sybr green master mix Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher sybr green master mix
    Downregulating UPF1 from HeLa or HepG2 cells results in an upregulation of the endogenous HFE transcripts which indicates that the physiological human HFE mRNA is a natural NMD-target. HeLa cells were transiently transfected with synthetic small-interfering RNA (siRNA) duplexes directed to human UPF1 or to a non-endogenous target (Luciferase; Luc) used as control. Twenty-four (24 h), forty-eight (48 h) and seventy-two hours (72 h) after siRNA treatment, cells were harvested and protein and RNA were isolated. (A) Representative Western blot analysis of the HeLa and HepG2 cells extracts transfected with human UPF1 siRNA. Immunoblotting was performed using a human UPF1 specific antibody and a α-tubulin specific antibody to control for variations in protein loading. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved by densitometric analysis using ImageJ software. (B) Relative changes in HFE mRNA levels were analyzed by quantitative reverse transcription-coupled real-time <t>PCR</t> (RT-qPCR), normalized to the levels of endogenous G protein pathway suppressor 1 ( GPS1 ) mRNA. For that, cDNA was synthesized from total RNA and then, each cDNA sample was used as template for qPCR, which was performed using the <t>SYBR</t> Green Master Mix (Applied Biosystems) and primers that specifically hybridize to the 5′-end of HFE exon seven. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed using Student's t test (unpaired, two tailed). Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. The double arrow represents the coordinates of the amplicon obtained in the qPCR. (C) Relative changes in HFE mRNA levels were quantified by RT-qPCR as in B but using primers that specifically hybridize to the 5′-end of the HFE exon six. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B . Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. Arrows represent the position of primers used in the qPCR. (D) Representative Western blot analysis of HeLa cells extracts transfected with human UPF1 siRNA or with a control Luciferase siRNA target. Twenty-four hours after siRNA treatment, cells were transfected with the β-globin reporter constructs (β39). Twenty-four hours post transfection, protein and RNA were isolated from the cells for analysis. Immunoblotting to confirm UPF1 knockdown was carried out with anti-UPF1 and with anti-α-tubulin antibodies (as a loading control). Identification of each band is on the right. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved as in A . (E) Relative β-globin mRNA levels in UPF1-depleted HeLa cells, normalized to the levels of puromycin resistance mRNA (plasmids carrying the reporter β-globin gene also contain the Puro r gene), were determined by quantitative RT-qPCR and compared to the corresponding β39 mRNA levels under control conditions (Luc siRNA-treated HeLa cells) (defined as 1; arbitrary units). Using the same RNA samples, relative changes in HFE mRNA levels were also quantified by RT-qPCR, using experimental conditions as in B . Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B .
    Sybr Green Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 28878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybr green master mix/product/Thermo Fisher
    Average 99 stars, based on 28878 article reviews
    Price from $9.99 to $1999.99
    sybr green master mix - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher sybr master mix
    HEV induces <t>ISG15</t> both in vitro and in vivo . (A) Replication kinetics of viral RNAs from the HEV P6 infectious cDNA clone and HEV P6GLuc replicon in Huh7-S10-3 liver cells. The cells were transfected with capped RNA transcripts from the genotype 3 HEV P6 infectious cDNA clone (HEV P6) or with the HEV P6GLuc replicon (HEV P6GLuc; P6 encoding Gaussia luciferase clone). The culture supernatants were collected at various time points and used to measure viral RNA replication levels by HEV real-time quantitative RT-PCR or by the Gaussia luciferase assay (GLuc assay). RLU, relative light units. (B) ISG15 mRNA expression levels in Huh7-S10-3 liver cells transfected with capped RNA transcripts from the HEV P6 infectious cDNA clone or the HEV P6GLuc replicon at 1 and 5 days posttransfection (dpt) were determined by <t>Sybr</t> green-based quantitative PCR (qPCR). The ISG15-mRNA fold change was calculated using the 2 −ΔΔ CT method for comparisons to untransfected control cells, and the RPS18 gene was used as the housekeeping gene. (C and D) ISG15 protein expression