lipid mediated transfection Search Results


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  • 99
    Thermo Fisher lipid mediated transfection
    a2V inhibition increases Notch signaling in TNBC A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after <t>transfection.</t> Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.
    Lipid Mediated Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Mirus Bio lipid mediated transfection
    a2V inhibition increases Notch signaling in TNBC A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after <t>transfection.</t> Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.
    Lipid Mediated Transfection, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipid mediated cell transfection lipofectamine 3000
    a2V inhibition increases Notch signaling in TNBC A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after <t>transfection.</t> Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.
    Lipid Mediated Cell Transfection Lipofectamine 3000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher lipid mediated transfection reagent rnaimax
    a2V inhibition increases Notch signaling in TNBC A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after <t>transfection.</t> Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.
    Lipid Mediated Transfection Reagent Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery lipid mediated dharmafect transfection reagent
    a2V inhibition increases Notch signaling in TNBC A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after <t>transfection.</t> Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.
    Lipid Mediated Dharmafect Transfection Reagent, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cationic lipid mediated plasmid dna transfection
    hSpry2 degradation is impaired in AR-treated cells HEK-293 cells were transiently transfected with cDNA encoding EGF-R (0.05 μ g), GFP–Cbl WT (4 μ g) and FLAG–hSprouty2 (2 μ g). At 48 h <t>post-transfection,</t> cells were serum-starved and incubated without (0 min) or with AR (136 nM) or EGF (17 nM) for 2, 5, 10, 40 or 90 min. ( A ) Immunoblotting results from a representative experiment. A 100 μ g cell lysate protein sample was loaded per lane, gel-resolved, transferred on to PVDF and immunoblotted as indicated. ( B ) Quantitative analysis of three independent degradation experiments, showing the average amount of total cellular hSprouty2 remaining after 90 min of receptor stimulation by ligand. Results are means ± S.D. A two-tailed Student’s t test with α =0.05 determined that a significantly different amount of hSprouty2 degradation was effected by AR compared with EGF (**).
    Cationic Lipid Mediated Plasmid Dna Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher cationic lipid mediated transient transfection
    hSpry2 degradation is impaired in AR-treated cells HEK-293 cells were transiently transfected with cDNA encoding EGF-R (0.05 μ g), GFP–Cbl WT (4 μ g) and FLAG–hSprouty2 (2 μ g). At 48 h <t>post-transfection,</t> cells were serum-starved and incubated without (0 min) or with AR (136 nM) or EGF (17 nM) for 2, 5, 10, 40 or 90 min. ( A ) Immunoblotting results from a representative experiment. A 100 μ g cell lysate protein sample was loaded per lane, gel-resolved, transferred on to PVDF and immunoblotted as indicated. ( B ) Quantitative analysis of three independent degradation experiments, showing the average amount of total cellular hSprouty2 remaining after 90 min of receptor stimulation by ligand. Results are means ± S.D. A two-tailed Student’s t test with α =0.05 determined that a significantly different amount of hSprouty2 degradation was effected by AR compared with EGF (**).
    Cationic Lipid Mediated Transient Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cationic lipid reagent mediated transfection technique
    (a) Overexpression of ADARs by cotransfection of either an ADAR1 (pSS151) or ADAR2 (pSKW005) plasmid with pNL4-3 enhanced HIV-1 p24 production, as shown by p24 ELISA of the culture supernatant of transfected COS-7 cells. The <t>transfection</t> efficiencies,
    Cationic Lipid Reagent Mediated Transfection Technique, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipid mediated dna transfection
    (a) Overexpression of ADARs by cotransfection of either an ADAR1 (pSS151) or ADAR2 (pSKW005) plasmid with pNL4-3 enhanced HIV-1 p24 production, as shown by p24 ELISA of the culture supernatant of transfected COS-7 cells. The <t>transfection</t> efficiencies,
    Lipid Mediated Dna Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipid mediated transfection protocol
    Differential expression is abrogated with <t>transfection,</t> time in culture, and age of DNA. Represents the fold difference in transcriptional activity after normalizing luciferase RLUs/ to −308G/GL3Luc, which was set at 1. ( a ) Jurkat cells were transiently
    Lipid Mediated Transfection Protocol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipid mediated transfection reagent cellfectin
    Differential expression is abrogated with <t>transfection,</t> time in culture, and age of DNA. Represents the fold difference in transcriptional activity after normalizing luciferase RLUs/ to −308G/GL3Luc, which was set at 1. ( a ) Jurkat cells were transiently
    Lipid Mediated Transfection Reagent Cellfectin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gemini Bio lipid mediated transfection reagent
    Differential expression is abrogated with <t>transfection,</t> time in culture, and age of DNA. Represents the fold difference in transcriptional activity after normalizing luciferase RLUs/ to −308G/GL3Luc, which was set at 1. ( a ) Jurkat cells were transiently
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    Horizon Discovery lipid mediated transfection
    Cas9 editing efficiencies at target sites in unsilenced, partially silenced, and fully silenced chromatin states. (a) A map of the Cas9/sgRNA-expressing plasmid. Cas9 and EGFP expression are both driven by the CBh promoter. The T2A signal allows EGFP to be translated as a separate peptide to avoid interference of Cas9 function. (b) Table of average frequencies of EGFP-expressing cells <t>(transfection</t> efficiencies) as determined by flow cytometry of triplicate samples for transfected Luc14, GAL4EED, and GAL4EED cells treated with doxycycline. (c) Mean editing frequencies normalized to transfection efficiency in Luc14, GAL4EED, or GAL4EED + dox cells for Cas9 targeted to sites sg046, sg055, sg032, sg034, sg054, sg031, sg025, sg044, and sg048. *Indicates significantly reduced editing efficiencies at fully silenced chromatin compared to unsilenced chromatin ( p
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    Mirus Bio long rna transfection lipid mediated transfections
    <t>Long-RNA</t> <t>transfection</t> yields ES-cell-level expression of reprogramming proteins in primary human fibroblasts. A. The transcribed strand of an Hbb -UTR-stabilized in vitro-transcription template encoding an arbitrary protein. The long arrow indicates the first transcribed base, and short arrows indicate restriction-enzyme cleavage sites. B. In vitro-transcribed RNA encoding reprogramming proteins. C. Western blots showing expression levels and lifetimes of Oct4, Sox2, Nanog, Lin28, and MyoD1 proteins in MRC-5 human fetal lung fibroblasts transfected with protein-encoding RNA, relative to levels in hES (H9) and rhabdomyosarcoma (Rh30) cells. β-actin was used as a loading control. Left panels: The amount of RNA per 50 µL electroporation volume was varied as indicated. Cells were lysed 6 hours after transfection. Right panels: Cells were transfected with 1 µg of RNA, and lysed at the indicated times. D. Expression and nuclear localization of Oct4, Sox2, Klf4, Utf1, Nanog, Lin28, and MyoD1 protein following long-RNA transfection. Cells were fixed and stained 6–12 hours after transfection. For each protein, identical camera settings and exposure times were used for the RNA-transfected and mock-transfected samples.
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    Image Search Results


    a2V inhibition increases Notch signaling in TNBC A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after transfection. Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.

    Journal: Oncotarget

    Article Title: The Vacuolar ATPase a2-subunit regulates Notch signaling in triple-negative breast cancer cells

    doi:

    Figure Lengend Snippet: a2V inhibition increases Notch signaling in TNBC A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after transfection. Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.

    Article Snippet: Cells were transfected with siRNA (final concentration, 10 nM) using lipid-mediated transfection with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

    Techniques: Inhibition, Multiple Displacement Amplification, Transfection, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Generated, Western Blot, Immunofluorescence, Construct, Luciferase, Recombinant, Binding Assay

    a2V-ATPase inhibition enhances Wnt signaling in TNBC A. MDA-MB-231 cells were transfected with scrambled control or a2V siRNA and harvested after 48 hrs of transfection. Fold change in mRNA expression levels of Wnt signaling genes WNT4, β-catenin (CTNNB1), C-MYC and Cyclin D1 (CYCD1) was assessed by qRT PCR. Prior to fold--change calculation, the values were normalized to signal generated from endogenous control 18srRNA. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01 compared to control siRNA. (B and C) TNBC cells were grown on chamber slides and treated with B. vehicle control or C. 0.1 μM Baf A1 for 4 hours. Cells were fixed, permeabilized and processed for immunofluorescence microscopy. Localization of β-catenin (green) is shown. Nucleus was stained with DAPI (blue). Scale bars: 10 μm.

    Journal: Oncotarget

    Article Title: The Vacuolar ATPase a2-subunit regulates Notch signaling in triple-negative breast cancer cells

    doi:

    Figure Lengend Snippet: a2V-ATPase inhibition enhances Wnt signaling in TNBC A. MDA-MB-231 cells were transfected with scrambled control or a2V siRNA and harvested after 48 hrs of transfection. Fold change in mRNA expression levels of Wnt signaling genes WNT4, β-catenin (CTNNB1), C-MYC and Cyclin D1 (CYCD1) was assessed by qRT PCR. Prior to fold--change calculation, the values were normalized to signal generated from endogenous control 18srRNA. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01 compared to control siRNA. (B and C) TNBC cells were grown on chamber slides and treated with B. vehicle control or C. 0.1 μM Baf A1 for 4 hours. Cells were fixed, permeabilized and processed for immunofluorescence microscopy. Localization of β-catenin (green) is shown. Nucleus was stained with DAPI (blue). Scale bars: 10 μm.

    Article Snippet: Cells were transfected with siRNA (final concentration, 10 nM) using lipid-mediated transfection with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

    Techniques: Inhibition, Multiple Displacement Amplification, Transfection, Expressing, Quantitative RT-PCR, Generated, Immunofluorescence, Microscopy, Staining

    hSpry2 degradation is impaired in AR-treated cells HEK-293 cells were transiently transfected with cDNA encoding EGF-R (0.05 μ g), GFP–Cbl WT (4 μ g) and FLAG–hSprouty2 (2 μ g). At 48 h post-transfection, cells were serum-starved and incubated without (0 min) or with AR (136 nM) or EGF (17 nM) for 2, 5, 10, 40 or 90 min. ( A ) Immunoblotting results from a representative experiment. A 100 μ g cell lysate protein sample was loaded per lane, gel-resolved, transferred on to PVDF and immunoblotted as indicated. ( B ) Quantitative analysis of three independent degradation experiments, showing the average amount of total cellular hSprouty2 remaining after 90 min of receptor stimulation by ligand. Results are means ± S.D. A two-tailed Student’s t test with α =0.05 determined that a significantly different amount of hSprouty2 degradation was effected by AR compared with EGF (**).

    Journal: The Biochemical journal

    Article Title: EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

    doi: 10.1042/BJ20071505

    Figure Lengend Snippet: hSpry2 degradation is impaired in AR-treated cells HEK-293 cells were transiently transfected with cDNA encoding EGF-R (0.05 μ g), GFP–Cbl WT (4 μ g) and FLAG–hSprouty2 (2 μ g). At 48 h post-transfection, cells were serum-starved and incubated without (0 min) or with AR (136 nM) or EGF (17 nM) for 2, 5, 10, 40 or 90 min. ( A ) Immunoblotting results from a representative experiment. A 100 μ g cell lysate protein sample was loaded per lane, gel-resolved, transferred on to PVDF and immunoblotted as indicated. ( B ) Quantitative analysis of three independent degradation experiments, showing the average amount of total cellular hSprouty2 remaining after 90 min of receptor stimulation by ligand. Results are means ± S.D. A two-tailed Student’s t test with α =0.05 determined that a significantly different amount of hSprouty2 degradation was effected by AR compared with EGF (**).

    Article Snippet: COS-7 cells grown to 80–90 % confluence on 100-mm tissue culture dishes received lipid-mediated transfection reagent (Lipofectamine™ 2000; Invitrogen).

    Techniques: Transfection, Incubation, Two Tailed Test

    AR is a partial agonist for site-specific EGF-R tyrosine phosphorylation HEK-293 cells were transiently transfected with cDNA encoding EGF-R (0.05 μ g) and GFP–Cbl WT (4 μ g) or GFP (3 μ g). At 48 h post-transfection, cells were serum-starved and incubated without (0) or with EGF (17 nM) or AR (17–136 nM) for 10 min. Cell lysate proteins (100 μ g per lane) were gel-resolved, transferred on to PVDF membrane and immunoblotted with the indicated antibodies. ( A ) Immunoblotting results from a representative experiment. ( B , C ) Quantitative analysis of phospho-Tyr 845 and phospho-Tyr 1045 levels in treated cells. In each case, the phosphotyrosine signal was normalized to the corresponding EGF-R level. Results are means ± S.D. for three independent experiments, except in ( C ), ● (two independent experiments).

    Journal: The Biochemical journal

    Article Title: EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

    doi: 10.1042/BJ20071505

    Figure Lengend Snippet: AR is a partial agonist for site-specific EGF-R tyrosine phosphorylation HEK-293 cells were transiently transfected with cDNA encoding EGF-R (0.05 μ g) and GFP–Cbl WT (4 μ g) or GFP (3 μ g). At 48 h post-transfection, cells were serum-starved and incubated without (0) or with EGF (17 nM) or AR (17–136 nM) for 10 min. Cell lysate proteins (100 μ g per lane) were gel-resolved, transferred on to PVDF membrane and immunoblotted with the indicated antibodies. ( A ) Immunoblotting results from a representative experiment. ( B , C ) Quantitative analysis of phospho-Tyr 845 and phospho-Tyr 1045 levels in treated cells. In each case, the phosphotyrosine signal was normalized to the corresponding EGF-R level. Results are means ± S.D. for three independent experiments, except in ( C ), ● (two independent experiments).

    Article Snippet: COS-7 cells grown to 80–90 % confluence on 100-mm tissue culture dishes received lipid-mediated transfection reagent (Lipofectamine™ 2000; Invitrogen).

    Techniques: Transfection, Incubation

    Relative to EGF treatment, AR treatment impairs EGFR down-regulation and degradation HEK-293 cells were transiently transfected with cDNA encoding EGF-R WT (0.05 μ g) and GFP (3.2 μ g), GFP–Cbl WT (4 μ g) or GFP–Cbl Y371F (4 μ g). ( A ) Down-regulation assay. At 48 h post-transfection, the cells were serum-starved and incubated at 37 °C without (0 min) or with EGF (17 nM) or AR (136 nM) for 10, 40 or 90 min. Cells were harvested intact on ice, plated in triplicate and stained with anti-EGF-R, anti-Syk (isotype-matched negative control) or anti-MHC Class I (isotype-matched positive control) antibodies. This was followed by incubation with phycoerythrin-conjugated secondary antibody and paraformaldehyde fixation. The surface phycoerythrin signals were analysed by flow cytometry for 5000 GFP-positive cells per sample. Surface receptor levels for each sample were determined by subtracting the mean fluorescence index of the anti-Syk-stained cells from the MFI of the matched anti-EGF-R-stained cells. For each transfection condition, the surface EGFR levels are expressed as a percentage of the EGF-R signal of unstimulated, matched transfection plates. Results are means ± S.D. for three independent experiments. ( B ) EGF-R degradation assay. A 100 μ g cell lysate protein sample was loaded per lane, gel-resolved, transferred on to PVDF and immunoblotted as indicated. Results shown are from a representative experiment employing 17 nM EGF and 136 nM AR. ( C ) Quantitative analysis of three independent degradation experiments, showing the average amount of total cellular EGF-R remaining after 90 min of receptor stimulation by ligand. Results are means ± S.D.

    Journal: The Biochemical journal

    Article Title: EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

    doi: 10.1042/BJ20071505

    Figure Lengend Snippet: Relative to EGF treatment, AR treatment impairs EGFR down-regulation and degradation HEK-293 cells were transiently transfected with cDNA encoding EGF-R WT (0.05 μ g) and GFP (3.2 μ g), GFP–Cbl WT (4 μ g) or GFP–Cbl Y371F (4 μ g). ( A ) Down-regulation assay. At 48 h post-transfection, the cells were serum-starved and incubated at 37 °C without (0 min) or with EGF (17 nM) or AR (136 nM) for 10, 40 or 90 min. Cells were harvested intact on ice, plated in triplicate and stained with anti-EGF-R, anti-Syk (isotype-matched negative control) or anti-MHC Class I (isotype-matched positive control) antibodies. This was followed by incubation with phycoerythrin-conjugated secondary antibody and paraformaldehyde fixation. The surface phycoerythrin signals were analysed by flow cytometry for 5000 GFP-positive cells per sample. Surface receptor levels for each sample were determined by subtracting the mean fluorescence index of the anti-Syk-stained cells from the MFI of the matched anti-EGF-R-stained cells. For each transfection condition, the surface EGFR levels are expressed as a percentage of the EGF-R signal of unstimulated, matched transfection plates. Results are means ± S.D. for three independent experiments. ( B ) EGF-R degradation assay. A 100 μ g cell lysate protein sample was loaded per lane, gel-resolved, transferred on to PVDF and immunoblotted as indicated. Results shown are from a representative experiment employing 17 nM EGF and 136 nM AR. ( C ) Quantitative analysis of three independent degradation experiments, showing the average amount of total cellular EGF-R remaining after 90 min of receptor stimulation by ligand. Results are means ± S.D.

    Article Snippet: COS-7 cells grown to 80–90 % confluence on 100-mm tissue culture dishes received lipid-mediated transfection reagent (Lipofectamine™ 2000; Invitrogen).

    Techniques: Transfection, Incubation, Staining, Negative Control, Positive Control, Flow Cytometry, Cytometry, Fluorescence, Degradation Assay

    AR treatment leads to the formation of EGF-R-positive vesicles that lack associated GFP–Cbl COS-7 cells were transiently transfected with cDNA encoding GFP–Cbl WT (4 μ g). At 48 h post-transfection, cells were serum-starved and incubated without ligand (0 min) or with AR (136 nM) or EGF (17 nM) for 5 or 25 min. Following stimulation, the cells were paraformaldehyde-fixed, permeabilized and stained with anti-EGF-R antibody. Scale bar, 20 μ m. Images shown are representative results from one experiment. Three independent experiments were performed, with evaluation of more than 20 cells per ligand condition per experiment.

    Journal: The Biochemical journal

    Article Title: EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

    doi: 10.1042/BJ20071505

    Figure Lengend Snippet: AR treatment leads to the formation of EGF-R-positive vesicles that lack associated GFP–Cbl COS-7 cells were transiently transfected with cDNA encoding GFP–Cbl WT (4 μ g). At 48 h post-transfection, cells were serum-starved and incubated without ligand (0 min) or with AR (136 nM) or EGF (17 nM) for 5 or 25 min. Following stimulation, the cells were paraformaldehyde-fixed, permeabilized and stained with anti-EGF-R antibody. Scale bar, 20 μ m. Images shown are representative results from one experiment. Three independent experiments were performed, with evaluation of more than 20 cells per ligand condition per experiment.

    Article Snippet: COS-7 cells grown to 80–90 % confluence on 100-mm tissue culture dishes received lipid-mediated transfection reagent (Lipofectamine™ 2000; Invitrogen).

    Techniques: Transfection, Incubation, Staining

    Equimolar concentrations of AR and EGF induce different levels of EGF-R ubiquitination, phosphorylation and degradation HEK-293 cells were transiently transfected with cDNA encoding EGF-R (0.05 μ g) and either GFP (3 μ g) or GFP–Cbl WT (4 μ g). At 48 h post-transfection, cells were serum-starved and incubated without (0 nM) or with AR (17 nM), BTC (8.5 nM) or EGF (17 nM) for 10, 40 or 90 min. Cell lysate proteins and immunoprecipitates (IP) were gel-resolved, transferred on to PVDF and immunoblotted (IB) with the antibodies indicated. ( A ) Immunoblotting results from a representative experiment. Top arrow: ubiquitinated EGF-R; middle arrow, non-ubiquitinated EGF-R; bottom arrow, GFP–Cbl; *, phosphorylated Hrs, a marker for EGF-R complex trafficking to early endosomes. ( B ) Quantitative analysis of three independent experiments, showing the amount of total cellular EGF-R remaining after receptor stimulation by the ligands (Amphi, amphiregulin). Results are means ± S.D. A two-tailed Student’s t test with α =0.05 determined that GFP–Cbl expression significantly affected EGF-R degradation only in the case of receptors activated by EGF (**).

    Journal: The Biochemical journal

    Article Title: EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

    doi: 10.1042/BJ20071505

    Figure Lengend Snippet: Equimolar concentrations of AR and EGF induce different levels of EGF-R ubiquitination, phosphorylation and degradation HEK-293 cells were transiently transfected with cDNA encoding EGF-R (0.05 μ g) and either GFP (3 μ g) or GFP–Cbl WT (4 μ g). At 48 h post-transfection, cells were serum-starved and incubated without (0 nM) or with AR (17 nM), BTC (8.5 nM) or EGF (17 nM) for 10, 40 or 90 min. Cell lysate proteins and immunoprecipitates (IP) were gel-resolved, transferred on to PVDF and immunoblotted (IB) with the antibodies indicated. ( A ) Immunoblotting results from a representative experiment. Top arrow: ubiquitinated EGF-R; middle arrow, non-ubiquitinated EGF-R; bottom arrow, GFP–Cbl; *, phosphorylated Hrs, a marker for EGF-R complex trafficking to early endosomes. ( B ) Quantitative analysis of three independent experiments, showing the amount of total cellular EGF-R remaining after receptor stimulation by the ligands (Amphi, amphiregulin). Results are means ± S.D. A two-tailed Student’s t test with α =0.05 determined that GFP–Cbl expression significantly affected EGF-R degradation only in the case of receptors activated by EGF (**).

    Article Snippet: COS-7 cells grown to 80–90 % confluence on 100-mm tissue culture dishes received lipid-mediated transfection reagent (Lipofectamine™ 2000; Invitrogen).

    Techniques: Transfection, Incubation, Marker, Two Tailed Test, Expressing

    (a) Overexpression of ADARs by cotransfection of either an ADAR1 (pSS151) or ADAR2 (pSKW005) plasmid with pNL4-3 enhanced HIV-1 p24 production, as shown by p24 ELISA of the culture supernatant of transfected COS-7 cells. The transfection efficiencies,

    Journal:

    Article Title: Double-Stranded RNA Adenosine Deaminases Enhance Expression of Human Immunodeficiency Virus Type 1 Proteins ▿

    doi: 10.1128/JVI.00238-08

    Figure Lengend Snippet: (a) Overexpression of ADARs by cotransfection of either an ADAR1 (pSS151) or ADAR2 (pSKW005) plasmid with pNL4-3 enhanced HIV-1 p24 production, as shown by p24 ELISA of the culture supernatant of transfected COS-7 cells. The transfection efficiencies,

    Article Snippet: pNL4-3 (1.5 μg) was cotransfected with 0.5 μg of either pSS43, pSS151, or pSKW005 into the appropriate cell line (2 × 105 cells of COS-7 or 293T) by using a cationic lipid reagent-mediated transfection technique (DMRIE-C reagent; Invitrogen).

    Techniques: Over Expression, Cotransfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Transfection

    Differential expression is abrogated with transfection, time in culture, and age of DNA. Represents the fold difference in transcriptional activity after normalizing luciferase RLUs/ to −308G/GL3Luc, which was set at 1. ( a ) Jurkat cells were transiently

    Journal: European Journal of Human Genetics

    Article Title: A critical assessment of the factors affecting reporter gene assays for promoter SNP function: a reassessment of -308 TNF polymorphism function using a novel integrated reporter system

    doi: 10.1038/ejhg.2009.80

    Figure Lengend Snippet: Differential expression is abrogated with transfection, time in culture, and age of DNA. Represents the fold difference in transcriptional activity after normalizing luciferase RLUs/ to −308G/GL3Luc, which was set at 1. ( a ) Jurkat cells were transiently

    Article Snippet: Mycoplasma-free Jurkat E6-1 cell lines were transiently transfected using either electroporation or when required, the lipid-mediated transfection protocol (Lipofectamine 2000, Invitrogen, Carlsbad, CA, USA).

    Techniques: Expressing, Transfection, Activity Assay, Luciferase

    Effects of co-transfection with pRL-TK control plasmid. Represents the fold difference in transcriptional activity of −308A/GL3Luc and −308G/GL3Luc after normalizing RLU/s to −308G/GL3Luc only, which was set at 1. Jurkat cells

    Journal: European Journal of Human Genetics

    Article Title: A critical assessment of the factors affecting reporter gene assays for promoter SNP function: a reassessment of -308 TNF polymorphism function using a novel integrated reporter system

    doi: 10.1038/ejhg.2009.80

    Figure Lengend Snippet: Effects of co-transfection with pRL-TK control plasmid. Represents the fold difference in transcriptional activity of −308A/GL3Luc and −308G/GL3Luc after normalizing RLU/s to −308G/GL3Luc only, which was set at 1. Jurkat cells

    Article Snippet: Mycoplasma-free Jurkat E6-1 cell lines were transiently transfected using either electroporation or when required, the lipid-mediated transfection protocol (Lipofectamine 2000, Invitrogen, Carlsbad, CA, USA).

    Techniques: Cotransfection, Plasmid Preparation, Activity Assay

    Cas9 editing efficiencies at target sites in unsilenced, partially silenced, and fully silenced chromatin states. (a) A map of the Cas9/sgRNA-expressing plasmid. Cas9 and EGFP expression are both driven by the CBh promoter. The T2A signal allows EGFP to be translated as a separate peptide to avoid interference of Cas9 function. (b) Table of average frequencies of EGFP-expressing cells (transfection efficiencies) as determined by flow cytometry of triplicate samples for transfected Luc14, GAL4EED, and GAL4EED cells treated with doxycycline. (c) Mean editing frequencies normalized to transfection efficiency in Luc14, GAL4EED, or GAL4EED + dox cells for Cas9 targeted to sites sg046, sg055, sg032, sg034, sg054, sg031, sg025, sg044, and sg048. *Indicates significantly reduced editing efficiencies at fully silenced chromatin compared to unsilenced chromatin ( p

    Journal: ACS synthetic biology

    Article Title: The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells

    doi: 10.1021/acssynbio.5b00299

    Figure Lengend Snippet: Cas9 editing efficiencies at target sites in unsilenced, partially silenced, and fully silenced chromatin states. (a) A map of the Cas9/sgRNA-expressing plasmid. Cas9 and EGFP expression are both driven by the CBh promoter. The T2A signal allows EGFP to be translated as a separate peptide to avoid interference of Cas9 function. (b) Table of average frequencies of EGFP-expressing cells (transfection efficiencies) as determined by flow cytometry of triplicate samples for transfected Luc14, GAL4EED, and GAL4EED cells treated with doxycycline. (c) Mean editing frequencies normalized to transfection efficiency in Luc14, GAL4EED, or GAL4EED + dox cells for Cas9 targeted to sites sg046, sg055, sg032, sg034, sg054, sg031, sg025, sg044, and sg048. *Indicates significantly reduced editing efficiencies at fully silenced chromatin compared to unsilenced chromatin ( p

    Article Snippet: The remaining wells were used for lipid-mediated transfection with 2.5 µL of 20 µM anti-SUZ12 siRNA duplex (Dharmacon)/well and 1.5 µL Oligofectamine (Life Technologies) per manufacturer’s protocol. siRNA sequence used was as follows: Sense: 5′ A.A.G.C.U.G.U.U.A.C.C.A.A.G.C.U.C.C.G.U.-G.U.U 3′; Antisense: 5′ C.A.C.G.G.A.G.C.U.U.G.G.U.A.A.C.A.G.-C.U.U.U.U 3′.

    Techniques: Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Cytometry

    Long-RNA transfection yields ES-cell-level expression of reprogramming proteins in primary human fibroblasts. A. The transcribed strand of an Hbb -UTR-stabilized in vitro-transcription template encoding an arbitrary protein. The long arrow indicates the first transcribed base, and short arrows indicate restriction-enzyme cleavage sites. B. In vitro-transcribed RNA encoding reprogramming proteins. C. Western blots showing expression levels and lifetimes of Oct4, Sox2, Nanog, Lin28, and MyoD1 proteins in MRC-5 human fetal lung fibroblasts transfected with protein-encoding RNA, relative to levels in hES (H9) and rhabdomyosarcoma (Rh30) cells. β-actin was used as a loading control. Left panels: The amount of RNA per 50 µL electroporation volume was varied as indicated. Cells were lysed 6 hours after transfection. Right panels: Cells were transfected with 1 µg of RNA, and lysed at the indicated times. D. Expression and nuclear localization of Oct4, Sox2, Klf4, Utf1, Nanog, Lin28, and MyoD1 protein following long-RNA transfection. Cells were fixed and stained 6–12 hours after transfection. For each protein, identical camera settings and exposure times were used for the RNA-transfected and mock-transfected samples.

    Journal: PLoS ONE

    Article Title: Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins

    doi: 10.1371/journal.pone.0011756

    Figure Lengend Snippet: Long-RNA transfection yields ES-cell-level expression of reprogramming proteins in primary human fibroblasts. A. The transcribed strand of an Hbb -UTR-stabilized in vitro-transcription template encoding an arbitrary protein. The long arrow indicates the first transcribed base, and short arrows indicate restriction-enzyme cleavage sites. B. In vitro-transcribed RNA encoding reprogramming proteins. C. Western blots showing expression levels and lifetimes of Oct4, Sox2, Nanog, Lin28, and MyoD1 proteins in MRC-5 human fetal lung fibroblasts transfected with protein-encoding RNA, relative to levels in hES (H9) and rhabdomyosarcoma (Rh30) cells. β-actin was used as a loading control. Left panels: The amount of RNA per 50 µL electroporation volume was varied as indicated. Cells were lysed 6 hours after transfection. Right panels: Cells were transfected with 1 µg of RNA, and lysed at the indicated times. D. Expression and nuclear localization of Oct4, Sox2, Klf4, Utf1, Nanog, Lin28, and MyoD1 protein following long-RNA transfection. Cells were fixed and stained 6–12 hours after transfection. For each protein, identical camera settings and exposure times were used for the RNA-transfected and mock-transfected samples.

    Article Snippet: Long-RNA Transfection Lipid-mediated transfections (TransIT-mRNA, Mirus) were performed according to the manufacturer's instructions.

    Techniques: Transfection, Expressing, In Vitro, Western Blot, Electroporation, Staining

    Innate immune suppression enables frequent long-RNA transfection. Combinatorial siRNA screening identifies siRNA cocktails that rescue cells from the innate immune response triggered by long-RNA transfection. A. Upregulation of innate immune genes following long-RNA transfection. MRC-5 fibroblasts were transfected with 0.4 µg of RNA per well of a 24-well plate using lipids. Expression of innate immune genes was measured by quantitative RT-PCR 24 hours after transfection. Gapdh was used as a loading control. Error bars indicate the standard deviation of replicate samples. B. Repeated long-RNA transfection causes cell death in human fibroblasts. MRC-5 fibroblasts were electroporated twice with 0.5 µg/50 µL of Lin28-encoding RNA at 48-hour intervals. Samples of cells transfected with RNA (black circles) and mock-transfected cells (gray squares) were trypsinized and counted at the indicated times. Data points and error bars indicate the mean and standard error of two independent experiments. Data points are connected for clarity. C. Combined knockdown of Ifnb1 , Eif2ak2 , and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. MRC-5 fibroblasts were transfected as in (B), but with the indicated siRNA on day 0, and 0.5 µg of Lin28-encoding RNA and additional siRNA on days 2 and 4 ( Table S1 ). Samples of cells were trypsinized and counted 24 hours after the second long-RNA transfection (day 5). Values indicate cell count relative to mock-transfected cells. † Standard error of replicate samples (n = 4). *p

    Journal: PLoS ONE

    Article Title: Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins

    doi: 10.1371/journal.pone.0011756

    Figure Lengend Snippet: Innate immune suppression enables frequent long-RNA transfection. Combinatorial siRNA screening identifies siRNA cocktails that rescue cells from the innate immune response triggered by long-RNA transfection. A. Upregulation of innate immune genes following long-RNA transfection. MRC-5 fibroblasts were transfected with 0.4 µg of RNA per well of a 24-well plate using lipids. Expression of innate immune genes was measured by quantitative RT-PCR 24 hours after transfection. Gapdh was used as a loading control. Error bars indicate the standard deviation of replicate samples. B. Repeated long-RNA transfection causes cell death in human fibroblasts. MRC-5 fibroblasts were electroporated twice with 0.5 µg/50 µL of Lin28-encoding RNA at 48-hour intervals. Samples of cells transfected with RNA (black circles) and mock-transfected cells (gray squares) were trypsinized and counted at the indicated times. Data points and error bars indicate the mean and standard error of two independent experiments. Data points are connected for clarity. C. Combined knockdown of Ifnb1 , Eif2ak2 , and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. MRC-5 fibroblasts were transfected as in (B), but with the indicated siRNA on day 0, and 0.5 µg of Lin28-encoding RNA and additional siRNA on days 2 and 4 ( Table S1 ). Samples of cells were trypsinized and counted 24 hours after the second long-RNA transfection (day 5). Values indicate cell count relative to mock-transfected cells. † Standard error of replicate samples (n = 4). *p

    Article Snippet: Long-RNA Transfection Lipid-mediated transfections (TransIT-mRNA, Mirus) were performed according to the manufacturer's instructions.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation, Cell Counting

    Repeated long-RNA transfection yields sustained, high-level expression of active proteins that modulate downstream targets. A. Sustaining high levels of Lin28 protein expression by frequent transfection with Lin28-encoding RNA. MRC-5 fibroblasts pre-transfected with a cocktail of siRNAs targeting Ifnb1 , Eif2ak2 , Stat2 , and Tlr3 were transfected five times with 0.5 µg of Lin28-encoding RNA and additional siRNA at 48-hour intervals. Cells were lysed at the indicated times, and the amount of Lin28 protein was analyzed by western blot. β-actin was used as a loading control. B. Sustained expression of Lin28 downregulates its target, mature let7 miRNA. Transfections were conducted as in (A). Data points indicate mature let7a levels in cells transfected once (circles), twice (squares), three times (diamonds), four times (triangles), or five times (crosses), relative to the level in mock-transfected cells. A solid smoothed line connects data points corresponding to cells transfected once, and a dashed smoothed line connects data points corresponding to cells transfected five times (dark symbols). U47 RNA was used as a loading control. Error bars indicate the standard error of replicate samples. C. let7a downregulation is Lin28-specific. Cells were transfected as in (A), but with MyoD1-encoding RNA. Error bars indicate the standard error of replicate samples. D. Expression of MyoD1 protein in fibroblasts. Fibroblasts cultured for three days with or without 2.5 µM 5-aza-dC (AZA) were electroporated with 1 µg/50 µL of MyoD1-encoding RNA. Cells were lysed at the indicated times, and the amount of MyoD1 protein in each sample was analyzed by western blot. E. Expression of MyoD1 in fibroblasts activates its normally silent targets, Cdh15 and Des in a methylation-dependent manner. Cells were transfected as in (D), and expression of Cdh15 and Des was measured by RT-PCR at the indicated times (squares, mock-transfected cells; circles, RNA-transfected cells). F. Regulation of Hmga2 expression by reprogramming proteins. Hmga2 expression in fibroblasts transfected with RNA encoding the indicated protein was measured by RT-PCR 24 hours after transfection. Values are given relative to mock-transfected cells. Gapdh was used as a loading control. *p

    Journal: PLoS ONE

    Article Title: Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins

    doi: 10.1371/journal.pone.0011756

    Figure Lengend Snippet: Repeated long-RNA transfection yields sustained, high-level expression of active proteins that modulate downstream targets. A. Sustaining high levels of Lin28 protein expression by frequent transfection with Lin28-encoding RNA. MRC-5 fibroblasts pre-transfected with a cocktail of siRNAs targeting Ifnb1 , Eif2ak2 , Stat2 , and Tlr3 were transfected five times with 0.5 µg of Lin28-encoding RNA and additional siRNA at 48-hour intervals. Cells were lysed at the indicated times, and the amount of Lin28 protein was analyzed by western blot. β-actin was used as a loading control. B. Sustained expression of Lin28 downregulates its target, mature let7 miRNA. Transfections were conducted as in (A). Data points indicate mature let7a levels in cells transfected once (circles), twice (squares), three times (diamonds), four times (triangles), or five times (crosses), relative to the level in mock-transfected cells. A solid smoothed line connects data points corresponding to cells transfected once, and a dashed smoothed line connects data points corresponding to cells transfected five times (dark symbols). U47 RNA was used as a loading control. Error bars indicate the standard error of replicate samples. C. let7a downregulation is Lin28-specific. Cells were transfected as in (A), but with MyoD1-encoding RNA. Error bars indicate the standard error of replicate samples. D. Expression of MyoD1 protein in fibroblasts. Fibroblasts cultured for three days with or without 2.5 µM 5-aza-dC (AZA) were electroporated with 1 µg/50 µL of MyoD1-encoding RNA. Cells were lysed at the indicated times, and the amount of MyoD1 protein in each sample was analyzed by western blot. E. Expression of MyoD1 in fibroblasts activates its normally silent targets, Cdh15 and Des in a methylation-dependent manner. Cells were transfected as in (D), and expression of Cdh15 and Des was measured by RT-PCR at the indicated times (squares, mock-transfected cells; circles, RNA-transfected cells). F. Regulation of Hmga2 expression by reprogramming proteins. Hmga2 expression in fibroblasts transfected with RNA encoding the indicated protein was measured by RT-PCR 24 hours after transfection. Values are given relative to mock-transfected cells. Gapdh was used as a loading control. *p

    Article Snippet: Long-RNA Transfection Lipid-mediated transfections (TransIT-mRNA, Mirus) were performed according to the manufacturer's instructions.

    Techniques: Transfection, Expressing, Western Blot, Cell Culture, Methylation, Reverse Transcription Polymerase Chain Reaction