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  • 99
    Thermo Fisher reference snrna specific dna oligonucleotide
    Reference Snrna Specific Dna Oligonucleotide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore gene specific sirnas
    LKB1 Is the Main Cargo Regulated by the Ran/RanBP1 Pathway during Axonogenesis (A and B) Rat cortical neuron transfected with control <t>siRNAs</t> and the eGFP -reporter (A) and siRNAs targeting LKB1 and the eGFP -reporter plasmid (B). (C) 72 hr cortical neuron overexpressing STK25-red fluorescent protein (RFP) and scrambled <t>siRNA</t> together with an eGFP -reporter plasmid. (D) 72 hr cortical neuron overexpressing STK25-RFP and ranBP1 -siRNA together with the eGFP -reporter plasmid. (E and F) 72 hr cortical neuron transfected with scrambled siRNA and the eGFP -reporter (E) and 72 hr cortical neuron transfected with siRNAs targeting ranBP1 and the eGFP -reporter plasmid (F). (G) 72 hr cortical neuron transfected with siRNAs ranBP1 , STK25-RFP , and an eGFP -reporter plasmid. (H) The mean percentage of 72 hr neurons with 0, 1, or ≥2 axons over three independent replicates were determined for each condition. The absence of axon in ranBP1 and LKB1 siRNA neurons is rescued by expressing STK25-RFP . At least 24 neurons were scored for the number of axons for each condition. (I) Sholl analysis of siRNA control (n = 15), ranBP1 -siRNAs neurons (n = 18), and ranBP1 -siRNAs neurons rescued by overexpressing STK25-RFP (n = 21) was performed 72 hr after transfection. A statistically significant decrease in intersection numbers was observed for the rescue experiment between 10 and 25 μm from the cell body (at 10, 15, 20, and 25 μm ∗ p
    Gene Specific Sirnas, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery gene specific sirna smart pools
    Loss of <t>αSNAP</t> triggers detachment of human intestinal epithelial cells. Control SK-CO15 cells ( SK-neo ) and cells stably expressing <t>siRNA-resistant</t> bovine αSNAP ( SK -α SNAP ) were transfected with either control or two human αSNAP-specific siRNA duplexes ( D1 and D2 ). A, expression of αSNAP and its binding partner NSF was determined 72 h post-transfection. B and C , cell detachment was examined by counting adhered and total number of cells using bright field microscopy 72 h post-transfection. Data are presented as the mean ± S.E. ( n = 3); *, p
    Gene Specific Sirna Smart Pools, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology gene specific sirna against rnf43
    Host factor <t>RNF43</t> decreases IAV replication. ( a–c ) HEK293T cells were transiently transfected with plasmid pCDNA3.1 (Mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) (1 and 2 μ g) followed by PR8 infection and incubated for 24 h. Total RNA was isolated and NP mRNA ( a ), NP vRNA ( b ) and RNF43 mRNA( c ) levels were estimated through qRT PCR. ( d ) HEK293T cells were treated with NT <t>siRNA</t> or RNF43 siRNA for 24 h followed by PR8 IAV infection for next 24 h. Cells were processed for qRT-PCR analysis of mRNA levels of NP and RNF43. ( e ) A549 cells, pretreated with either NT or RNF43 siRNA were transfected with plasmids-encoding polymerase complex components (PA, PB1, PB2 and NP) derived from PR8 (H1N1 virus) were co-transfected alongside a reporter plasmid containing noncoding sequence from the NS1 segment of influenza A virus and luciferase gene driven by the Pol 1 promoter. The relative luciferase units were calculated after normalization with plasmid pRL-TK (Promega, Madison, WI, USA), which expresses Renilla luciferase that was transfected along. Results are shown as mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P
    Gene Specific Sirna Against Rnf43, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology gene specific sirnas against api5
    Host factor <t>RNF43</t> decreases IAV replication. ( a–c ) HEK293T cells were transiently transfected with plasmid pCDNA3.1 (Mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) (1 and 2 μ g) followed by PR8 infection and incubated for 24 h. Total RNA was isolated and NP mRNA ( a ), NP vRNA ( b ) and RNF43 mRNA( c ) levels were estimated through qRT PCR. ( d ) HEK293T cells were treated with NT <t>siRNA</t> or RNF43 siRNA for 24 h followed by PR8 IAV infection for next 24 h. Cells were processed for qRT-PCR analysis of mRNA levels of NP and RNF43. ( e ) A549 cells, pretreated with either NT or RNF43 siRNA were transfected with plasmids-encoding polymerase complex components (PA, PB1, PB2 and NP) derived from PR8 (H1N1 virus) were co-transfected alongside a reporter plasmid containing noncoding sequence from the NS1 segment of influenza A virus and luciferase gene driven by the Pol 1 promoter. The relative luciferase units were calculated after normalization with plasmid pRL-TK (Promega, Madison, WI, USA), which expresses Renilla luciferase that was transfected along. Results are shown as mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P
    Gene Specific Sirnas Against Api5, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology gene specific sirnas against actinin 4
    Host factor <t>RNF43</t> decreases IAV replication. ( a–c ) HEK293T cells were transiently transfected with plasmid pCDNA3.1 (Mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) (1 and 2 μ g) followed by PR8 infection and incubated for 24 h. Total RNA was isolated and NP mRNA ( a ), NP vRNA ( b ) and RNF43 mRNA( c ) levels were estimated through qRT PCR. ( d ) HEK293T cells were treated with NT <t>siRNA</t> or RNF43 siRNA for 24 h followed by PR8 IAV infection for next 24 h. Cells were processed for qRT-PCR analysis of mRNA levels of NP and RNF43. ( e ) A549 cells, pretreated with either NT or RNF43 siRNA were transfected with plasmids-encoding polymerase complex components (PA, PB1, PB2 and NP) derived from PR8 (H1N1 virus) were co-transfected alongside a reporter plasmid containing noncoding sequence from the NS1 segment of influenza A virus and luciferase gene driven by the Pol 1 promoter. The relative luciferase units were calculated after normalization with plasmid pRL-TK (Promega, Madison, WI, USA), which expresses Renilla luciferase that was transfected along. Results are shown as mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P
    Gene Specific Sirnas Against Actinin 4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery gene specific on targetplus smartpool sirnas
    Host factor <t>RNF43</t> decreases IAV replication. ( a–c ) HEK293T cells were transiently transfected with plasmid pCDNA3.1 (Mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) (1 and 2 μ g) followed by PR8 infection and incubated for 24 h. Total RNA was isolated and NP mRNA ( a ), NP vRNA ( b ) and RNF43 mRNA( c ) levels were estimated through qRT PCR. ( d ) HEK293T cells were treated with NT <t>siRNA</t> or RNF43 siRNA for 24 h followed by PR8 IAV infection for next 24 h. Cells were processed for qRT-PCR analysis of mRNA levels of NP and RNF43. ( e ) A549 cells, pretreated with either NT or RNF43 siRNA were transfected with plasmids-encoding polymerase complex components (PA, PB1, PB2 and NP) derived from PR8 (H1N1 virus) were co-transfected alongside a reporter plasmid containing noncoding sequence from the NS1 segment of influenza A virus and luciferase gene driven by the Pol 1 promoter. The relative luciferase units were calculated after normalization with plasmid pRL-TK (Promega, Madison, WI, USA), which expresses Renilla luciferase that was transfected along. Results are shown as mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P
    Gene Specific On Targetplus Smartpool Sirnas, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology gene specific small interfering rna sirna sequences against ascl1
    <t>ASCL1</t> suppression reduces neuroendocrine marker production and induces cell-cycle arrest in BON and H727 cells Treatment of BON and H727 cells with an ASCL1 -specific <t>siRNA</t> reduced chromogranin A (CgA) and synaptophysin (SYP) levels, shown by Western blotting. Cells receiving the nonspecific-siRNA (NS-siRNA), exhibited no visible change in ASCL1, CgA or SYP levels relative to cells treated with Lipofectamine 2000 ® alone (no siRNA) (A). Concurrent with ASCL1 suppression following ASCL1 -siRNA treatment, BON and H727 cell lines exhibited a reduction in cyclin B1 and D1 expression, and an increase in p21 Waf1/Cip1 and p27 Kip1 expression, as demonstrated by Western blotting, therefore suggesting the occurrence of cell-cycle arrest. Cells receiving the NS-siRNA experienced no effect on levels of any of these markers (B).
    Gene Specific Small Interfering Rna Sirna Sequences Against Ascl1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Horizon Discovery sirna
    (A) Dot blots analysis of the interaction of APR-1 protein with TNFR1 human <t>p75NTR,</t> mouse p75NTR, FADD and rat p75NTR. A total of 5 μg of glutathione S -transferase (GST)-TNFR1 (100.0 pmol), TNFR2 (111.0 pmol), human p75NTR (106.4 pmol), mouse p75NTR (106 pmol), FADD (92.6 pmol), TrKA (113.6 pmol), TrKB (113.6 pmol), TrKC (113.6 pmol), Fas 142.8 pmol), death domain (142.8 pmol), APR-1 (100 pmol), rat p75NTR (106 pmol) or GST (192.0 pmol) were diluted in PBS and blotted onto nitrocellulose membrane and subsequently incubated overnight with in vitro transcribed and translated [ 35 S] APR-1 protein. (B) Interaction of APR-1 with P75NTR. The total cell lysates prepared from A375-APR-1 and BLM-APR-1 before and after the induction of APR-1 protein were subjected for either electrophoresis (for the detection of APR-1 and P75NTR) or for co-immunoprecipitation (IP) with either anti-P75NTR antibody or with anti-APR-1 antibody. Western blotting of IP: p75NTR for APR-1 revealed the interaction of APR-1 to P75NTR, whereas Western blotting of IP: APR-1 for P75NTR revealed the interaction of P75NTR to APR-1. β-actin was used as internal control for loading and transfer. (C) Schematic diagram of the extracellular and intracellular domains of p75NTR. Transmembrane domain, JMD and death domain. (D) GST-P75NTR recombinant proteins 1–427aa (106.4 pmol), 1–341aa (135.1 pmol), 1–311aa (147 pmol), 1–274 aa (166 pmol), 275–340aa (694.3) and 341–427aa (526.2 pmol), were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with the P75NTR domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount and the position of P75NTR recombinant proteins (left panel). (E) GST-JMD and death domain of P75NTR were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with both domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount of both JMD and death domains (left panel). (F) Western blot analysis demonstrates the expression of APR-1 by the addition of Dox to the culture medium of BLM-APR- 1, the knockdown of p75NTR by its specific <t>siRNA</t> and the suppression of APR-1-induced cleavage of PARP by the p75NTR siRNA. β-actin was used as internal control for loading and transfer. (G) Analysis of cell viability by counting using trypan blue staining. Rescue of APR-1-induced reduction of cell viability by the knockdown of p75NTR by siRNA for 24 or 48 hrs. Data are mean of three experiments performed separately.
    Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 94/100, based on 16150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology tlr4 sirna
    (A) Dot blots analysis of the interaction of APR-1 protein with TNFR1 human <t>p75NTR,</t> mouse p75NTR, FADD and rat p75NTR. A total of 5 μg of glutathione S -transferase (GST)-TNFR1 (100.0 pmol), TNFR2 (111.0 pmol), human p75NTR (106.4 pmol), mouse p75NTR (106 pmol), FADD (92.6 pmol), TrKA (113.6 pmol), TrKB (113.6 pmol), TrKC (113.6 pmol), Fas 142.8 pmol), death domain (142.8 pmol), APR-1 (100 pmol), rat p75NTR (106 pmol) or GST (192.0 pmol) were diluted in PBS and blotted onto nitrocellulose membrane and subsequently incubated overnight with in vitro transcribed and translated [ 35 S] APR-1 protein. (B) Interaction of APR-1 with P75NTR. The total cell lysates prepared from A375-APR-1 and BLM-APR-1 before and after the induction of APR-1 protein were subjected for either electrophoresis (for the detection of APR-1 and P75NTR) or for co-immunoprecipitation (IP) with either anti-P75NTR antibody or with anti-APR-1 antibody. Western blotting of IP: p75NTR for APR-1 revealed the interaction of APR-1 to P75NTR, whereas Western blotting of IP: APR-1 for P75NTR revealed the interaction of P75NTR to APR-1. β-actin was used as internal control for loading and transfer. (C) Schematic diagram of the extracellular and intracellular domains of p75NTR. Transmembrane domain, JMD and death domain. (D) GST-P75NTR recombinant proteins 1–427aa (106.4 pmol), 1–341aa (135.1 pmol), 1–311aa (147 pmol), 1–274 aa (166 pmol), 275–340aa (694.3) and 341–427aa (526.2 pmol), were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with the P75NTR domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount and the position of P75NTR recombinant proteins (left panel). (E) GST-JMD and death domain of P75NTR were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with both domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount of both JMD and death domains (left panel). (F) Western blot analysis demonstrates the expression of APR-1 by the addition of Dox to the culture medium of BLM-APR- 1, the knockdown of p75NTR by its specific <t>siRNA</t> and the suppression of APR-1-induced cleavage of PARP by the p75NTR siRNA. β-actin was used as internal control for loading and transfer. (G) Analysis of cell viability by counting using trypan blue staining. Rescue of APR-1-induced reduction of cell viability by the knockdown of p75NTR by siRNA for 24 or 48 hrs. Data are mean of three experiments performed separately.
    Tlr4 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Horizon Discovery human genes
    (A) Dot blots analysis of the interaction of APR-1 protein with TNFR1 human <t>p75NTR,</t> mouse p75NTR, FADD and rat p75NTR. A total of 5 μg of glutathione S -transferase (GST)-TNFR1 (100.0 pmol), TNFR2 (111.0 pmol), human p75NTR (106.4 pmol), mouse p75NTR (106 pmol), FADD (92.6 pmol), TrKA (113.6 pmol), TrKB (113.6 pmol), TrKC (113.6 pmol), Fas 142.8 pmol), death domain (142.8 pmol), APR-1 (100 pmol), rat p75NTR (106 pmol) or GST (192.0 pmol) were diluted in PBS and blotted onto nitrocellulose membrane and subsequently incubated overnight with in vitro transcribed and translated [ 35 S] APR-1 protein. (B) Interaction of APR-1 with P75NTR. The total cell lysates prepared from A375-APR-1 and BLM-APR-1 before and after the induction of APR-1 protein were subjected for either electrophoresis (for the detection of APR-1 and P75NTR) or for co-immunoprecipitation (IP) with either anti-P75NTR antibody or with anti-APR-1 antibody. Western blotting of IP: p75NTR for APR-1 revealed the interaction of APR-1 to P75NTR, whereas Western blotting of IP: APR-1 for P75NTR revealed the interaction of P75NTR to APR-1. β-actin was used as internal control for loading and transfer. (C) Schematic diagram of the extracellular and intracellular domains of p75NTR. Transmembrane domain, JMD and death domain. (D) GST-P75NTR recombinant proteins 1–427aa (106.4 pmol), 1–341aa (135.1 pmol), 1–311aa (147 pmol), 1–274 aa (166 pmol), 275–340aa (694.3) and 341–427aa (526.2 pmol), were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with the P75NTR domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount and the position of P75NTR recombinant proteins (left panel). (E) GST-JMD and death domain of P75NTR were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with both domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount of both JMD and death domains (left panel). (F) Western blot analysis demonstrates the expression of APR-1 by the addition of Dox to the culture medium of BLM-APR- 1, the knockdown of p75NTR by its specific <t>siRNA</t> and the suppression of APR-1-induced cleavage of PARP by the p75NTR siRNA. β-actin was used as internal control for loading and transfer. (G) Analysis of cell viability by counting using trypan blue staining. Rescue of APR-1-induced reduction of cell viability by the knockdown of p75NTR by siRNA for 24 or 48 hrs. Data are mean of three experiments performed separately.
    Human Genes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 91/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery hmgb1
    <t>HMGB1</t> accumulated in LCM induced microglial activation. (A) HMGB1 levels were determined in whole cell lysates of primary microglia cultures 12 hrs after transfection with HMGB1 siRNA (HMGB1) or nonspecific (NS) siRNA (40 pM). (B) LPS (100 ng/ml) was treated 12 hrs after HMGB1 siRNA or nonspecific siRNA (40 pM) transfection. After 18 hrs of LPS treatment, culture media were replaced with fresh medium, and LCM was collected at 24 hrs after medium replacement. Naïve primary microglia were then cultured in this LCM for 24 hrs and nitrite levels were measured. (C) Nitrite levels were measured at 24 hrs after LCM treatment in the presence of HMGB1 A box domain (10, 50, 100 ng/ml) or anti-HMGB1 antibody (100 or 500 ng/ml). HMGB1 B box domain (50, 100 ng/ml) and anti-human IgG (500 ng/ml) were used as negative controls. Data are presented as means±SEMs (n=4), ** p
    Hmgb1, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology tim 1 specific sirna
    Role of <t>Tim-1</t> in EBOV-induced activation of CD4 + T cells. (A and B) Disabling of Tim-1 reduces activation of EBOV-exposed T cells. Jurkat cells were transfected with control <t>siRNA</t> or siRNA targeting Tim-1, incubated for 48 h, stimulated with EBOV for an additional 48 h, stained, and analyzed for CD25 (A) and CD69 (B). (C) The EBOV-mediated activation of T cells depends on Lck kinase. Primary CD4 + T cells were incubated with Src inhibitor Ly294 for 1 h, stimulated with EBOV for 48 h, and analyzed for CD25 and CD69. (D) The EBOV-mediated activation of T cells depends on the PI3K pathway. Primary CD4 + T cells were incubated with PI3K inhibitor PP2 and EBOV and analyzed as described for panel F. (E) Binding of EBOV GP and EBOV-mediated downregulation of CD3 depend on Lck kinase and PI3K. Primary CD4 + T cells were incubated with Ly294 or PP2, and the percentages of binding and the CD4 Hi CD3 Low population were determined. (F) EBOV-induced phosphorylation of Akt depends on Src kinase and PI3K. CD4 + T cells were treated with Ly294 and PP2. Akt and its phosphorylated form were analyzed by Western blotting. *, P
    Tim 1 Specific Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery apoe specific sirnas
    Effect of <t>ApoE</t> knockdown on HIV-1 production in MDMs. (A) MDMs were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), which is a mixture (SMARTpool) of 4 <t>siRNAs</t> (“4-pool”), cultured for 2 days, and analyzed for their ApoE levels by western blot. Anti-β-actin blot was used as a loading control. Data shown are representative of experiments obtained from 4 different donors with similar results. (B) MDMs were transfected as in (A), cultured for 2 days, and analyzed for their cell surface CD4 (left panel) and CCR5 (right panel) expression by flow cytometry. The mean fluorescence intensity (MFI) values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (C) MDMs were transfected as in (A), cultured for 3 (left panel) or 6 days (right panel), and analyzed for their viability by the MTT assay. The viabilities of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (D) MDMs (4 donors) were transfected as in (A), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). The culture supernatants (“sup”) were collected as indicated (dpi, days of post-infection), and analyzed for their levels of p24 concentration by ELISA. The left panel in each donor set shows the overall kinetics of viral production. The right panel in each donor set shows the p24 concentrations in an earlier phase (3 dpi for donors 1 and 2, or 2 dpi for donors 3 and 4). (E) MDMs were transfected as in (A), infected as in (D), and analyzed for their levels of p24 concentration in the supernatants by ELISA. The p24 values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 4 donors are summarized. * p
    Apoe Specific Sirnas, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Horizon Discovery small interfering rna sirna oligonucleotides
    Effect of <t>ApoE</t> knockdown on HIV-1 production in MDMs. (A) MDMs were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), which is a mixture (SMARTpool) of 4 <t>siRNAs</t> (“4-pool”), cultured for 2 days, and analyzed for their ApoE levels by western blot. Anti-β-actin blot was used as a loading control. Data shown are representative of experiments obtained from 4 different donors with similar results. (B) MDMs were transfected as in (A), cultured for 2 days, and analyzed for their cell surface CD4 (left panel) and CCR5 (right panel) expression by flow cytometry. The mean fluorescence intensity (MFI) values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (C) MDMs were transfected as in (A), cultured for 3 (left panel) or 6 days (right panel), and analyzed for their viability by the MTT assay. The viabilities of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (D) MDMs (4 donors) were transfected as in (A), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). The culture supernatants (“sup”) were collected as indicated (dpi, days of post-infection), and analyzed for their levels of p24 concentration by ELISA. The left panel in each donor set shows the overall kinetics of viral production. The right panel in each donor set shows the p24 concentrations in an earlier phase (3 dpi for donors 1 and 2, or 2 dpi for donors 3 and 4). (E) MDMs were transfected as in (A), infected as in (D), and analyzed for their levels of p24 concentration in the supernatants by ELISA. The p24 values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 4 donors are summarized. * p
    Small Interfering Rna Sirna Oligonucleotides, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 90/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of <t>ApoE</t> knockdown on HIV-1 production in MDMs. (A) MDMs were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), which is a mixture (SMARTpool) of 4 <t>siRNAs</t> (“4-pool”), cultured for 2 days, and analyzed for their ApoE levels by western blot. Anti-β-actin blot was used as a loading control. Data shown are representative of experiments obtained from 4 different donors with similar results. (B) MDMs were transfected as in (A), cultured for 2 days, and analyzed for their cell surface CD4 (left panel) and CCR5 (right panel) expression by flow cytometry. The mean fluorescence intensity (MFI) values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (C) MDMs were transfected as in (A), cultured for 3 (left panel) or 6 days (right panel), and analyzed for their viability by the MTT assay. The viabilities of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (D) MDMs (4 donors) were transfected as in (A), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). The culture supernatants (“sup”) were collected as indicated (dpi, days of post-infection), and analyzed for their levels of p24 concentration by ELISA. The left panel in each donor set shows the overall kinetics of viral production. The right panel in each donor set shows the p24 concentrations in an earlier phase (3 dpi for donors 1 and 2, or 2 dpi for donors 3 and 4). (E) MDMs were transfected as in (A), infected as in (D), and analyzed for their levels of p24 concentration in the supernatants by ELISA. The p24 values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 4 donors are summarized. * p
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    Horizon Discovery sirnas
    Effect of <t>ApoE</t> knockdown on HIV-1 production in MDMs. (A) MDMs were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), which is a mixture (SMARTpool) of 4 <t>siRNAs</t> (“4-pool”), cultured for 2 days, and analyzed for their ApoE levels by western blot. Anti-β-actin blot was used as a loading control. Data shown are representative of experiments obtained from 4 different donors with similar results. (B) MDMs were transfected as in (A), cultured for 2 days, and analyzed for their cell surface CD4 (left panel) and CCR5 (right panel) expression by flow cytometry. The mean fluorescence intensity (MFI) values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (C) MDMs were transfected as in (A), cultured for 3 (left panel) or 6 days (right panel), and analyzed for their viability by the MTT assay. The viabilities of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (D) MDMs (4 donors) were transfected as in (A), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). The culture supernatants (“sup”) were collected as indicated (dpi, days of post-infection), and analyzed for their levels of p24 concentration by ELISA. The left panel in each donor set shows the overall kinetics of viral production. The right panel in each donor set shows the p24 concentrations in an earlier phase (3 dpi for donors 1 and 2, or 2 dpi for donors 3 and 4). (E) MDMs were transfected as in (A), infected as in (D), and analyzed for their levels of p24 concentration in the supernatants by ELISA. The p24 values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 4 donors are summarized. * p
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    Effect of <t>ApoE</t> knockdown on HIV-1 production in MDMs. (A) MDMs were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), which is a mixture (SMARTpool) of 4 <t>siRNAs</t> (“4-pool”), cultured for 2 days, and analyzed for their ApoE levels by western blot. Anti-β-actin blot was used as a loading control. Data shown are representative of experiments obtained from 4 different donors with similar results. (B) MDMs were transfected as in (A), cultured for 2 days, and analyzed for their cell surface CD4 (left panel) and CCR5 (right panel) expression by flow cytometry. The mean fluorescence intensity (MFI) values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (C) MDMs were transfected as in (A), cultured for 3 (left panel) or 6 days (right panel), and analyzed for their viability by the MTT assay. The viabilities of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (D) MDMs (4 donors) were transfected as in (A), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). The culture supernatants (“sup”) were collected as indicated (dpi, days of post-infection), and analyzed for their levels of p24 concentration by ELISA. The left panel in each donor set shows the overall kinetics of viral production. The right panel in each donor set shows the p24 concentrations in an earlier phase (3 dpi for donors 1 and 2, or 2 dpi for donors 3 and 4). (E) MDMs were transfected as in (A), infected as in (D), and analyzed for their levels of p24 concentration in the supernatants by ELISA. The p24 values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 4 donors are summarized. * p
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    Image Search Results