levels in Huh7-S10-3 cells transfected with capped RNA from the HEV P6 infectious cDNA clone or the HEV P6GLuc replicon (C and D) or the HEV P6 replication-deficient infectious cDNA clone (P6GAD) (D). CC, untransfected cell control. The ISG15 protein expression, at 5 days posttransfection, in cell lysates (40 μg/lane) was analyzed by Western blotting using anti-ISG15 antibody (1:1,000 dilution). Fold change in band density, as calculated via normalization of ISG15 with β-actin (loading control), was measured using ImageJ software (NIH, Bethesda, MD). (E) ISG15 and USP18 mRNA expression levels in swine liver tissue samples obtained from animals experimentally infected with HEV ( n = 3) at 3 weeks postinfection. The fold changes in swine ISG15 and USP18 mRNA levels were calculated using the 2 −ΔΔ CT method compared to the uninfected control animals ( n = 2), and the RPS18 gene was used as the housekeeping gene. The data represent means ± standard errors of the means (SEM) of results from three independent transfection experiments (A, B, and C), one experiment representative of two independent transfection experiments (D), and HEV-infected animals ( n = 3) compared to uninfected animals ( n = 2) (E). **, P ≤ 0.01.
    Sybr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1058 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybr master mix/product/Thermo Fisher
    Average 99 stars, based on 1058 article reviews
    Price from $9.99 to $1999.99
    sybr master mix - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    Downregulating UPF1 from HeLa or HepG2 cells results in an upregulation of the endogenous HFE transcripts which indicates that the physiological human HFE mRNA is a natural NMD-target. HeLa cells were transiently transfected with synthetic small-interfering RNA (siRNA) duplexes directed to human UPF1 or to a non-endogenous target (Luciferase; Luc) used as control. Twenty-four (24 h), forty-eight (48 h) and seventy-two hours (72 h) after siRNA treatment, cells were harvested and protein and RNA were isolated. (A) Representative Western blot analysis of the HeLa and HepG2 cells extracts transfected with human UPF1 siRNA. Immunoblotting was performed using a human UPF1 specific antibody and a α-tubulin specific antibody to control for variations in protein loading. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved by densitometric analysis using ImageJ software. (B) Relative changes in HFE mRNA levels were analyzed by quantitative reverse transcription-coupled real-time PCR (RT-qPCR), normalized to the levels of endogenous G protein pathway suppressor 1 ( GPS1 ) mRNA. For that, cDNA was synthesized from total RNA and then, each cDNA sample was used as template for qPCR, which was performed using the SYBR Green Master Mix (Applied Biosystems) and primers that specifically hybridize to the 5′-end of HFE exon seven. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed using Student's t test (unpaired, two tailed). Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. The double arrow represents the coordinates of the amplicon obtained in the qPCR. (C) Relative changes in HFE mRNA levels were quantified by RT-qPCR as in B but using primers that specifically hybridize to the 5′-end of the HFE exon six. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B . Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. Arrows represent the position of primers used in the qPCR. (D) Representative Western blot analysis of HeLa cells extracts transfected with human UPF1 siRNA or with a control Luciferase siRNA target. Twenty-four hours after siRNA treatment, cells were transfected with the β-globin reporter constructs (β39). Twenty-four hours post transfection, protein and RNA were isolated from the cells for analysis. Immunoblotting to confirm UPF1 knockdown was carried out with anti-UPF1 and with anti-α-tubulin antibodies (as a loading control). Identification of each band is on the right. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved as in A . (E) Relative β-globin mRNA levels in UPF1-depleted HeLa cells, normalized to the levels of puromycin resistance mRNA (plasmids carrying the reporter β-globin gene also contain the Puro r gene), were determined by quantitative RT-qPCR and compared to the corresponding β39 mRNA levels under control conditions (Luc siRNA-treated HeLa cells) (defined as 1; arbitrary units). Using the same RNA samples, relative changes in HFE mRNA levels were also quantified by RT-qPCR, using experimental conditions as in B . Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B .

    Journal: PLoS ONE

    Article Title: Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

    doi: 10.1371/journal.pone.0035461

    Figure Lengend Snippet: Downregulating UPF1 from HeLa or HepG2 cells results in an upregulation of the endogenous HFE transcripts which indicates that the physiological human HFE mRNA is a natural NMD-target. HeLa cells were transiently transfected with synthetic small-interfering RNA (siRNA) duplexes directed to human UPF1 or to a non-endogenous target (Luciferase; Luc) used as control. Twenty-four (24 h), forty-eight (48 h) and seventy-two hours (72 h) after siRNA treatment, cells were harvested and protein and RNA were isolated. (A) Representative Western blot analysis of the HeLa and HepG2 cells extracts transfected with human UPF1 siRNA. Immunoblotting was performed using a human UPF1 specific antibody and a α-tubulin specific antibody to control for variations in protein loading. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved by densitometric analysis using ImageJ software. (B) Relative changes in HFE mRNA levels were analyzed by quantitative reverse transcription-coupled real-time PCR (RT-qPCR), normalized to the levels of endogenous G protein pathway suppressor 1 ( GPS1 ) mRNA. For that, cDNA was synthesized from total RNA and then, each cDNA sample was used as template for qPCR, which was performed using the SYBR Green Master Mix (Applied Biosystems) and primers that specifically hybridize to the 5′-end of HFE exon seven. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed using Student's t test (unpaired, two tailed). Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. The double arrow represents the coordinates of the amplicon obtained in the qPCR. (C) Relative changes in HFE mRNA levels were quantified by RT-qPCR as in B but using primers that specifically hybridize to the 5′-end of the HFE exon six. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B . Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. Arrows represent the position of primers used in the qPCR. (D) Representative Western blot analysis of HeLa cells extracts transfected with human UPF1 siRNA or with a control Luciferase siRNA target. Twenty-four hours after siRNA treatment, cells were transfected with the β-globin reporter constructs (β39). Twenty-four hours post transfection, protein and RNA were isolated from the cells for analysis. Immunoblotting to confirm UPF1 knockdown was carried out with anti-UPF1 and with anti-α-tubulin antibodies (as a loading control). Identification of each band is on the right. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved as in A . (E) Relative β-globin mRNA levels in UPF1-depleted HeLa cells, normalized to the levels of puromycin resistance mRNA (plasmids carrying the reporter β-globin gene also contain the Puro r gene), were determined by quantitative RT-qPCR and compared to the corresponding β39 mRNA levels under control conditions (Luc siRNA-treated HeLa cells) (defined as 1; arbitrary units). Using the same RNA samples, relative changes in HFE mRNA levels were also quantified by RT-qPCR, using experimental conditions as in B . Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B .

    Article Snippet: Real-time PCR was performed in an ABI Prism 7000 Sequence Detection System using SYBR Green Master Mix (Applied Biosystems).

    Techniques: Transfection, Small Interfering RNA, Luciferase, Isolation, Western Blot, Software, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Synthesized, SYBR Green Assay, Two Tailed Test, Amplification, Construct

    Extracellular DNA contributes to inter-kingdom pathogenicity. Mono- and dual-species biofilms were seeded at 1 × 10 6 CFU/mL in black 96-well plates and eDNA release at 1.5, 6, 12, and 24 h measured using a SYBR ® Green 1 based microplate fluorescence assay (MFA) in comparison to a standard curve (A) . Biofilms were washed with 0.2 M EDTA to remove the ECM and resulting eDNA quantified using the MFA described above in comparison to a standard curve (B) . ECM associated DNA was then precipitated from matrix extracts and species contributions were analyzed using qPCR (C) . C. albicans only biofilms were grown for 24 h in black 96-well plates. After washing biofilms were then treated with either 130 or 650 mg/L of DNase for 4 h. SYTO9 ® stained S. aureus cells (1 × 10 6 CFU/mL) were then added to the biofilm and incubated for 90 mins before being fluorescently quantified in comparison to an vehicle control treated biofilm (D). Data represents duplicate samples from three independent experiments ( ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Candida albicans Mycofilms Support Staphylococcus aureus Colonization and Enhances Miconazole Resistance in Dual-Species Interactions

    doi: 10.3389/fmicb.2017.00258

    Figure Lengend Snippet: Extracellular DNA contributes to inter-kingdom pathogenicity. Mono- and dual-species biofilms were seeded at 1 × 10 6 CFU/mL in black 96-well plates and eDNA release at 1.5, 6, 12, and 24 h measured using a SYBR ® Green 1 based microplate fluorescence assay (MFA) in comparison to a standard curve (A) . Biofilms were washed with 0.2 M EDTA to remove the ECM and resulting eDNA quantified using the MFA described above in comparison to a standard curve (B) . ECM associated DNA was then precipitated from matrix extracts and species contributions were analyzed using qPCR (C) . C. albicans only biofilms were grown for 24 h in black 96-well plates. After washing biofilms were then treated with either 130 or 650 mg/L of DNase for 4 h. SYTO9 ® stained S. aureus cells (1 × 10 6 CFU/mL) were then added to the biofilm and incubated for 90 mins before being fluorescently quantified in comparison to an vehicle control treated biofilm (D). Data represents duplicate samples from three independent experiments ( ∗ p

    Article Snippet: Briefly, 1 μL of extracted DNA was added to a PCR mastermix containing Fast SYBR® Green Master Mix, 10 μM species-specific forward and reverse primers ( Table ), and RNase free water. qPCR was then carried out using the Step-One plus real time PCR machine (Life Technologies, Paisley, UK) using the following thermal profile: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. Colony forming equivalents (CFE) were calculated compared to a standard curve of serially diluted DNA of each species as previously described ( ).

    Techniques: SYBR Green Assay, Fluorescence, Real-time Polymerase Chain Reaction, Staining, Incubation

    Genotypic levels of total RpL 14 mRNA expression in groups of whole individuals. Amount of RpL 14 mRNA in adults and larvae relative to three normalization genes. Height of bars indicate total amount of RpL 14 mRNA based on SYBR green-based quantitative reverse-transcription PCR and each bar is split to represent the proportion of total RpL 14 expressed from the RpL 14 [r] gene in { Ud }86 (white) and the endogenous RpL 14 [+] gene (gray), based on gene-specific TaqMan probes. Error bars represent 1 standard error for three biological replicates.

    Journal: PLoS ONE

    Article Title: First Steps towards Underdominant Genetic Transformation of Insect Populations

    doi: 10.1371/journal.pone.0097557

    Figure Lengend Snippet: Genotypic levels of total RpL 14 mRNA expression in groups of whole individuals. Amount of RpL 14 mRNA in adults and larvae relative to three normalization genes. Height of bars indicate total amount of RpL 14 mRNA based on SYBR green-based quantitative reverse-transcription PCR and each bar is split to represent the proportion of total RpL 14 expressed from the RpL 14 [r] gene in { Ud }86 (white) and the endogenous RpL 14 [+] gene (gray), based on gene-specific TaqMan probes. Error bars represent 1 standard error for three biological replicates.

    Article Snippet: 1 µL of the resulting reaction was used as a template for qPCR, using TaqMan Fast Master Mix (Applied Biosystems) for determining the relative ratios of wild-type and rescue transcripts or SYBR Green Fast Master Mix (Applied Biosystems) for total RpL14 mRNA levels.

    Techniques: Expressing, SYBR Green Assay, Polymerase Chain Reaction

    The BAC NEUROG3-SeAP/EGFP transgenes are expressed in the developing pancreas. (A) Homologous recombination ( Yang et al., 1997 ) was used to replace the human neurogenin-3 coding sequence in the NEUROG3 BAC (RP11-343J3T) with two reporter genes, SeAP and EGFP, flanked on the 5′ end by the human β-globulin intron and the 3′ end by the SV40 polyadenylation signal, and separated by a viral IRES. (B) Levels of neurogenin-3 mRNA in mouse pancreas were measured by real-time TaqMan RT-PCR at the embryonic dates shown and in adult islets, and are expressed relative to levels of histone H3.3a mRNA. (C) Levels of the SeAP / EGF P mRNA in mouse pancreas were measured by real-time SYBR Green RT-PCR at the embryonic dates shown and are expressed relative to levels of mouse β-actin mRNA. (D) Tissue SeAP activity was measured in pancreas homogenates at the embryonic dates shown and is expressed relative to total protein. All data represent mean + s.e.m. from at least three independent experiments.

    Journal: Disease Models & Mechanisms

    Article Title: A mouse model for monitoring islet cell genesis and developing therapies for diabetes

    doi: 10.1242/dmm.002998

    Figure Lengend Snippet: The BAC NEUROG3-SeAP/EGFP transgenes are expressed in the developing pancreas. (A) Homologous recombination ( Yang et al., 1997 ) was used to replace the human neurogenin-3 coding sequence in the NEUROG3 BAC (RP11-343J3T) with two reporter genes, SeAP and EGFP, flanked on the 5′ end by the human β-globulin intron and the 3′ end by the SV40 polyadenylation signal, and separated by a viral IRES. (B) Levels of neurogenin-3 mRNA in mouse pancreas were measured by real-time TaqMan RT-PCR at the embryonic dates shown and in adult islets, and are expressed relative to levels of histone H3.3a mRNA. (C) Levels of the SeAP / EGF P mRNA in mouse pancreas were measured by real-time SYBR Green RT-PCR at the embryonic dates shown and are expressed relative to levels of mouse β-actin mRNA. (D) Tissue SeAP activity was measured in pancreas homogenates at the embryonic dates shown and is expressed relative to total protein. All data represent mean + s.e.m. from at least three independent experiments.

    Article Snippet: Quantification of SeAP /EGFP cDNA was performed with SYBR Green using Fast SYBR Master Mix (Applied Biosystems) and reported relative to levels of the cDNA encoding mouse β-actin.

    Techniques: BAC Assay, Homologous Recombination, Sequencing, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Activity Assay

    Src homology 2 domain–containing adaptor protein B (SHB) expression in bone marrow (BM)–derived dendritic cell (BMDC) development. BM cells from C57BL/6 mice were cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor (10 ng/mL) to generate BMDCs. (A) Total RNA was isolated from immature dendritic cells (imDCs) and lipopolysaccharide (LPS)-treated mature dendritic cells (mDCs), and SHB mRNA was assessed from each sample by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR) with the Maxime RT-PCR PreMix (iNtRON) and Fast SYBR Green Master Mix (Life Technologies) kits, respectively. (B) The amount of SHB protein expressed in imDCs and mDCs was assessed by Western blot (WB) assay. (C) SHB expression in imDCs and mDCs was assessed by fluorescence-activated cell sorting after intracellular staining. (D, E) Splenic DCs (spDCs) were isolated from mice using a CD11c + isolation kit (Miltenyi Biotech) and treated or not with LPS (200 ng/mL) for 24 hours. Intracellular SHB protein expression in spDCs was assessed by fluorescence-activated cell sorter (D) and WB assay (E). RT-PCR data are shown as the mean±standard deviation of nine samples pooled from three independent experiments.

    Journal: Clinical and Experimental Vaccine Research

    Article Title: SH2 domain–containing adaptor protein B expressed in dendritic cells is involved in T-cell homeostasis by regulating dendritic cell–mediated Th2 immunity

    doi: 10.7774/cevr.2017.6.1.50

    Figure Lengend Snippet: Src homology 2 domain–containing adaptor protein B (SHB) expression in bone marrow (BM)–derived dendritic cell (BMDC) development. BM cells from C57BL/6 mice were cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor (10 ng/mL) to generate BMDCs. (A) Total RNA was isolated from immature dendritic cells (imDCs) and lipopolysaccharide (LPS)-treated mature dendritic cells (mDCs), and SHB mRNA was assessed from each sample by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR) with the Maxime RT-PCR PreMix (iNtRON) and Fast SYBR Green Master Mix (Life Technologies) kits, respectively. (B) The amount of SHB protein expressed in imDCs and mDCs was assessed by Western blot (WB) assay. (C) SHB expression in imDCs and mDCs was assessed by fluorescence-activated cell sorting after intracellular staining. (D, E) Splenic DCs (spDCs) were isolated from mice using a CD11c + isolation kit (Miltenyi Biotech) and treated or not with LPS (200 ng/mL) for 24 hours. Intracellular SHB protein expression in spDCs was assessed by fluorescence-activated cell sorter (D) and WB assay (E). RT-PCR data are shown as the mean±standard deviation of nine samples pooled from three independent experiments.

    Article Snippet: Quantitative PCR was performed using the Fast SYBR Green Master Mix kit (Life Technologies).

    Techniques: Expressing, Derivative Assay, Mouse Assay, Cell Culture, Isolation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay, Western Blot, Fluorescence, FACS, Staining, Standard Deviation

    HEV induces ISG15 both in vitro and in vivo . (A) Replication kinetics of viral RNAs from the HEV P6 infectious cDNA clone and HEV P6GLuc replicon in Huh7-S10-3 liver cells. The cells were transfected with capped RNA transcripts from the genotype 3 HEV P6 infectious cDNA clone (HEV P6) or with the HEV P6GLuc replicon (HEV P6GLuc; P6 encoding Gaussia luciferase clone). The culture supernatants were collected at various time points and used to measure viral RNA replication levels by HEV real-time quantitative RT-PCR or by the Gaussia luciferase assay (GLuc assay). RLU, relative light units. (B) ISG15 mRNA expression levels in Huh7-S10-3 liver cells transfected with capped RNA transcripts from the HEV P6 infectious cDNA clone or the HEV P6GLuc replicon at 1 and 5 days posttransfection (dpt) were determined by Sybr green-based quantitative PCR (qPCR). The ISG15-mRNA fold change was calculated using the 2 −ΔΔ CT method for comparisons to untransfected control cells, and the RPS18 gene was used as the housekeeping gene. (C and D) ISG15 protein expression levels in Huh7-S10-3 cells transfected with capped RNA from the HEV P6 infectious cDNA clone or the HEV P6GLuc replicon (C and D) or the HEV P6 replication-deficient infectious cDNA clone (P6GAD) (D). CC, untransfected cell control. The ISG15 protein expression, at 5 days posttransfection, in cell lysates (40 μg/lane) was analyzed by Western blotting using anti-ISG15 antibody (1:1,000 dilution). Fold change in band density, as calculated via normalization of ISG15 with β-actin (loading control), was measured using ImageJ software (NIH, Bethesda, MD). (E) ISG15 and USP18 mRNA expression levels in swine liver tissue samples obtained from animals experimentally infected with HEV ( n = 3) at 3 weeks postinfection. The fold changes in swine ISG15 and USP18 mRNA levels were calculated using the 2 −ΔΔ CT method compared to the uninfected control animals ( n = 2), and the RPS18 gene was used as the housekeeping gene. The data represent means ± standard errors of the means (SEM) of results from three independent transfection experiments (A, B, and C), one experiment representative of two independent transfection experiments (D), and HEV-infected animals ( n = 3) compared to uninfected animals ( n = 2) (E). **, P ≤ 0.01.

    Journal: Journal of Virology

    Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

    doi: 10.1128/JVI.00621-17

    Figure Lengend Snippet: HEV induces ISG15 both in vitro and in vivo . (A) Replication kinetics of viral RNAs from the HEV P6 infectious cDNA clone and HEV P6GLuc replicon in Huh7-S10-3 liver cells. The cells were transfected with capped RNA transcripts from the genotype 3 HEV P6 infectious cDNA clone (HEV P6) or with the HEV P6GLuc replicon (HEV P6GLuc; P6 encoding Gaussia luciferase clone). The culture supernatants were collected at various time points and used to measure viral RNA replication levels by HEV real-time quantitative RT-PCR or by the Gaussia luciferase assay (GLuc assay). RLU, relative light units. (B) ISG15 mRNA expression levels in Huh7-S10-3 liver cells transfected with capped RNA transcripts from the HEV P6 infectious cDNA clone or the HEV P6GLuc replicon at 1 and 5 days posttransfection (dpt) were determined by Sybr green-based quantitative PCR (qPCR). The ISG15-mRNA fold change was calculated using the 2 −ΔΔ CT method for comparisons to untransfected control cells, and the RPS18 gene was used as the housekeeping gene. (C and D) ISG15 protein expression levels in Huh7-S10-3 cells transfected with capped RNA from the HEV P6 infectious cDNA clone or the HEV P6GLuc replicon (C and D) or the HEV P6 replication-deficient infectious cDNA clone (P6GAD) (D). CC, untransfected cell control. The ISG15 protein expression, at 5 days posttransfection, in cell lysates (40 μg/lane) was analyzed by Western blotting using anti-ISG15 antibody (1:1,000 dilution). Fold change in band density, as calculated via normalization of ISG15 with β-actin (loading control), was measured using ImageJ software (NIH, Bethesda, MD). (E) ISG15 and USP18 mRNA expression levels in swine liver tissue samples obtained from animals experimentally infected with HEV ( n = 3) at 3 weeks postinfection. The fold changes in swine ISG15 and USP18 mRNA levels were calculated using the 2 −ΔΔ CT method compared to the uninfected control animals ( n = 2), and the RPS18 gene was used as the housekeeping gene. The data represent means ± standard errors of the means (SEM) of results from three independent transfection experiments (A, B, and C), one experiment representative of two independent transfection experiments (D), and HEV-infected animals ( n = 3) compared to uninfected animals ( n = 2) (E). **, P ≤ 0.01.

    Article Snippet: The mRNA levels of ISG15, OAS1, PKR, Mx1, USP18, and RPS18 (housekeeping control) were determined using SYBR master mix (Applied Biosystems, Grand Island, NY) with gene-specific primer sets ( ) and a Bio-Rad IQ5 system.

    Techniques: In Vitro, In Vivo, Transfection, Luciferase, Quantitative RT-PCR, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Western Blot, Software, Infection

    ISG15 regulates type I IFN signaling. (A) Relative pSTAT1 levels. Huh7-S10-3 liver cells were transfected with 20 nM control siRNA (siCnt) or ISG15-siRNA (siISG15) or UBE1L-siRNA plus UBE2L6-siRNA (siE1E2). At 24 hpt, cells were treated with IFN-α (100 IU/ml) for 30 min to 7 h. Cell lysate (20 μg/lane) was analyzed by Western blotting with the indicated antibody, anti-pSTAT1 (1:1,000 dilution), anti-STAT1 (1:1,000 dilution), and anti-β-actin (1:1,000 dilution). Fold change in band intensity was determined using ImageJ software (NIH, Bethesda, MD). The data represent means ± SEM of results from five independent experiments. a, P ≤ 0.05; aa, P ≤ 0.01 (compared with siCnt plus IFN-α at the given time point). (B) IFN-stimulated response element (ISRE) promoter activity levels. Huh7-S10-3 liver cells were cotransfected with siCnt/siISG15/siE1E2 along with pGL4.45[luc2P/ISRE/Hygro] (firefly luciferase) and pGL4.74[hRluc/TK] (renilla luciferase). At 24 hpt, cells were treated with various concentrations of IFN-α. Relative levels of fold induction of the cell-associated firefly luciferase activity, compared to the corresponding untreated cell control levels, at 18 h post-IFN-α treatment were estimated using a dual-luciferase assay kit and were normalized with renilla luciferase expression levels. The data represent means ± SEM of results from triplicate sample experiments. aa, P ≤ 0.01 (compared to siCnt plus IFN-α). (C to F) ISG mRNA levels in siRNA-transfected and IFN-α-treated samples were measured for Mx1 (C), OAS1 (D), PKR (E), and ISG15 (F) using Sybr green qPCR. Fold change in mRNA levels compared to the untransfected control was calculated using the 2 −ΔΔ CT method, and the RPS18 gene was used as the housekeeping gene. The data represent means ± SEM of results from three independent experiments. a, P ≤ 0.05; aa, P ≤ 0.01; aaa, P ≤ 0.001 (compared to siCnt plus IFN-α).

    Journal: Journal of Virology

    Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

    doi: 10.1128/JVI.00621-17

    Figure Lengend Snippet: ISG15 regulates type I IFN signaling. (A) Relative pSTAT1 levels. Huh7-S10-3 liver cells were transfected with 20 nM control siRNA (siCnt) or ISG15-siRNA (siISG15) or UBE1L-siRNA plus UBE2L6-siRNA (siE1E2). At 24 hpt, cells were treated with IFN-α (100 IU/ml) for 30 min to 7 h. Cell lysate (20 μg/lane) was analyzed by Western blotting with the indicated antibody, anti-pSTAT1 (1:1,000 dilution), anti-STAT1 (1:1,000 dilution), and anti-β-actin (1:1,000 dilution). Fold change in band intensity was determined using ImageJ software (NIH, Bethesda, MD). The data represent means ± SEM of results from five independent experiments. a, P ≤ 0.05; aa, P ≤ 0.01 (compared with siCnt plus IFN-α at the given time point). (B) IFN-stimulated response element (ISRE) promoter activity levels. Huh7-S10-3 liver cells were cotransfected with siCnt/siISG15/siE1E2 along with pGL4.45[luc2P/ISRE/Hygro] (firefly luciferase) and pGL4.74[hRluc/TK] (renilla luciferase). At 24 hpt, cells were treated with various concentrations of IFN-α. Relative levels of fold induction of the cell-associated firefly luciferase activity, compared to the corresponding untreated cell control levels, at 18 h post-IFN-α treatment were estimated using a dual-luciferase assay kit and were normalized with renilla luciferase expression levels. The data represent means ± SEM of results from triplicate sample experiments. aa, P ≤ 0.01 (compared to siCnt plus IFN-α). (C to F) ISG mRNA levels in siRNA-transfected and IFN-α-treated samples were measured for Mx1 (C), OAS1 (D), PKR (E), and ISG15 (F) using Sybr green qPCR. Fold change in mRNA levels compared to the untransfected control was calculated using the 2 −ΔΔ CT method, and the RPS18 gene was used as the housekeeping gene. The data represent means ± SEM of results from three independent experiments. a, P ≤ 0.05; aa, P ≤ 0.01; aaa, P ≤ 0.001 (compared to siCnt plus IFN-α).

    Article Snippet: The mRNA levels of ISG15, OAS1, PKR, Mx1, USP18, and RPS18 (housekeeping control) were determined using SYBR master mix (Applied Biosystems, Grand Island, NY) with gene-specific primer sets ( ) and a Bio-Rad IQ5 system.

    Techniques: Transfection, Western Blot, Software, Activity Assay, Luciferase, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction