28a expression in differentiated cells. Tubulin served as a loading control. ( b ) Western blot analysis of the Lin28a levels in P19 stable cell lines expressing GFP-tagged human Lin28a. Lanes 1 and 2 are d0 and d9 P19 cells with integrated GFP, respectively. Lanes 3 and 4 are P19 Lin28a-GFP cell lines with stable expression of GFP-tagged human Lin28a in undifferentiated and differentiated cells, respectively. Tubulin served as a loading control. ( c ) Bright-field microscopy images showing representative images of P19 embryonic bodies at day 4 of differentiation. ( d ) Quantification of P19 embryonic bodies sizes at day 4 of differentiation represented as box plots. Statistical significance was calculated using the Mann-Whitney U -test (**) - P ≤ 0.01, (***) - P ≤ 0.001. ( e ) Real-time qRT-PCR analysis of mature miR-9, let-7a and miR-101 on day 9 of P19 FRT (white bars), P19 GFP (light grey bars), P19 GFP-Lin28a (black bars), P19 FRT/Lin28a 3# (horizontal lines bars) and P19 FRT/Lin28a 4# (dark grey bars). The values were normalized to miR-16 levels. The fold change was plotted relative to values derived from the corresponding control cell lines P19 FRT and P19 GFP, which were set to 1. Mean values and standard deviations (SD) of three independent biological replicates are shown. Statistical significance was calculated using t -test (***) −P ≤ 0.001, (****) - P ≤ 0.0001.

    Article Snippet: Real time qRT–PCR was performed using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Life Technologies) following the manufacturer’s instructions on a Roche 480 LightCycler.

    Techniques: Expressing, Cell Differentiation, Western Blot, Stable Transfection, Clone Assay, Microscopy, MANN-WHITNEY, Quantitative RT-PCR, Derivative Assay

    The level of brain-specific miR-9 is transcriptionally and post-transcriptionally regulated during RA-induced neuronal differentiation of P19 cells ( a, c, d ) Real-time qRT-PCR analysis of mature miR-9, let-7a and miR-302a levels at different stages of RA-induced neuronal differentiation (Day – d). The values were normalized to miR-16 levels. The fold change was plotted relative to values derived from undifferentiated cells (d0), which were set to 1. Mean values and standard deviations (SD) of three independent biological replicates are shown. ( b ) Real-time qRT–PCR of miR-9 primary transcripts (pri-miR) at different stages of RA-induced P19 cell neuronal differentiation. The fold changes of the corresponding pri-miRNA abundance, pri-miR-9-1 (black bars), pri-miR-9-2 (grey bars) and pri-miR-9-3 (white bars), were plotted relative to the d0 values, which were set to 1. The values were normalized to the cyclophilin A mRNA levels. Mean values and standard deviations (SD) of three independent experiments are shown.

    Journal: Nature communications

    Article Title: Lin28a regulates neuronal differentiation and controls miR-9 production

    doi: 10.1038/ncomms4687

    Figure Lengend Snippet: The level of brain-specific miR-9 is transcriptionally and post-transcriptionally regulated during RA-induced neuronal differentiation of P19 cells ( a, c, d ) Real-time qRT-PCR analysis of mature miR-9, let-7a and miR-302a levels at different stages of RA-induced neuronal differentiation (Day – d). The values were normalized to miR-16 levels. The fold change was plotted relative to values derived from undifferentiated cells (d0), which were set to 1. Mean values and standard deviations (SD) of three independent biological replicates are shown. ( b ) Real-time qRT–PCR of miR-9 primary transcripts (pri-miR) at different stages of RA-induced P19 cell neuronal differentiation. The fold changes of the corresponding pri-miRNA abundance, pri-miR-9-1 (black bars), pri-miR-9-2 (grey bars) and pri-miR-9-3 (white bars), were plotted relative to the d0 values, which were set to 1. The values were normalized to the cyclophilin A mRNA levels. Mean values and standard deviations (SD) of three independent experiments are shown.

    Article Snippet: Real time qRT–PCR was performed using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Life Technologies) following the manufacturer’s instructions on a Roche 480 LightCycler.

    Techniques: Quantitative RT-PCR, Derivative Assay

    Effect of LPA treatment on pro-migratory, pro-invasive, and pro-angiogenic gene expression. PMM were seeded onto 24-well plates, serum-starved overnight, and treated with 0.1% BSA (control) or LPA (1 μM). At the indicated time points, RNA was isolated, reverse-transcribed, and analyzed by qPCR. Expression ratios were normalized to HPRT. Results of three separate experiments in triplicate are expressed as mean + SD (* p

    Journal: Journal of Neuroinflammation

    Article Title: Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

    doi: 10.1186/s12974-017-1024-1

    Figure Lengend Snippet: Effect of LPA treatment on pro-migratory, pro-invasive, and pro-angiogenic gene expression. PMM were seeded onto 24-well plates, serum-starved overnight, and treated with 0.1% BSA (control) or LPA (1 μM). At the indicated time points, RNA was isolated, reverse-transcribed, and analyzed by qPCR. Expression ratios were normalized to HPRT. Results of three separate experiments in triplicate are expressed as mean + SD (* p

    Article Snippet: RNA was reverse-transcribed using the SuperScript® III reverse transcription kit (Invitrogen, Waltham, MA, USA). qPCR was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System using the QuantiTect SYBR® Green PCR kit (Qiagen, Hilden, Germany).

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

    LPA controls the expression of pro-migratory, pro-invasive, and pro-angiogenic genes via the LPA/LPAR5/PKD axis. PMM were cultured in 24-well plates, serum-starved overnight, and treated with DMSO, DMSO plus LPA (1 μM), and LPA (1 μM) plus TCLPA5 (5 μM) or CRT (1 μM). At the indicated time points, cells were scraped, RNA was isolated and reverse-transcribed, and the gene products indicated were analyzed by qPCR. Expression ratios were normalized to HPRT expression. Results of three separate experiments performed in triplicate are expressed as mean + SD (* p

    Journal: Journal of Neuroinflammation

    Article Title: Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

    doi: 10.1186/s12974-017-1024-1

    Figure Lengend Snippet: LPA controls the expression of pro-migratory, pro-invasive, and pro-angiogenic genes via the LPA/LPAR5/PKD axis. PMM were cultured in 24-well plates, serum-starved overnight, and treated with DMSO, DMSO plus LPA (1 μM), and LPA (1 μM) plus TCLPA5 (5 μM) or CRT (1 μM). At the indicated time points, cells were scraped, RNA was isolated and reverse-transcribed, and the gene products indicated were analyzed by qPCR. Expression ratios were normalized to HPRT expression. Results of three separate experiments performed in triplicate are expressed as mean + SD (* p

    Article Snippet: RNA was reverse-transcribed using the SuperScript® III reverse transcription kit (Invitrogen, Waltham, MA, USA). qPCR was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System using the QuantiTect SYBR® Green PCR kit (Qiagen, Hilden, Germany).

    Techniques: Expressing, Cell Culture, Isolation, Real-time Polymerase Chain Reaction

    PKD isoform expression in BV-2 cells and PMM. Gene expression of PKD1–3 in a BV-2 and b PMM was analyzed by qPCR and normalized to HPRT. Values are expressed as mean + SD. PKD2 expression in BV-2 and PKD1 in PMM was arbitrarily set to 1. n.d. not detectable. c Protein expression of PKD isoforms was determined by western blotting. Samples from the whole brain and cortex were used as controls. One representative blot out of three is shown. β-Tubulin was used as loading control

    Journal: Journal of Neuroinflammation

    Article Title: Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

    doi: 10.1186/s12974-017-1024-1

    Figure Lengend Snippet: PKD isoform expression in BV-2 cells and PMM. Gene expression of PKD1–3 in a BV-2 and b PMM was analyzed by qPCR and normalized to HPRT. Values are expressed as mean + SD. PKD2 expression in BV-2 and PKD1 in PMM was arbitrarily set to 1. n.d. not detectable. c Protein expression of PKD isoforms was determined by western blotting. Samples from the whole brain and cortex were used as controls. One representative blot out of three is shown. β-Tubulin was used as loading control

    Article Snippet: RNA was reverse-transcribed using the SuperScript® III reverse transcription kit (Invitrogen, Waltham, MA, USA). qPCR was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System using the QuantiTect SYBR® Green PCR kit (Qiagen, Hilden, Germany).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    PKD2 is mainly responsible for a motile PMM phenotype. PMM were cultured in PDL-coated 24-well plates and transduced by shPKD1 and shPKD2. Control vectors expressing scrambled shRNA were used as control. a Silencing efficacy was assessed 72 h post transduction by qPCR. b – d Cells were serum-starved overnight and treated with 0.1% BSA or 1 μM LPA for 24 h. Velocity, accumulated distance, and Euclidean distance were analyzed. e Serum-starved PMM incubated with 0.1% BSA or 1 μM LPA were lysed, RNA was isolated and reverse-transcribed, and the gene products indicated were analyzed by qPCR. Expression ratios are normalized to HPRT expression. Results of three separate experiments in triplicate are presented as mean + SD (* p

    Journal: Journal of Neuroinflammation

    Article Title: Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

    doi: 10.1186/s12974-017-1024-1

    Figure Lengend Snippet: PKD2 is mainly responsible for a motile PMM phenotype. PMM were cultured in PDL-coated 24-well plates and transduced by shPKD1 and shPKD2. Control vectors expressing scrambled shRNA were used as control. a Silencing efficacy was assessed 72 h post transduction by qPCR. b – d Cells were serum-starved overnight and treated with 0.1% BSA or 1 μM LPA for 24 h. Velocity, accumulated distance, and Euclidean distance were analyzed. e Serum-starved PMM incubated with 0.1% BSA or 1 μM LPA were lysed, RNA was isolated and reverse-transcribed, and the gene products indicated were analyzed by qPCR. Expression ratios are normalized to HPRT expression. Results of three separate experiments in triplicate are presented as mean + SD (* p

    Article Snippet: RNA was reverse-transcribed using the SuperScript® III reverse transcription kit (Invitrogen, Waltham, MA, USA). qPCR was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System using the QuantiTect SYBR® Green PCR kit (Qiagen, Hilden, Germany).

    Techniques: Cell Culture, Expressing, shRNA, Transduction, Real-time Polymerase Chain Reaction, Incubation, Isolation

    Effects of AAs, 20E, insulin and HR3 on YPP genes. (A) Relative expression of gene—AAEL006563, Carboxypeptidase, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (E) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids and Insulin (AA+INS), Insulin and 20E (INS+20E), amino acids plus 20E (AA+20E), and amino acids plus 20E and Insulin (AA+20E+INS). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E, insulin and HR3 on YPP genes. (A) Relative expression of gene—AAEL006563, Carboxypeptidase, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (E) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids and Insulin (AA+INS), Insulin and 20E (INS+20E), amino acids plus 20E (AA+20E), and amino acids plus 20E and Insulin (AA+20E+INS). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Effects of AAs, 20E and JH on representative LGs. (A) Relative expression of gene—AALE004328 –Origin recognition complex, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (C) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiment. All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E and JH on representative LGs. (A) Relative expression of gene—AALE004328 –Origin recognition complex, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (C) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiment. All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Effects of AAs, 20E and HR3 on EMGs. (A) Relative expression of gene—AAEL001433, fgf receptor activating protein detected by qRT-PCR, in tissues subjected to in- vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E and HR3 on EMGs. (A) Relative expression of gene—AAEL001433, fgf receptor activating protein detected by qRT-PCR, in tissues subjected to in- vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Genes cyclically activated by JH through Met—Functional group enrichment and the effects of AAs, 20E and JH. (A) Venn diagram showing genes that are up regulated in late post eclosion (LPE) and late post blood meal (LGs) periods and that are down regulated in Met knocked-down (imet down) fat body tissues. (B-D) Comparison of functional categories viz. (B) Information storage and Processing, (C) Metabolism and (D) Cellular Processes and Signalling, that constitute the late genes (LGs) and cyclical genes (CGs) using the inNOG database. (E-F) Relative expression of gene—AAEL001171, tRNA-dihydrouridine synthase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media, (E) without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E); (F) without (JH-) and with (JH+) juvenile hormone. (G) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet); injecting double stranded RNA for the Luciferase gene (iluc) served as the control. (H) Expression profile of the gene after the first blood meal. (I) Expression profile of the gene after the completion of the first reproductive cycle (egg laying) and post second blood meal. All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Genes cyclically activated by JH through Met—Functional group enrichment and the effects of AAs, 20E and JH. (A) Venn diagram showing genes that are up regulated in late post eclosion (LPE) and late post blood meal (LGs) periods and that are down regulated in Met knocked-down (imet down) fat body tissues. (B-D) Comparison of functional categories viz. (B) Information storage and Processing, (C) Metabolism and (D) Cellular Processes and Signalling, that constitute the late genes (LGs) and cyclical genes (CGs) using the inNOG database. (E-F) Relative expression of gene—AAEL001171, tRNA-dihydrouridine synthase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media, (E) without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E); (F) without (JH-) and with (JH+) juvenile hormone. (G) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet); injecting double stranded RNA for the Luciferase gene (iluc) served as the control. (H) Expression profile of the gene after the first blood meal. (I) Expression profile of the gene after the completion of the first reproductive cycle (egg laying) and post second blood meal. All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Functional Assay, Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Effects of AAs, 20E, JH and HR3 on representative LMGs. (A) Relative expression of genes—AAEL003568, Threonine dehydratase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (D) Relative expression in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (E) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B , C and E ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E, JH and HR3 on representative LMGs. (A) Relative expression of genes—AAEL003568, Threonine dehydratase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (D) Relative expression in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (E) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B , C and E ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Effects of AAs, 20E and JH on representative EGs. (A) Relative expression of AAEL002269, Purine nucleoside phosphorylase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes after knock-down of the JH receptor Met (imet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E and JH on representative EGs. (A) Relative expression of AAEL002269, Purine nucleoside phosphorylase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes after knock-down of the JH receptor Met (imet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Ribosome bound mRNA abundance in tobacco organs. Abundance of mRNA bound to ribosomes in young leaves, fully expanded leaves, roots, flowers and floral buds of tobacco. Values represent relative polysomal RNA levels per µg of ribosome-bound RNA (Additional file 7 : Dataset 2). Data represent mean values of four biological replicates for young leaves and fully expanded leaves, three biological replicates for roots and flowers and two biological replicates for floral buds. Significance was estimated with two way ANOVA; P

    Journal: Plant Methods

    Article Title: Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

    doi: 10.1186/s13007-017-0257-4

    Figure Lengend Snippet: Ribosome bound mRNA abundance in tobacco organs. Abundance of mRNA bound to ribosomes in young leaves, fully expanded leaves, roots, flowers and floral buds of tobacco. Values represent relative polysomal RNA levels per µg of ribosome-bound RNA (Additional file 7 : Dataset 2). Data represent mean values of four biological replicates for young leaves and fully expanded leaves, three biological replicates for roots and flowers and two biological replicates for floral buds. Significance was estimated with two way ANOVA; P

    Article Snippet: Five micrograms of RNA were reverse transcribed using the Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, USA).

    Techniques:

    Mitochondrial RNA abundance in tobacco organs. RNA levels of mitochondrial transcripts in young leaves, fully expanded leaves, roots, flowers and floral buds. Values represent relative transcript levels per µg of total RNA (Additional file 5 : Dataset 1). Data represent mean values of four biological replicates for young leaves and fully expanded leaves, three biological replicates for roots and flowers, and two biological replicates for floral buds. Significant changes were calculated using Two way ANOVA ( p

    Journal: Plant Methods

    Article Title: Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

    doi: 10.1186/s13007-017-0257-4

    Figure Lengend Snippet: Mitochondrial RNA abundance in tobacco organs. RNA levels of mitochondrial transcripts in young leaves, fully expanded leaves, roots, flowers and floral buds. Values represent relative transcript levels per µg of total RNA (Additional file 5 : Dataset 1). Data represent mean values of four biological replicates for young leaves and fully expanded leaves, three biological replicates for roots and flowers, and two biological replicates for floral buds. Significant changes were calculated using Two way ANOVA ( p

    Article Snippet: Five micrograms of RNA were reverse transcribed using the Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, USA).

    Techniques:

    Determination of mitochondrial DNA content per nuclear DNA and the expression analysis of RpoT genes in tobacco organs using qRT-PCR analysis. Young tobacco leaves, fully expanded leaves, roots, flowers and floral buds were assayed for mitochondrial DNA content relative to nuclear DNA content ( A ). Primer combination was chosen from mitochondrial intergenic region orf112b to cob (position 34,443–40,865) and from a nuclear non-coding region from RpoTm. Data were determined by calculating the difference of mitochondrial to nuclear CT values. Bars in the figure represent standard deviations for three biological replicates. B RpoTm, and C RpoTmp messenger RNA abundance was determined by quantitative real-time PCR. Data were normalized to cytoplasmic EF1α and GAPDH levels as an internal control. RpoT gene expression is presented as 40-ΔCT values. Given values were derived from three different biological replicates; standard deviations are indicated. Different letters mark mean values that are significantly different from each other (one way ANOVA; p

    Journal: Plant Methods

    Article Title: Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

    doi: 10.1186/s13007-017-0257-4

    Figure Lengend Snippet: Determination of mitochondrial DNA content per nuclear DNA and the expression analysis of RpoT genes in tobacco organs using qRT-PCR analysis. Young tobacco leaves, fully expanded leaves, roots, flowers and floral buds were assayed for mitochondrial DNA content relative to nuclear DNA content ( A ). Primer combination was chosen from mitochondrial intergenic region orf112b to cob (position 34,443–40,865) and from a nuclear non-coding region from RpoTm. Data were determined by calculating the difference of mitochondrial to nuclear CT values. Bars in the figure represent standard deviations for three biological replicates. B RpoTm, and C RpoTmp messenger RNA abundance was determined by quantitative real-time PCR. Data were normalized to cytoplasmic EF1α and GAPDH levels as an internal control. RpoT gene expression is presented as 40-ΔCT values. Given values were derived from three different biological replicates; standard deviations are indicated. Different letters mark mean values that are significantly different from each other (one way ANOVA; p

    Article Snippet: Five micrograms of RNA were reverse transcribed using the Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, USA).

    Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Derivative Assay

    Northern blot analysis of mitochondrial RNA abundance in tobacco organs. a The analyzed tissues are indicated above the blots. Three mitochondrial genes (cox1, atp9 and rps10) were analyzed to confirm the pattern of mitochondrial mRNA abundance observed through mitochondrial microarray analysis. Equal loading of RNA was checked through 18S rRNA hybridization. The blots are representatives for three independent replicates. b Signal intensities relative to the 18s rRNA control from the Northern blots presented in part a of this figure. Pixel intensity values as determined using ImageJ were used to calculate the ratios

    Journal: Plant Methods

    Article Title: Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

    doi: 10.1186/s13007-017-0257-4

    Figure Lengend Snippet: Northern blot analysis of mitochondrial RNA abundance in tobacco organs. a The analyzed tissues are indicated above the blots. Three mitochondrial genes (cox1, atp9 and rps10) were analyzed to confirm the pattern of mitochondrial mRNA abundance observed through mitochondrial microarray analysis. Equal loading of RNA was checked through 18S rRNA hybridization. The blots are representatives for three independent replicates. b Signal intensities relative to the 18s rRNA control from the Northern blots presented in part a of this figure. Pixel intensity values as determined using ImageJ were used to calculate the ratios

    Article Snippet: Five micrograms of RNA were reverse transcribed using the Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, USA).

    Techniques: Northern Blot, Microarray, Hybridization

    A charge-rebalance approach via reducing acidic residues of HBc. (A) A cartoon illustrates the charge rebalance approach. (B) The dimeric structure of HBc (PDB ID:1QGT) with each monomer in green and orange ribbon modes. The side chains of four glutamic acids are located close to the capsid interior: E40 (cyan), E46 (yellow), E113 (white) and E117 (blue) 18 70 71 90 . (C) The schematic presentation of a 40-mer HBc in the context of a 240-mer icosahedral particle (T = 4) is illustrated using PyMol (DeLano Scientific LLC, Palo Alto, CA, USA). Upper panel : E40 (cyan), E46 (yellow), E113 (white) and E117 (blue). Lower panel : all four acidic residues are in black. (D) Parental HBc mutant ARD-I + II + III + IV exhibited a very short DNA phenotype by Southern blot, which can be partially rescued by E-to-A single or double mutations at E46 or E113. (E) The Southern result in ( D ) can be confirmed by Whole Genome PCR analysis using core particle-associated viral DNAs as a template. (F) Upper panel : Northern blot analysis of HBc parental mutant ARD-I + II + III + IV and its charge rebalanced derivatives. Rescued viral RNAs with significantly increased size (between two dotted lines) were observed in HBc mutant E46A + E113A (last lane). Lower panels : Total cytoplasmic HBV RNA and GAPDH RNA were included as controls. (G) The Northern results in ( F ) was confirmed by RT-PCR analysis using core particle-associated viral RNAs. (H) Size distributions of encapsidated viral DNAs in WT, mutant ARD-I + II + III + IV, and its double-rescued mutant. Each clone was characterized by sequencing as described in Fig. 2D . The horizontal line represents the medium size of encapsidated viral DNA species. ***p

    Journal: Scientific Reports

    Article Title: HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids

    doi: 10.1038/srep38959

    Figure Lengend Snippet: A charge-rebalance approach via reducing acidic residues of HBc. (A) A cartoon illustrates the charge rebalance approach. (B) The dimeric structure of HBc (PDB ID:1QGT) with each monomer in green and orange ribbon modes. The side chains of four glutamic acids are located close to the capsid interior: E40 (cyan), E46 (yellow), E113 (white) and E117 (blue) 18 70 71 90 . (C) The schematic presentation of a 40-mer HBc in the context of a 240-mer icosahedral particle (T = 4) is illustrated using PyMol (DeLano Scientific LLC, Palo Alto, CA, USA). Upper panel : E40 (cyan), E46 (yellow), E113 (white) and E117 (blue). Lower panel : all four acidic residues are in black. (D) Parental HBc mutant ARD-I + II + III + IV exhibited a very short DNA phenotype by Southern blot, which can be partially rescued by E-to-A single or double mutations at E46 or E113. (E) The Southern result in ( D ) can be confirmed by Whole Genome PCR analysis using core particle-associated viral DNAs as a template. (F) Upper panel : Northern blot analysis of HBc parental mutant ARD-I + II + III + IV and its charge rebalanced derivatives. Rescued viral RNAs with significantly increased size (between two dotted lines) were observed in HBc mutant E46A + E113A (last lane). Lower panels : Total cytoplasmic HBV RNA and GAPDH RNA were included as controls. (G) The Northern results in ( F ) was confirmed by RT-PCR analysis using core particle-associated viral RNAs. (H) Size distributions of encapsidated viral DNAs in WT, mutant ARD-I + II + III + IV, and its double-rescued mutant. Each clone was characterized by sequencing as described in Fig. 2D . The horizontal line represents the medium size of encapsidated viral DNA species. ***p

    Article Snippet: For RT-PCR, 55 μM of oligo (dT)18 and 5 μg of core-associated RNAs were used for reverse transcription reaction by SuperScript III RT kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Mutagenesis, Southern Blot, Polymerase Chain Reaction, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Arginine-deficient HBc mutants preferentially encapsidated spliced viral RNA and DNA. (A) Core particle -associated viral RNAs of HBc ARD mutants were analyzed by RT-PCR and agarose gel with ethidium bromide staining. The positions of the RT-PCR primers are shown as black arrows in Fig. 2E. Compared to WT, mutant ARD-III + IV encapsidated shorter-sized viral RNAs, while mutant ARD-I + II + III + IV encapsidated the shortest RNA species. (B) The diagram summarized the statistics of RT-PCR sequencing results of core particle -associated viral RNAs of WT, mutants ARD-III + IV and I + II + III + IV. Each symbol represents one independently isolated clone from E. coli . The horizontal line represents the medium size of encapsidated RNA species. ***p

    Journal: Scientific Reports

    Article Title: HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids

    doi: 10.1038/srep38959

    Figure Lengend Snippet: Arginine-deficient HBc mutants preferentially encapsidated spliced viral RNA and DNA. (A) Core particle -associated viral RNAs of HBc ARD mutants were analyzed by RT-PCR and agarose gel with ethidium bromide staining. The positions of the RT-PCR primers are shown as black arrows in Fig. 2E. Compared to WT, mutant ARD-III + IV encapsidated shorter-sized viral RNAs, while mutant ARD-I + II + III + IV encapsidated the shortest RNA species. (B) The diagram summarized the statistics of RT-PCR sequencing results of core particle -associated viral RNAs of WT, mutants ARD-III + IV and I + II + III + IV. Each symbol represents one independently isolated clone from E. coli . The horizontal line represents the medium size of encapsidated RNA species. ***p

    Article Snippet: For RT-PCR, 55 μM of oligo (dT)18 and 5 μg of core-associated RNAs were used for reverse transcription reaction by SuperScript III RT kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Mutagenesis, Sequencing, Isolation

    Gradual reduction in the positive charge contents of HBc correlated with the gradual reduction in the size and amount of viral particle-associated HBV DNA and RNA. ( A ) HBc consists of two distinct domains responsible for capsid assembly and viral RNA encapsidation. Three major phosphorylation sites at serine (S) 155, 162 and 170 are shown in blue 34 . Arginines (R) are shown in red. ( B ) The charge balance hypothesis (electrostatic homeostasis) highlights the electrostatic interactions between the positive charge from HBc arginine-rich domains (ARD) and the negative charge from encapsidated nucleic acids. Such a balanced or imbalanced electrostatic interaction in the capsid interior could regulate viral RNA encapsidation, DNA replication, capsid conformation, stability and assembly 40 48 49 . ( C ) Construction of 15 different ARD mutants with different R-to-A substitutions at different positions. Viral DNA and RNA from transfected culture were analyzed. ( D ) Upper panel: Both numbers and positions of positive-charged arginine could strongly influence the size and intensity of HBV DNA replicative intermediates by Southern blot analysis. RC: full-length relaxed circle DNA (4.0 kb). Heat-denatured HBV DNAs (100 °C, 5 min) banded as smearing signals starting from the 1.5 kb SS (single-strand) DNA position (lane 18). Smearing signals represent HBV DNA replicative intermediates with different MW. Lower panel : Intracellular capsid particles were assayed by native agarose gel electrophoresis and Western blot using an anti-HBc antibody. ( E ) The core particle-associated DNAs were analyzed by Whole Genome PCR (M M 10 ) and agarose gel electrophoresis with ethidium bromide staining. The mainstream population of encapsidated viral DNAs decreased in size when the arginine content was decreased. #Shorter form HBV DNA; *Full-length 3.2 kb HBV DNA (lane 17); FL-HBV: 3.2 kb full-length DNA amplified from an HBV plasmid (lane 19). (F) Upper panel : HBV core-associated viral RNAs were analyzed by Northern blot. Consistent with the gradual reduction in viral DNA sizes in ( D , E ), the size reduction of encapsidated RNA was associated with the gradual reduction of the positive charge content of HBc ARD. RI and Sp RNA: replicative intermediates of reverse-transcribing viral RNAs and spliced RNAs. Lower panel : total cytoplasmic HBV RNA and GAPDH were included as controls.

    Journal: Scientific Reports

    Article Title: HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids

    doi: 10.1038/srep38959

    Figure Lengend Snippet: Gradual reduction in the positive charge contents of HBc correlated with the gradual reduction in the size and amount of viral particle-associated HBV DNA and RNA. ( A ) HBc consists of two distinct domains responsible for capsid assembly and viral RNA encapsidation. Three major phosphorylation sites at serine (S) 155, 162 and 170 are shown in blue 34 . Arginines (R) are shown in red. ( B ) The charge balance hypothesis (electrostatic homeostasis) highlights the electrostatic interactions between the positive charge from HBc arginine-rich domains (ARD) and the negative charge from encapsidated nucleic acids. Such a balanced or imbalanced electrostatic interaction in the capsid interior could regulate viral RNA encapsidation, DNA replication, capsid conformation, stability and assembly 40 48 49 . ( C ) Construction of 15 different ARD mutants with different R-to-A substitutions at different positions. Viral DNA and RNA from transfected culture were analyzed. ( D ) Upper panel: Both numbers and positions of positive-charged arginine could strongly influence the size and intensity of HBV DNA replicative intermediates by Southern blot analysis. RC: full-length relaxed circle DNA (4.0 kb). Heat-denatured HBV DNAs (100 °C, 5 min) banded as smearing signals starting from the 1.5 kb SS (single-strand) DNA position (lane 18). Smearing signals represent HBV DNA replicative intermediates with different MW. Lower panel : Intracellular capsid particles were assayed by native agarose gel electrophoresis and Western blot using an anti-HBc antibody. ( E ) The core particle-associated DNAs were analyzed by Whole Genome PCR (M M 10 ) and agarose gel electrophoresis with ethidium bromide staining. The mainstream population of encapsidated viral DNAs decreased in size when the arginine content was decreased. #Shorter form HBV DNA; *Full-length 3.2 kb HBV DNA (lane 17); FL-HBV: 3.2 kb full-length DNA amplified from an HBV plasmid (lane 19). (F) Upper panel : HBV core-associated viral RNAs were analyzed by Northern blot. Consistent with the gradual reduction in viral DNA sizes in ( D , E ), the size reduction of encapsidated RNA was associated with the gradual reduction of the positive charge content of HBc ARD. RI and Sp RNA: replicative intermediates of reverse-transcribing viral RNAs and spliced RNAs. Lower panel : total cytoplasmic HBV RNA and GAPDH were included as controls.

    Article Snippet: For RT-PCR, 55 μM of oligo (dT)18 and 5 μg of core-associated RNAs were used for reverse transcription reaction by SuperScript III RT kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Transfection, Southern Blot, Agarose Gel Electrophoresis, Western Blot, Polymerase Chain Reaction, Staining, Amplification, Plasmid Preparation, Northern Blot

    A cartoon summary of three major approaches used here in testing the HBV charge balance hypothesis. (A) The short RNA phenotype in arginine-deficient HBc mutants (e.g., ARD-III + IV) can be rescued successfully by using a charge rebalance approach ( Fig. 3A ). Both E-to-A and S-to-A mutations can restore the efficient packaging of longer-sized viral RNA, suggesting that serine phosphorylation at the ARD could contribute negative charge to electrostatic homeostasis required in the capsid interior ( Figs 3 and 4 , and Supplementary Figure S3 ). Similarly, mutant ARD-I + II + III + IV contains the most severe arginine deficiency and encapsidates predominantly spliced mini-RNAs of the shortest size ( Figs 1 and 2 ). ( B ) Hyper-phosphorylation of HBc is responsible for the biogenesis and maintenance of empty capsids (without encapsidated RNA) in both insect and human cell systems ( Figs 5 and 6 ). Empty capsids can also be expressed in E. coli by engineering a mutant S7D bearing 7 phosphorylation-mimicking aspartic acids at HBc ARD ( Fig. 5 ). ( C ) WT HBV with double phosphorylations at S162 and S170 of HBc is believed to preferentially encapsidated 3.5 kb pgRNA 37 38 41 . In contrast, mutant S3E, containing three S-to-E mutations at the major phosphorylation sites (S155E, S162E and S170E) preferentially encapsidated spliced viral RNA 39 . Mutant S3A contains three de-phosphorylation mimicking mutations at three major phosphorylation sites (S155A, S162A, and S170A), and is known to be deficient in viral RNA encapsidation 37 38 41 . Interestingly, in the replicon context in HuH-7 cells, mutants S3A, S5A, and S7A can package increasing amounts of cellular RNA ( Fig. 6F ). Furthermore, we predict here that when all three major sites are phosphorylated, the 2.2 kb spliced RNA (1959 nt in Fig. 2E ) will be more fitting for encapsidation than the non-spliced 3.5 kb pgRNA. Grey: wild type serine; black: phosphorylated serine; orange: S-to-E mutations; green: S-to-A mutations.

    Journal: Scientific Reports

    Article Title: HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids

    doi: 10.1038/srep38959

    Figure Lengend Snippet: A cartoon summary of three major approaches used here in testing the HBV charge balance hypothesis. (A) The short RNA phenotype in arginine-deficient HBc mutants (e.g., ARD-III + IV) can be rescued successfully by using a charge rebalance approach ( Fig. 3A ). Both E-to-A and S-to-A mutations can restore the efficient packaging of longer-sized viral RNA, suggesting that serine phosphorylation at the ARD could contribute negative charge to electrostatic homeostasis required in the capsid interior ( Figs 3 and 4 , and Supplementary Figure S3 ). Similarly, mutant ARD-I + II + III + IV contains the most severe arginine deficiency and encapsidates predominantly spliced mini-RNAs of the shortest size ( Figs 1 and 2 ). ( B ) Hyper-phosphorylation of HBc is responsible for the biogenesis and maintenance of empty capsids (without encapsidated RNA) in both insect and human cell systems ( Figs 5 and 6 ). Empty capsids can also be expressed in E. coli by engineering a mutant S7D bearing 7 phosphorylation-mimicking aspartic acids at HBc ARD ( Fig. 5 ). ( C ) WT HBV with double phosphorylations at S162 and S170 of HBc is believed to preferentially encapsidated 3.5 kb pgRNA 37 38 41 . In contrast, mutant S3E, containing three S-to-E mutations at the major phosphorylation sites (S155E, S162E and S170E) preferentially encapsidated spliced viral RNA 39 . Mutant S3A contains three de-phosphorylation mimicking mutations at three major phosphorylation sites (S155A, S162A, and S170A), and is known to be deficient in viral RNA encapsidation 37 38 41 . Interestingly, in the replicon context in HuH-7 cells, mutants S3A, S5A, and S7A can package increasing amounts of cellular RNA ( Fig. 6F ). Furthermore, we predict here that when all three major sites are phosphorylated, the 2.2 kb spliced RNA (1959 nt in Fig. 2E ) will be more fitting for encapsidation than the non-spliced 3.5 kb pgRNA. Grey: wild type serine; black: phosphorylated serine; orange: S-to-E mutations; green: S-to-A mutations.

    Article Snippet: For RT-PCR, 55 μM of oligo (dT)18 and 5 μg of core-associated RNAs were used for reverse transcription reaction by SuperScript III RT kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Mutagenesis, De-Phosphorylation Assay

    MTase-defective rVSV (mtdVSV)-based vaccine induces high levels of ZIKV-specific antibody in A129 mice. A129 mice were immunized intramuscularly with pCI, pCI-prM-E, or pCI-NS1 at a dose of 50 µg DNA per mouse, and were boosted with the same plasmid at the same dose two weeks later. For VSV-based vaccines, mice were administered intramuscularly using a single dose (1 × 10 5 PFU). The body weight for each mouse was evaluated at indicated time points ( a ). The average body weights of five mice were shown. At day 7, two out of five mice in rVSV-prM-E-NS1 group were dead and the other three terminated at day 10. After immunization, blood samples were collected at weeks 1 and 3. ZIKV E-specific antibody was measured by ELISA at weeks 1 ( b ) and 3 ( c ) post-immunization. ZIKV-specific neutralizing Ab was measured at weeks 1 ( d ) and 3 ( e ) post-immunization. ZIKV NS1-specific Ab was measured by ELISA at weeks 1 ( f ) and 3 ( g ) post-immunization. ELISA titers shown are GMT of 5 mice ± standard deviation. At the termination of this experiment, brains were collected and the presence of the VSV RNA was quantified by real-time RT-PCR using primers annealing to the VSV L gene ( h ). Antibody and viral load data are expressed as the GMT of five mice (black bars). Exact P value (by Student’s t -test) in each panel: b **** P = 1.36 × 10 −6 ; d **** P = 5.44 × 10 −5 ; g **** P = 6.70 × 10 −5 ; h **** P = 4.32 × 10 −6 , N.S., not significant

    Journal: Nature Communications

    Article Title: A Zika virus vaccine expressing premembrane-envelope-NS1 polyprotein

    doi: 10.1038/s41467-018-05276-4

    Figure Lengend Snippet: MTase-defective rVSV (mtdVSV)-based vaccine induces high levels of ZIKV-specific antibody in A129 mice. A129 mice were immunized intramuscularly with pCI, pCI-prM-E, or pCI-NS1 at a dose of 50 µg DNA per mouse, and were boosted with the same plasmid at the same dose two weeks later. For VSV-based vaccines, mice were administered intramuscularly using a single dose (1 × 10 5 PFU). The body weight for each mouse was evaluated at indicated time points ( a ). The average body weights of five mice were shown. At day 7, two out of five mice in rVSV-prM-E-NS1 group were dead and the other three terminated at day 10. After immunization, blood samples were collected at weeks 1 and 3. ZIKV E-specific antibody was measured by ELISA at weeks 1 ( b ) and 3 ( c ) post-immunization. ZIKV-specific neutralizing Ab was measured at weeks 1 ( d ) and 3 ( e ) post-immunization. ZIKV NS1-specific Ab was measured by ELISA at weeks 1 ( f ) and 3 ( g ) post-immunization. ELISA titers shown are GMT of 5 mice ± standard deviation. At the termination of this experiment, brains were collected and the presence of the VSV RNA was quantified by real-time RT-PCR using primers annealing to the VSV L gene ( h ). Antibody and viral load data are expressed as the GMT of five mice (black bars). Exact P value (by Student’s t -test) in each panel: b **** P = 1.36 × 10 −6 ; d **** P = 5.44 × 10 −5 ; g **** P = 6.70 × 10 −5 ; h **** P = 4.32 × 10 −6 , N.S., not significant

    Article Snippet: Reverse transcription (RT) was conducted using a primer (5′-CTCGTCTCTTCTTCTCCTTCCTAGCATTGA-3′) targeting the capsid (C) gene of ZIKV and the Superscript III transcriptase kit (Invitrogen, Carlsbad, CA).

    Techniques: Mouse Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Quantitative RT-PCR

    Recombinant rVSV expressing ZIKV antigens are immunogenic in mice. a Expression of ZIKV E truncations by VSV vector. BSRT7 cells were infected with each recombinant virus expressing ZIKV antigen at an MOI of 3.0. At 16 h post-infection, cells were lysed in 500 μl of lysis buffer, and 10 μl of lysate was analyzed by SDS-PAGE and were blotted with anti-ZIKV E protein monoclonal antibody. b Expression of full-length ZIKV E protein by VSV vector. BSRT7 cells were infected with the indicated recombinant virus expressing ZIKV antigen at an MOI of 3.0. Cell lysates were harvested at 16 h post-infection, and analyzed by western blot. c Expression of NS1 protein by VSV vector. Same cell lysates from b were subjected to western blot analysis using anti-ZIKV NS1 antibody. d Comparison of the expression of ZIKV E truncations with or without anchor C signal peptide by VSV vector. BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. Cell lysates were harvested at 16 h post-infection, and analyzed by western blot. e Kinetics of ZIKV E protein expression by the VSV vector. Top panel: BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. Cytoplasmic extracts were harvested at the indicated time points. Equal amounts of total cytoplasmic lysate were analyzed by SDS-PAGE, followed by western blot analysis. Bottom panel: Equal amounts of total cytoplasmic lysate were blotted with anti-β-actin antibody. f Electron microscopy analysis of ZIKV virus-like particles (VLPs). ZIKV VLPs were negatively stained with 1% ammonium molybdate and visualized by a transmission electron microscope. rVSV-prM-E crude indicates a mixture of ZIKV VLPs and VSV virions from supernatant harvested from BSRT7 cells infected by rVSV-prM-E. ZIKV VLPs were further purified from rVSV-prM-E or rVSV-prM-E-NS1-infected cells. No VLPs were found in rVSV-E. ZIKV Cambodian strain was grown in Vero cells, purified, and used as a control. The yellow arrow indicates a VSV particle; white arrows indicate ZIKV VLPs; and yellow stars indicate ZIKV virions. g Kinetics of ZIKV-specific ELISA antibody induced by rVSV expressing ZIKV antigens. Groups of five female BALB/c mice were inoculated intranasally with a single dose (10 6 PFU) of rVSV or rVSV expressing ZIKV antigens. For DNA vaccine, mice were immunized intramuscularly with 50 µg of pCI-prM-E, and boosted with same dose two weeks later. Serum samples were collected weekly and analyzed by ELISA for ZIKV-specific serum IgG Ab. Data are expressed as the geometric mean titers (GMT) of five mice ± standard deviation. Western blots shown are the representatives of three independent experiments

    Journal: Nature Communications

    Article Title: A Zika virus vaccine expressing premembrane-envelope-NS1 polyprotein

    doi: 10.1038/s41467-018-05276-4

    Figure Lengend Snippet: Recombinant rVSV expressing ZIKV antigens are immunogenic in mice. a Expression of ZIKV E truncations by VSV vector. BSRT7 cells were infected with each recombinant virus expressing ZIKV antigen at an MOI of 3.0. At 16 h post-infection, cells were lysed in 500 μl of lysis buffer, and 10 μl of lysate was analyzed by SDS-PAGE and were blotted with anti-ZIKV E protein monoclonal antibody. b Expression of full-length ZIKV E protein by VSV vector. BSRT7 cells were infected with the indicated recombinant virus expressing ZIKV antigen at an MOI of 3.0. Cell lysates were harvested at 16 h post-infection, and analyzed by western blot. c Expression of NS1 protein by VSV vector. Same cell lysates from b were subjected to western blot analysis using anti-ZIKV NS1 antibody. d Comparison of the expression of ZIKV E truncations with or without anchor C signal peptide by VSV vector. BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. Cell lysates were harvested at 16 h post-infection, and analyzed by western blot. e Kinetics of ZIKV E protein expression by the VSV vector. Top panel: BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. Cytoplasmic extracts were harvested at the indicated time points. Equal amounts of total cytoplasmic lysate were analyzed by SDS-PAGE, followed by western blot analysis. Bottom panel: Equal amounts of total cytoplasmic lysate were blotted with anti-β-actin antibody. f Electron microscopy analysis of ZIKV virus-like particles (VLPs). ZIKV VLPs were negatively stained with 1% ammonium molybdate and visualized by a transmission electron microscope. rVSV-prM-E crude indicates a mixture of ZIKV VLPs and VSV virions from supernatant harvested from BSRT7 cells infected by rVSV-prM-E. ZIKV VLPs were further purified from rVSV-prM-E or rVSV-prM-E-NS1-infected cells. No VLPs were found in rVSV-E. ZIKV Cambodian strain was grown in Vero cells, purified, and used as a control. The yellow arrow indicates a VSV particle; white arrows indicate ZIKV VLPs; and yellow stars indicate ZIKV virions. g Kinetics of ZIKV-specific ELISA antibody induced by rVSV expressing ZIKV antigens. Groups of five female BALB/c mice were inoculated intranasally with a single dose (10 6 PFU) of rVSV or rVSV expressing ZIKV antigens. For DNA vaccine, mice were immunized intramuscularly with 50 µg of pCI-prM-E, and boosted with same dose two weeks later. Serum samples were collected weekly and analyzed by ELISA for ZIKV-specific serum IgG Ab. Data are expressed as the geometric mean titers (GMT) of five mice ± standard deviation. Western blots shown are the representatives of three independent experiments

    Article Snippet: Reverse transcription (RT) was conducted using a primer (5′-CTCGTCTCTTCTTCTCCTTCCTAGCATTGA-3′) targeting the capsid (C) gene of ZIKV and the Superscript III transcriptase kit (Invitrogen, Carlsbad, CA).

    Techniques: Recombinant, Expressing, Mouse Assay, Plasmid Preparation, Infection, Lysis, SDS Page, Western Blot, Electron Microscopy, Staining, Transmission Assay, Microscopy, Purification, Enzyme-linked Immunosorbent Assay, Standard Deviation

    ZIKV antigen expression and antibody response by MTase-defective rVSV (mtdVSV) vector. a Single-step growth curve of mtdVSVs. Confluent BSRT7 cells were infected with individual viruses at an MOI of 3.0. After 1 h of incubation, the inoculum was removed, the cells were washed with DMEM, and fresh medium (containing 2% fetal bovine serum) was added, followed by incubation at 37 °C. Samples of supernatant were harvested at the indicated intervals over a 48-h time period, and the viral titer was determined by plaque assay. Data are the GMT of three independent experiments ± standard deviation. b Expression of ZIKV antigens by the mtdVSV in cell lysates. BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. At 24 h post-infection, cell lysates were harvested and analyzed by western blot using antibody against ZIKV E or NS1 protein. c Kinetics of ZIKV E protein expression in cell lysates. Top panel: BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. At 12, 24, and 36 h post-infection, cell lysates were harvested and analyzed by western blot using antibody against ZIKV E protein. Bottom panel: Equal amounts of total cytoplasmic lysate were blotted with anti-β-actin antibody. d Kinetics of ZIKV E protein release into cell culture supernatants. Cell culture supernatants were harvested from virus-infected cells at the indicated time points, and 10 µl of supernatant was analyzed by western blot using E-specific antibody. e ZIKV NS1 protein released into the cell culture supernatant. Cell culture supernatants were harvested from virus-infected cells at 36 h post-infection, and 10 µl of supernatant was analyzed by western blot using ZIKV serum antibody. f Dynamics of mouse body weight after inoculation with mtdVSV. Five six-week-old female BALB/c mice in each group were intranasally inoculated with DMEM or 10 6 PFU of rVSV or mtdVSV expressing ZIKV antigens. The body weight for each mouse was evaluated at indicated time points. The average body weights of five mice were shown. All mice in rVSV group were dead and euthanized at day 7. g Kinetics of ZIKV specific antibody induced by mtdVSV expressing ZIKV antigen. Serum samples were collected weekly and analyzed by ELISA for ZIKV-specific serum IgG Ab. The titers are expressed as the GMT of five mice ± standard deviation. h ZIKV specific neutralizing antibody titer at week 5 post-inoculation. i ZIKV NS1-specific antibody detected by ELISA at week 5 post-inoculation. The western blot gels presented are a representative of three independent experiments. Mouse body weights are mean of five mice ± standard deviation

    Journal: Nature Communications

    Article Title: A Zika virus vaccine expressing premembrane-envelope-NS1 polyprotein

    doi: 10.1038/s41467-018-05276-4

    Figure Lengend Snippet: ZIKV antigen expression and antibody response by MTase-defective rVSV (mtdVSV) vector. a Single-step growth curve of mtdVSVs. Confluent BSRT7 cells were infected with individual viruses at an MOI of 3.0. After 1 h of incubation, the inoculum was removed, the cells were washed with DMEM, and fresh medium (containing 2% fetal bovine serum) was added, followed by incubation at 37 °C. Samples of supernatant were harvested at the indicated intervals over a 48-h time period, and the viral titer was determined by plaque assay. Data are the GMT of three independent experiments ± standard deviation. b Expression of ZIKV antigens by the mtdVSV in cell lysates. BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. At 24 h post-infection, cell lysates were harvested and analyzed by western blot using antibody against ZIKV E or NS1 protein. c Kinetics of ZIKV E protein expression in cell lysates. Top panel: BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. At 12, 24, and 36 h post-infection, cell lysates were harvested and analyzed by western blot using antibody against ZIKV E protein. Bottom panel: Equal amounts of total cytoplasmic lysate were blotted with anti-β-actin antibody. d Kinetics of ZIKV E protein release into cell culture supernatants. Cell culture supernatants were harvested from virus-infected cells at the indicated time points, and 10 µl of supernatant was analyzed by western blot using E-specific antibody. e ZIKV NS1 protein released into the cell culture supernatant. Cell culture supernatants were harvested from virus-infected cells at 36 h post-infection, and 10 µl of supernatant was analyzed by western blot using ZIKV serum antibody. f Dynamics of mouse body weight after inoculation with mtdVSV. Five six-week-old female BALB/c mice in each group were intranasally inoculated with DMEM or 10 6 PFU of rVSV or mtdVSV expressing ZIKV antigens. The body weight for each mouse was evaluated at indicated time points. The average body weights of five mice were shown. All mice in rVSV group were dead and euthanized at day 7. g Kinetics of ZIKV specific antibody induced by mtdVSV expressing ZIKV antigen. Serum samples were collected weekly and analyzed by ELISA for ZIKV-specific serum IgG Ab. The titers are expressed as the GMT of five mice ± standard deviation. h ZIKV specific neutralizing antibody titer at week 5 post-inoculation. i ZIKV NS1-specific antibody detected by ELISA at week 5 post-inoculation. The western blot gels presented are a representative of three independent experiments. Mouse body weights are mean of five mice ± standard deviation

    Article Snippet: Reverse transcription (RT) was conducted using a primer (5′-CTCGTCTCTTCTTCTCCTTCCTAGCATTGA-3′) targeting the capsid (C) gene of ZIKV and the Superscript III transcriptase kit (Invitrogen, Carlsbad, CA).

    Techniques: Expressing, Plasmid Preparation, Infection, Incubation, Plaque Assay, Standard Deviation, Recombinant, Western Blot, Cell Culture, Mouse Assay, Enzyme-linked Immunosorbent Assay

    MTase-defective rVSV (mtdVSV)-based vaccine induces ZIKV-specific T helper cell responses. Six-week-old BALB/c mice were immunized with each vaccine candidate (5 mice per group). Mice were euthanized at day 35 post-immunization, the spleen was isolated from each mouse, homogenized, a cell suspension prepared, split into three wells (triplicate per mouse) and cultured in 96-well microtiter plates in the presence of 20 µg/ml of ZIKV E protein for 5 days. a Proliferation of CD4+ T cells. The frequencies of ZIKV-specific Th1 (IFN-γ + CD4 + and TNF-α + CD4 + ) ( b ), Th2 cells (IL-4 + CD4 + , IL-5 + CD4 + ) ( c ), Th17 (IL-17A + CD4 + ) ( d ), and Tfh (IL-21 + CD4 + ) ( e ) cells were determined by flow cytometry after intracellular staining with the corresponding anti-cytokine. Data were expressed as mean % positive cells (the mean of 15 samples: 3 wells × 5 mice) ± standard deviation. Asterisk indicates that the group was statistically different with unstimulated and DMEM groups. P -value in from left to right for each panel by Student’s t -test: a **** P = 3.55 × 10 −9 , **** P = 4.10 × 10 −6 , **** P = 4.21 × 10 −7 . b ** P = 0.00676, ** P = 0.00394, **** P = 7.58 × 10 −6 , **** P = 3.32 × 10 −5 . c * P = 0.0243, ** P = 0.00180, * P = 0.0304, * P = 0.0149, *** P = 0.000409, **** P = 7.72 × 10 −6 , * P = 0.0102. d ** P = 0.00749, **** P = 2.52 × 10 −6 , *** P = 0.000907. e *** P = 0.000313, *** P = 0.000162

    Journal: Nature Communications

    Article Title: A Zika virus vaccine expressing premembrane-envelope-NS1 polyprotein

    doi: 10.1038/s41467-018-05276-4

    Figure Lengend Snippet: MTase-defective rVSV (mtdVSV)-based vaccine induces ZIKV-specific T helper cell responses. Six-week-old BALB/c mice were immunized with each vaccine candidate (5 mice per group). Mice were euthanized at day 35 post-immunization, the spleen was isolated from each mouse, homogenized, a cell suspension prepared, split into three wells (triplicate per mouse) and cultured in 96-well microtiter plates in the presence of 20 µg/ml of ZIKV E protein for 5 days. a Proliferation of CD4+ T cells. The frequencies of ZIKV-specific Th1 (IFN-γ + CD4 + and TNF-α + CD4 + ) ( b ), Th2 cells (IL-4 + CD4 + , IL-5 + CD4 + ) ( c ), Th17 (IL-17A + CD4 + ) ( d ), and Tfh (IL-21 + CD4 + ) ( e ) cells were determined by flow cytometry after intracellular staining with the corresponding anti-cytokine. Data were expressed as mean % positive cells (the mean of 15 samples: 3 wells × 5 mice) ± standard deviation. Asterisk indicates that the group was statistically different with unstimulated and DMEM groups. P -value in from left to right for each panel by Student’s t -test: a **** P = 3.55 × 10 −9 , **** P = 4.10 × 10 −6 , **** P = 4.21 × 10 −7 . b ** P = 0.00676, ** P = 0.00394, **** P = 7.58 × 10 −6 , **** P = 3.32 × 10 −5 . c * P = 0.0243, ** P = 0.00180, * P = 0.0304, * P = 0.0149, *** P = 0.000409, **** P = 7.72 × 10 −6 , * P = 0.0102. d ** P = 0.00749, **** P = 2.52 × 10 −6 , *** P = 0.000907. e *** P = 0.000313, *** P = 0.000162

    Article Snippet: Reverse transcription (RT) was conducted using a primer (5′-CTCGTCTCTTCTTCTCCTTCCTAGCATTGA-3′) targeting the capsid (C) gene of ZIKV and the Superscript III transcriptase kit (Invitrogen, Carlsbad, CA).

    Techniques: Mouse Assay, Isolation, Cell Culture, Flow Cytometry, Cytometry, Staining, Standard Deviation

    The MCMV m152 protein specifically targets STING‐dependent signaling 293T cells were co‐transfected with expression plasmids for Cherry‐STING, the murine IFNβ‐luciferase reporter (IFNβ‐Luc), a Renilla luciferase normalization control (pRL‐TK), and the indicated expression plasmids or empty vector (ev). Cells were additionally co‐transfected with expression plasmids for cGAS‐GFP (stimulated) or IRES‐GFP (unstimulated). 20 hours post‐transfection, cells were lysed and a dual‐luciferase assay was performed. An expression plasmid for RIG‐I N (stimulated) or ev (unstimulated) was co‐transfected with IFNβ‐Luc, pRL‐TK and the indicated expression plasmids in 293T cells and analyzed as in (A). An expression plasmid for TBK1 (stimulated) or ev (unstimulated) was co‐transfected together with IFNβ‐Luc, pRL‐TK and the indicated expression plasmids and analyzed as in (A). 293T cells were co‐transfected with a plasmid expressing constitutively active IRF3 (IRF3‐5D; stimulated) or IRES‐GFP (unstimulated) together with IFNβ‐Luc, pRL‐TK and the indicated expression plasmids and analyzed as in (A). The ISG56‐luciferase reporter, pRL‐TK, and the indicated expression plasmids were co‐transfected in 293T cells. 24 hours post‐transfection, cells were stimulated with 0.1 ng/μl human IFNβ or mock stimulated and analyzed 16 h later as described in (A). iMEF gt/gt stably expressing Cherry‐STING and either ev or V5‐tagged m152 were stimulated with 5 μg/ml ISD (F), 10 μg/ml poly(I:C) (G), or mock stimulated with Lipofectamine. 4 hours post‐stimulation, RNA was extracted to determine IFNβ mRNA transcripts by qRT–PCR. iBMDM stably expressing ev or m152‐V5 were stimulated in duplicates with 10 μg/ml cGAMP (H), 5 μg/ml ISD (I), Newcastle disease virus (NDV) infection (J), or 1 μM CpG DNA (K). 6 (H) or 16 (I‐K) hours later, secreted IFNβ (H‐J) or TNFα (K) levels were determined by ELISA. Data information: (A‐G) Data are combined from three independent experiments. (H‐K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t ‐test (unpaired, two‐tailed), n.s. not significant, * P

    Journal: The EMBO Journal

    Article Title: The herpesviral antagonist m152 reveals differential activation of STING‐dependent IRF and NF‐κB signaling and STING's dual role during MCMV infection

    doi: 10.15252/embj.2018100983

    Figure Lengend Snippet: The MCMV m152 protein specifically targets STING‐dependent signaling 293T cells were co‐transfected with expression plasmids for Cherry‐STING, the murine IFNβ‐luciferase reporter (IFNβ‐Luc), a Renilla luciferase normalization control (pRL‐TK), and the indicated expression plasmids or empty vector (ev). Cells were additionally co‐transfected with expression plasmids for cGAS‐GFP (stimulated) or IRES‐GFP (unstimulated). 20 hours post‐transfection, cells were lysed and a dual‐luciferase assay was performed. An expression plasmid for RIG‐I N (stimulated) or ev (unstimulated) was co‐transfected with IFNβ‐Luc, pRL‐TK and the indicated expression plasmids in 293T cells and analyzed as in (A). An expression plasmid for TBK1 (stimulated) or ev (unstimulated) was co‐transfected together with IFNβ‐Luc, pRL‐TK and the indicated expression plasmids and analyzed as in (A). 293T cells were co‐transfected with a plasmid expressing constitutively active IRF3 (IRF3‐5D; stimulated) or IRES‐GFP (unstimulated) together with IFNβ‐Luc, pRL‐TK and the indicated expression plasmids and analyzed as in (A). The ISG56‐luciferase reporter, pRL‐TK, and the indicated expression plasmids were co‐transfected in 293T cells. 24 hours post‐transfection, cells were stimulated with 0.1 ng/μl human IFNβ or mock stimulated and analyzed 16 h later as described in (A). iMEF gt/gt stably expressing Cherry‐STING and either ev or V5‐tagged m152 were stimulated with 5 μg/ml ISD (F), 10 μg/ml poly(I:C) (G), or mock stimulated with Lipofectamine. 4 hours post‐stimulation, RNA was extracted to determine IFNβ mRNA transcripts by qRT–PCR. iBMDM stably expressing ev or m152‐V5 were stimulated in duplicates with 10 μg/ml cGAMP (H), 5 μg/ml ISD (I), Newcastle disease virus (NDV) infection (J), or 1 μM CpG DNA (K). 6 (H) or 16 (I‐K) hours later, secreted IFNβ (H‐J) or TNFα (K) levels were determined by ELISA. Data information: (A‐G) Data are combined from three independent experiments. (H‐K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t ‐test (unpaired, two‐tailed), n.s. not significant, * P

    Article Snippet: For synthesis of cDNA and quantification of gene transcripts, 100 ng of RNA was used per sample and quantitative RT‐PCR was performed using the EXPRESS One‐Step Superscript™ qRT‐PCR Kit (Invitrogen #11781200) on a LightCycler 96 instrument (Roche).

    Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Stable Transfection, Quantitative RT-PCR, Infection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    MCMV lacking m152 induces an elevated type I IFN response leading to lower levels of viral transcripts in vitro Schematic representation of the recombinant MCMV m152stop virus constructed on the MCMV Δm157 backbone (here referred to as parental MCMV and MCMV m152stop for simplicity). Shown is the transcriptional coding region of m152. STOP indicates the introduced 16 base pair (bp) stop cassette. iMEF were infected by centrifugal enhancement with either parental MCMV (par.) or MCMV m152stop at an MOI of 0.5 or mock infected. Three hours post‐infection (hpi), cells were lysed and subjected to immunoblotting (IB) with the specified antibodies. iMEF and iMEF gt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop, or parental MCMV alone, respectively (MOI 0.5). Three hpi lysates were subjected to an anti‐m152 IP, and samples were analyzed by IB with indicated antibodies. IB shown is representative of two independent experiments. Primary BMDM were infected with parental MCMV (par.) or MCMV m152stop at an MOI of 0.1 or mock infected. At 16 hpi, secreted levels of IFNα or IFNβ were quantified by ELISA. Data are representative of two independent experiments. iMEF gt/gt stably expressing Cherry‐STING were infected with parental MCMV or MCMV m152stop (MOI 0.5). Live cell imaging was performed and STING translocation quantified 120 and 180 min post‐stimulation. Data shown are representative of two independent experiments. iMEF or iMEF gt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). At 6 hpi, total RNA was extracted to determine MCMV IE1 and MCMV E1 (F), IFNb1 and IL6 (G) transcripts by qRT–PCR. Data shown are combined from two out of three independent experiments. 293T cells were co‐transfected with Cherry‐STING, the pNF‐κB luciferase reporter, pRL‐TK, cGAS‐GFP (stimulated), or IRES‐GFP (unstimulated) and either ev or m152. Cells were lysed and analyzed as described in Fig 1 . Data are combined from three independent experiments. Data information: Student's t ‐test (unpaired, two‐tailed), n.s. not significant, * P

    Journal: The EMBO Journal

    Article Title: The herpesviral antagonist m152 reveals differential activation of STING‐dependent IRF and NF‐κB signaling and STING's dual role during MCMV infection

    doi: 10.15252/embj.2018100983

    Figure Lengend Snippet: MCMV lacking m152 induces an elevated type I IFN response leading to lower levels of viral transcripts in vitro Schematic representation of the recombinant MCMV m152stop virus constructed on the MCMV Δm157 backbone (here referred to as parental MCMV and MCMV m152stop for simplicity). Shown is the transcriptional coding region of m152. STOP indicates the introduced 16 base pair (bp) stop cassette. iMEF were infected by centrifugal enhancement with either parental MCMV (par.) or MCMV m152stop at an MOI of 0.5 or mock infected. Three hours post‐infection (hpi), cells were lysed and subjected to immunoblotting (IB) with the specified antibodies. iMEF and iMEF gt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop, or parental MCMV alone, respectively (MOI 0.5). Three hpi lysates were subjected to an anti‐m152 IP, and samples were analyzed by IB with indicated antibodies. IB shown is representative of two independent experiments. Primary BMDM were infected with parental MCMV (par.) or MCMV m152stop at an MOI of 0.1 or mock infected. At 16 hpi, secreted levels of IFNα or IFNβ were quantified by ELISA. Data are representative of two independent experiments. iMEF gt/gt stably expressing Cherry‐STING were infected with parental MCMV or MCMV m152stop (MOI 0.5). Live cell imaging was performed and STING translocation quantified 120 and 180 min post‐stimulation. Data shown are representative of two independent experiments. iMEF or iMEF gt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). At 6 hpi, total RNA was extracted to determine MCMV IE1 and MCMV E1 (F), IFNb1 and IL6 (G) transcripts by qRT–PCR. Data shown are combined from two out of three independent experiments. 293T cells were co‐transfected with Cherry‐STING, the pNF‐κB luciferase reporter, pRL‐TK, cGAS‐GFP (stimulated), or IRES‐GFP (unstimulated) and either ev or m152. Cells were lysed and analyzed as described in Fig 1 . Data are combined from three independent experiments. Data information: Student's t ‐test (unpaired, two‐tailed), n.s. not significant, * P

    Article Snippet: For synthesis of cDNA and quantification of gene transcripts, 100 ng of RNA was used per sample and quantitative RT‐PCR was performed using the EXPRESS One‐Step Superscript™ qRT‐PCR Kit (Invitrogen #11781200) on a LightCycler 96 instrument (Roche).

    Techniques: In Vitro, Recombinant, Construct, Infection, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Live Cell Imaging, Translocation Assay, Quantitative RT-PCR, Transfection, Luciferase, Two Tailed Test

    The STING‐mediated NF‐κB response is activated from the ER and specifically pro‐viral for MCMV transcription 293T cells were co‐transfected with Cherry‐STING, pRL‐TK, cGAS‐GFP (stimulated), or IRES‐GFP (unstimulated) and either the pNF‐κB, p55‐CIB, p125, or p125AA luciferase reporter. Data are representative of two independent experiments. 293T cells were co‐transfected with expression plasmids for cGAS‐GFP (stimulated) or ev (unstimulated) together with either cherry‐tagged WT STING or cherry‐tagged K288R STING. Cells were fixed for imaging 24 h post‐transfection. Scale bar represents 10 μm. iMEF gt/gt stably expressing cherry‐tagged WT STING or K288R STING and either ev or V5‐tagged m152 were stimulated with ISD (10 μg/ml) or mock stimulated. At 4 hpi, total RNA was extracted to determine IFNb1 (C) and IL6 (D) transcripts by qRT–PCR. Data shown are combined from three (C) or two (D) out of three independent experiments. iMEF gt/gt (‐) and iMEF gt/gt stably expressing either cherry‐tagged WT STING or K288R STING were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). Six hpi total RNA was extracted to determine MCMV E1 transcripts by qRT–PCR. Data shown are combined from three independent experiments. Data information: Student's t ‐test (unpaired, two‐tailed), n.s. not significant, * P

    Journal: The EMBO Journal

    Article Title: The herpesviral antagonist m152 reveals differential activation of STING‐dependent IRF and NF‐κB signaling and STING's dual role during MCMV infection

    doi: 10.15252/embj.2018100983

    Figure Lengend Snippet: The STING‐mediated NF‐κB response is activated from the ER and specifically pro‐viral for MCMV transcription 293T cells were co‐transfected with Cherry‐STING, pRL‐TK, cGAS‐GFP (stimulated), or IRES‐GFP (unstimulated) and either the pNF‐κB, p55‐CIB, p125, or p125AA luciferase reporter. Data are representative of two independent experiments. 293T cells were co‐transfected with expression plasmids for cGAS‐GFP (stimulated) or ev (unstimulated) together with either cherry‐tagged WT STING or cherry‐tagged K288R STING. Cells were fixed for imaging 24 h post‐transfection. Scale bar represents 10 μm. iMEF gt/gt stably expressing cherry‐tagged WT STING or K288R STING and either ev or V5‐tagged m152 were stimulated with ISD (10 μg/ml) or mock stimulated. At 4 hpi, total RNA was extracted to determine IFNb1 (C) and IL6 (D) transcripts by qRT–PCR. Data shown are combined from three (C) or two (D) out of three independent experiments. iMEF gt/gt (‐) and iMEF gt/gt stably expressing either cherry‐tagged WT STING or K288R STING were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). Six hpi total RNA was extracted to determine MCMV E1 transcripts by qRT–PCR. Data shown are combined from three independent experiments. Data information: Student's t ‐test (unpaired, two‐tailed), n.s. not significant, * P

    Article Snippet: For synthesis of cDNA and quantification of gene transcripts, 100 ng of RNA was used per sample and quantitative RT‐PCR was performed using the EXPRESS One‐Step Superscript™ qRT‐PCR Kit (Invitrogen #11781200) on a LightCycler 96 instrument (Roche).

    Techniques: Transfection, Luciferase, Expressing, Imaging, Stable Transfection, Quantitative RT-PCR, Infection, Two Tailed Test

    STING trafficking and downstream signaling is delayed in iMEF infected with parental MCMV compared to infection with MCMV m152stop iMEF were infected by centrifugal enhancement with parental MCMV at an MOI of 0.5 or mock infected. Cells were lysed at the indicated time points, and lysates were subjected to immunoblotting with antibodies specific to the MCMV proteins immediate‐early protein 1 (IE1) and m152. Tubulin levels were determined with a tubulin antibody. Representative still images from live cell imaging experiments with iMEF gt/gt stably expressing Cherry‐STING infected with the parental MCMV (upper panel) or MCMV m152stop (lower panel) at 120 min post‐infection. iMEF were infected by centrifugal enhancement with parental MCMV or MCMV m152stop at an MOI of 0.1 or mock infected. Cells were lysed at the indicated time points, and lysates were subjected to immunoblotting with specified antibodies. iMEF or iMEF gt/gt were infected by centrifugal enhancement with parental MCMV at an MOI of 0.01. Six hpi, total RNA was extracted and m152 transcript levels were determined by qRT–PCR. Data were normalized to 10 7 cellular β‐actin transcripts and are shown as mean ± SD. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: The herpesviral antagonist m152 reveals differential activation of STING‐dependent IRF and NF‐κB signaling and STING's dual role during MCMV infection

    doi: 10.15252/embj.2018100983

    Figure Lengend Snippet: STING trafficking and downstream signaling is delayed in iMEF infected with parental MCMV compared to infection with MCMV m152stop iMEF were infected by centrifugal enhancement with parental MCMV at an MOI of 0.5 or mock infected. Cells were lysed at the indicated time points, and lysates were subjected to immunoblotting with antibodies specific to the MCMV proteins immediate‐early protein 1 (IE1) and m152. Tubulin levels were determined with a tubulin antibody. Representative still images from live cell imaging experiments with iMEF gt/gt stably expressing Cherry‐STING infected with the parental MCMV (upper panel) or MCMV m152stop (lower panel) at 120 min post‐infection. iMEF were infected by centrifugal enhancement with parental MCMV or MCMV m152stop at an MOI of 0.1 or mock infected. Cells were lysed at the indicated time points, and lysates were subjected to immunoblotting with specified antibodies. iMEF or iMEF gt/gt were infected by centrifugal enhancement with parental MCMV at an MOI of 0.01. Six hpi, total RNA was extracted and m152 transcript levels were determined by qRT–PCR. Data were normalized to 10 7 cellular β‐actin transcripts and are shown as mean ± SD. Source data are available online for this figure.

    Article Snippet: For synthesis of cDNA and quantification of gene transcripts, 100 ng of RNA was used per sample and quantitative RT‐PCR was performed using the EXPRESS One‐Step Superscript™ qRT‐PCR Kit (Invitrogen #11781200) on a LightCycler 96 instrument (Roche).

    Techniques: Infection, Live Cell Imaging, Stable Transfection, Expressing, Quantitative RT-PCR

    In the absence of STING, the impairment of MCMV m152stop in vivo is rescued Type I IFN levels in spleen homogenates (A) and serum (B) and IL‐6 levels in spleen homogenates (C) of C57BL/6J (B6J) or STING −/− mice following i.v. infection with 4 × 10 5 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA. IFNβ levels of STING −/− mice were below the detection limit (n.d.). B6J or STING −/− mice were i.v. infected with 4 × 10 5 PFU parental MCMV or MCMV m152stop. Six hpi, RNA was extracted from spleen homogenates and expression of MCMV IE1 and E1 transcripts was determined by qRT–PCR. B6J or STING −/− mice were i.v. infected with 4 × 10 5 PFU parental MCMV. Six hpi, RNA was extracted from spleen homogenates and m152 transcript levels were determined by qRT–PCR. B6J or STING −/− mice were depleted of NK cells via treatment with anti‐NK1.1 (right panel) or left untreated (left panel). One day after NK cell depletion, B6J or STING −/− mice were i.v. infected with 4 × 10 5 PFU parental MCMV or MCMV m152stop. Sixteen hpi, RNA was extracted from spleen homogenates and MCMV E1 transcript levels were determined by qRT–PCR. Data were normalized to 10 7 cellular β‐actin transcripts (D–F). Data information: n = 5–6 mice per group; Student's t ‐test (unpaired, two‐tailed), n.s. not significant, ** P

    Journal: The EMBO Journal

    Article Title: The herpesviral antagonist m152 reveals differential activation of STING‐dependent IRF and NF‐κB signaling and STING's dual role during MCMV infection

    doi: 10.15252/embj.2018100983

    Figure Lengend Snippet: In the absence of STING, the impairment of MCMV m152stop in vivo is rescued Type I IFN levels in spleen homogenates (A) and serum (B) and IL‐6 levels in spleen homogenates (C) of C57BL/6J (B6J) or STING −/− mice following i.v. infection with 4 × 10 5 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA. IFNβ levels of STING −/− mice were below the detection limit (n.d.). B6J or STING −/− mice were i.v. infected with 4 × 10 5 PFU parental MCMV or MCMV m152stop. Six hpi, RNA was extracted from spleen homogenates and expression of MCMV IE1 and E1 transcripts was determined by qRT–PCR. B6J or STING −/− mice were i.v. infected with 4 × 10 5 PFU parental MCMV. Six hpi, RNA was extracted from spleen homogenates and m152 transcript levels were determined by qRT–PCR. B6J or STING −/− mice were depleted of NK cells via treatment with anti‐NK1.1 (right panel) or left untreated (left panel). One day after NK cell depletion, B6J or STING −/− mice were i.v. infected with 4 × 10 5 PFU parental MCMV or MCMV m152stop. Sixteen hpi, RNA was extracted from spleen homogenates and MCMV E1 transcript levels were determined by qRT–PCR. Data were normalized to 10 7 cellular β‐actin transcripts (D–F). Data information: n = 5–6 mice per group; Student's t ‐test (unpaired, two‐tailed), n.s. not significant, ** P

    Article Snippet: For synthesis of cDNA and quantification of gene transcripts, 100 ng of RNA was used per sample and quantitative RT‐PCR was performed using the EXPRESS One‐Step Superscript™ qRT‐PCR Kit (Invitrogen #11781200) on a LightCycler 96 instrument (Roche).

    Techniques: In Vivo, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Two Tailed Test

    Modulatory effect of m152 on the type I IFN response is present in Balb/c mice Primary MEF from B6J or Balb/c mice were infected by centrifugal enhancement with MCMV WT (MW97.01) or MCMV Δm152 at an MOI of 0.1. Four hpi total RNA was extracted to determine IFNb1 and IL6 transcripts by qRT–PCR. Data were normalized to 10 7 cellular β‐actin transcripts and combined from three (left panel) or two (right panel) independent experiments. Balb/c mice were i.v. infected with 1 × 10 6 PFU MCMV WT (MW97.01) or MCMV Δm152. IFNα (left panel) and IL‐6 (right panel) levels in spleen organ homogenates were analyzed 6 hpi by ELISA. Balb/c mice were i.v. infected with 1 × 10 6 PFU MCMV WT (MW97.01) or MCMV Δm152. Six hpi, RNA was extracted from spleen homogenates and MCMV IE1 (left panel) and MCMV E1 (right panel) transcript levels were determined by qRT–PCR. Data were normalized to 10 7 cellular β‐actin transcripts. Data information: (B‐C) n = 6 mice per group; n.s. not significant, * P

    Journal: The EMBO Journal

    Article Title: The herpesviral antagonist m152 reveals differential activation of STING‐dependent IRF and NF‐κB signaling and STING's dual role during MCMV infection

    doi: 10.15252/embj.2018100983

    Figure Lengend Snippet: Modulatory effect of m152 on the type I IFN response is present in Balb/c mice Primary MEF from B6J or Balb/c mice were infected by centrifugal enhancement with MCMV WT (MW97.01) or MCMV Δm152 at an MOI of 0.1. Four hpi total RNA was extracted to determine IFNb1 and IL6 transcripts by qRT–PCR. Data were normalized to 10 7 cellular β‐actin transcripts and combined from three (left panel) or two (right panel) independent experiments. Balb/c mice were i.v. infected with 1 × 10 6 PFU MCMV WT (MW97.01) or MCMV Δm152. IFNα (left panel) and IL‐6 (right panel) levels in spleen organ homogenates were analyzed 6 hpi by ELISA. Balb/c mice were i.v. infected with 1 × 10 6 PFU MCMV WT (MW97.01) or MCMV Δm152. Six hpi, RNA was extracted from spleen homogenates and MCMV IE1 (left panel) and MCMV E1 (right panel) transcript levels were determined by qRT–PCR. Data were normalized to 10 7 cellular β‐actin transcripts. Data information: (B‐C) n = 6 mice per group; n.s. not significant, * P

    Article Snippet: For synthesis of cDNA and quantification of gene transcripts, 100 ng of RNA was used per sample and quantitative RT‐PCR was performed using the EXPRESS One‐Step Superscript™ qRT‐PCR Kit (Invitrogen #11781200) on a LightCycler 96 instrument (Roche).

    Techniques: Mouse Assay, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    In vivo ASO administration was well tolerated a, Left, body weight of individual WT C57BL/6 female mice (2 months old) treated with PBS or ASO measured weekly for 4 wk post-treatment. Right, Change in body weight at each time point relative to body weight at time of treatment. n =4 per group, mean ± s.e.m. b, Percent change in body weight of Pat YFP mice 4 wk post-treatment relative to pre-treatment. n =3-4, mean ± s.e.m. c, Microglial activation was measured by Aif1 qRT-PCR 4 wk post-treatment. CTX, cortex; HIP, hippocampus; SC, thoracic spinal cord. * P

    Journal: Nature

    Article Title: Towards a therapy for Angelman syndrome by reduction of a long non-coding RNA

    doi: 10.1038/nature13975

    Figure Lengend Snippet: In vivo ASO administration was well tolerated a, Left, body weight of individual WT C57BL/6 female mice (2 months old) treated with PBS or ASO measured weekly for 4 wk post-treatment. Right, Change in body weight at each time point relative to body weight at time of treatment. n =4 per group, mean ± s.e.m. b, Percent change in body weight of Pat YFP mice 4 wk post-treatment relative to pre-treatment. n =3-4, mean ± s.e.m. c, Microglial activation was measured by Aif1 qRT-PCR 4 wk post-treatment. CTX, cortex; HIP, hippocampus; SC, thoracic spinal cord. * P

    Article Snippet: For qRT-PCR, approximately 10 ng RNA was added to EXPRESS One-Step SuperScript qRT-PCR Kit (Life Technologies) with Taqman primer and probe sets or EXPRESS One-Step SYBR GreenER Kit (Life Technologies) with SYBR primer sets (see for sequences).

    Techniques: In Vivo, Allele-specific Oligonucleotide, Mouse Assay, Activation Assay, Quantitative RT-PCR

    ASOs targeting Snord116 reduced Ube3a-ATS pre-mRNA a, Upper panel , schematic of the ASO binding sites and location of qRT-PCR primer and probe sets. Lower panel , qRT-PCR from WT primary neurons treated with ASO A or ASO116 (72 h) using primer and probe sets to the indicated regions of Ube3a-ATS pre-mRNA and mRNA. b, Nascent transcripts were isolated from WT primary neurons incubated with 5-ethynyl uridine (see Methods ) for the indicated time. qRT-PCR for pre-mRNA and mature mRNA (HG, host gene) within the Snord116 region. The red line indicates the 30 min delay between the appearance of pre-mRNA and mature mRNA. Assuming a transcription elongation rate of 4 kb/min, it would take RNAPII 80 min to transcribe the 332 kb distance from the last copy of Snord116 to the ASO binding site. n =2 per group, mean ± absolute deviation.

    Journal: Nature

    Article Title: Towards a therapy for Angelman syndrome by reduction of a long non-coding RNA

    doi: 10.1038/nature13975

    Figure Lengend Snippet: ASOs targeting Snord116 reduced Ube3a-ATS pre-mRNA a, Upper panel , schematic of the ASO binding sites and location of qRT-PCR primer and probe sets. Lower panel , qRT-PCR from WT primary neurons treated with ASO A or ASO116 (72 h) using primer and probe sets to the indicated regions of Ube3a-ATS pre-mRNA and mRNA. b, Nascent transcripts were isolated from WT primary neurons incubated with 5-ethynyl uridine (see Methods ) for the indicated time. qRT-PCR for pre-mRNA and mature mRNA (HG, host gene) within the Snord116 region. The red line indicates the 30 min delay between the appearance of pre-mRNA and mature mRNA. Assuming a transcription elongation rate of 4 kb/min, it would take RNAPII 80 min to transcribe the 332 kb distance from the last copy of Snord116 to the ASO binding site. n =2 per group, mean ± absolute deviation.

    Article Snippet: For qRT-PCR, approximately 10 ng RNA was added to EXPRESS One-Step SuperScript qRT-PCR Kit (Life Technologies) with Taqman primer and probe sets or EXPRESS One-Step SYBR GreenER Kit (Life Technologies) with SYBR primer sets (see for sequences).

    Techniques: Allele-specific Oligonucleotide, Binding Assay, Quantitative RT-PCR, Isolation, Incubation

    ASOs complementary to two regions of Ube3a-ATS differed in their ability to unsilence paternal Ube3a Pat YFP primary neurons were treated with ASOs that bind Ube3a-ATS 5′ of Ube3a (non-overlap ASOs, n =15) or that bind to the gene body region (overlap ASOs, n =12) for 72 h. The level of Ube3a YFP -ATS reduction and Ube3a YFP up-regulation was analyzed by qRT-PCR and normalized to untreated control (UTC) neurons. Mean ± s.e.m.

    Journal: Nature

    Article Title: Towards a therapy for Angelman syndrome by reduction of a long non-coding RNA

    doi: 10.1038/nature13975

    Figure Lengend Snippet: ASOs complementary to two regions of Ube3a-ATS differed in their ability to unsilence paternal Ube3a Pat YFP primary neurons were treated with ASOs that bind Ube3a-ATS 5′ of Ube3a (non-overlap ASOs, n =15) or that bind to the gene body region (overlap ASOs, n =12) for 72 h. The level of Ube3a YFP -ATS reduction and Ube3a YFP up-regulation was analyzed by qRT-PCR and normalized to untreated control (UTC) neurons. Mean ± s.e.m.

    Article Snippet: For qRT-PCR, approximately 10 ng RNA was added to EXPRESS One-Step SuperScript qRT-PCR Kit (Life Technologies) with Taqman primer and probe sets or EXPRESS One-Step SYBR GreenER Kit (Life Technologies) with SYBR primer sets (see for sequences).

    Techniques: Quantitative RT-PCR

    ASO treatment in AS mice up-regulated Ube3a a, RNA levels of Ube3a-ATS and Ube3a were determined by qRT-PCR in WT mice treated with PBS and AS mice treated with non-targeting control ASO (ctl ASO), or ASO A. n =2-3 per group, mean ± s.e.m. b, UBE3A immunofluorescence on brain sections was performed 2 to 8 wk post-treatment. c-h, ASO treatment in adult AS mice did not reverse some disease-associated phenotypes. c, Total distance traveled in the open field assay. d, Vertical activity in the open field assay. e, Stereotype activity in the open field assay. f, Marble burying test. The y axis represents the number of marbles at least 50% buried. g, Accelerating rotarod test during eight trials. h, Post-shock and cued response measured during the fear conditioning assay. n =13-15 per group * P

    Journal: Nature

    Article Title: Towards a therapy for Angelman syndrome by reduction of a long non-coding RNA

    doi: 10.1038/nature13975

    Figure Lengend Snippet: ASO treatment in AS mice up-regulated Ube3a a, RNA levels of Ube3a-ATS and Ube3a were determined by qRT-PCR in WT mice treated with PBS and AS mice treated with non-targeting control ASO (ctl ASO), or ASO A. n =2-3 per group, mean ± s.e.m. b, UBE3A immunofluorescence on brain sections was performed 2 to 8 wk post-treatment. c-h, ASO treatment in adult AS mice did not reverse some disease-associated phenotypes. c, Total distance traveled in the open field assay. d, Vertical activity in the open field assay. e, Stereotype activity in the open field assay. f, Marble burying test. The y axis represents the number of marbles at least 50% buried. g, Accelerating rotarod test during eight trials. h, Post-shock and cued response measured during the fear conditioning assay. n =13-15 per group * P

    Article Snippet: For qRT-PCR, approximately 10 ng RNA was added to EXPRESS One-Step SuperScript qRT-PCR Kit (Life Technologies) with Taqman primer and probe sets or EXPRESS One-Step SYBR GreenER Kit (Life Technologies) with SYBR primer sets (see for sequences).

    Techniques: Allele-specific Oligonucleotide, Mouse Assay, Quantitative RT-PCR, CTL Assay, Immunofluorescence, Activity Assay

    Unsilencing of the Ube3a paternal allele by Ube3a-ATS targeted ASOs in cultured mouse neurons a, Schematic mouse Ube3a genomic locus. IC, imprinting center. b, UBE3A YFP fluorescence (arbitrary units, a.u.) in ASO-treated primary neurons relative to untreated control. Ctl ASO, non-targeting control ASO. c, YFP fluorescent imaging of treated Pat YFP neurons. d, Normalized mRNA levels in Pat YFP neurons treated with increasing dose (upper panel) or for increasing time (lower panel). e, Northern blot of Snord116 expression. Snord116 intensity relative to 5.8S rRNA is quantified. f, Normalized mRNA levels of long genes. g, Western blot (upper) and qRT-PCR (lower) from Pat YFP neurons. ASO, inactive is a sequence-matched RNase H inactive ASO. * P

    Journal: Nature

    Article Title: Towards a therapy for Angelman syndrome by reduction of a long non-coding RNA

    doi: 10.1038/nature13975

    Figure Lengend Snippet: Unsilencing of the Ube3a paternal allele by Ube3a-ATS targeted ASOs in cultured mouse neurons a, Schematic mouse Ube3a genomic locus. IC, imprinting center. b, UBE3A YFP fluorescence (arbitrary units, a.u.) in ASO-treated primary neurons relative to untreated control. Ctl ASO, non-targeting control ASO. c, YFP fluorescent imaging of treated Pat YFP neurons. d, Normalized mRNA levels in Pat YFP neurons treated with increasing dose (upper panel) or for increasing time (lower panel). e, Northern blot of Snord116 expression. Snord116 intensity relative to 5.8S rRNA is quantified. f, Normalized mRNA levels of long genes. g, Western blot (upper) and qRT-PCR (lower) from Pat YFP neurons. ASO, inactive is a sequence-matched RNase H inactive ASO. * P

    Article Snippet: For qRT-PCR, approximately 10 ng RNA was added to EXPRESS One-Step SuperScript qRT-PCR Kit (Life Technologies) with Taqman primer and probe sets or EXPRESS One-Step SYBR GreenER Kit (Life Technologies) with SYBR primer sets (see for sequences).

    Techniques: Cell Culture, Fluorescence, Allele-specific Oligonucleotide, CTL Assay, Imaging, Northern Blot, Expressing, Western Blot, Quantitative RT-PCR, Sequencing

    Snord116 was not reduced in the hypothalamus qRT-PCR on RNA isolated from Pat YFP mice 4 wk post-treatment of PBS or ASO B.

    Journal: Nature

    Article Title: Towards a therapy for Angelman syndrome by reduction of a long non-coding RNA

    doi: 10.1038/nature13975

    Figure Lengend Snippet: Snord116 was not reduced in the hypothalamus qRT-PCR on RNA isolated from Pat YFP mice 4 wk post-treatment of PBS or ASO B.

    Article Snippet: For qRT-PCR, approximately 10 ng RNA was added to EXPRESS One-Step SuperScript qRT-PCR Kit (Life Technologies) with Taqman primer and probe sets or EXPRESS One-Step SYBR GreenER Kit (Life Technologies) with SYBR primer sets (see for sequences).

    Techniques: Quantitative RT-PCR, Isolation, Mouse Assay, Allele-specific Oligonucleotide

    Expression of TLR2 and TLR4 in LA-4 murine lung epithelial cells following B. pseudomallei infection using (A) qRT-PCR and (B) flow cytometry. LA-4 cells were infected with KHW for 2 h and control cells were treated with F12K medium. The expression of TLR2 (▪) and TLR4 (▪) genes at 1 h, 4 h and 24 h post infection was quantified by qRT-PCR. Results are normalized to both β-actin and uninfected control cells, and were expressed as normalized fold change (ΔΔC t ) ± standard deviation of experimental triplicates. ** P

    Journal: PLoS ONE

    Article Title: Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection

    doi: 10.1371/journal.pone.0007308

    Figure Lengend Snippet: Expression of TLR2 and TLR4 in LA-4 murine lung epithelial cells following B. pseudomallei infection using (A) qRT-PCR and (B) flow cytometry. LA-4 cells were infected with KHW for 2 h and control cells were treated with F12K medium. The expression of TLR2 (▪) and TLR4 (▪) genes at 1 h, 4 h and 24 h post infection was quantified by qRT-PCR. Results are normalized to both β-actin and uninfected control cells, and were expressed as normalized fold change (ΔΔC t ) ± standard deviation of experimental triplicates. ** P

    Article Snippet: The RNA was then reverse transcribed to cDNA using the SuperScript III platinum two-step qRT-PCR kit (Invitrogen, Carlsbad, California).

    Techniques: Expressing, Infection, Quantitative RT-PCR, Flow Cytometry, Cytometry, Standard Deviation

    Differential expression of antimicrobial peptide genes in the lung of BALB/c mice following B. pseudomallei , KHW infection. Groups of 6 BALB/c mice were treated intranasally with 34 CFU of KHW. Mice in the control group were treated with 1X PBS. At 4 h, 24 h, 48 h and 72 h post KHW infection, RNA was extracted from the lungs of infected and control mice and expression of lysozyme (□), CCL20 (▪) and SLPI (▪) was then determined using qRT-PCR. Results are normalized to both β-actin and uninfected control cells, and were expressed as normalized fold change (ΔΔC t ) ± standard deviation of six mice. All results shown are representation of two independent experiments. ** P

    Journal: PLoS ONE

    Article Title: Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection

    doi: 10.1371/journal.pone.0007308

    Figure Lengend Snippet: Differential expression of antimicrobial peptide genes in the lung of BALB/c mice following B. pseudomallei , KHW infection. Groups of 6 BALB/c mice were treated intranasally with 34 CFU of KHW. Mice in the control group were treated with 1X PBS. At 4 h, 24 h, 48 h and 72 h post KHW infection, RNA was extracted from the lungs of infected and control mice and expression of lysozyme (□), CCL20 (▪) and SLPI (▪) was then determined using qRT-PCR. Results are normalized to both β-actin and uninfected control cells, and were expressed as normalized fold change (ΔΔC t ) ± standard deviation of six mice. All results shown are representation of two independent experiments. ** P

    Article Snippet: The RNA was then reverse transcribed to cDNA using the SuperScript III platinum two-step qRT-PCR kit (Invitrogen, Carlsbad, California).

    Techniques: Expressing, Mouse Assay, Infection, Quantitative RT-PCR, Standard Deviation

    Differential expression of antimicrobial peptide genes in (A) LA-4 murine lung epithelial cell line and primary lung epithelial cells from (B) BALB/c and (C) C57Bl/6 mice following KHW infection. Every 4 bars represent 0 h, 2 h, 6 h and 24 h post infection. Lung epithelial cells were infected with KHW. RNA was extracted from the cells at 0 h, 2 h, 6 h and 24 h post infection and the expression of lysozyme (□), CCL20 (▪) and SLPI (▪) genes was quantified by qRT-PCR. Results are normalized to both β-actin and uninfected control cells, and were expressed as normalized fold change (ΔΔC t ) ± standard deviation of experimental triplicates. All results shown are representation of three independent experiments. ** P

    Journal: PLoS ONE

    Article Title: Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection

    doi: 10.1371/journal.pone.0007308

    Figure Lengend Snippet: Differential expression of antimicrobial peptide genes in (A) LA-4 murine lung epithelial cell line and primary lung epithelial cells from (B) BALB/c and (C) C57Bl/6 mice following KHW infection. Every 4 bars represent 0 h, 2 h, 6 h and 24 h post infection. Lung epithelial cells were infected with KHW. RNA was extracted from the cells at 0 h, 2 h, 6 h and 24 h post infection and the expression of lysozyme (□), CCL20 (▪) and SLPI (▪) genes was quantified by qRT-PCR. Results are normalized to both β-actin and uninfected control cells, and were expressed as normalized fold change (ΔΔC t ) ± standard deviation of experimental triplicates. All results shown are representation of three independent experiments. ** P

    Article Snippet: The RNA was then reverse transcribed to cDNA using the SuperScript III platinum two-step qRT-PCR kit (Invitrogen, Carlsbad, California).

    Techniques: Expressing, Mouse Assay, Infection, Quantitative RT-PCR, Standard Deviation

    Regulation of lysozyme, CCL20 and SLPI by NF-κB and p38 MAPK pathways. LA-4 cells were pre-incubated with or without BAY11-7082 (10 µM) and SB203580 (10 µM) for 1 h prior to KHW infection; without treatment (▪), BAY11-7082 pretreatment (□), SB203580 pretreatment (▪). Total RNA was extracted from the cells at 24 h post KHW infection and the expression of lysozyme, CCL20 and SLPI was determined using qRT-PCR. Results are normalized to both β-actin and uninfected control cells, and were expressed as normalized fold change (ΔΔC t ) ± standard deviation of experimental triplicates. All results shown are representation of three independent experiments. ++ P

    Journal: PLoS ONE

    Article Title: Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection

    doi: 10.1371/journal.pone.0007308

    Figure Lengend Snippet: Regulation of lysozyme, CCL20 and SLPI by NF-κB and p38 MAPK pathways. LA-4 cells were pre-incubated with or without BAY11-7082 (10 µM) and SB203580 (10 µM) for 1 h prior to KHW infection; without treatment (▪), BAY11-7082 pretreatment (□), SB203580 pretreatment (▪). Total RNA was extracted from the cells at 24 h post KHW infection and the expression of lysozyme, CCL20 and SLPI was determined using qRT-PCR. Results are normalized to both β-actin and uninfected control cells, and were expressed as normalized fold change (ΔΔC t ) ± standard deviation of experimental triplicates. All results shown are representation of three independent experiments. ++ P

    Article Snippet: The RNA was then reverse transcribed to cDNA using the SuperScript III platinum two-step qRT-PCR kit (Invitrogen, Carlsbad, California).

    Techniques: Incubation, Infection, Expressing, Quantitative RT-PCR, Standard Deviation

    HCV induces IFN during the first 12 hrs of infection and inhibits it thereafter. Huh7.25.CD81 and Huh7.5 cells were transfected with the pGL2-IFNβ-FLUC/pRL-TK-RLUC reporter plasmids together with plasmids expressing HA-TRIM25 (Huh7.25.CD81; A and B) or RIG-I (Huh7.5; C and D). 24 h post-transfection, the cells were infected with SeV (40 HAU/ml) or JFH1 (m.o.i = 0.2). A and C : 24 hrs post-transfection, the cells were infected with SeV (40 HAU/ml) or JFH1 (m.o.i = 0.2). At the times indicated, cell lysates were prepared and analysed for IFN induction as described in Materials and Methods . The graphs represent the levels of F-luc activity normalized to R-luc RNA expressed as IFN-β fold-induction over control cells that were simply transfected with pGL2-IFNβ-FLUC/pRL-TK-RLUC. Error bars represent the mean ± S.D. for triplicates. In addition, cell lysates from JFH1-infected cells were pooled and analysed for the presence of NS3 as a marker of HCV infection. B and D : 24 hrs post-transfection, Huh7.25.CD81 and Huh7.5 cells were infected with JFH1 (m.o.i = 0.2). At the times indicated, cells were processed for RNA extraction and HCV or IFNβ RNA were quantified by qRT-PCR respectively, and normalized against RNA from GAPDH. Error bars represent the mean ± S.D. for triplicates.

    Journal: PLoS ONE

    Article Title: Hepatitis C Virus Controls Interferon Production through PKR Activation

    doi: 10.1371/journal.pone.0010575

    Figure Lengend Snippet: HCV induces IFN during the first 12 hrs of infection and inhibits it thereafter. Huh7.25.CD81 and Huh7.5 cells were transfected with the pGL2-IFNβ-FLUC/pRL-TK-RLUC reporter plasmids together with plasmids expressing HA-TRIM25 (Huh7.25.CD81; A and B) or RIG-I (Huh7.5; C and D). 24 h post-transfection, the cells were infected with SeV (40 HAU/ml) or JFH1 (m.o.i = 0.2). A and C : 24 hrs post-transfection, the cells were infected with SeV (40 HAU/ml) or JFH1 (m.o.i = 0.2). At the times indicated, cell lysates were prepared and analysed for IFN induction as described in Materials and Methods . The graphs represent the levels of F-luc activity normalized to R-luc RNA expressed as IFN-β fold-induction over control cells that were simply transfected with pGL2-IFNβ-FLUC/pRL-TK-RLUC. Error bars represent the mean ± S.D. for triplicates. In addition, cell lysates from JFH1-infected cells were pooled and analysed for the presence of NS3 as a marker of HCV infection. B and D : 24 hrs post-transfection, Huh7.25.CD81 and Huh7.5 cells were infected with JFH1 (m.o.i = 0.2). At the times indicated, cells were processed for RNA extraction and HCV or IFNβ RNA were quantified by qRT-PCR respectively, and normalized against RNA from GAPDH. Error bars represent the mean ± S.D. for triplicates.

    Article Snippet: Reverse-transcription, amplification and real-time detection of PCR products were performed with 5 µl total RNA samples, using the SuperScript III Platinum one-step qRT-PCR kit (Invitrogen) and an AbiPrism 7700 machine.

    Techniques: Infection, Transfection, Expressing, Hemagglutination Assay, Activity Assay, Marker, RNA Extraction, Quantitative RT-PCR

    PKR positively controls HCV yield through inhibition of the IFN induction pathway. Huh7.25.CD81 cells were first transfected with 25 nM of siRNA directed against PKR or 25 nM of control siRNA and then transfected 24 hrs later with either empty vector or the TRIM25 expressing plasmid. 24 hrs post-transfection, the cells were infected with JFH1 at an m.o.i. of 0.2. At the indicated times, cells were processed for RNA extraction and HCV RNAs were quantified by qRT-PCR and normalized against RNA from GAPDH. Error bars represent the mean ± S.D. for triplicates.

    Journal: PLoS ONE

    Article Title: Hepatitis C Virus Controls Interferon Production through PKR Activation

    doi: 10.1371/journal.pone.0010575

    Figure Lengend Snippet: PKR positively controls HCV yield through inhibition of the IFN induction pathway. Huh7.25.CD81 cells were first transfected with 25 nM of siRNA directed against PKR or 25 nM of control siRNA and then transfected 24 hrs later with either empty vector or the TRIM25 expressing plasmid. 24 hrs post-transfection, the cells were infected with JFH1 at an m.o.i. of 0.2. At the indicated times, cells were processed for RNA extraction and HCV RNAs were quantified by qRT-PCR and normalized against RNA from GAPDH. Error bars represent the mean ± S.D. for triplicates.

    Article Snippet: Reverse-transcription, amplification and real-time detection of PCR products were performed with 5 µl total RNA samples, using the SuperScript III Platinum one-step qRT-PCR kit (Invitrogen) and an AbiPrism 7700 machine.

    Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, Infection, RNA Extraction, Quantitative RT-PCR

    HCV triggers a transient inhibition of protein translation. Huh7.25.CD81 cells were transfected with 400 ng of CAT-IRES HCV -LUC ( A and B ) or 50 ng of CAT-IRES EMCV -LUC ( C and D ), together with the pRL-TK-RLUC plasmid (40 ng) and the HA-TRIM25 expressing plasmid (100 ng). 24 hrs post-transfection, cells were infected with JFH1 at an m.o.i of 0.2. A and C : At the indicated times, total cellular RNA was extracted and F-luc and GAPDH RNA were quantified by qRT-PCR. The graphs represent the number of copies of the IRES HCV -luc (A) or of the IRES EMCV -luc (C) RNA normalized to the number of copies of GAPDH RNA. Error bars represent the mean ± S.D. for triplicates. B and D : At the indicated times, cell lysates were prepared and the IRES HCV -luc or IRES EMCV -luc activity was analysed by a reporter assay. For normalization, the levels of firefly luciferase activity were divided in each case by the ratio R-luc RNA/GAPDH RNA that was calculated after measurement of the R-luc RNA by RTqPCR using the total cellular RNA extracted for A and C. Error bars represent the mean ± S.D. for triplicates.

    Journal: PLoS ONE

    Article Title: Hepatitis C Virus Controls Interferon Production through PKR Activation

    doi: 10.1371/journal.pone.0010575

    Figure Lengend Snippet: HCV triggers a transient inhibition of protein translation. Huh7.25.CD81 cells were transfected with 400 ng of CAT-IRES HCV -LUC ( A and B ) or 50 ng of CAT-IRES EMCV -LUC ( C and D ), together with the pRL-TK-RLUC plasmid (40 ng) and the HA-TRIM25 expressing plasmid (100 ng). 24 hrs post-transfection, cells were infected with JFH1 at an m.o.i of 0.2. A and C : At the indicated times, total cellular RNA was extracted and F-luc and GAPDH RNA were quantified by qRT-PCR. The graphs represent the number of copies of the IRES HCV -luc (A) or of the IRES EMCV -luc (C) RNA normalized to the number of copies of GAPDH RNA. Error bars represent the mean ± S.D. for triplicates. B and D : At the indicated times, cell lysates were prepared and the IRES HCV -luc or IRES EMCV -luc activity was analysed by a reporter assay. For normalization, the levels of firefly luciferase activity were divided in each case by the ratio R-luc RNA/GAPDH RNA that was calculated after measurement of the R-luc RNA by RTqPCR using the total cellular RNA extracted for A and C. Error bars represent the mean ± S.D. for triplicates.

    Article Snippet: Reverse-transcription, amplification and real-time detection of PCR products were performed with 5 µl total RNA samples, using the SuperScript III Platinum one-step qRT-PCR kit (Invitrogen) and an AbiPrism 7700 machine.

    Techniques: Inhibition, Transfection, Plasmid Preparation, Hemagglutination Assay, Expressing, Infection, Quantitative RT-PCR, Activity Assay, Reporter Assay, Luciferase

    Pharmacological inhibitors of PKR increase IFN induction and inhibit HCV infection. A : Huh7.25.CD81 cells were first transfected with the pGL2-IFNβ-FLUC/pRL-TK-RLUC reporter plasmids and the TRIM25 expressing plasmid. 24 hrs post-transfection, the cells were infected with JFH1 at an m.o.i of 0.2. At 11 hrs post-infection, cells were exposed to 200 µM of C16 or 30 µM of the PRI peptide as described in Materials and Methods . At the indicated times, one set of cells was treated for RNA extraction and the other for reporter assay as described in the legend to Figure 3 . IFN expression was expressed as fold-induction over control cells that were simply transfected with pGL2-IFNβ-FLUC/pRL-TK-RLUC. The graph represents the level of firefly luciferase activity normalized to the ratio R-luc RNA/GAPDH RNA. Error bars represent the mean +/- S.D. for triplicates. B : Huh7.25.CD81 cells were first transfected with the TRIM25 expressing plasmid or an empty plasmid. 24 hrs post-transfection, the cells were infected with JFH1 at an m.o.i of 0.2. At 11 hrs post-infection, cells were exposed to 200 µM of C16 or 30 µM of the PRI peptide as described in Materials and Methods . At the indicated times, cell lysates were processed for RNA extraction and HCV RNAs were quantified by qRT-PCR and normalized against RNA from GAPDH. Error bars represent the mean +/- S.D. for duplicates.

    Journal: PLoS ONE

    Article Title: Hepatitis C Virus Controls Interferon Production through PKR Activation

    doi: 10.1371/journal.pone.0010575

    Figure Lengend Snippet: Pharmacological inhibitors of PKR increase IFN induction and inhibit HCV infection. A : Huh7.25.CD81 cells were first transfected with the pGL2-IFNβ-FLUC/pRL-TK-RLUC reporter plasmids and the TRIM25 expressing plasmid. 24 hrs post-transfection, the cells were infected with JFH1 at an m.o.i of 0.2. At 11 hrs post-infection, cells were exposed to 200 µM of C16 or 30 µM of the PRI peptide as described in Materials and Methods . At the indicated times, one set of cells was treated for RNA extraction and the other for reporter assay as described in the legend to Figure 3 . IFN expression was expressed as fold-induction over control cells that were simply transfected with pGL2-IFNβ-FLUC/pRL-TK-RLUC. The graph represents the level of firefly luciferase activity normalized to the ratio R-luc RNA/GAPDH RNA. Error bars represent the mean +/- S.D. for triplicates. B : Huh7.25.CD81 cells were first transfected with the TRIM25 expressing plasmid or an empty plasmid. 24 hrs post-transfection, the cells were infected with JFH1 at an m.o.i of 0.2. At 11 hrs post-infection, cells were exposed to 200 µM of C16 or 30 µM of the PRI peptide as described in Materials and Methods . At the indicated times, cell lysates were processed for RNA extraction and HCV RNAs were quantified by qRT-PCR and normalized against RNA from GAPDH. Error bars represent the mean +/- S.D. for duplicates.

    Article Snippet: Reverse-transcription, amplification and real-time detection of PCR products were performed with 5 µl total RNA samples, using the SuperScript III Platinum one-step qRT-PCR kit (Invitrogen) and an AbiPrism 7700 machine.

    Techniques: Infection, Transfection, Expressing, Plasmid Preparation, RNA Extraction, Reporter Assay, Luciferase, Activity Assay, Quantitative RT-PCR