    LKB1 Is the Main Cargo Regulated by the Ran/RanBP1 Pathway during Axonogenesis (A and B) Rat cortical neuron transfected with control siRNAs and the eGFP -reporter (A) and siRNAs targeting LKB1 and the eGFP -reporter plasmid (B). (C) 72 hr cortical neuron overexpressing STK25-red fluorescent protein (RFP) and scrambled siRNA together with an eGFP -reporter plasmid. (D) 72 hr cortical neuron overexpressing STK25-RFP and ranBP1 -siRNA together with the eGFP -reporter plasmid. (E and F) 72 hr cortical neuron transfected with scrambled siRNA and the eGFP -reporter (E) and 72 hr cortical neuron transfected with siRNAs targeting ranBP1 and the eGFP -reporter plasmid (F). (G) 72 hr cortical neuron transfected with siRNAs ranBP1 , STK25-RFP , and an eGFP -reporter plasmid. (H) The mean percentage of 72 hr neurons with 0, 1, or ≥2 axons over three independent replicates were determined for each condition. The absence of axon in ranBP1 and LKB1 siRNA neurons is rescued by expressing STK25-RFP . At least 24 neurons were scored for the number of axons for each condition. (I) Sholl analysis of siRNA control (n = 15), ranBP1 -siRNAs neurons (n = 18), and ranBP1 -siRNAs neurons rescued by overexpressing STK25-RFP (n = 21) was performed 72 hr after transfection. A statistically significant decrease in intersection numbers was observed for the rescue experiment between 10 and 25 μm from the cell body (at 10, 15, 20, and 25 μm ∗ p

