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  • 99
    Thermo Fisher sirna
    Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 93491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore shrna
    Shrna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1724 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taqman microrna reverse transcription kit
    Taqman Microrna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 32040 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Horizon Discovery sirna
    Localization of MX2 and NUP214 following transfection with Nup/NTR targeting <t>siRNA</t> continued. Deconvolution microscopic images (single optical sections) of <t>HeLa</t> cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.
    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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    95
    Santa Cruz Biotechnology sirna
    TLR and CD36 mediate <t>PKCδ</t> and IRAK1 activation and IL-1β production in THP1 cells. THP1 cells were treated with control, TLR6, TLR4, TLR2, or CD36 <t>siRNA</t> for 18 h. Total and phosphorylated PKCδ (A) and IRAK1 (B) were measured after 15 min of Ox-LDL stimulation by immunoblotting (n = 3). C: IL-1β level was measured in the supernatant after Ox-LDL treatment for 48 h (in triplicate, n = 3). Blots represent one of three similar experiments. Values represent mean ± SE. # P
    Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 9855 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher sirnas
    SiRNA-mediated knockdown of TMEM97 increases NPC1 protein levels, ameliorates cholesterol accumulation and restores cholesterol delivery to the ER in NPC1 -deficient HeLa cells. ( A ) Cultured HeLa cells were transfected for 48h with either control siRNA or indicated <t>siRNAs</t> targeting NPC1 , TMEM97 or <t>NPC2</t> . Whole cell lysates were subjected to Western blotting and probed with antibodies against NPC1 and β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels of control siRNA treated cells ( n = 3 independent experiments per condition; ** P
    Sirnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 47833 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher control sirna
    SiRNA-mediated knockdown of TMEM97 increases NPC1 protein levels, ameliorates cholesterol accumulation and restores cholesterol delivery to the ER in NPC1 -deficient HeLa cells. ( A ) Cultured HeLa cells were transfected for 48h with either control siRNA or indicated <t>siRNAs</t> targeting NPC1 , TMEM97 or <t>NPC2</t> . Whole cell lysates were subjected to Western blotting and probed with antibodies against NPC1 and β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels of control siRNA treated cells ( n = 3 independent experiments per condition; ** P
    Control Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Horizon Discovery sirnas
    <t>MED19</t> and MED26 are not essential for the apparent integrity of Mediator. Nuclear lysates from HeLa cells transfected with control ( CNTL ), MED19-, MED26-, or MED19 and 26-specific <t>siRNAs</t> were subjected to immunoprecipitation ( IP ) using antibodies specific for MED4. Immunoprecipitates were extensively washed prior to resolution by SDS-10% PAGE and processing by Western blot ( WB ) analysis using the specified antibodies. Input , 10% of the nuclear lysates used for IP reactions. Structural domains to which individual Mediator subunits may be relegated are indicated (Head, Middle, Tail, Kinase, or Unassigned ( Unass. )). Asterisks mark MED19 and MED26 Mediator subunits targeted for RNAi-mediated depletion.
    Sirnas, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 94/100, based on 11504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GenePharma Company sirna
    FFAR2 positively regulates IAV replication in A549 cells. (A to D) FFAR2 <t>siRNA-</t> or scrambled <t>siRNA-transfected</t> A549 cells were infected with AH05 (H5N1) (MOI = 0.1) (A), WSN (H1N1) (MOI = 0.01) (B), SH13 (H9N2) (MOI = 0.1) (C), or VSV-EGFP (100 TCID 50 ) (D) virus. Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells (A to C) or by determining the TCID 50 on HEK293T cells (D). **, P
    Sirna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 94/100, based on 4882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen allstars negative control sirna
    FFAR2 positively regulates IAV replication in A549 cells. (A to D) FFAR2 <t>siRNA-</t> or scrambled <t>siRNA-transfected</t> A549 cells were infected with AH05 (H5N1) (MOI = 0.1) (A), WSN (H1N1) (MOI = 0.01) (B), SH13 (H9N2) (MOI = 0.1) (C), or VSV-EGFP (100 TCID 50 ) (D) virus. Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells (A to C) or by determining the TCID 50 on HEK293T cells (D). **, P
    Allstars Negative Control Sirna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 5667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Qiagen sirna
    FFAR2 positively regulates IAV replication in A549 cells. (A to D) FFAR2 <t>siRNA-</t> or scrambled <t>siRNA-transfected</t> A549 cells were infected with AH05 (H5N1) (MOI = 0.1) (A), WSN (H1N1) (MOI = 0.01) (B), SH13 (H9N2) (MOI = 0.1) (C), or VSV-EGFP (100 TCID 50 ) (D) virus. Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells (A to C) or by determining the TCID 50 on HEK293T cells (D). **, P
    Sirna, supplied by Qiagen, used in various techniques. Bioz Stars score: 86/100, based on 15050 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher negative control sirna
    E2f1-mediated transcriptional induction of Cdc25a may induce liver cysts in LKO mice. A : Renilla luciferase activity (LUC2) produced from wild-type or mutant Cdc25a 3′-UTR reporter plasmids or empty vector normalized to firefly luciferase activity (LUC1) produced from the same plasmid after transfection into Hepa cells together with negative control (NC) RNA or miR-122 mimic. Error bars represent standard deviations derived from three independent experiments. B : Both pre-mRNA (hnRNA) and mRNA are up-regulated at similar level in DEN-injected LKO livers. Real-time RT-qPCR analysis of Cdc25a mRNA and hnRNA in livers ( n = 5) was performed. The data were normalized to Gapdh RNA level. C : Western blot analysis of liver extracts with specific antibodies at week 25 post-DEN injection. D : Quantification of the protein signals. mRNA ( E ) and protein levels ( F ) of Cdc25a and E2f1 in Hepa cells <t>transfected</t> with 60 nmol/L negative control or E2f1 <t>siRNA</t> ( n = 3). The results were normalized to Gapdh level. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, and ∗∗∗ P ≤ 0.001.
    Negative Control Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 7824 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher silencer select pre designed sirna
    E2f1-mediated transcriptional induction of Cdc25a may induce liver cysts in LKO mice. A : Renilla luciferase activity (LUC2) produced from wild-type or mutant Cdc25a 3′-UTR reporter plasmids or empty vector normalized to firefly luciferase activity (LUC1) produced from the same plasmid after transfection into Hepa cells together with negative control (NC) RNA or miR-122 mimic. Error bars represent standard deviations derived from three independent experiments. B : Both pre-mRNA (hnRNA) and mRNA are up-regulated at similar level in DEN-injected LKO livers. Real-time RT-qPCR analysis of Cdc25a mRNA and hnRNA in livers ( n = 5) was performed. The data were normalized to Gapdh RNA level. C : Western blot analysis of liver extracts with specific antibodies at week 25 post-DEN injection. D : Quantification of the protein signals. mRNA ( E ) and protein levels ( F ) of Cdc25a and E2f1 in Hepa cells <t>transfected</t> with 60 nmol/L negative control or E2f1 <t>siRNA</t> ( n = 3). The results were normalized to Gapdh level. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, and ∗∗∗ P ≤ 0.001.
    Silencer Select Pre Designed Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher silencer negative control no 1 sirna
    E2f1-mediated transcriptional induction of Cdc25a may induce liver cysts in LKO mice. A : Renilla luciferase activity (LUC2) produced from wild-type or mutant Cdc25a 3′-UTR reporter plasmids or empty vector normalized to firefly luciferase activity (LUC1) produced from the same plasmid after transfection into Hepa cells together with negative control (NC) RNA or miR-122 mimic. Error bars represent standard deviations derived from three independent experiments. B : Both pre-mRNA (hnRNA) and mRNA are up-regulated at similar level in DEN-injected LKO livers. Real-time RT-qPCR analysis of Cdc25a mRNA and hnRNA in livers ( n = 5) was performed. The data were normalized to Gapdh RNA level. C : Western blot analysis of liver extracts with specific antibodies at week 25 post-DEN injection. D : Quantification of the protein signals. mRNA ( E ) and protein levels ( F ) of Cdc25a and E2f1 in Hepa cells <t>transfected</t> with 60 nmol/L negative control or E2f1 <t>siRNA</t> ( n = 3). The results were normalized to Gapdh level. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, and ∗∗∗ P ≤ 0.001.
    Silencer Negative Control No 1 Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3053 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sirna oligos
    E2f1-mediated transcriptional induction of Cdc25a may induce liver cysts in LKO mice. A : Renilla luciferase activity (LUC2) produced from wild-type or mutant Cdc25a 3′-UTR reporter plasmids or empty vector normalized to firefly luciferase activity (LUC1) produced from the same plasmid after transfection into Hepa cells together with negative control (NC) RNA or miR-122 mimic. Error bars represent standard deviations derived from three independent experiments. B : Both pre-mRNA (hnRNA) and mRNA are up-regulated at similar level in DEN-injected LKO livers. Real-time RT-qPCR analysis of Cdc25a mRNA and hnRNA in livers ( n = 5) was performed. The data were normalized to Gapdh RNA level. C : Western blot analysis of liver extracts with specific antibodies at week 25 post-DEN injection. D : Quantification of the protein signals. mRNA ( E ) and protein levels ( F ) of Cdc25a and E2f1 in Hepa cells <t>transfected</t> with 60 nmol/L negative control or E2f1 <t>siRNA</t> ( n = 3). The results were normalized to Gapdh level. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, and ∗∗∗ P ≤ 0.001.
    Sirna Oligos, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1972 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effect of Nup and NTR depletion on primate lentivirus infection and MX2 sensitivity. Infectivity of (A) HIV-2 or (B) SIVmac GFP reporter virus in HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on primate lentivirus infection and MX2 sensitivity. Infectivity of (A) HIV-2 or (B) SIVmac GFP reporter virus in HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Stable Transfection, Transduction, Transfection

    Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effect of Nup and NTR depletion on HIV-1 infection and MX2 sensitivity in the presence of CsA. ( A–B ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). ( C–D ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 infection and MX2 sensitivity in the presence of CsA. ( A–B ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). ( C–D ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Stable Transfection, Transduction, Transfection

    Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Knockdown of Nups and NTRs in HOS cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HOS cells 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in HOS cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HOS cells 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Transfection, Generated, Infection

    Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Effect of Nup and NTR depletion on HIV-1 CA mutant infection and MX2 sensitivity. Infectivity of HIV-1 CA mutant GFP reporter viruses in HOS cells or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 CA mutant infection and MX2 sensitivity. Infectivity of HIV-1 CA mutant GFP reporter viruses in HOS cells or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Mutagenesis, Infection, Stable Transfection, Transduction, Transfection

    Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Knockdown of Nups and NTRs in HT1080 cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HT1080 cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. (n/a – not tested since HT1080 cells do not express NUP210, see Figure 2—figure supplement 1 ). Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in HT1080 cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HT1080 cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. (n/a – not tested since HT1080 cells do not express NUP210, see Figure 2—figure supplement 1 ). Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Stable Transfection, Transduction, Transfection, Generated, Infection

    Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Knockdown of Nups and NTRs in non-dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot is generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown in . The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in non-dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot is generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown in . The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Stable Transfection, Transduction, Transfection, Generated, Infection

    Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Cell-cycle profile of HOS cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HOS cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye DRAQ5. For each plot, 3000–12000 events were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Cell-cycle profile of HOS cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HOS cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye DRAQ5. For each plot, 3000–12000 events were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Flow Cytometry, Cytometry, Staining

    Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Effect of ARFAPTIN2 fusion protein expression on lentivirus and HIV-1 CA mutant infection. ( A ) Infection of HeLa cells stably transduced with doxycycline-inducible arfaptin2 myc-tagged fusion proteins with various GFP reporter viruses in the presence (white bars) or absence (black bars) of doxycycline. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. ( B ) Infectivity of HIV-1 N57S mutant GFP reporter virus in HT1080 cells stably transduced with arfaptin2 fusion proteins in the presence (white bars) or absence (black bars) of doxycycline and the presence or absence of CsA. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. (C) Infectivity of HIV-1-GFP reporter virus in dividing or non-dividing (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible arfaptin2 or MX2(N91)-arfaptin2 in the presence (open circles) or absence (filled circles) of doxycycline. Titers are mean ± sem, n = 3 technical replicates and are representative of three independent experiments. ( D ) Infectivity of HIV-1 GFP reporter virus in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2(N91)-ARFAPTIN2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of ARFAPTIN2 fusion protein expression on lentivirus and HIV-1 CA mutant infection. ( A ) Infection of HeLa cells stably transduced with doxycycline-inducible arfaptin2 myc-tagged fusion proteins with various GFP reporter viruses in the presence (white bars) or absence (black bars) of doxycycline. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. ( B ) Infectivity of HIV-1 N57S mutant GFP reporter virus in HT1080 cells stably transduced with arfaptin2 fusion proteins in the presence (white bars) or absence (black bars) of doxycycline and the presence or absence of CsA. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. (C) Infectivity of HIV-1-GFP reporter virus in dividing or non-dividing (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible arfaptin2 or MX2(N91)-arfaptin2 in the presence (open circles) or absence (filled circles) of doxycycline. Titers are mean ± sem, n = 3 technical replicates and are representative of three independent experiments. ( D ) Infectivity of HIV-1 GFP reporter virus in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2(N91)-ARFAPTIN2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Expressing, Mutagenesis, Infection, Stable Transfection, Transduction, Transfection

    Effect of Nup and NTR depletion on HIV-1 infection in HOS cells. ( A ) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ±sem, n = 3 technical replicates, representative of three independent experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 infection in HOS cells. ( A ) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ±sem, n = 3 technical replicates, representative of three independent experiments.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Transfection

    Knockdown of Nups and NTRs in dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Stable Transfection, Transduction, Transfection, Generated, Infection

    Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HOS cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 6 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HOS cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 6 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing

    Cell-cycle profile of HeLa cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HeLa cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye propidium iodide (PI). For each plot, 3000–12000 events per sample were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Cell-cycle profile of HeLa cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HeLa cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye propidium iodide (PI). For each plot, 3000–12000 events per sample were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Flow Cytometry, Cytometry, Staining

    Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HT1080 cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HT1080 cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 5 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HT1080 cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HT1080 cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 5 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing

    Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effects of depleting individual Nups on NPC integrity and function, and MX2 localization. Summary of the localization of Nups, MX2-RFP, CPSF6-RFP, or NLS-GFP-LacZ fusions upon siRNA transfection of HeLa or HT1080 cells shown in Figure 6 , and Figure 6—figure supplements 1 – 18 . Aberrant localization following siRNA transfection in ≥~80% of cells is indicated by an ‘x’ and normal localization is indicated by a dot.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effects of depleting individual Nups on NPC integrity and function, and MX2 localization. Summary of the localization of Nups, MX2-RFP, CPSF6-RFP, or NLS-GFP-LacZ fusions upon siRNA transfection of HeLa or HT1080 cells shown in Figure 6 , and Figure 6—figure supplements 1 – 18 . Aberrant localization following siRNA transfection in ≥~80% of cells is indicated by an ‘x’ and normal localization is indicated by a dot.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection

    Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effect of Nup and NTR depletion on HIV-1 A92E and N57S CA mutant infection in the presence of CsA. ( A–C ) Infectivity of HIV-1 A92E or N57S GFP reporter viruses in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of 5 μM CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). X-axis legend for A-B is below graph B, x-axis legend for C-D is below graph D. ( D ) Infectivity of HIV-1 N57S GFP reporter virus in HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 A92E and N57S CA mutant infection in the presence of CsA. ( A–C ) Infectivity of HIV-1 A92E or N57S GFP reporter viruses in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of 5 μM CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). X-axis legend for A-B is below graph B, x-axis legend for C-D is below graph D. ( D ) Infectivity of HIV-1 N57S GFP reporter virus in HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Mutagenesis, Infection, Stable Transfection, Transduction, Transfection

    Effect of Nup and NTR depletion on non-primate lentivirus infection and MX2 sensitivity. Infectivity of ( A ) EIAV or ( B-D ) FIV GFP reporter virus in ( A-B ) HeLa or ( C ) HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline, or ( D ) HOS cells. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on non-primate lentivirus infection and MX2 sensitivity. Infectivity of ( A ) EIAV or ( B-D ) FIV GFP reporter virus in ( A-B ) HeLa or ( C ) HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline, or ( D ) HOS cells. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: RNA interference HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Stable Transfection, Transduction, Transfection

    Magnetic resonance imaging (MRI) findings after small interfering RNA (siRNA) oligonucleotide injection in the rabbit anular puncture model of disc degeneration. MRI examinations were performed on all spinal columns isolated from the rabbits ex vivo at death 8 weeks after the siRNA oligonucleotide injection. In these representative MRIs, the T 2 signal intensity in the nucleus pulposus of the ADAMTS5 siRNA-injected discs was stronger than that in the control siRNA-injected discs.

    Journal: Arthritis Research & Therapy

    Article Title: Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model

    doi: 10.1186/ar2851

    Figure Lengend Snippet: Magnetic resonance imaging (MRI) findings after small interfering RNA (siRNA) oligonucleotide injection in the rabbit anular puncture model of disc degeneration. MRI examinations were performed on all spinal columns isolated from the rabbits ex vivo at death 8 weeks after the siRNA oligonucleotide injection. In these representative MRIs, the T 2 signal intensity in the nucleus pulposus of the ADAMTS5 siRNA-injected discs was stronger than that in the control siRNA-injected discs.

    Article Snippet: One week after the initial puncture, the discs were exposed again from the contralateral side, and either control siRNA or ADAMTS5 siRNA oligonucleotide (Dharmacon, was injected into the center of the NP by using a 26-gauge needle (10 μg in 10 μl phosphate-buffered saline (PBS) per disc).

    Techniques: Magnetic Resonance Imaging, Small Interfering RNA, Injection, Isolation, Ex Vivo

    Histologic assessment after ADAMTS5 small interfering RNA (siRNA) or control siRNA injection in the rabbit anular puncture model of disc degeneration. In ADAMTS5 siRNA-injected discs, the anulus fibrosus (AF) and the border between the AF and nucleus pulposus (NP) showed a tendency to have a lower (better) histologic score than the control siRNA-injected discs (Mann-Whitney test; P

    Journal: Arthritis Research & Therapy

    Article Title: Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model

    doi: 10.1186/ar2851

    Figure Lengend Snippet: Histologic assessment after ADAMTS5 small interfering RNA (siRNA) or control siRNA injection in the rabbit anular puncture model of disc degeneration. In ADAMTS5 siRNA-injected discs, the anulus fibrosus (AF) and the border between the AF and nucleus pulposus (NP) showed a tendency to have a lower (better) histologic score than the control siRNA-injected discs (Mann-Whitney test; P

    Article Snippet: One week after the initial puncture, the discs were exposed again from the contralateral side, and either control siRNA or ADAMTS5 siRNA oligonucleotide (Dharmacon, was injected into the center of the NP by using a 26-gauge needle (10 μg in 10 μl phosphate-buffered saline (PBS) per disc).