    Journal: Cell Reports

    Article Title: RanBP1 Couples Nuclear Export and Golgi Regulation through LKB1 to Promote Cortical Neuron Polarity

    doi: 10.1016/j.celrep.2018.07.107

    Figure Lengend Snippet: LKB1 Is the Main Cargo Regulated by the Ran/RanBP1 Pathway during Axonogenesis (A and B) Rat cortical neuron transfected with control siRNAs and the eGFP -reporter (A) and siRNAs targeting LKB1 and the eGFP -reporter plasmid (B). (C) 72 hr cortical neuron overexpressing STK25-red fluorescent protein (RFP) and scrambled siRNA together with an eGFP -reporter plasmid. (D) 72 hr cortical neuron overexpressing STK25-RFP and ranBP1 -siRNA together with the eGFP -reporter plasmid. (E and F) 72 hr cortical neuron transfected with scrambled siRNA and the eGFP -reporter (E) and 72 hr cortical neuron transfected with siRNAs targeting ranBP1 and the eGFP -reporter plasmid (F). (G) 72 hr cortical neuron transfected with siRNAs ranBP1 , STK25-RFP , and an eGFP -reporter plasmid. (H) The mean percentage of 72 hr neurons with 0, 1, or ≥2 axons over three independent replicates were determined for each condition. The absence of axon in ranBP1 and LKB1 siRNA neurons is rescued by expressing STK25-RFP . At least 24 neurons were scored for the number of axons for each condition. (I) Sholl analysis of siRNA control (n = 15), ranBP1 -siRNAs neurons (n = 18), and ranBP1 -siRNAs neurons rescued by overexpressing STK25-RFP (n = 21) was performed 72 hr after transfection. A statistically significant decrease in intersection numbers was observed for the rescue experiment between 10 and 25 μm from the cell body (at 10, 15, 20, and 25 μm ∗ p

    Article Snippet: Briefly, 1 × 106 cells were resuspended in 100 μL of Nucleofector solution containing 3 μg of an expression vector or a pool of three gene-specific siRNAs (10 nM of each siRNA, 30 nM total), or a combination of expression vectors plus siRNAs. siRNAs targeting ran (NM_053439), ranBP1 (NM_001108324), rcc1 (NM_001128189) and LKB1 (NM_001108069), and a siRNA negative control (MISSION® siRNA Universal Negative Control, SIC001) (Sigma).

    Techniques: Transfection, Plasmid Preparation, Expressing

    The Ran Pathway Is Required for Rat Cortical Neuron Polarization (A) 72 hr cortical neuron co-transfected with a control siRNA and an eGFP reporter plasmid. (B) 72 hr cortical neuron co-transfected with ran siRNAs and an eGFP -reporter plasmid (scale bar, 10 μm). (C) 72 hr cortical neuron co-transfected with rcc1 siRNAs and an eGFP -reporter plasmid (scale bar, 10 μm). (D) Neuron polarity was scored at 72 hr after transfection using the axonal marker Tau-1. The mean percentage of neurons with 0 or 1 axon over three independent replicates was determined for each condition: control siRNA (n = 31 neurons), ran siRNA (n = 32 neurons), and rcc1 siRNA (n = 28 neurons). After converting percentages to arcsin values, two-tailed unpaired t tests were performed, comparing control siRNA to ran siRNA (p = 0.0019) and Rcc1 siRNA (p = 0.0014). (E) Sholl analysis of ran siRNAs neurons (n = 32 neurons), rcc1 siRNAs neurons (n = 28 neurons), and control scrambled siRNA neurons (n = 31 neurons) was performed 72 hr after transfection. A statistically significant increase in intersection numbers was observed for ran knockdown between 10 and 20 μm from the cell body (at 10 and 15 μm ∗ p

    Journal: Cell Reports

    Article Title: RanBP1 Couples Nuclear Export and Golgi Regulation through LKB1 to Promote Cortical Neuron Polarity

    doi: 10.1016/j.celrep.2018.07.107

    Figure Lengend Snippet: The Ran Pathway Is Required for Rat Cortical Neuron Polarization (A) 72 hr cortical neuron co-transfected with a control siRNA and an eGFP reporter plasmid. (B) 72 hr cortical neuron co-transfected with ran siRNAs and an eGFP -reporter plasmid (scale bar, 10 μm). (C) 72 hr cortical neuron co-transfected with rcc1 siRNAs and an eGFP -reporter plasmid (scale bar, 10 μm). (D) Neuron polarity was scored at 72 hr after transfection using the axonal marker Tau-1. The mean percentage of neurons with 0 or 1 axon over three independent replicates was determined for each condition: control siRNA (n = 31 neurons), ran siRNA (n = 32 neurons), and rcc1 siRNA (n = 28 neurons). After converting percentages to arcsin values, two-tailed unpaired t tests were performed, comparing control siRNA to ran siRNA (p = 0.0019) and Rcc1 siRNA (p = 0.0014). (E) Sholl analysis of ran siRNAs neurons (n = 32 neurons), rcc1 siRNAs neurons (n = 28 neurons), and control scrambled siRNA neurons (n = 31 neurons) was performed 72 hr after transfection. A statistically significant increase in intersection numbers was observed for ran knockdown between 10 and 20 μm from the cell body (at 10 and 15 μm ∗ p

    Article Snippet: Briefly, 1 × 106 cells were resuspended in 100 μL of Nucleofector solution containing 3 μg of an expression vector or a pool of three gene-specific siRNAs (10 nM of each siRNA, 30 nM total), or a combination of expression vectors plus siRNAs. siRNAs targeting ran (NM_053439), ranBP1 (NM_001108324), rcc1 (NM_001128189) and LKB1 (NM_001108069), and a siRNA negative control (MISSION® siRNA Universal Negative Control, SIC001) (Sigma).

    Techniques: Transfection, Plasmid Preparation, Marker, Two Tailed Test

    Loss of αSNAP triggers detachment of human intestinal epithelial cells. Control SK-CO15 cells ( SK-neo ) and cells stably expressing siRNA-resistant bovine αSNAP ( SK -α SNAP ) were transfected with either control or two human αSNAP-specific siRNA duplexes ( D1 and D2 ). A, expression of αSNAP and its binding partner NSF was determined 72 h post-transfection. B and C , cell detachment was examined by counting adhered and total number of cells using bright field microscopy 72 h post-transfection. Data are presented as the mean ± S.E. ( n = 3); *, p

    Journal: The Journal of Biological Chemistry

    Article Title: N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells *

    doi: 10.1074/jbc.M113.498691

    Figure Lengend Snippet: Loss of αSNAP triggers detachment of human intestinal epithelial cells. Control SK-CO15 cells ( SK-neo ) and cells stably expressing siRNA-resistant bovine αSNAP ( SK -α SNAP ) were transfected with either control or two human αSNAP-specific siRNA duplexes ( D1 and D2 ). A, expression of αSNAP and its binding partner NSF was determined 72 h post-transfection. B and C , cell detachment was examined by counting adhered and total number of cells using bright field microscopy 72 h post-transfection. Data are presented as the mean ± S.E. ( n = 3); *, p

    Article Snippet: Individual siRNA duplexes, GAAGGUGGCUGGUUACGCU (duplex 1) and CAGAGUUGGUGGACAUCGA (duplex 2; Dharmacon, Lafayette, CO), were used to down-regulate αSNAP expression, whereas knockdown of other targets was performed by using gene-specific siRNA pools purchased either from Dharmacon or Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Stable Transfection, Expressing, Transfection, Binding Assay, Microscopy

    Effect of αSNAP depletion on ECM adhesion and integrin processing are independent from induction of apoptosis. SK-CO15 cells were transfected with either control or two αSNAP-specific siRNA duplexes. 24 h later these cells were treated for an additional 48 h with either vehicle or a pan-caspase inhibitor, Q-VAD-OPH. Cells were examined for apoptosis induction and β1 integrin maturation ( A ), β1 integrin localization ( B ), and cell attachment to ECM ( C ). Arrows indicate plasma membrane labeling of β1 integrin in control SK-CO15 cells, whereas arrowheads show intracellular localization of β1 integrin in αSNAP-depleted cells treated and untreated with pan-caspase inhibitor. Data are presented as the mean ± S.E. ( n = 3); *, p