    Techniques: Small Interfering RNA, Injection, MANN-WHITNEY

    Safranin-O-stained sections reflecting typical histologic changes after injection of control small interfering RNA (siRNA) or ADAMTS5 siRNA in the rabbit anular puncture model of disc degeneration. Eight weeks after the control or ADAMTS5 siRNA injections, the control siRNA group displayed a complete loss of nucleus pulposus (NP) tissues, which had been replaced by a fibrocartilaginous tissue (a, c) . The severely degenerated discs that had received the control siRNA showed a loss of proteoglycans and the collapsed, wavy fibrocartilage lamellae typical of the anulus fibrosus (AF), with associated fibrochondrocytes (e, g) . In the ADAMTS5 siRNA-injected discs, safranin-O staining demonstrated the maintenance of intervertebral disc structure with a lightly stained matrix and large cells (b, d) ; the NP was rounded and bloated looking, and consisted of numerous large, vacuolated cells and smaller chondrocyte-like cells (f, h) . A clear demarcation was seen between the NP and inner anulus in the ADAMTS5 siRNA-injected discs. (Magnification ×20 (a-d), ×100 (e-h)). The level in a, b, e, and f is L2/3, and in c, d, g, and h is L4/5.

    Journal: Arthritis Research & Therapy

    Article Title: Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model

    doi: 10.1186/ar2851

    Figure Lengend Snippet: Safranin-O-stained sections reflecting typical histologic changes after injection of control small interfering RNA (siRNA) or ADAMTS5 siRNA in the rabbit anular puncture model of disc degeneration. Eight weeks after the control or ADAMTS5 siRNA injections, the control siRNA group displayed a complete loss of nucleus pulposus (NP) tissues, which had been replaced by a fibrocartilaginous tissue (a, c) . The severely degenerated discs that had received the control siRNA showed a loss of proteoglycans and the collapsed, wavy fibrocartilage lamellae typical of the anulus fibrosus (AF), with associated fibrochondrocytes (e, g) . In the ADAMTS5 siRNA-injected discs, safranin-O staining demonstrated the maintenance of intervertebral disc structure with a lightly stained matrix and large cells (b, d) ; the NP was rounded and bloated looking, and consisted of numerous large, vacuolated cells and smaller chondrocyte-like cells (f, h) . A clear demarcation was seen between the NP and inner anulus in the ADAMTS5 siRNA-injected discs. (Magnification ×20 (a-d), ×100 (e-h)). The level in a, b, e, and f is L2/3, and in c, d, g, and h is L4/5.

    Article Snippet: One week after the initial puncture, the discs were exposed again from the contralateral side, and either control siRNA or ADAMTS5 siRNA oligonucleotide (Dharmacon, was injected into the center of the NP by using a 26-gauge needle (10 μg in 10 μl phosphate-buffered saline (PBS) per disc).

    Techniques: Staining, Injection, Small Interfering RNA

    Effect of interleukin-1β (IL-1β) stimulation on ADAMTS5 mRNA expression in rabbit nucleus pulposus (NP) cells. After real-time polymerase chain reaction (PCR), the ADAMTS5 mRNA expression level after IL-1β stimulation (24 hours) in rabbit NP cells is shown (a) . IL-1β at 10 ng/ml induced the highest level of increased expression of mRNA for ADAMTS5 (about 12-fold); that concentration was chosen for subsequent studies. (b) NP cells seeded in a 12-well plate at a density of 1 × 10 5 cells/well. After the 48-hour preculture period, cells were cultured in serum-free media in the presence of IL-1β (10 ng/ml) for 24 hours. After the 24-hour treatment with IL-1β, NP cells were transiently transfected with the anti- ADAMTS5 oligonucleotide or control oligonucleotide by adding oligonucleotide directly to the culture media. Twenty-four hours later, NP cells were collected, and the expression of ADAMTS5 was analyzed with real-time PCR. ADAMTS5 mRNA expression was knocked down by about 70% in rabbit NP cells that were transfected with ADAMTS5 siRNA and stimulated with IL-1β (10 ng/ml). The results are reported normalized to GAPDH .

    Journal: Arthritis Research & Therapy

    Article Title: Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model

    doi: 10.1186/ar2851

    Figure Lengend Snippet: Effect of interleukin-1β (IL-1β) stimulation on ADAMTS5 mRNA expression in rabbit nucleus pulposus (NP) cells. After real-time polymerase chain reaction (PCR), the ADAMTS5 mRNA expression level after IL-1β stimulation (24 hours) in rabbit NP cells is shown (a) . IL-1β at 10 ng/ml induced the highest level of increased expression of mRNA for ADAMTS5 (about 12-fold); that concentration was chosen for subsequent studies. (b) NP cells seeded in a 12-well plate at a density of 1 × 10 5 cells/well. After the 48-hour preculture period, cells were cultured in serum-free media in the presence of IL-1β (10 ng/ml) for 24 hours. After the 24-hour treatment with IL-1β, NP cells were transiently transfected with the anti- ADAMTS5 oligonucleotide or control oligonucleotide by adding oligonucleotide directly to the culture media. Twenty-four hours later, NP cells were collected, and the expression of ADAMTS5 was analyzed with real-time PCR. ADAMTS5 mRNA expression was knocked down by about 70% in rabbit NP cells that were transfected with ADAMTS5 siRNA and stimulated with IL-1β (10 ng/ml). The results are reported normalized to GAPDH .

    Article Snippet: One week after the initial puncture, the discs were exposed again from the contralateral side, and either control siRNA or ADAMTS5 siRNA oligonucleotide (Dharmacon, was injected into the center of the NP by using a 26-gauge needle (10 μg in 10 μl phosphate-buffered saline (PBS) per disc).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Concentration Assay, Cell Culture, Transfection

    Radiographic assessment in the rabbit anular puncture model of disc degeneration. An anular puncture model was established in 5-month-old New Zealand white rabbits. Under general anesthesia, lumbar intervertebral discs were exposed, and the initial puncture with an 18-gauge needle at a defined depth of puncture (5 mm) was performed on two noncontiguous discs (L2/3 and L4/5), with the disc (L3/4) between the punctured discs left intact as a control. One week after the initial puncture, either control small interfering RNA (siRNA) or ADAMTS5 siRNA oligonucleotide (10 μg in 10-μl phosphate-buffered saline (PBS) per disc) was injected into the center of the nucleus pulposus by using a 26-gauge needle. Nine weeks after the initial anular puncture (8 weeks after the injection), all rabbits were killed. Radiographs were taken at time 0 and at weeks 1, 2, 3, 5, 7, and 9 after the puncture to quantity changes in the disc-height index (DHI). The %DHI was calculated as [%DHI = (Postoperative DHI/Preoperative DHI) × 100]. At 8 weeks after the ADAMTS5 siRNA injection, no difference was found in the mean %DHI of the ADAMTS5 siRNA-injected punctured discs compared with the punctured discs that received the control siRNA injection ( P > 0.05, repeated ANOVA).

    Journal: Arthritis Research & Therapy

    Article Title: Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model

    doi: 10.1186/ar2851

    Figure Lengend Snippet: Radiographic assessment in the rabbit anular puncture model of disc degeneration. An anular puncture model was established in 5-month-old New Zealand white rabbits. Under general anesthesia, lumbar intervertebral discs were exposed, and the initial puncture with an 18-gauge needle at a defined depth of puncture (5 mm) was performed on two noncontiguous discs (L2/3 and L4/5), with the disc (L3/4) between the punctured discs left intact as a control. One week after the initial puncture, either control small interfering RNA (siRNA) or ADAMTS5 siRNA oligonucleotide (10 μg in 10-μl phosphate-buffered saline (PBS) per disc) was injected into the center of the nucleus pulposus by using a 26-gauge needle. Nine weeks after the initial anular puncture (8 weeks after the injection), all rabbits were killed. Radiographs were taken at time 0 and at weeks 1, 2, 3, 5, 7, and 9 after the puncture to quantity changes in the disc-height index (DHI). The %DHI was calculated as [%DHI = (Postoperative DHI/Preoperative DHI) × 100]. At 8 weeks after the ADAMTS5 siRNA injection, no difference was found in the mean %DHI of the ADAMTS5 siRNA-injected punctured discs compared with the punctured discs that received the control siRNA injection ( P > 0.05, repeated ANOVA).

    Article Snippet: One week after the initial puncture, the discs were exposed again from the contralateral side, and either control siRNA or ADAMTS5 siRNA oligonucleotide (Dharmacon, was injected into the center of the NP by using a 26-gauge needle (10 μg in 10 μl phosphate-buffered saline (PBS) per disc).

    Techniques: Small Interfering RNA, Injection

    Magnetic resonance imaging (MRI) assessment 8 weeks after small interfering RNA (siRNA) injection in the rabbit anular puncture model of disc degeneration. An observer blinded to the study assessed the MRIs by using the modified Thompson scale, based on changes in the degree and area of signal intensity from grades 1 to 4. After assessment of the MRI grades, a significantly lower (better) MRI grade in the ADAMTS5 siRNA-injected discs was observed compared with the control siRNA-injected discs ( P = 0.02, Mann-Whitney test).

    Journal: Arthritis Research & Therapy

    Article Title: Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model

    doi: 10.1186/ar2851

    Figure Lengend Snippet: Magnetic resonance imaging (MRI) assessment 8 weeks after small interfering RNA (siRNA) injection in the rabbit anular puncture model of disc degeneration. An observer blinded to the study assessed the MRIs by using the modified Thompson scale, based on changes in the degree and area of signal intensity from grades 1 to 4. After assessment of the MRI grades, a significantly lower (better) MRI grade in the ADAMTS5 siRNA-injected discs was observed compared with the control siRNA-injected discs ( P = 0.02, Mann-Whitney test).

    Article Snippet: One week after the initial puncture, the discs were exposed again from the contralateral side, and either control siRNA or ADAMTS5 siRNA oligonucleotide (Dharmacon, was injected into the center of the NP by using a 26-gauge needle (10 μg in 10 μl phosphate-buffered saline (PBS) per disc).

    Techniques: Magnetic Resonance Imaging, Small Interfering RNA, Injection, Modification, MANN-WHITNEY

    Establishment of small interfering RNA (siRNA) oligonucleotide for ADAMTS5 in rabbit nucleus pulposus (NP) cells. After the 48-hour preculture period, rabbit NP cells were transfected with the siRNA oligonucleotide specific for either the control or ADAMTS5 . At 48 hours after transfection in NP cells, the ADAMTS5 siRNA-transfected cells showed approximately a 75% knock-down of ADAMTS5 mRNA compared with the control siRNA. The results are reported normalized to glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ).

    Journal: Arthritis Research & Therapy

    Article Title: Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model

    doi: 10.1186/ar2851

    Figure Lengend Snippet: Establishment of small interfering RNA (siRNA) oligonucleotide for ADAMTS5 in rabbit nucleus pulposus (NP) cells. After the 48-hour preculture period, rabbit NP cells were transfected with the siRNA oligonucleotide specific for either the control or ADAMTS5 . At 48 hours after transfection in NP cells, the ADAMTS5 siRNA-transfected cells showed approximately a 75% knock-down of ADAMTS5 mRNA compared with the control siRNA. The results are reported normalized to glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ).

    Article Snippet: One week after the initial puncture, the discs were exposed again from the contralateral side, and either control siRNA or ADAMTS5 siRNA oligonucleotide (Dharmacon, was injected into the center of the NP by using a 26-gauge needle (10 μg in 10 μl phosphate-buffered saline (PBS) per disc).

    Techniques: Small Interfering RNA, Transfection

    TLR and CD36 mediate PKCδ and IRAK1 activation and IL-1β production in THP1 cells. THP1 cells were treated with control, TLR6, TLR4, TLR2, or CD36 siRNA for 18 h. Total and phosphorylated PKCδ (A) and IRAK1 (B) were measured after 15 min of Ox-LDL stimulation by immunoblotting (n = 3). C: IL-1β level was measured in the supernatant after Ox-LDL treatment for 48 h (in triplicate, n = 3). Blots represent one of three similar experiments. Values represent mean ± SE. # P

    Journal: Journal of Lipid Research

    Article Title: PKCδ-IRAK1 axis regulates oxidized LDL-induced IL-1β production in monocytes [S]

    doi: 10.1194/jlr.M045658

    Figure Lengend Snippet: TLR and CD36 mediate PKCδ and IRAK1 activation and IL-1β production in THP1 cells. THP1 cells were treated with control, TLR6, TLR4, TLR2, or CD36 siRNA for 18 h. Total and phosphorylated PKCδ (A) and IRAK1 (B) were measured after 15 min of Ox-LDL stimulation by immunoblotting (n = 3). C: IL-1β level was measured in the supernatant after Ox-LDL treatment for 48 h (in triplicate, n = 3). Blots represent one of three similar experiments. Values represent mean ± SE. # P

    Article Snippet: Human anti-CD36 (SMϕ); anti-TLR2, -4, and -6; Tanshinone IIa; PKCδ siRNA; TLR2, -4, and -6 siRNA; and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Activation Assay

    IRAK1 mediates PKCδ-induced IL-1β production. A: Secretory IL-1β was measured in culture supernatant of THP1 monocytes at 48 h of Ox-LDL (40 μg/ml) stimulation after pretreatment with Go6976 (20 nM), Ro-31-8220 (1 μM), and Rottlerin (2 μM) (in triplicate, n = 4). B: Phosphorylation of PKCδ in THP1 cells was measured at various time points by Western blotting. Cell extracts were resolved on SDS-PAGE and, after transfer to PVDF membrane, were probed with a phospho antibody that recognizes activated PKCδ. At the same time, expression of total PKCδ and β-actin were monitored by probing the blots with specific antibodies (n = 3). C: Bar graph representing IL-1β level in the supernatant obtained from Ox-LDL-stimulated THP1 monocytes treated with control siRNA or PKCδ siRNA (3 μg, in triplicate, n = 4). D: IRAK1 kinase activity measured by an in vitro kinase assay at 30 min of Ox-LDL stimulation after pretreatment with Go6976, Ro-31-8220, and Rottlerin. Cells were lysed and immunoprecipitated. IRAK1 was subjected to kinase assay in the presence of γ32 PATP and MBP as substrate (n = 3). E: Bar graph represents fold change in the expression of phospho-IRAK1 in Ox-LDL-stimulated THP1 monocytes with control siRNA or PKCδ siRNA treatment (n = 3). Blots represent one of three similar experiments. Values represent mean ± SE. * P

    Journal: Journal of Lipid Research

    Article Title: PKCδ-IRAK1 axis regulates oxidized LDL-induced IL-1β production in monocytes [S]

    doi: 10.1194/jlr.M045658

    Figure Lengend Snippet: IRAK1 mediates PKCδ-induced IL-1β production. A: Secretory IL-1β was measured in culture supernatant of THP1 monocytes at 48 h of Ox-LDL (40 μg/ml) stimulation after pretreatment with Go6976 (20 nM), Ro-31-8220 (1 μM), and Rottlerin (2 μM) (in triplicate, n = 4). B: Phosphorylation of PKCδ in THP1 cells was measured at various time points by Western blotting. Cell extracts were resolved on SDS-PAGE and, after transfer to PVDF membrane, were probed with a phospho antibody that recognizes activated PKCδ. At the same time, expression of total PKCδ and β-actin were monitored by probing the blots with specific antibodies (n = 3). C: Bar graph representing IL-1β level in the supernatant obtained from Ox-LDL-stimulated THP1 monocytes treated with control siRNA or PKCδ siRNA (3 μg, in triplicate, n = 4). D: IRAK1 kinase activity measured by an in vitro kinase assay at 30 min of Ox-LDL stimulation after pretreatment with Go6976, Ro-31-8220, and Rottlerin. Cells were lysed and immunoprecipitated. IRAK1 was subjected to kinase assay in the presence of γ32 PATP and MBP as substrate (n = 3). E: Bar graph represents fold change in the expression of phospho-IRAK1 in Ox-LDL-stimulated THP1 monocytes with control siRNA or PKCδ siRNA treatment (n = 3). Blots represent one of three similar experiments. Values represent mean ± SE. * P

    Article Snippet: Human anti-CD36 (SMϕ); anti-TLR2, -4, and -6; Tanshinone IIa; PKCδ siRNA; TLR2, -4, and -6 siRNA; and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Western Blot, SDS Page, Expressing, Activity Assay, In Vitro, Kinase Assay, Immunoprecipitation

    TLR and CD36 mediate PKCδ and IRAK1 activation and IL-1β production in primary human monocytes. Primary human monocytes were treated with control, TLR6, TLR4, TLR2, or CD36 siRNA for 18 h. Total and phosphorylated PKCδ (A) and IRAK1 (B) were measured after 15 min of plasma containing high Ox-LDL (from SIRS patients) stimulation by immunoblotting (n = 3). C: Secreted IL-1β was measured in the supernatant after high Ox-LDL-containing plasma (from SIRS patients) treatment for 48 h (in triplicate, n = 3). Blots represent one of three similar experiments. Values represent mean ± SE. # P

    Journal: Journal of Lipid Research

    Article Title: PKCδ-IRAK1 axis regulates oxidized LDL-induced IL-1β production in monocytes [S]

    doi: 10.1194/jlr.M045658

    Figure Lengend Snippet: TLR and CD36 mediate PKCδ and IRAK1 activation and IL-1β production in primary human monocytes. Primary human monocytes were treated with control, TLR6, TLR4, TLR2, or CD36 siRNA for 18 h. Total and phosphorylated PKCδ (A) and IRAK1 (B) were measured after 15 min of plasma containing high Ox-LDL (from SIRS patients) stimulation by immunoblotting (n = 3). C: Secreted IL-1β was measured in the supernatant after high Ox-LDL-containing plasma (from SIRS patients) treatment for 48 h (in triplicate, n = 3). Blots represent one of three similar experiments. Values represent mean ± SE. # P

    Article Snippet: Human anti-CD36 (SMϕ); anti-TLR2, -4, and -6; Tanshinone IIa; PKCδ siRNA; TLR2, -4, and -6 siRNA; and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Activation Assay

    PKCδ and IRAK1 operate upstream of JNK-AP-1 during IL-1β production. Phosphorylation of JNK was measured in Rottlerin and IRAK1/4 INH (A) or PKCδ siRNA-pretreated THP1 monocytes (B) by phospho blotting after 15 min of Ox-LDL (40 μg/ml) treatment. Membranes were also probed with total JNK antibody (n = 4). C: THP1 cells were pretreated with IRAK1/4 INH, JNK INH II, Rottlerin, and PKCδ siRNA; and subsequently AP-1 activation was measured in nuclear extract at 30 min of Ox-LDL stimulation (in triplicate, n = 3). D: Bar diagram and Western blot representing fold increase of phospho-PKCδ expression at indicated time points in the human monocytes incubated with Ox-LDL (n = 3). E: Bar graph represents secretory IL-1β in the supernatant obtained from control and Ox-LDL-stimulated monocytes for 48 h in the presence or absence of Rottlerin (2 μM, n = 4, in triplicate). F: Secretory IL-1β in PKCδ siRNA-treated cells. Blots represent one of three to four similar experiments. Values represent mean ± SE. * P

    Journal: Journal of Lipid Research

    Article Title: PKCδ-IRAK1 axis regulates oxidized LDL-induced IL-1β production in monocytes [S]

    doi: 10.1194/jlr.M045658

    Figure Lengend Snippet: PKCδ and IRAK1 operate upstream of JNK-AP-1 during IL-1β production. Phosphorylation of JNK was measured in Rottlerin and IRAK1/4 INH (A) or PKCδ siRNA-pretreated THP1 monocytes (B) by phospho blotting after 15 min of Ox-LDL (40 μg/ml) treatment. Membranes were also probed with total JNK antibody (n = 4). C: THP1 cells were pretreated with IRAK1/4 INH, JNK INH II, Rottlerin, and PKCδ siRNA; and subsequently AP-1 activation was measured in nuclear extract at 30 min of Ox-LDL stimulation (in triplicate, n = 3). D: Bar diagram and Western blot representing fold increase of phospho-PKCδ expression at indicated time points in the human monocytes incubated with Ox-LDL (n = 3). E: Bar graph represents secretory IL-1β in the supernatant obtained from control and Ox-LDL-stimulated monocytes for 48 h in the presence or absence of Rottlerin (2 μM, n = 4, in triplicate). F: Secretory IL-1β in PKCδ siRNA-treated cells. Blots represent one of three to four similar experiments. Values represent mean ± SE. * P

    Article Snippet: Human anti-CD36 (SMϕ); anti-TLR2, -4, and -6; Tanshinone IIa; PKCδ siRNA; TLR2, -4, and -6 siRNA; and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Activation Assay, Western Blot, Expressing, Incubation

    Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Immunofluorescence, Transfection, Imaging

    Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Transfection

    Changes in autophagy gene expression in U-138 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-138 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Changes in autophagy gene expression in U-138 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-138 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Expressing, Transfection

    Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Activation Assay, Transfection, Construct

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Transfection

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Transfection

    Changes in autophagy gene expression in U-118 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-118 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Changes in autophagy gene expression in U-118 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-118 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Expressing, Transfection

    Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Transfection, Microscopy, Expressing

    Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Transfection

    The effect of PRKCE silencing on U-138 MG and U-118 MG cells. Cells were harvested 72 h after transfection with non-targeting siRNA (Control siRNA) or PKCε siRNA. The downregulation of PRKCE mRNA expression ( a ) and PKCε protein level ( b ) are shown. The bar graphs present average values of three independent experiments. ** P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: The effect of PRKCE silencing on U-138 MG and U-118 MG cells. Cells were harvested 72 h after transfection with non-targeting siRNA (Control siRNA) or PKCε siRNA. The downregulation of PRKCE mRNA expression ( a ) and PKCε protein level ( b ) are shown. The bar graphs present average values of three independent experiments. ** P

    Article Snippet: Immunodetection U-118 MG and U-138 MG cells were treated with PKCε siRNA (25 nM) or control siRNA (25 nM) (both from Santa Cruz Biotechnology, CA, USA) for 72 h and rapamycin (300 nM) or 3-methyladenine (5 mM) (both from Sigma, St Louis, MO, USA) for 24 h. Whole cell extracts were prepared using a modified RIPA lysis buffer [ ].

    Techniques: Transfection, Expressing

    SiRNA-mediated knockdown of TMEM97 increases NPC1 protein levels, ameliorates cholesterol accumulation and restores cholesterol delivery to the ER in NPC1 -deficient HeLa cells. ( A ) Cultured HeLa cells were transfected for 48h with either control siRNA or indicated siRNAs targeting NPC1 , TMEM97 or NPC2 . Whole cell lysates were subjected to Western blotting and probed with antibodies against NPC1 and β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels of control siRNA treated cells ( n = 3 independent experiments per condition; ** P

    Journal: Human Molecular Genetics

    Article Title: Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells

    doi: 10.1093/hmg/ddw204

    Figure Lengend Snippet: SiRNA-mediated knockdown of TMEM97 increases NPC1 protein levels, ameliorates cholesterol accumulation and restores cholesterol delivery to the ER in NPC1 -deficient HeLa cells. ( A ) Cultured HeLa cells were transfected for 48h with either control siRNA or indicated siRNAs targeting NPC1 , TMEM97 or NPC2 . Whole cell lysates were subjected to Western blotting and probed with antibodies against NPC1 and β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels of control siRNA treated cells ( n = 3 independent experiments per condition; ** P

    Article Snippet: For RNA interference the following siRNAs were used: For knockdown of TMEM97 : siRNAs #140160 (I) (Ambion) and #SI03198314 (II) (QIAGEN); for knockdown of NPC1 : siRNAs #114041 (I) and #106017 (II) (both from Ambion); for knockdown of NPC2 : siRNAs #135801 (I) and #135802 (II) (both from Ambion); as a non-silencing negative control: scrambled-siRNA #4611 (Ambion).

    Techniques: Cell Culture, Transfection, Western Blot

    Reduction of TMEM97 elevates residual mutant NPC1 protein levels and restores cholesterol trafficking in NPC1 -mutant fibroblasts. (A) Primary human skin fibroblasts homozygous or compound-heterozygous for indicated NPC1 alleles (in brackets, see Supplementary Material, Table S1 for details) were transfected with NPC1 or TMEM97 siRNAs. Ninety-six hours after transfection, whole cell lysates were subjected to Western blotting and analyzed for NPC1 or β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels in control siRNA treated cells ( n = 2 independent experiments; mean±SEM, * P

    Journal: Human Molecular Genetics

    Article Title: Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells

    doi: 10.1093/hmg/ddw204

    Figure Lengend Snippet: Reduction of TMEM97 elevates residual mutant NPC1 protein levels and restores cholesterol trafficking in NPC1 -mutant fibroblasts. (A) Primary human skin fibroblasts homozygous or compound-heterozygous for indicated NPC1 alleles (in brackets, see Supplementary Material, Table S1 for details) were transfected with NPC1 or TMEM97 siRNAs. Ninety-six hours after transfection, whole cell lysates were subjected to Western blotting and analyzed for NPC1 or β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels in control siRNA treated cells ( n = 2 independent experiments; mean±SEM, * P

    Article Snippet: For RNA interference the following siRNAs were used: For knockdown of TMEM97 : siRNAs #140160 (I) (Ambion) and #SI03198314 (II) (QIAGEN); for knockdown of NPC1 : siRNAs #114041 (I) and #106017 (II) (both from Ambion); for knockdown of NPC2 : siRNAs #135801 (I) and #135802 (II) (both from Ambion); as a non-silencing negative control: scrambled-siRNA #4611 (Ambion).

    Techniques: Mutagenesis, Transfection, Western Blot

    Knockdown of TMEM97 increases NPC1 protein levels in lysosomal and extra-lysosomal compartments. ( A ) Confocal images from HeLa cells treated with indicated siRNAs for 48h and stained for NPC1 with an NPC1-specific antibody ( 32 ) (scale bar = 10µm). Box plots show medians (lines), lower and upper quartiles (boxes), 10th and 90th percentiles (whiskers) and outliers (circles) of NPC1 signal intensities per cell as quantified automatically from stacks of confocal images using CellProfiler software ( n = 5 independent experiments; *** P

    Journal: Human Molecular Genetics

    Article Title: Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells

    doi: 10.1093/hmg/ddw204

    Figure Lengend Snippet: Knockdown of TMEM97 increases NPC1 protein levels in lysosomal and extra-lysosomal compartments. ( A ) Confocal images from HeLa cells treated with indicated siRNAs for 48h and stained for NPC1 with an NPC1-specific antibody ( 32 ) (scale bar = 10µm). Box plots show medians (lines), lower and upper quartiles (boxes), 10th and 90th percentiles (whiskers) and outliers (circles) of NPC1 signal intensities per cell as quantified automatically from stacks of confocal images using CellProfiler software ( n = 5 independent experiments; *** P

    Article Snippet: For RNA interference the following siRNAs were used: For knockdown of TMEM97 : siRNAs #140160 (I) (Ambion) and #SI03198314 (II) (QIAGEN); for knockdown of NPC1 : siRNAs #114041 (I) and #106017 (II) (both from Ambion); for knockdown of NPC2 : siRNAs #135801 (I) and #135802 (II) (both from Ambion); as a non-silencing negative control: scrambled-siRNA #4611 (Ambion).

    Techniques: Staining, Software

    MED19 and MED26 are not essential for the apparent integrity of Mediator. Nuclear lysates from HeLa cells transfected with control ( CNTL ), MED19-, MED26-, or MED19 and 26-specific siRNAs were subjected to immunoprecipitation ( IP ) using antibodies specific for MED4. Immunoprecipitates were extensively washed prior to resolution by SDS-10% PAGE and processing by Western blot ( WB ) analysis using the specified antibodies. Input , 10% of the nuclear lysates used for IP reactions. Structural domains to which individual Mediator subunits may be relegated are indicated (Head, Middle, Tail, Kinase, or Unassigned ( Unass. )). Asterisks mark MED19 and MED26 Mediator subunits targeted for RNAi-mediated depletion.

    Journal: The Journal of Biological Chemistry

    Article Title: MED19 and MED26 Are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression *MED19 and MED26 Are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression * S⃞

    doi: 10.1074/jbc.M806514200

    Figure Lengend Snippet: MED19 and MED26 are not essential for the apparent integrity of Mediator. Nuclear lysates from HeLa cells transfected with control ( CNTL ), MED19-, MED26-, or MED19 and 26-specific siRNAs were subjected to immunoprecipitation ( IP ) using antibodies specific for MED4. Immunoprecipitates were extensively washed prior to resolution by SDS-10% PAGE and processing by Western blot ( WB ) analysis using the specified antibodies. Input , 10% of the nuclear lysates used for IP reactions. Structural domains to which individual Mediator subunits may be relegated are indicated (Head, Middle, Tail, Kinase, or Unassigned ( Unass. )). Asterisks mark MED19 and MED26 Mediator subunits targeted for RNAi-mediated depletion.

    Article Snippet: For siRNA transfections, cells (∼60% confluent) were transfected with siRNAs at a final concentration of 20 n m for 3 days before further analyses. siRNAs (Dharmacon) correspond to the following: MED19 (J-016056-11); MED26 (J-011948-09); control non-target siRNA (D-001210-01).

    Techniques: Transfection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot

    MED19 and MED26 synergistically mediate the interaction between REST and Mediator. Nuclear lysates from HeLa cells transfected with control (CNTL), MED19-, MED26-, or MED19 and 26-specific siRNAs as indicated were incubated with immobilized GST-REST-(141–600). Specifically bound proteins were resolved by SDS, 10% PAGE prior to WB analysis using the specified antibodies. Input , 10% of the nuclear lysates used in binding reactions.

    Journal: The Journal of Biological Chemistry

    Article Title: MED19 and MED26 Are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression *MED19 and MED26 Are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression * S⃞

    doi: 10.1074/jbc.M806514200

    Figure Lengend Snippet: MED19 and MED26 synergistically mediate the interaction between REST and Mediator. Nuclear lysates from HeLa cells transfected with control (CNTL), MED19-, MED26-, or MED19 and 26-specific siRNAs as indicated were incubated with immobilized GST-REST-(141–600). Specifically bound proteins were resolved by SDS, 10% PAGE prior to WB analysis using the specified antibodies. Input , 10% of the nuclear lysates used in binding reactions.

    Article Snippet: For siRNA transfections, cells (∼60% confluent) were transfected with siRNAs at a final concentration of 20 n m for 3 days before further analyses. siRNAs (Dharmacon) correspond to the following: MED19 (J-016056-11); MED26 (J-011948-09); control non-target siRNA (D-001210-01).

    Techniques: Transfection, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Binding Assay

    MED19 and MED26 are synergistically required for recruitment of Mediator and G9a-dependent H3K9me2 by RE1-bound REST. Soluble chromatin prepared from HeLa cells transfected with control, MED19-, MED26-, or MED19 and 26-specific siRNAs as indicated was subjected to IP using the indicated antibodies. Immunoprecipitated chromatin was analyzed by quantitative PCR using primers flanking RE1 elements within the M4, SNAP25, and Synapsin1 (Syn1) genes. The level of RE1 site occupancy for each protein is expressed relative to its level of occupancy in control siRNA-transfected cells, which was arbitrarily assigned a value of 100%. Data represent the mean ± S.E. of at least three independent experiments performed in triplicate. Asterisks denote statistically significant values relative to control siRNA (Student's t test, **, p

    Journal: The Journal of Biological Chemistry

    Article Title: MED19 and MED26 Are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression *MED19 and MED26 Are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression * S⃞

    doi: 10.1074/jbc.M806514200

    Figure Lengend Snippet: MED19 and MED26 are synergistically required for recruitment of Mediator and G9a-dependent H3K9me2 by RE1-bound REST. Soluble chromatin prepared from HeLa cells transfected with control, MED19-, MED26-, or MED19 and 26-specific siRNAs as indicated was subjected to IP using the indicated antibodies. Immunoprecipitated chromatin was analyzed by quantitative PCR using primers flanking RE1 elements within the M4, SNAP25, and Synapsin1 (Syn1) genes. The level of RE1 site occupancy for each protein is expressed relative to its level of occupancy in control siRNA-transfected cells, which was arbitrarily assigned a value of 100%. Data represent the mean ± S.E. of at least three independent experiments performed in triplicate. Asterisks denote statistically significant values relative to control siRNA (Student's t test, **, p

    Article Snippet: For siRNA transfections, cells (∼60% confluent) were transfected with siRNAs at a final concentration of 20 n m for 3 days before further analyses. siRNAs (Dharmacon) correspond to the following: MED19 (J-016056-11); MED26 (J-011948-09); control non-target siRNA (D-001210-01).

    Techniques: Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction

    MED19 and MED26 are synergistically required for REST-directed transcriptional repression. A , HeLa cells were transfected with control, MED19-, MED26-, or MED19 and 26-specific siRNAs as indicated 48 h prior to co-transfection with pG5TK-Luc along with Gal4 or Gal4-REST derivatives as indicated and subsequent assay of transfected whole cell lysates for normalized luciferase activities. Luciferase activities are expressed relative to the luciferase activity obtained in cells transfected with control siRNA and Gal4. Data represent the mean ± S.E. of at least three independent transfections performed in duplicate. Asterisks denote statistically significant values relative to control siRNA (Student's t test; **, p

    Journal: The Journal of Biological Chemistry

    Article Title: MED19 and MED26 Are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression *MED19 and MED26 Are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression * S⃞

    doi: 10.1074/jbc.M806514200

    Figure Lengend Snippet: MED19 and MED26 are synergistically required for REST-directed transcriptional repression. A , HeLa cells were transfected with control, MED19-, MED26-, or MED19 and 26-specific siRNAs as indicated 48 h prior to co-transfection with pG5TK-Luc along with Gal4 or Gal4-REST derivatives as indicated and subsequent assay of transfected whole cell lysates for normalized luciferase activities. Luciferase activities are expressed relative to the luciferase activity obtained in cells transfected with control siRNA and Gal4. Data represent the mean ± S.E. of at least three independent transfections performed in duplicate. Asterisks denote statistically significant values relative to control siRNA (Student's t test; **, p

    Article Snippet: For siRNA transfections, cells (∼60% confluent) were transfected with siRNAs at a final concentration of 20 n m for 3 days before further analyses. siRNAs (Dharmacon) correspond to the following: MED19 (J-016056-11); MED26 (J-011948-09); control non-target siRNA (D-001210-01).

    Techniques: Transfection, Cotransfection, Subsequent Assay, Luciferase, Activity Assay

    FFAR2 positively regulates IAV replication in A549 cells. (A to D) FFAR2 siRNA- or scrambled siRNA-transfected A549 cells were infected with AH05 (H5N1) (MOI = 0.1) (A), WSN (H1N1) (MOI = 0.01) (B), SH13 (H9N2) (MOI = 0.1) (C), or VSV-EGFP (100 TCID 50 ) (D) virus. Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells (A to C) or by determining the TCID 50 on HEK293T cells (D). **, P

    Journal: Journal of Virology

    Article Title: The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

    doi: 10.1128/JVI.01707-19

    Figure Lengend Snippet: FFAR2 positively regulates IAV replication in A549 cells. (A to D) FFAR2 siRNA- or scrambled siRNA-transfected A549 cells were infected with AH05 (H5N1) (MOI = 0.1) (A), WSN (H1N1) (MOI = 0.01) (B), SH13 (H9N2) (MOI = 0.1) (C), or VSV-EGFP (100 TCID 50 ) (D) virus. Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells (A to C) or by determining the TCID 50 on HEK293T cells (D). **, P

    Article Snippet: A549 cells were seeded into 96-well plates and then transfected with FFAR2 siRNA (5′-GCCUCUGUAUGGAGUGAUU-3′) or with a scrambled siRNA (GenePharma, Shanghai, China) at a concentration of 30 nM by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA).

    Techniques: Transfection, Infection

    Ffar2 enhances IAV replication in mouse RAW 264.7 cells. (A) Mouse RAW 264.7 cells were transfected with mouse Ffar2 siRNA or scrambled siRNA for 48 h, and the knockdown of Ffar2 was confirmed by Western blotting with a rabbit anti-FFAR2 pAb. (B) Mouse RAW 264.7 cells were treated with mouse Ffar2 siRNA or scrambled siRNA and cultured at 37°C for 48 h. Cell viability was determined by using a CellTiter-Glo assay. (C and D) Ffar2 siRNA- or scrambled siRNA-transfected RAW 264.7 cells were infected with AH05 (H5N1) virus at an MOI of 0.1 (C) or 5 (D). Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells. **, P

    Journal: Journal of Virology

    Article Title: The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

    doi: 10.1128/JVI.01707-19

    Figure Lengend Snippet: Ffar2 enhances IAV replication in mouse RAW 264.7 cells. (A) Mouse RAW 264.7 cells were transfected with mouse Ffar2 siRNA or scrambled siRNA for 48 h, and the knockdown of Ffar2 was confirmed by Western blotting with a rabbit anti-FFAR2 pAb. (B) Mouse RAW 264.7 cells were treated with mouse Ffar2 siRNA or scrambled siRNA and cultured at 37°C for 48 h. Cell viability was determined by using a CellTiter-Glo assay. (C and D) Ffar2 siRNA- or scrambled siRNA-transfected RAW 264.7 cells were infected with AH05 (H5N1) virus at an MOI of 0.1 (C) or 5 (D). Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells. **, P

    Article Snippet: A549 cells were seeded into 96-well plates and then transfected with FFAR2 siRNA (5′-GCCUCUGUAUGGAGUGAUU-3′) or with a scrambled siRNA (GenePharma, Shanghai, China) at a concentration of 30 nM by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA).

    Techniques: Transfection, Western Blot, Cell Culture, Glo Assay, Infection

    The FFAR2 downstream effectors β-arrestin1 and AP2B1 promote the replication of IAV. (A) A549 cells were transfected with siRNA targeting β- arrestin1 or β- arrestin2 or with scrambled siRNA for 48 h. The levels of β- arrestin1 or β- arrestin2 mRNA in β- arrestin1 or β- arrestin2 siRNA-treated cells were determined by qRT-PCR in cell lysates and standardized to those in the scrambled siRNA-treated cells. ***, P

    Journal: Journal of Virology

    Article Title: The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

    doi: 10.1128/JVI.01707-19

    Figure Lengend Snippet: The FFAR2 downstream effectors β-arrestin1 and AP2B1 promote the replication of IAV. (A) A549 cells were transfected with siRNA targeting β- arrestin1 or β- arrestin2 or with scrambled siRNA for 48 h. The levels of β- arrestin1 or β- arrestin2 mRNA in β- arrestin1 or β- arrestin2 siRNA-treated cells were determined by qRT-PCR in cell lysates and standardized to those in the scrambled siRNA-treated cells. ***, P

    Article Snippet: A549 cells were seeded into 96-well plates and then transfected with FFAR2 siRNA (5′-GCCUCUGUAUGGAGUGAUU-3′) or with a scrambled siRNA (GenePharma, Shanghai, China) at a concentration of 30 nM by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA).

    Techniques: Transfection, Quantitative RT-PCR

    FFAR2 is important for the internalization of IAV. (A) A549 cells were treated with FFAR2 siRNA or scrambled siRNA for 48 h, stained with a wheat germ agglutinin (WGA)-Alexa Fluor 488 conjugate, and analyzed by flow cytometry. (B and C) A549 cells treated with FFAR2 siRNA or scrambled siRNA were stained with lectins that have specificity for α-2,3-sialic acids (MAL) (B) or α-2,6-sialic acids (SNA) (C) and analyzed by flow cytometry. (D and E) A549 cells were treated with FFAR2 siRNA or scrambled siRNA for 48 h and then infected with AH05 (H5N1) virus on ice at 4°C for 1 h. Cells were fixed, stained with a mouse anti-HA mAb (D) or a mouse anti-NP mAb (E) and Alexa Fluor 488 goat anti-mouse IgG(H+L), and analyzed by flow cytometry. (F) A549 cells were transfected with FFAR2 siRNA or scrambled siRNA for 48 h and infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h. After unbound virus was washed away, the cells were collected and titrated for infectious virus by means of plaque assays on MDCK cells. (G) A549 cells were infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h, followed by a neutral wash (ice-cold PBS, pH 7.2) or an acidic wash (ice-cold PBS-HCl, pH 1.3) before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit NP pAb. (H) FFAR2 siRNA- or scrambled siRNA-treated A549 cells were infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h, followed by a culture temperature shift to 37°C for 30 min to allow for internalization. The cells were then washed with ice-cold PBS-HCl (pH 1.3) before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit NP pAb. (I) A549 cells were pretreated with 100 μM 4-CMTB, 10 μM Cmp58, or DMSO for 3 h and then infected with AH05 (H5N1) (MOI = 5) at 37°C. The infected cells were washed with ice-cold PBS-HCl (pH 1.3) at the indicated time points before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit NP pAb. A549 cells pretreated with neuraminidase (Neu) were stained with WGA (A), MAL (B), and SNA (C), respectively, and used as negative controls. The band intensities of the Western blots were quantified by using ImageJ software and are expressed as relative NP/GAPDH ratios (G to I).

    Journal: Journal of Virology

    Article Title: The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

    doi: 10.1128/JVI.01707-19

    Figure Lengend Snippet: FFAR2 is important for the internalization of IAV. (A) A549 cells were treated with FFAR2 siRNA or scrambled siRNA for 48 h, stained with a wheat germ agglutinin (WGA)-Alexa Fluor 488 conjugate, and analyzed by flow cytometry. (B and C) A549 cells treated with FFAR2 siRNA or scrambled siRNA were stained with lectins that have specificity for α-2,3-sialic acids (MAL) (B) or α-2,6-sialic acids (SNA) (C) and analyzed by flow cytometry. (D and E) A549 cells were treated with FFAR2 siRNA or scrambled siRNA for 48 h and then infected with AH05 (H5N1) virus on ice at 4°C for 1 h. Cells were fixed, stained with a mouse anti-HA mAb (D) or a mouse anti-NP mAb (E) and Alexa Fluor 488 goat anti-mouse IgG(H+L), and analyzed by flow cytometry. (F) A549 cells were transfected with FFAR2 siRNA or scrambled siRNA for 48 h and infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h. After unbound virus was washed away, the cells were collected and titrated for infectious virus by means of plaque assays on MDCK cells. (G) A549 cells were infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h, followed by a neutral wash (ice-cold PBS, pH 7.2) or an acidic wash (ice-cold PBS-HCl, pH 1.3) before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit NP pAb. (H) FFAR2 siRNA- or scrambled siRNA-treated A549 cells were infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h, followed by a culture temperature shift to 37°C for 30 min to allow for internalization. The cells were then washed with ice-cold PBS-HCl (pH 1.3) before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit NP pAb. (I) A549 cells were pretreated with 100 μM 4-CMTB, 10 μM Cmp58, or DMSO for 3 h and then infected with AH05 (H5N1) (MOI = 5) at 37°C. The infected cells were washed with ice-cold PBS-HCl (pH 1.3) at the indicated time points before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit NP pAb. A549 cells pretreated with neuraminidase (Neu) were stained with WGA (A), MAL (B), and SNA (C), respectively, and used as negative controls. The band intensities of the Western blots were quantified by using ImageJ software and are expressed as relative NP/GAPDH ratios (G to I).

    Article Snippet: A549 cells were seeded into 96-well plates and then transfected with FFAR2 siRNA (5′-GCCUCUGUAUGGAGUGAUU-3′) or with a scrambled siRNA (GenePharma, Shanghai, China) at a concentration of 30 nM by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA).

    Techniques: Staining, Whole Genome Amplification, Flow Cytometry, Cytometry, Infection, Transfection, Lysis, Western Blot, Software

    FFAR2 knockdown does not stimulate IFN pathways. (A and B) FFAR2 siRNA- or scrambled siRNA-transfected A549 cells were left untreated or were treated with IFN-α (A) or IFN-β (B) for 24 h. The cell lysates were then subjected to Western blotting with a rabbit anti-Mx1 pAb for the detection of Mx1 protein. (C) HEK293T cells were treated with FFAR2 siRNA or scrambled siRNA for 24 h and then transfected with the ISRE-Luc reporter plasmid and the pRL-TK control plasmid. Twenty-four hours later, a dual-luciferase reporter assay was performed. After normalization with cotransfected Renilla luciferase activity, the relative firefly luciferase activity of the FFAR2 knockdown cells was expressed as the fold induction of the ISRE firefly luciferase activity compared to control cells.

    Journal: Journal of Virology

    Article Title: The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

    doi: 10.1128/JVI.01707-19

    Figure Lengend Snippet: FFAR2 knockdown does not stimulate IFN pathways. (A and B) FFAR2 siRNA- or scrambled siRNA-transfected A549 cells were left untreated or were treated with IFN-α (A) or IFN-β (B) for 24 h. The cell lysates were then subjected to Western blotting with a rabbit anti-Mx1 pAb for the detection of Mx1 protein. (C) HEK293T cells were treated with FFAR2 siRNA or scrambled siRNA for 24 h and then transfected with the ISRE-Luc reporter plasmid and the pRL-TK control plasmid. Twenty-four hours later, a dual-luciferase reporter assay was performed. After normalization with cotransfected Renilla luciferase activity, the relative firefly luciferase activity of the FFAR2 knockdown cells was expressed as the fold induction of the ISRE firefly luciferase activity compared to control cells.

    Article Snippet: A549 cells were seeded into 96-well plates and then transfected with FFAR2 siRNA (5′-GCCUCUGUAUGGAGUGAUU-3′) or with a scrambled siRNA (GenePharma, Shanghai, China) at a concentration of 30 nM by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA).

    Techniques: Transfection, Western Blot, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay

    Identification of FFAR2 as a host factor involved in the replication of IAV by using H1N1 NA-Venus, H5N1 NA-Venus, and H9N2 NA-Venus reporter viruses. (A) A549 cells were transfected with siRNA targeting FFAR2 or with scrambled siRNA for 48 h, and the knockdown of FFAR2 was detected by Western blotting. (B) A549 cells were treated with FFAR2 siRNA or scrambled siRNA and cultured at 37°C for 48 h. Cell viability was determined by using a CellTiter-Glo assay. (C to E) A549 cells were seeded into 96-well plates containing FFAR2 siRNA or scrambled siRNA and cultured at 37°C for 48 h. They were then infected with one of the Venus reporter viruses, H1N1 NA-Venus (MOI = 0.1) (C), H5N1 NA-Venus (MOI = 0.1) (D), or H9N2 NA-Venus (MOI = 0.1) (E), and cultured at 37°C for 24 h. Venus expression was visualized by using the Operetta high-content imaging system. The effect of FFAR2 knockdown by siRNA interference on virus replication was calculated by normalizing the average fluorescence intensity of the FFAR2 siRNA-treated wells with that of the scrambled siRNA-treated cells. The data are presented as means ± standard deviations (SD) for triplicate samples. ***, P

    Journal: Journal of Virology

    Article Title: The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

    doi: 10.1128/JVI.01707-19

    Figure Lengend Snippet: Identification of FFAR2 as a host factor involved in the replication of IAV by using H1N1 NA-Venus, H5N1 NA-Venus, and H9N2 NA-Venus reporter viruses. (A) A549 cells were transfected with siRNA targeting FFAR2 or with scrambled siRNA for 48 h, and the knockdown of FFAR2 was detected by Western blotting. (B) A549 cells were treated with FFAR2 siRNA or scrambled siRNA and cultured at 37°C for 48 h. Cell viability was determined by using a CellTiter-Glo assay. (C to E) A549 cells were seeded into 96-well plates containing FFAR2 siRNA or scrambled siRNA and cultured at 37°C for 48 h. They were then infected with one of the Venus reporter viruses, H1N1 NA-Venus (MOI = 0.1) (C), H5N1 NA-Venus (MOI = 0.1) (D), or H9N2 NA-Venus (MOI = 0.1) (E), and cultured at 37°C for 24 h. Venus expression was visualized by using the Operetta high-content imaging system. The effect of FFAR2 knockdown by siRNA interference on virus replication was calculated by normalizing the average fluorescence intensity of the FFAR2 siRNA-treated wells with that of the scrambled siRNA-treated cells. The data are presented as means ± standard deviations (SD) for triplicate samples. ***, P

    Article Snippet: A549 cells were seeded into 96-well plates and then transfected with FFAR2 siRNA (5′-GCCUCUGUAUGGAGUGAUU-3′) or with a scrambled siRNA (GenePharma, Shanghai, China) at a concentration of 30 nM by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA).

    Techniques: Transfection, Western Blot, Cell Culture, Glo Assay, Infection, Expressing, Imaging, Fluorescence

    FFAR2 affects the early stage of the IAV replication cycle. (A) FFAR2 siRNA- or scrambled siRNA-treated A549 cells were infected with AH05 (H5N1) virus (MOI = 5). Whole-cell lysates were collected at the indicated time points and subjected to Western blotting with a rabbit anti-NP pAb. (B) A549 cells were pretreated with 100 μM 4-CMTB or DMSO for 3 h and then infected with AH05 (H5N1) virus (MOI = 5). Whole-cell lysates were collected at the indicated time points and subjected to Western blotting with a rabbit anti-NP pAb. (C) A549 cells were treated with FFAR2 siRNA or scrambled siRNA for 48 h and then infected with AH05 (H5N1) virus (MOI = 5). At 2, 3, and 4 h p.i., the infected cells were fixed and stained with a rabbit anti-NP pAb, followed by incubation with Alexa Fluor 633 goat anti-rabbit IgG(H+L) (red). The nuclei were stained with DAPI (blue). The ratio of virus-infected cells showing nuclear localization of viral NP was calculated from more than 150 cells and is indicated at the bottom of each panel of three images. (D) A549 cells were pretreated with 100 μM 4-CMTB, 10 μM Cmp58, or DMSO for 3 h and then infected with AH05 (H5N1) virus (MOI = 5). At 3 and 4 h p.i., the infected cells were fixed and stained as described above for panel C. (E) FFAR2 siRNA- or scrambled siRNA-transfected A549 cells were infected with AH05 (H5N1) virus (MOI = 5). At 2 and 3 h p.i., the cells were separated into nuclear and cytoplasmic fractions. Each fraction was subjected to Western blotting with a rabbit anti-NP pAb, a rabbit anti-GAPDH pAb, and a rabbit anti-LaminB1 pAb. (F) HEK293T cells treated with FFAR2 siRNA or scrambled siRNA for 24 h were transfected with the four viral RNP protein expression plasmids (PB2, PB1, PA, and NP), together with pHH21-SC09NS F-Luc and pRL-TK. Thirty-six hours later, the knockdown of FFAR2 was confirmed by Western blotting, and a dual-luciferase assay was performed in which the firefly luciferase activity was normalized to the activity of the internal control, Renilla luciferase. The band intensities of the Western blots were quantified by using ImageJ software and are expressed as relative NP/GAPDH ratios (A and B).

    Journal: Journal of Virology

    Article Title: The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

    doi: 10.1128/JVI.01707-19

    Figure Lengend Snippet: FFAR2 affects the early stage of the IAV replication cycle. (A) FFAR2 siRNA- or scrambled siRNA-treated A549 cells were infected with AH05 (H5N1) virus (MOI = 5). Whole-cell lysates were collected at the indicated time points and subjected to Western blotting with a rabbit anti-NP pAb. (B) A549 cells were pretreated with 100 μM 4-CMTB or DMSO for 3 h and then infected with AH05 (H5N1) virus (MOI = 5). Whole-cell lysates were collected at the indicated time points and subjected to Western blotting with a rabbit anti-NP pAb. (C) A549 cells were treated with FFAR2 siRNA or scrambled siRNA for 48 h and then infected with AH05 (H5N1) virus (MOI = 5). At 2, 3, and 4 h p.i., the infected cells were fixed and stained with a rabbit anti-NP pAb, followed by incubation with Alexa Fluor 633 goat anti-rabbit IgG(H+L) (red). The nuclei were stained with DAPI (blue). The ratio of virus-infected cells showing nuclear localization of viral NP was calculated from more than 150 cells and is indicated at the bottom of each panel of three images. (D) A549 cells were pretreated with 100 μM 4-CMTB, 10 μM Cmp58, or DMSO for 3 h and then infected with AH05 (H5N1) virus (MOI = 5). At 3 and 4 h p.i., the infected cells were fixed and stained as described above for panel C. (E) FFAR2 siRNA- or scrambled siRNA-transfected A549 cells were infected with AH05 (H5N1) virus (MOI = 5). At 2 and 3 h p.i., the cells were separated into nuclear and cytoplasmic fractions. Each fraction was subjected to Western blotting with a rabbit anti-NP pAb, a rabbit anti-GAPDH pAb, and a rabbit anti-LaminB1 pAb. (F) HEK293T cells treated with FFAR2 siRNA or scrambled siRNA for 24 h were transfected with the four viral RNP protein expression plasmids (PB2, PB1, PA, and NP), together with pHH21-SC09NS F-Luc and pRL-TK. Thirty-six hours later, the knockdown of FFAR2 was confirmed by Western blotting, and a dual-luciferase assay was performed in which the firefly luciferase activity was normalized to the activity of the internal control, Renilla luciferase. The band intensities of the Western blots were quantified by using ImageJ software and are expressed as relative NP/GAPDH ratios (A and B).

    Article Snippet: A549 cells were seeded into 96-well plates and then transfected with FFAR2 siRNA (5′-GCCUCUGUAUGGAGUGAUU-3′) or with a scrambled siRNA (GenePharma, Shanghai, China) at a concentration of 30 nM by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA).

    Techniques: Infection, Western Blot, Staining, Incubation, Transfection, Expressing, Luciferase, Activity Assay, Software

    G protein-coupled receptor kinases (GRKs) are required for IAV replication. (A) A549 cells were transfected with siRNA targeting GRK2 , GRK3 , GRK5 , or GRK6 for 48 h. The levels of mRNA of the indicated gene in the cell lysates were determined by qRT-PCR and standardized to those of the scrambled siRNA-treated cells. ***, P

    Journal: Journal of Virology

    Article Title: The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

    doi: 10.1128/JVI.01707-19

    Figure Lengend Snippet: G protein-coupled receptor kinases (GRKs) are required for IAV replication. (A) A549 cells were transfected with siRNA targeting GRK2 , GRK3 , GRK5 , or GRK6 for 48 h. The levels of mRNA of the indicated gene in the cell lysates were determined by qRT-PCR and standardized to those of the scrambled siRNA-treated cells. ***, P

    Article Snippet: A549 cells were seeded into 96-well plates and then transfected with FFAR2 siRNA (5′-GCCUCUGUAUGGAGUGAUU-3′) or with a scrambled siRNA (GenePharma, Shanghai, China) at a concentration of 30 nM by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA).

    Techniques: Transfection, Quantitative RT-PCR

    E2f1-mediated transcriptional induction of Cdc25a may induce liver cysts in LKO mice. A : Renilla luciferase activity (LUC2) produced from wild-type or mutant Cdc25a 3′-UTR reporter plasmids or empty vector normalized to firefly luciferase activity (LUC1) produced from the same plasmid after transfection into Hepa cells together with negative control (NC) RNA or miR-122 mimic. Error bars represent standard deviations derived from three independent experiments. B : Both pre-mRNA (hnRNA) and mRNA are up-regulated at similar level in DEN-injected LKO livers. Real-time RT-qPCR analysis of Cdc25a mRNA and hnRNA in livers ( n = 5) was performed. The data were normalized to Gapdh RNA level. C : Western blot analysis of liver extracts with specific antibodies at week 25 post-DEN injection. D : Quantification of the protein signals. mRNA ( E ) and protein levels ( F ) of Cdc25a and E2f1 in Hepa cells transfected with 60 nmol/L negative control or E2f1 siRNA ( n = 3). The results were normalized to Gapdh level. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, and ∗∗∗ P ≤ 0.001.

    Journal: The American Journal of Pathology

    Article Title: Hepatic Loss of miR-122 Predisposes Mice to Hepatobiliary Cyst and Hepatocellular Carcinoma upon Diethylnitrosamine Exposure

    doi: 10.1016/j.ajpath.2013.08.004

    Figure Lengend Snippet: E2f1-mediated transcriptional induction of Cdc25a may induce liver cysts in LKO mice. A : Renilla luciferase activity (LUC2) produced from wild-type or mutant Cdc25a 3′-UTR reporter plasmids or empty vector normalized to firefly luciferase activity (LUC1) produced from the same plasmid after transfection into Hepa cells together with negative control (NC) RNA or miR-122 mimic. Error bars represent standard deviations derived from three independent experiments. B : Both pre-mRNA (hnRNA) and mRNA are up-regulated at similar level in DEN-injected LKO livers. Real-time RT-qPCR analysis of Cdc25a mRNA and hnRNA in livers ( n = 5) was performed. The data were normalized to Gapdh RNA level. C : Western blot analysis of liver extracts with specific antibodies at week 25 post-DEN injection. D : Quantification of the protein signals. mRNA ( E ) and protein levels ( F ) of Cdc25a and E2f1 in Hepa cells transfected with 60 nmol/L negative control or E2f1 siRNA ( n = 3). The results were normalized to Gapdh level. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, and ∗∗∗ P ≤ 0.001.

    Article Snippet: Hepa cells cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum were transfected with 60 nmol/L E2f1 siRNA (M-044993-03) or negative control siRNA (D-001206-13-05) obtained from Thermo Fisher Scientific (Waltham, MA) using Lipofectamine 2000 (Invitrogen, Grand Island, NY) for 4 hours.

    Techniques: Mouse Assay, Luciferase, Activity Assay, Produced, Mutagenesis, Plasmid Preparation, Transfection, Negative Control, Derivative Assay, Injection, Quantitative RT-PCR, Western Blot