    Journal: The Journal of Biological Chemistry

    Article Title: N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells *

    doi: 10.1074/jbc.M113.498691

    Figure Lengend Snippet: Effect of αSNAP depletion on ECM adhesion and integrin processing are independent from induction of apoptosis. SK-CO15 cells were transfected with either control or two αSNAP-specific siRNA duplexes. 24 h later these cells were treated for an additional 48 h with either vehicle or a pan-caspase inhibitor, Q-VAD-OPH. Cells were examined for apoptosis induction and β1 integrin maturation ( A ), β1 integrin localization ( B ), and cell attachment to ECM ( C ). Arrows indicate plasma membrane labeling of β1 integrin in control SK-CO15 cells, whereas arrowheads show intracellular localization of β1 integrin in αSNAP-depleted cells treated and untreated with pan-caspase inhibitor. Data are presented as the mean ± S.E. ( n = 3); *, p

    Article Snippet: Individual siRNA duplexes, GAAGGUGGCUGGUUACGCU (duplex 1) and CAGAGUUGGUGGACAUCGA (duplex 2; Dharmacon, Lafayette, CO), were used to down-regulate αSNAP expression, whereas knockdown of other targets was performed by using gene-specific siRNA pools purchased either from Dharmacon or Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Transfection, Cell Attachment Assay, Labeling

    Inhibitory anti- β 1 integrin antibody, but not siRNA-mediated integrin depletion, inhibits epithelial ECM adhesion and invasion. A and B , SK-CO15 cells were transfected with control, β1 integrin, β4 integrin siRNAs, or co-transfected with siRNAs against these integrins and αSNAP. Expression of adhesion proteins and cell attachment to the substrate were examined 72 h post-transfection. C–E , control ( SK-neo ) and αSNAP-overexpressing ( SK -α SNAP ) epithelial cells were preincubated with either β1 integrin-inhibitory antibody, MAB13, or the isotype-matched control antibody. Cell adhesion to collagen I ( C ) and invasion into Matrigel ( D and E ) was examined as described under “Experimental Procedures.” Data are presented as the mean ± S.E. ( n = 3); *, p

    Journal: The Journal of Biological Chemistry

    Article Title: N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells *

    doi: 10.1074/jbc.M113.498691

    Figure Lengend Snippet: Inhibitory anti- β 1 integrin antibody, but not siRNA-mediated integrin depletion, inhibits epithelial ECM adhesion and invasion. A and B , SK-CO15 cells were transfected with control, β1 integrin, β4 integrin siRNAs, or co-transfected with siRNAs against these integrins and αSNAP. Expression of adhesion proteins and cell attachment to the substrate were examined 72 h post-transfection. C–E , control ( SK-neo ) and αSNAP-overexpressing ( SK -α SNAP ) epithelial cells were preincubated with either β1 integrin-inhibitory antibody, MAB13, or the isotype-matched control antibody. Cell adhesion to collagen I ( C ) and invasion into Matrigel ( D and E ) was examined as described under “Experimental Procedures.” Data are presented as the mean ± S.E. ( n = 3); *, p

    Article Snippet: Individual siRNA duplexes, GAAGGUGGCUGGUUACGCU (duplex 1) and CAGAGUUGGUGGACAUCGA (duplex 2; Dharmacon, Lafayette, CO), were used to down-regulate αSNAP expression, whereas knockdown of other targets was performed by using gene-specific siRNA pools purchased either from Dharmacon or Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Transfection, Expressing, Cell Attachment Assay

    Loss of αSNAP disrupts structure of focal adhesions and affects expression and processing of key FA proteins. Control and αSNAP siRNA-transfected SK-CO15 cells were fixed 72 h post-transfection and FA were visualized by immunofluorescence labeling of vinculin and confocal microscopy ( A ). Arrows show intact vinculin-based FA structures in control cells, whereas arrowheads indicate diffuse vinculin labeling in αSNAP-depleted cells. Scale bar , 20 μm. Expression and phosphorylation of major transmembrane, scaffolding, and signaling FA proteins in control and αSNAP-depleted epithelial cells was determined by immunoblotting analysis ( B ) with densitometric quantification ( C ). Data are presented as the mean ± S.E. ( n = 3); *, p

    Journal: The Journal of Biological Chemistry

    Article Title: N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells *

    doi: 10.1074/jbc.M113.498691

    Figure Lengend Snippet: Loss of αSNAP disrupts structure of focal adhesions and affects expression and processing of key FA proteins. Control and αSNAP siRNA-transfected SK-CO15 cells were fixed 72 h post-transfection and FA were visualized by immunofluorescence labeling of vinculin and confocal microscopy ( A ). Arrows show intact vinculin-based FA structures in control cells, whereas arrowheads indicate diffuse vinculin labeling in αSNAP-depleted cells. Scale bar , 20 μm. Expression and phosphorylation of major transmembrane, scaffolding, and signaling FA proteins in control and αSNAP-depleted epithelial cells was determined by immunoblotting analysis ( B ) with densitometric quantification ( C ). Data are presented as the mean ± S.E. ( n = 3); *, p

    Article Snippet: Individual siRNA duplexes, GAAGGUGGCUGGUUACGCU (duplex 1) and CAGAGUUGGUGGACAUCGA (duplex 2; Dharmacon, Lafayette, CO), were used to down-regulate αSNAP expression, whereas knockdown of other targets was performed by using gene-specific siRNA pools purchased either from Dharmacon or Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Expressing, Transfection, Immunofluorescence, Labeling, Confocal Microscopy, Scaffolding

    The effects of αSNAP depletion on β 1 integrin and other FA proteins can be rescued by expression of siRNA-resistant αSNAP. SK-CO15 cells stably expressing siRNA-resistant bovine αSNAP ( SK -α SNAP ) and their empty vector controls ( SK-neo ) were transfected with either control or αSNAP-specific siRNAs. A, localization of β1 integrin was determined by immunofluorescence labeling and confocal microscopy 72 h post-transfection. B, expression and processing of different FA proteins was determined by immunoblotting. Arrows indicate plasma membrane labeling of β1 integrin in control or αSNAP-siRNA-transfected and rescued cells. Arrowheads show intracellular localization of β1 integrin in αSNAP-depleted cells without rescue. Scale bar , 20 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells *

    doi: 10.1074/jbc.M113.498691

    Figure Lengend Snippet: The effects of αSNAP depletion on β 1 integrin and other FA proteins can be rescued by expression of siRNA-resistant αSNAP. SK-CO15 cells stably expressing siRNA-resistant bovine αSNAP ( SK -α SNAP ) and their empty vector controls ( SK-neo ) were transfected with either control or αSNAP-specific siRNAs. A, localization of β1 integrin was determined by immunofluorescence labeling and confocal microscopy 72 h post-transfection. B, expression and processing of different FA proteins was determined by immunoblotting. Arrows indicate plasma membrane labeling of β1 integrin in control or αSNAP-siRNA-transfected and rescued cells. Arrowheads show intracellular localization of β1 integrin in αSNAP-depleted cells without rescue. Scale bar , 20 μm.

    Article Snippet: Individual siRNA duplexes, GAAGGUGGCUGGUUACGCU (duplex 1) and CAGAGUUGGUGGACAUCGA (duplex 2; Dharmacon, Lafayette, CO), were used to down-regulate αSNAP expression, whereas knockdown of other targets was performed by using gene-specific siRNA pools purchased either from Dharmacon or Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Expressing, Stable Transfection, Plasmid Preparation, Transfection, Immunofluorescence, Labeling, Confocal Microscopy

    A) Cytotoxic effects of transfection with Lipofectamine 2000 (L2000) and magnetofection with PolyMAG (MF) on primary fibroblasts. The percentage of PI-positive cells is presented as increase above “baseline cytotoxicity”; “baseline cytotoxicity” experiments were performed at least in triplicates for each transfection procedure. A ratio of 1 μg siRNA to 5 μl L2000 was used for lipofection, and 2 μg siRNA to 2.5 μl PolyMAG was used for magnetofection. Mock controls were exposed to transfection reagents, but not to FITC-labeled siRNA. Cytotoxicity results, after subtraction of the mean of baseline results, are presented as mean ± SD (n = 5 experimental transfections); ** p = 0.0079. B) Downregulation of ACAD-9 in primary fibroblasts by siRNAs delivered using both magnetofection with PolyMag (MF) and lipofection with Lipofectamine 2000 (L2000). For a direct comparison, both procedures were carried out at the same time. mRNA abundance is shown relative to expression of transcript in mock transfected cells for each transfection procedure. A nonspecific siRNA without homology to any known murine genes served as negative control.

    Journal: Biotechnic & histochemistry : official publication of the Biological Stain Commission

    Article Title: Efficient and gentle siRNA delivery by magnetofection

    doi: 10.3109/10520291003675485

    Figure Lengend Snippet: A) Cytotoxic effects of transfection with Lipofectamine 2000 (L2000) and magnetofection with PolyMAG (MF) on primary fibroblasts. The percentage of PI-positive cells is presented as increase above “baseline cytotoxicity”; “baseline cytotoxicity” experiments were performed at least in triplicates for each transfection procedure. A ratio of 1 μg siRNA to 5 μl L2000 was used for lipofection, and 2 μg siRNA to 2.5 μl PolyMAG was used for magnetofection. Mock controls were exposed to transfection reagents, but not to FITC-labeled siRNA. Cytotoxicity results, after subtraction of the mean of baseline results, are presented as mean ± SD (n = 5 experimental transfections); ** p = 0.0079. B) Downregulation of ACAD-9 in primary fibroblasts by siRNAs delivered using both magnetofection with PolyMag (MF) and lipofection with Lipofectamine 2000 (L2000). For a direct comparison, both procedures were carried out at the same time. mRNA abundance is shown relative to expression of transcript in mock transfected cells for each transfection procedure. A nonspecific siRNA without homology to any known murine genes served as negative control.

    Article Snippet: Immunofluorescence images were taken with a confocal microscope from Olympus (Fluoview FV 1000). mRNA expression levels using a gene-specific siRNA pool (SMARTpool, Dharmacon Inc., Lafayette, CO) were quantitatively assessed by real-time PCR 48 h after magnetofection with PolyMAG and transfection with Lipofectamine 2000, respectively.

    Techniques: Transfection, Magnetofection, Labeling, Expressing, Negative Control

    h MAK3 , h MAK10 , or h MAK31 knockdown induces apoptosis in HeLa cells. (A) Cells cultured in six-well plates were transfected with 50 nM h MAK3 , h MAK10 , or h MAK31 SMART pool siRNAs or control siRNA (si GAPDH or nontargeting siRNA). After 48 h, total RNA was isolated and processed by RT-PCR with specific primers against the genes of interest. (B) At 48 h posttransfection the cell proliferation rate was determined using a BrdU assay, measuring the amount of bromodeoxyuridine incorporated into nuclear DNA. The results are given as percent mitogenic activity. Error bars (B and C) represent standard deviations. P values for independent t tests for samples versus control are indicated (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Knockdown of Human Nα-Terminal Acetyltransferase Complex C Leads to p53-Dependent Apoptosis and Aberrant Human Arl8b Localization ▿

    doi: 10.1128/MCB.01909-08

    Figure Lengend Snippet: h MAK3 , h MAK10 , or h MAK31 knockdown induces apoptosis in HeLa cells. (A) Cells cultured in six-well plates were transfected with 50 nM h MAK3 , h MAK10 , or h MAK31 SMART pool siRNAs or control siRNA (si GAPDH or nontargeting siRNA). After 48 h, total RNA was isolated and processed by RT-PCR with specific primers against the genes of interest. (B) At 48 h posttransfection the cell proliferation rate was determined using a BrdU assay, measuring the amount of bromodeoxyuridine incorporated into nuclear DNA. The results are given as percent mitogenic activity. Error bars (B and C) represent standard deviations. P values for independent t tests for samples versus control are indicated (*, P

    Article Snippet: Gene-specific SMART pool siRNAs were purchased from Dharmacon and used at a final concentration of 20 to 50 nM to silence the h MAK3 ( NAT12 ), h MAK10 , and h MAK31 ( LSMD1 ) genes (sih MAK3 [ NAT12 ], catalog no. M-009961-02; sih MAK10 , catalog no. L-021268-01; sih MAK31 [ LSMD1 ], catalog no. L-014876-01).

    Techniques: Cell Culture, Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, BrdU Staining, Activity Assay

    Gga1 knockdown impaired the formation of large myotubes and expression of MYH3. ( A ) Morphological analysis of Golgi associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1)-depleted myotubes. C2C12 cells transfected with a control short interfering RNA (siRNA) (control: a-c and a′-c′) and Gga1 siRNA (GGA1kd: d-f and d′-f′) were subjected to immunofluorescence microscopy before (day 0) or after differentiation for four days (day 4) using anti-GGA1 (a, d, a′, and d′: green) and anti-myosin heavy chain 3 (MYH3) (b, e, b′, and e′: red) antibodies. The merged images are indicated in the right column. ( B ) Quantitative analysis of the MYH3 signal in the control and Gga1 kd myotubes at day 4. The signal density is plotted. Error bars indicate the SD (n = 8). ( C ) Quantitative analysis of the average cell width in the control and Gga1 kd myotubes at day 4. The value is plotted. Error bars indicate the SD (n = 4). *. P

    Journal: PLoS ONE

    Article Title: Clathrin adaptor GGA1 modulates myogenesis of C2C12 myoblasts

    doi: 10.1371/journal.pone.0207533

    Figure Lengend Snippet: Gga1 knockdown impaired the formation of large myotubes and expression of MYH3. ( A ) Morphological analysis of Golgi associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1)-depleted myotubes. C2C12 cells transfected with a control short interfering RNA (siRNA) (control: a-c and a′-c′) and Gga1 siRNA (GGA1kd: d-f and d′-f′) were subjected to immunofluorescence microscopy before (day 0) or after differentiation for four days (day 4) using anti-GGA1 (a, d, a′, and d′: green) and anti-myosin heavy chain 3 (MYH3) (b, e, b′, and e′: red) antibodies. The merged images are indicated in the right column. ( B ) Quantitative analysis of the MYH3 signal in the control and Gga1 kd myotubes at day 4. The signal density is plotted. Error bars indicate the SD (n = 8). ( C ) Quantitative analysis of the average cell width in the control and Gga1 kd myotubes at day 4. The value is plotted. Error bars indicate the SD (n = 4). *. P

    Article Snippet: To knock down Gga gene expression, C2C12 cells were transfected with 20 nM of gene-specific small interfering RNA (siRNA, ON-TARGETplus SMART pool, Dharmacon) using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Binding Assay, Transfection, Small Interfering RNA, Immunofluorescence, Microscopy

    Preparation of specific antibodies to mGGA1 and mGGA2. ( A ) Purified His6-tagged mouse Golgi associated, gamma-adaptin ear containing, ARF binding protein (GGA)1 (mGGA1) and GGA2 (mGGA2) full-length protein expressed in bacteria. Approximately 2 mg of each recombinant protein was subjected to SDS-PAGE and visualized by Coomassie brilliant blue staining. ( B and C ) Endogenous GGA1 ( B ) and GGA2 ( C ) were detected using specific antibodies. Lysates from untreated (Con) or GGA1 depleted (GGA1kd), or GGA2 depleted (GGA2kd) C2C12 cells were subjected to immunoblotting with anti-GGA1 (B) or anti-GGA2 (C) antibodies. ( D ) C2C12 cells were treated with control short interfering RNA (siRNA) (a, c) or siRNAs to Gga1 (b) and Gga2 (d), and the cells were subjected to immunofluorescence microscopy with anti-GGA1 (a and b: green) and anti-GGA2 (c and d: green) antibodies. Nuclei were counterstained with Hoechst33342 (blue). Scale bar: 20 mm.

    Journal: PLoS ONE

    Article Title: Clathrin adaptor GGA1 modulates myogenesis of C2C12 myoblasts

    doi: 10.1371/journal.pone.0207533

    Figure Lengend Snippet: Preparation of specific antibodies to mGGA1 and mGGA2. ( A ) Purified His6-tagged mouse Golgi associated, gamma-adaptin ear containing, ARF binding protein (GGA)1 (mGGA1) and GGA2 (mGGA2) full-length protein expressed in bacteria. Approximately 2 mg of each recombinant protein was subjected to SDS-PAGE and visualized by Coomassie brilliant blue staining. ( B and C ) Endogenous GGA1 ( B ) and GGA2 ( C ) were detected using specific antibodies. Lysates from untreated (Con) or GGA1 depleted (GGA1kd), or GGA2 depleted (GGA2kd) C2C12 cells were subjected to immunoblotting with anti-GGA1 (B) or anti-GGA2 (C) antibodies. ( D ) C2C12 cells were treated with control short interfering RNA (siRNA) (a, c) or siRNAs to Gga1 (b) and Gga2 (d), and the cells were subjected to immunofluorescence microscopy with anti-GGA1 (a and b: green) and anti-GGA2 (c and d: green) antibodies. Nuclei were counterstained with Hoechst33342 (blue). Scale bar: 20 mm.

    Article Snippet: To knock down Gga gene expression, C2C12 cells were transfected with 20 nM of gene-specific small interfering RNA (siRNA, ON-TARGETplus SMART pool, Dharmacon) using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Purification, Binding Assay, Recombinant, SDS Page, Staining, Small Interfering RNA, Immunofluorescence, Microscopy

    GGA1 is involved in myogenesis of C2C12 cells. ( A ) Knockdown of Gga1 (Golgi associated, gamma adaptin ear containing, ARF binding protein 1) affects myogenesis of C2C12 cells. Short interfering RNA (siRNA) for non-target (a), Gga1 (b), and Gga2 (c) were transfected into C2C12 cells and muscle differentiation was induced for four days. Gga1 kd cells showed fewer myotubes compared with those formed by the other cells. ( B ) The fusion index in (A) was calculated as described in the Materials and methods. The population of the nuclei in myotubes was indicated by the closed bars and that of unfused mononuclear myoblasts was indicated by the open bars. Error bars indicate SD (n = 6). ( C ) Quantification of the percentage of nuclei in control and Gga1 kd (siGGA1) myotubes. Error bars indicate SD (n = 3). *. P

    Journal: PLoS ONE

    Article Title: Clathrin adaptor GGA1 modulates myogenesis of C2C12 myoblasts

    doi: 10.1371/journal.pone.0207533

    Figure Lengend Snippet: GGA1 is involved in myogenesis of C2C12 cells. ( A ) Knockdown of Gga1 (Golgi associated, gamma adaptin ear containing, ARF binding protein 1) affects myogenesis of C2C12 cells. Short interfering RNA (siRNA) for non-target (a), Gga1 (b), and Gga2 (c) were transfected into C2C12 cells and muscle differentiation was induced for four days. Gga1 kd cells showed fewer myotubes compared with those formed by the other cells. ( B ) The fusion index in (A) was calculated as described in the Materials and methods. The population of the nuclei in myotubes was indicated by the closed bars and that of unfused mononuclear myoblasts was indicated by the open bars. Error bars indicate SD (n = 6). ( C ) Quantification of the percentage of nuclei in control and Gga1 kd (siGGA1) myotubes. Error bars indicate SD (n = 3). *. P

    Article Snippet: To knock down Gga gene expression, C2C12 cells were transfected with 20 nM of gene-specific small interfering RNA (siRNA, ON-TARGETplus SMART pool, Dharmacon) using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Binding Assay, Small Interfering RNA, Transfection

    Host factor RNF43 decreases IAV replication. ( a–c ) HEK293T cells were transiently transfected with plasmid pCDNA3.1 (Mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) (1 and 2 μ g) followed by PR8 infection and incubated for 24 h. Total RNA was isolated and NP mRNA ( a ), NP vRNA ( b ) and RNF43 mRNA( c ) levels were estimated through qRT PCR. ( d ) HEK293T cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by PR8 IAV infection for next 24 h. Cells were processed for qRT-PCR analysis of mRNA levels of NP and RNF43. ( e ) A549 cells, pretreated with either NT or RNF43 siRNA were transfected with plasmids-encoding polymerase complex components (PA, PB1, PB2 and NP) derived from PR8 (H1N1 virus) were co-transfected alongside a reporter plasmid containing noncoding sequence from the NS1 segment of influenza A virus and luciferase gene driven by the Pol 1 promoter. The relative luciferase units were calculated after normalization with plasmid pRL-TK (Promega, Madison, WI, USA), which expresses Renilla luciferase that was transfected along. Results are shown as mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P

    Journal: Cell Death & Disease

    Article Title: The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43

    doi: 10.1038/cddis.2015.131

    Figure Lengend Snippet: Host factor RNF43 decreases IAV replication. ( a–c ) HEK293T cells were transiently transfected with plasmid pCDNA3.1 (Mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) (1 and 2 μ g) followed by PR8 infection and incubated for 24 h. Total RNA was isolated and NP mRNA ( a ), NP vRNA ( b ) and RNF43 mRNA( c ) levels were estimated through qRT PCR. ( d ) HEK293T cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by PR8 IAV infection for next 24 h. Cells were processed for qRT-PCR analysis of mRNA levels of NP and RNF43. ( e ) A549 cells, pretreated with either NT or RNF43 siRNA were transfected with plasmids-encoding polymerase complex components (PA, PB1, PB2 and NP) derived from PR8 (H1N1 virus) were co-transfected alongside a reporter plasmid containing noncoding sequence from the NS1 segment of influenza A virus and luciferase gene driven by the Pol 1 promoter. The relative luciferase units were calculated after normalization with plasmid pRL-TK (Promega, Madison, WI, USA), which expresses Renilla luciferase that was transfected along. Results are shown as mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P

    Article Snippet: Pool of gene specific siRNA against RNF43 was purchased from Santa Cruz, CA, USA.

    Techniques: Transfection, Plasmid Preparation, Infection, Incubation, Isolation, Quantitative RT-PCR, Derivative Assay, Sequencing, Luciferase

    RNF43 attenuates IAV NP-induced cell death. ( a ) A549 cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by X-31 IAV infection at 1 MOI. Cells were harvested at 24 h.p.i, stained with Annexin V FITC and subjected to flow cytometry. The percentage of Annexin V positive population is plotted on the graph. ( b ) A549 cells were transiently transfected with the indicated combinations of plasmids and 48 h post-transfection cells were harvested and processed for flow cytometric analysis of Annexin V FITC stained population which is plotted on the graph. ( c ) Same experiment was conducted in p53 −/− HCT116 cells. All graphs represent mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P

    Journal: Cell Death & Disease

    Article Title: The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43

    doi: 10.1038/cddis.2015.131

    Figure Lengend Snippet: RNF43 attenuates IAV NP-induced cell death. ( a ) A549 cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by X-31 IAV infection at 1 MOI. Cells were harvested at 24 h.p.i, stained with Annexin V FITC and subjected to flow cytometry. The percentage of Annexin V positive population is plotted on the graph. ( b ) A549 cells were transiently transfected with the indicated combinations of plasmids and 48 h post-transfection cells were harvested and processed for flow cytometric analysis of Annexin V FITC stained population which is plotted on the graph. ( c ) Same experiment was conducted in p53 −/− HCT116 cells. All graphs represent mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P

    Article Snippet: Pool of gene specific siRNA against RNF43 was purchased from Santa Cruz, CA, USA.

    Techniques: Infection, Staining, Flow Cytometry, Cytometry, Transfection

    RNF43 decreases NP driven increased p53 transcriptional activity and signaling in the cells. ( a ) A549 cells were transfected with p21-Luc reporter plasmid, with or without growing amounts of pEGFP-NP (500 and 750 ng, and 1 μg) in conjunction with pcDNA-p53 and a control plasmid, Renilla luciferase pRL-TK. Luciferase activity of cell lysates of transfectants was analyzed. ( b ) A549 cells were transfected with p21-Luc reporter plasmid, pcDNA-p53 and a control plasmid, Renilla luciferase pRL-TK along with plasmids pEGFPN1 (mock), pEGFP-NP (NP-GFP) and pCDNA3.1-RNF43-Flag-HA (RNF43-HA) in the indicated combinations. Luciferase activity was measured. ( c ) HEK293T cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by PR8 IAV infection at 1 MOI. Cells were harvested at 24-h post infection and total RNA was extracted followed by p21 mRNA estimation with qRT-PCR. ( d ) A549 cells were transiently transfected with pEGFP-NP with or without pCDNA3.1-RNF43-Flag-HA and were harvested at 48 h followed by SDS-PAGE. Western blotting was done using indicated antibodies. ( e ) A549 cells were seeded in a 6-well plate and were infected with PR8 virus at 1 MOI. Cells were harvested at 0, 4, 8, 16 and 24-h post infection, and subjected to Western blotting with indicated antibodies. ( f ) A549 cells were transfected with pCDNA3.1 (mock) and pCDNA3.1-RNF43-Flag-HA (RNF43-HA) plasmids followed by PR8 infection, 24-h post transfection. The cells were harvested after 24 h followed by Western blot analysis with anti-acetyl p53, HA, NP and β -actin antibodies. ( g ) A549 cells were transiently transfected with pCDNA3.1 (mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) plasmid constructs and after 24 h of incubation, cells were infected with PR8 virus at an MOI of 1 for 24 h. ( h ) Similarly, A549 cells were transiently transfected with pEGFP-NP with or without pCDNA3.1-RNF43-Flag-HA and were incubated for 48 h. ( g and h ) Cells were harvested and processed for Western blot analysis with indicated antibodies. Results in a – c are shown as mean±S.D. three independent experiments. * and # indicate statistically significant difference at P

    Journal: Cell Death & Disease

    Article Title: The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43

    doi: 10.1038/cddis.2015.131

    Figure Lengend Snippet: RNF43 decreases NP driven increased p53 transcriptional activity and signaling in the cells. ( a ) A549 cells were transfected with p21-Luc reporter plasmid, with or without growing amounts of pEGFP-NP (500 and 750 ng, and 1 μg) in conjunction with pcDNA-p53 and a control plasmid, Renilla luciferase pRL-TK. Luciferase activity of cell lysates of transfectants was analyzed. ( b ) A549 cells were transfected with p21-Luc reporter plasmid, pcDNA-p53 and a control plasmid, Renilla luciferase pRL-TK along with plasmids pEGFPN1 (mock), pEGFP-NP (NP-GFP) and pCDNA3.1-RNF43-Flag-HA (RNF43-HA) in the indicated combinations. Luciferase activity was measured. ( c ) HEK293T cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by PR8 IAV infection at 1 MOI. Cells were harvested at 24-h post infection and total RNA was extracted followed by p21 mRNA estimation with qRT-PCR. ( d ) A549 cells were transiently transfected with pEGFP-NP with or without pCDNA3.1-RNF43-Flag-HA and were harvested at 48 h followed by SDS-PAGE. Western blotting was done using indicated antibodies. ( e ) A549 cells were seeded in a 6-well plate and were infected with PR8 virus at 1 MOI. Cells were harvested at 0, 4, 8, 16 and 24-h post infection, and subjected to Western blotting with indicated antibodies. ( f ) A549 cells were transfected with pCDNA3.1 (mock) and pCDNA3.1-RNF43-Flag-HA (RNF43-HA) plasmids followed by PR8 infection, 24-h post transfection. The cells were harvested after 24 h followed by Western blot analysis with anti-acetyl p53, HA, NP and β -actin antibodies. ( g ) A549 cells were transiently transfected with pCDNA3.1 (mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) plasmid constructs and after 24 h of incubation, cells were infected with PR8 virus at an MOI of 1 for 24 h. ( h ) Similarly, A549 cells were transiently transfected with pEGFP-NP with or without pCDNA3.1-RNF43-Flag-HA and were incubated for 48 h. ( g and h ) Cells were harvested and processed for Western blot analysis with indicated antibodies. Results in a – c are shown as mean±S.D. three independent experiments. * and # indicate statistically significant difference at P

    Article Snippet: Pool of gene specific siRNA against RNF43 was purchased from Santa Cruz, CA, USA.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Quantitative RT-PCR, SDS Page, Western Blot, Construct, Incubation

    ASCL1 suppression reduces neuroendocrine marker production and induces cell-cycle arrest in BON and H727 cells Treatment of BON and H727 cells with an ASCL1 -specific siRNA reduced chromogranin A (CgA) and synaptophysin (SYP) levels, shown by Western blotting. Cells receiving the nonspecific-siRNA (NS-siRNA), exhibited no visible change in ASCL1, CgA or SYP levels relative to cells treated with Lipofectamine 2000 ® alone (no siRNA) (A). Concurrent with ASCL1 suppression following ASCL1 -siRNA treatment, BON and H727 cell lines exhibited a reduction in cyclin B1 and D1 expression, and an increase in p21 Waf1/Cip1 and p27 Kip1 expression, as demonstrated by Western blotting, therefore suggesting the occurrence of cell-cycle arrest. Cells receiving the NS-siRNA experienced no effect on levels of any of these markers (B).

    Journal: Cancer gene therapy

    Article Title: Chrysin suppresses the achaete-scute complex-like1 and alters the neuroendocrine phenotype of carcinoids

    doi: 10.1038/cgt.2015.49

    Figure Lengend Snippet: ASCL1 suppression reduces neuroendocrine marker production and induces cell-cycle arrest in BON and H727 cells Treatment of BON and H727 cells with an ASCL1 -specific siRNA reduced chromogranin A (CgA) and synaptophysin (SYP) levels, shown by Western blotting. Cells receiving the nonspecific-siRNA (NS-siRNA), exhibited no visible change in ASCL1, CgA or SYP levels relative to cells treated with Lipofectamine 2000 ® alone (no siRNA) (A). Concurrent with ASCL1 suppression following ASCL1 -siRNA treatment, BON and H727 cell lines exhibited a reduction in cyclin B1 and D1 expression, and an increase in p21 Waf1/Cip1 and p27 Kip1 expression, as demonstrated by Western blotting, therefore suggesting the occurrence of cell-cycle arrest. Cells receiving the NS-siRNA experienced no effect on levels of any of these markers (B).

    Article Snippet: A pool of four gene-specific small interfering RNA (siRNA) sequences against ASCL1 (catalog no. sc-37692, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and a nonspecific-siRNA (catalog no. sc-37007, Santa Cruz Biotechnology, Inc.) as a negative control were dissolved in media containing Lipofectamine 2000® (Life Technologies, Grand Island, NY, USA) as per manufacturer instructions, and added dropwise to plated cells.

    Techniques: Marker, Western Blot, Expressing

    ASCL1 suppression inhibits BON cell proliferation and CgA expression Treatment with an ASCL1 -specific siRNA resulted in an inhibition in BON cell proliferation over a 6 day period. A MTT cell viability assay was performed every 2 days, and optical densities were normalized to values starting from the day of transfection. No effect was observed on cell proliferation among cells treated with the nonspecific-siRNA (NS-siRNA) relative to cells treated with Lipofectamine 2000 ® alone (no siRNA). Data is graphed ± SEM (*p

    Journal: Cancer gene therapy

    Article Title: Chrysin suppresses the achaete-scute complex-like1 and alters the neuroendocrine phenotype of carcinoids

    doi: 10.1038/cgt.2015.49

    Figure Lengend Snippet: ASCL1 suppression inhibits BON cell proliferation and CgA expression Treatment with an ASCL1 -specific siRNA resulted in an inhibition in BON cell proliferation over a 6 day period. A MTT cell viability assay was performed every 2 days, and optical densities were normalized to values starting from the day of transfection. No effect was observed on cell proliferation among cells treated with the nonspecific-siRNA (NS-siRNA) relative to cells treated with Lipofectamine 2000 ® alone (no siRNA). Data is graphed ± SEM (*p

    Article Snippet: A pool of four gene-specific small interfering RNA (siRNA) sequences against ASCL1 (catalog no. sc-37692, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and a nonspecific-siRNA (catalog no. sc-37007, Santa Cruz Biotechnology, Inc.) as a negative control were dissolved in media containing Lipofectamine 2000® (Life Technologies, Grand Island, NY, USA) as per manufacturer instructions, and added dropwise to plated cells.

    Techniques: Expressing, Inhibition, MTT Assay, Viability Assay, Transfection

    (A) Dot blots analysis of the interaction of APR-1 protein with TNFR1 human p75NTR, mouse p75NTR, FADD and rat p75NTR. A total of 5 μg of glutathione S -transferase (GST)-TNFR1 (100.0 pmol), TNFR2 (111.0 pmol), human p75NTR (106.4 pmol), mouse p75NTR (106 pmol), FADD (92.6 pmol), TrKA (113.6 pmol), TrKB (113.6 pmol), TrKC (113.6 pmol), Fas 142.8 pmol), death domain (142.8 pmol), APR-1 (100 pmol), rat p75NTR (106 pmol) or GST (192.0 pmol) were diluted in PBS and blotted onto nitrocellulose membrane and subsequently incubated overnight with in vitro transcribed and translated [ 35 S] APR-1 protein. (B) Interaction of APR-1 with P75NTR. The total cell lysates prepared from A375-APR-1 and BLM-APR-1 before and after the induction of APR-1 protein were subjected for either electrophoresis (for the detection of APR-1 and P75NTR) or for co-immunoprecipitation (IP) with either anti-P75NTR antibody or with anti-APR-1 antibody. Western blotting of IP: p75NTR for APR-1 revealed the interaction of APR-1 to P75NTR, whereas Western blotting of IP: APR-1 for P75NTR revealed the interaction of P75NTR to APR-1. β-actin was used as internal control for loading and transfer. (C) Schematic diagram of the extracellular and intracellular domains of p75NTR. Transmembrane domain, JMD and death domain. (D) GST-P75NTR recombinant proteins 1–427aa (106.4 pmol), 1–341aa (135.1 pmol), 1–311aa (147 pmol), 1–274 aa (166 pmol), 275–340aa (694.3) and 341–427aa (526.2 pmol), were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with the P75NTR domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount and the position of P75NTR recombinant proteins (left panel). (E) GST-JMD and death domain of P75NTR were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with both domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount of both JMD and death domains (left panel). (F) Western blot analysis demonstrates the expression of APR-1 by the addition of Dox to the culture medium of BLM-APR- 1, the knockdown of p75NTR by its specific siRNA and the suppression of APR-1-induced cleavage of PARP by the p75NTR siRNA. β-actin was used as internal control for loading and transfer. (G) Analysis of cell viability by counting using trypan blue staining. Rescue of APR-1-induced reduction of cell viability by the knockdown of p75NTR by siRNA for 24 or 48 hrs. Data are mean of three experiments performed separately.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Apoptosis related protein-1 triggers melanoma cell death via interaction with the juxtamembrane region of p75 neurotrophin receptor

    doi: 10.1111/j.1582-4934.2011.01304.x

    Figure Lengend Snippet: (A) Dot blots analysis of the interaction of APR-1 protein with TNFR1 human p75NTR, mouse p75NTR, FADD and rat p75NTR. A total of 5 μg of glutathione S -transferase (GST)-TNFR1 (100.0 pmol), TNFR2 (111.0 pmol), human p75NTR (106.4 pmol), mouse p75NTR (106 pmol), FADD (92.6 pmol), TrKA (113.6 pmol), TrKB (113.6 pmol), TrKC (113.6 pmol), Fas 142.8 pmol), death domain (142.8 pmol), APR-1 (100 pmol), rat p75NTR (106 pmol) or GST (192.0 pmol) were diluted in PBS and blotted onto nitrocellulose membrane and subsequently incubated overnight with in vitro transcribed and translated [ 35 S] APR-1 protein. (B) Interaction of APR-1 with P75NTR. The total cell lysates prepared from A375-APR-1 and BLM-APR-1 before and after the induction of APR-1 protein were subjected for either electrophoresis (for the detection of APR-1 and P75NTR) or for co-immunoprecipitation (IP) with either anti-P75NTR antibody or with anti-APR-1 antibody. Western blotting of IP: p75NTR for APR-1 revealed the interaction of APR-1 to P75NTR, whereas Western blotting of IP: APR-1 for P75NTR revealed the interaction of P75NTR to APR-1. β-actin was used as internal control for loading and transfer. (C) Schematic diagram of the extracellular and intracellular domains of p75NTR. Transmembrane domain, JMD and death domain. (D) GST-P75NTR recombinant proteins 1–427aa (106.4 pmol), 1–341aa (135.1 pmol), 1–311aa (147 pmol), 1–274 aa (166 pmol), 275–340aa (694.3) and 341–427aa (526.2 pmol), were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with the P75NTR domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount and the position of P75NTR recombinant proteins (left panel). (E) GST-JMD and death domain of P75NTR were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with both domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount of both JMD and death domains (left panel). (F) Western blot analysis demonstrates the expression of APR-1 by the addition of Dox to the culture medium of BLM-APR- 1, the knockdown of p75NTR by its specific siRNA and the suppression of APR-1-induced cleavage of PARP by the p75NTR siRNA. β-actin was used as internal control for loading and transfer. (G) Analysis of cell viability by counting using trypan blue staining. Rescue of APR-1-induced reduction of cell viability by the knockdown of p75NTR by siRNA for 24 or 48 hrs. Data are mean of three experiments performed separately.

    Article Snippet: Application of siRNAs The knockdown of p75NTR was performed by its specific siRNA (D-009340–03; Dharmacon RNA Technology, Lafayette, CO, USA) as recommended by manufacturer’s protocol.

    Techniques: Incubation, In Vitro, Electrophoresis, Immunoprecipitation, Western Blot, Recombinant, SDS Page, Staining, Expressing

    HMGB1 accumulated in LCM induced microglial activation. (A) HMGB1 levels were determined in whole cell lysates of primary microglia cultures 12 hrs after transfection with HMGB1 siRNA (HMGB1) or nonspecific (NS) siRNA (40 pM). (B) LPS (100 ng/ml) was treated 12 hrs after HMGB1 siRNA or nonspecific siRNA (40 pM) transfection. After 18 hrs of LPS treatment, culture media were replaced with fresh medium, and LCM was collected at 24 hrs after medium replacement. Naïve primary microglia were then cultured in this LCM for 24 hrs and nitrite levels were measured. (C) Nitrite levels were measured at 24 hrs after LCM treatment in the presence of HMGB1 A box domain (10, 50, 100 ng/ml) or anti-HMGB1 antibody (100 or 500 ng/ml). HMGB1 B box domain (50, 100 ng/ml) and anti-human IgG (500 ng/ml) were used as negative controls. Data are presented as means±SEMs (n=4), ** p

    Journal: Experimental Neurobiology

    Article Title: HMGB1-Binding Heptamer Confers Anti-Inflammatory Effects in Primary Microglia Culture

    doi: 10.5607/en.2013.22.4.301

    Figure Lengend Snippet: HMGB1 accumulated in LCM induced microglial activation. (A) HMGB1 levels were determined in whole cell lysates of primary microglia cultures 12 hrs after transfection with HMGB1 siRNA (HMGB1) or nonspecific (NS) siRNA (40 pM). (B) LPS (100 ng/ml) was treated 12 hrs after HMGB1 siRNA or nonspecific siRNA (40 pM) transfection. After 18 hrs of LPS treatment, culture media were replaced with fresh medium, and LCM was collected at 24 hrs after medium replacement. Naïve primary microglia were then cultured in this LCM for 24 hrs and nitrite levels were measured. (C) Nitrite levels were measured at 24 hrs after LCM treatment in the presence of HMGB1 A box domain (10, 50, 100 ng/ml) or anti-HMGB1 antibody (100 or 500 ng/ml). HMGB1 B box domain (50, 100 ng/ml) and anti-human IgG (500 ng/ml) were used as negative controls. Data are presented as means±SEMs (n=4), ** p

    Article Snippet: SMART pool siRNA specifically targeting HMGB1 (ON-TARGET plus SMART pool siRNA L-114889, accession no.NM_ 001109373; Dharmacon, Lafayette, CO, USA) and non-specific siRNA (on-TARGET plus Non-targeting pool D-001810, Dharmacon, Lafayette, CO, USA) were used.

    Techniques: Laser Capture Microdissection, Activation Assay, Transfection, Cell Culture

    HMGB1 accumulation in LPS-treated primary microglial culture media (LCM). (A, B) Primary microglial cultures (1.5×10 5 cells/well) were incubated with LPS (100 ng/ml) for 1, 3, 6, 12, 18, or 24 hrs and HMGB1 levels in culture media (A) and in whole cell lysates (B) were determined by immunoblotting. α-Tubulin was used as a control for whole cell lysates (B). (C) After treating cells with LPS (100 ng/ml) for 18 hrs, primary microglial culture medium was replaced with fresh LPS-free medium and LCM was collected at 12 or 24 hrs after media replacement. HMGB1 levels were determined by immunoblotting. (D) LCM was concentrated, and treated to fresh primary microglial cultures. Nitrite levels were measured 24 hrs later. Data are presented as means±SEMs (n=4), * p

    Journal: Experimental Neurobiology

    Article Title: HMGB1-Binding Heptamer Confers Anti-Inflammatory Effects in Primary Microglia Culture

    doi: 10.5607/en.2013.22.4.301

    Figure Lengend Snippet: HMGB1 accumulation in LPS-treated primary microglial culture media (LCM). (A, B) Primary microglial cultures (1.5×10 5 cells/well) were incubated with LPS (100 ng/ml) for 1, 3, 6, 12, 18, or 24 hrs and HMGB1 levels in culture media (A) and in whole cell lysates (B) were determined by immunoblotting. α-Tubulin was used as a control for whole cell lysates (B). (C) After treating cells with LPS (100 ng/ml) for 18 hrs, primary microglial culture medium was replaced with fresh LPS-free medium and LCM was collected at 12 or 24 hrs after media replacement. HMGB1 levels were determined by immunoblotting. (D) LCM was concentrated, and treated to fresh primary microglial cultures. Nitrite levels were measured 24 hrs later. Data are presented as means±SEMs (n=4), * p

    Article Snippet: SMART pool siRNA specifically targeting HMGB1 (ON-TARGET plus SMART pool siRNA L-114889, accession no.NM_ 001109373; Dharmacon, Lafayette, CO, USA) and non-specific siRNA (on-TARGET plus Non-targeting pool D-001810, Dharmacon, Lafayette, CO, USA) were used.

    Techniques: Laser Capture Microdissection, Incubation

    HBHP bound to HMGB1 in LCM. (A) LCM was incubated with biotinylated-HBHP (Bt-HBHP; 1 or 5 µg/ml) or with biotinylated-scrambled peptide (Bt-HBHP-sc; 5 µg/ml) for 4 hrs and pull-down assays were performed using streptavidin agarose beads. Amounts of HMGB1 were determined by immunoblotting using anti-HMGB1 antibody. (B) LCM was collected from HMGB1 siRNA (HMGB1-; 40 pM)- or nonspecific siRNA (NS-; 40 pM)-transfected microglia cultures and pull-down assays were performed after incubating LCMs with biotinylated-HBHP. (C) Biotinylated-HBHP (5 µg/ml) was incubated with LCM in the presence of 1, 5, or 10 µg/ml of HMGB1 A box or 5 or 10 µg/ml of HMGB1 B box and pull-down assays were performed. Input controls before immunoprecipitations were presented by immunoblotting with anti-HMGB1. Results are representative of three independent experiments.

    Journal: Experimental Neurobiology

    Article Title: HMGB1-Binding Heptamer Confers Anti-Inflammatory Effects in Primary Microglia Culture

    doi: 10.5607/en.2013.22.4.301

    Figure Lengend Snippet: HBHP bound to HMGB1 in LCM. (A) LCM was incubated with biotinylated-HBHP (Bt-HBHP; 1 or 5 µg/ml) or with biotinylated-scrambled peptide (Bt-HBHP-sc; 5 µg/ml) for 4 hrs and pull-down assays were performed using streptavidin agarose beads. Amounts of HMGB1 were determined by immunoblotting using anti-HMGB1 antibody. (B) LCM was collected from HMGB1 siRNA (HMGB1-; 40 pM)- or nonspecific siRNA (NS-; 40 pM)-transfected microglia cultures and pull-down assays were performed after incubating LCMs with biotinylated-HBHP. (C) Biotinylated-HBHP (5 µg/ml) was incubated with LCM in the presence of 1, 5, or 10 µg/ml of HMGB1 A box or 5 or 10 µg/ml of HMGB1 B box and pull-down assays were performed. Input controls before immunoprecipitations were presented by immunoblotting with anti-HMGB1. Results are representative of three independent experiments.

    Article Snippet: SMART pool siRNA specifically targeting HMGB1 (ON-TARGET plus SMART pool siRNA L-114889, accession no.NM_ 001109373; Dharmacon, Lafayette, CO, USA) and non-specific siRNA (on-TARGET plus Non-targeting pool D-001810, Dharmacon, Lafayette, CO, USA) were used.

    Techniques: Laser Capture Microdissection, Incubation, Transfection, Liquid Chromatography with Mass Spectroscopy

    Role of Tim-1 in EBOV-induced activation of CD4 + T cells. (A and B) Disabling of Tim-1 reduces activation of EBOV-exposed T cells. Jurkat cells were transfected with control siRNA or siRNA targeting Tim-1, incubated for 48 h, stimulated with EBOV for an additional 48 h, stained, and analyzed for CD25 (A) and CD69 (B). (C) The EBOV-mediated activation of T cells depends on Lck kinase. Primary CD4 + T cells were incubated with Src inhibitor Ly294 for 1 h, stimulated with EBOV for 48 h, and analyzed for CD25 and CD69. (D) The EBOV-mediated activation of T cells depends on the PI3K pathway. Primary CD4 + T cells were incubated with PI3K inhibitor PP2 and EBOV and analyzed as described for panel F. (E) Binding of EBOV GP and EBOV-mediated downregulation of CD3 depend on Lck kinase and PI3K. Primary CD4 + T cells were incubated with Ly294 or PP2, and the percentages of binding and the CD4 Hi CD3 Low population were determined. (F) EBOV-induced phosphorylation of Akt depends on Src kinase and PI3K. CD4 + T cells were treated with Ly294 and PP2. Akt and its phosphorylated form were analyzed by Western blotting. *, P

    Journal: mBio

    Article Title: Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm

    doi: 10.1128/mBio.00845-17

    Figure Lengend Snippet: Role of Tim-1 in EBOV-induced activation of CD4 + T cells. (A and B) Disabling of Tim-1 reduces activation of EBOV-exposed T cells. Jurkat cells were transfected with control siRNA or siRNA targeting Tim-1, incubated for 48 h, stimulated with EBOV for an additional 48 h, stained, and analyzed for CD25 (A) and CD69 (B). (C) The EBOV-mediated activation of T cells depends on Lck kinase. Primary CD4 + T cells were incubated with Src inhibitor Ly294 for 1 h, stimulated with EBOV for 48 h, and analyzed for CD25 and CD69. (D) The EBOV-mediated activation of T cells depends on the PI3K pathway. Primary CD4 + T cells were incubated with PI3K inhibitor PP2 and EBOV and analyzed as described for panel F. (E) Binding of EBOV GP and EBOV-mediated downregulation of CD3 depend on Lck kinase and PI3K. Primary CD4 + T cells were incubated with Ly294 or PP2, and the percentages of binding and the CD4 Hi CD3 Low population were determined. (F) EBOV-induced phosphorylation of Akt depends on Src kinase and PI3K. CD4 + T cells were treated with Ly294 and PP2. Akt and its phosphorylated form were analyzed by Western blotting. *, P

    Article Snippet: Gene knockdown experiments were performed using pooled control or Tim-1-specific siRNA (Santa Cruz Biotech).

    Techniques: Activation Assay, Transfection, Incubation, Staining, Binding Assay, Western Blot

    Effect of ApoE knockdown on HIV-1 production in MDMs. (A) MDMs were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), which is a mixture (SMARTpool) of 4 siRNAs (“4-pool”), cultured for 2 days, and analyzed for their ApoE levels by western blot. Anti-β-actin blot was used as a loading control. Data shown are representative of experiments obtained from 4 different donors with similar results. (B) MDMs were transfected as in (A), cultured for 2 days, and analyzed for their cell surface CD4 (left panel) and CCR5 (right panel) expression by flow cytometry. The mean fluorescence intensity (MFI) values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (C) MDMs were transfected as in (A), cultured for 3 (left panel) or 6 days (right panel), and analyzed for their viability by the MTT assay. The viabilities of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (D) MDMs (4 donors) were transfected as in (A), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). The culture supernatants (“sup”) were collected as indicated (dpi, days of post-infection), and analyzed for their levels of p24 concentration by ELISA. The left panel in each donor set shows the overall kinetics of viral production. The right panel in each donor set shows the p24 concentrations in an earlier phase (3 dpi for donors 1 and 2, or 2 dpi for donors 3 and 4). (E) MDMs were transfected as in (A), infected as in (D), and analyzed for their levels of p24 concentration in the supernatants by ELISA. The p24 values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 4 donors are summarized. * p

    Journal: PLoS Pathogens

    Article Title: Apolipoprotein E is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages

    doi: 10.1371/journal.ppat.1007372

    Figure Lengend Snippet: Effect of ApoE knockdown on HIV-1 production in MDMs. (A) MDMs were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), which is a mixture (SMARTpool) of 4 siRNAs (“4-pool”), cultured for 2 days, and analyzed for their ApoE levels by western blot. Anti-β-actin blot was used as a loading control. Data shown are representative of experiments obtained from 4 different donors with similar results. (B) MDMs were transfected as in (A), cultured for 2 days, and analyzed for their cell surface CD4 (left panel) and CCR5 (right panel) expression by flow cytometry. The mean fluorescence intensity (MFI) values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (C) MDMs were transfected as in (A), cultured for 3 (left panel) or 6 days (right panel), and analyzed for their viability by the MTT assay. The viabilities of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (D) MDMs (4 donors) were transfected as in (A), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). The culture supernatants (“sup”) were collected as indicated (dpi, days of post-infection), and analyzed for their levels of p24 concentration by ELISA. The left panel in each donor set shows the overall kinetics of viral production. The right panel in each donor set shows the p24 concentrations in an earlier phase (3 dpi for donors 1 and 2, or 2 dpi for donors 3 and 4). (E) MDMs were transfected as in (A), infected as in (D), and analyzed for their levels of p24 concentration in the supernatants by ELISA. The p24 values of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 4 donors are summarized. * p

    Article Snippet: Both the control siRNAs (non-targeting siRNA pool; D-001206-13, non-targeting siRNA #1; D-001210-01) and the ApoE-specific siRNAs (ApoE siRNA pool; M-006470-00, ApoE siRNA #1; D-006470-01, ApoE siRNA #2; D-006470-02) were purchased from Dharmacon.

    Techniques: Transfection, Cell Culture, Western Blot, Expressing, Flow Cytometry, Cytometry, Fluorescence, MTT Assay, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Effects of ApoE knockdown on infectivity of produced HIV-1 and localization of HIV-1 Env in MDMs. (A) MDMs were transfected with either ApoE siRNA #1 or pooled non-targeting siRNAs, cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). In the left panel, the supernatants were collected at 2 dpi, and the infectivity of the produced viruses was analyzed by the luciferase (Luc) reporter gene assay with TZM-bl cells, by changing the viral input (p24 amount) as indicated. RLU, relative light units. In the right panel, the infectivity of the viruses produced by ApoE siRNA-transfected cells are represented as percentages relative to that by control siRNA-transfected cells, and results for MDMs obtained from 3 donors are summarized. * p

    Journal: PLoS Pathogens

    Article Title: Apolipoprotein E is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages

    doi: 10.1371/journal.ppat.1007372

    Figure Lengend Snippet: Effects of ApoE knockdown on infectivity of produced HIV-1 and localization of HIV-1 Env in MDMs. (A) MDMs were transfected with either ApoE siRNA #1 or pooled non-targeting siRNAs, cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). In the left panel, the supernatants were collected at 2 dpi, and the infectivity of the produced viruses was analyzed by the luciferase (Luc) reporter gene assay with TZM-bl cells, by changing the viral input (p24 amount) as indicated. RLU, relative light units. In the right panel, the infectivity of the viruses produced by ApoE siRNA-transfected cells are represented as percentages relative to that by control siRNA-transfected cells, and results for MDMs obtained from 3 donors are summarized. * p

    Article Snippet: Both the control siRNAs (non-targeting siRNA pool; D-001206-13, non-targeting siRNA #1; D-001210-01) and the ApoE-specific siRNAs (ApoE siRNA pool; M-006470-00, ApoE siRNA #1; D-006470-01, ApoE siRNA #2; D-006470-02) were purchased from Dharmacon.

    Techniques: Infection, Produced, Transfection, Cell Culture, Luciferase, Reporter Gene Assay

    Effects of ApoE knockdown on MX2 expression and HIV-1 production in MDMs. (A) MDMs (3 donors) were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), which is a mixture (SMARTpool) of 4 siRNAs (“4-pool”), cultured for 2 days, and analyzed for their MX2 mRNA levels by qRT-PCR followed by the normalization to the mRNA level of β-actin . Each mRNA level was calculated relative to the control siRNA-transfected MDMs (fold). Error bars in these panels indicate standard deviation of triplicate PCR assays. MDMs left untreated or treated with IFN-α for 24 h were added as a reference for MX2 expression (see donor 3). (B) MDMs were transfected and analyzed as in (A). The MX2 mRNA expression levels of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (C) MDMs were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), cultured for 2 days, and analyzed for their ApoE levels by western blot. In addition to the pool of 4 (#1, #2, #3 and #4) ApoE-targeting siRNAs (“4-pool”), #1 and #2 siRNAs were used. Anti-β-actin blot was used as a loading control. Data shown are representative of experiments obtained from 4 different donors with similar results. (D) MDMs (3 donors) were transfected with the indicated siRNAs (ApoE-targeting or non-targeting siRNA), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). The culture supernatants were collected as indicated, and analyzed for their levels of p24 concentration by ELISA. (E) MDMs were transfected with either ApoE-targeting siRNA (#1) or non-targeting siRNAs (SMART pool), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24) for another 2 days. Then, their intracellular Gag levels were determined by flow cytometry. In the right panel, the frequencies of intracellular Gag-positive MDMs in the ApoE siRNA transfection are represented as percentages relative to those in the control siRNA transfection, and results for MDMs obtained from 3 donors are summarized. * p

    Journal: PLoS Pathogens

    Article Title: Apolipoprotein E is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages

    doi: 10.1371/journal.ppat.1007372

    Figure Lengend Snippet: Effects of ApoE knockdown on MX2 expression and HIV-1 production in MDMs. (A) MDMs (3 donors) were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), which is a mixture (SMARTpool) of 4 siRNAs (“4-pool”), cultured for 2 days, and analyzed for their MX2 mRNA levels by qRT-PCR followed by the normalization to the mRNA level of β-actin . Each mRNA level was calculated relative to the control siRNA-transfected MDMs (fold). Error bars in these panels indicate standard deviation of triplicate PCR assays. MDMs left untreated or treated with IFN-α for 24 h were added as a reference for MX2 expression (see donor 3). (B) MDMs were transfected and analyzed as in (A). The MX2 mRNA expression levels of ApoE siRNA-transfected cells are represented as percentages relative to those of control siRNA-transfected cells. Results for MDMs obtained from 3 different donors are summarized. n . s ., not significant. (C) MDMs were transfected with either ApoE-targeting siRNA (“si-ApoE”) or non-targeting siRNA as a control (“si-Cr”), cultured for 2 days, and analyzed for their ApoE levels by western blot. In addition to the pool of 4 (#1, #2, #3 and #4) ApoE-targeting siRNAs (“4-pool”), #1 and #2 siRNAs were used. Anti-β-actin blot was used as a loading control. Data shown are representative of experiments obtained from 4 different donors with similar results. (D) MDMs (3 donors) were transfected with the indicated siRNAs (ApoE-targeting or non-targeting siRNA), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24). The culture supernatants were collected as indicated, and analyzed for their levels of p24 concentration by ELISA. (E) MDMs were transfected with either ApoE-targeting siRNA (#1) or non-targeting siRNAs (SMART pool), cultured for 2 days, and infected with HIV-1 JR-FL (100 ng/mL p24) for another 2 days. Then, their intracellular Gag levels were determined by flow cytometry. In the right panel, the frequencies of intracellular Gag-positive MDMs in the ApoE siRNA transfection are represented as percentages relative to those in the control siRNA transfection, and results for MDMs obtained from 3 donors are summarized. * p

    Article Snippet: Both the control siRNAs (non-targeting siRNA pool; D-001206-13, non-targeting siRNA #1; D-001210-01) and the ApoE-specific siRNAs (ApoE siRNA pool; M-006470-00, ApoE siRNA #1; D-006470-01, ApoE siRNA #2; D-006470-02) were purchased from Dharmacon.

    Techniques: Expressing, Transfection, Cell Culture, Quantitative RT-PCR, Standard Deviation, Polymerase Chain Reaction, Western Blot, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry