lipid transfections Search Results


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  • 99
    Thermo Fisher cationic lipid lipofectamine 2000
    Viral dsRNA complexed with cationic lipids activates mesangial cells to produce high amounts of Il-6 and type I IFN via a Trif-independent pathway. Primary mesangial cells were isolated from wild-type C56BL/6 mice and Trif -mutant mice as described in Materials and Methods . Cells were stimulated with increasing doses of poly I:C (pI:C) RNA alone or complexed with the cationic lipid (CL) <t>Lipofectamine.</t> Supernatants were harvested after 24 hours and analyzed by ELISA for the following mediators: Il-6 ( A ), IFN-α ( B ), and IFN-β ( C ). Data are means ± SEM from three experiments each analyzed in duplicate and presented in a logarithmic scale. ∗ P
    Cationic Lipid Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher siport lipid transfection agent
    Viral dsRNA complexed with cationic lipids activates mesangial cells to produce high amounts of Il-6 and type I IFN via a Trif-independent pathway. Primary mesangial cells were isolated from wild-type C56BL/6 mice and Trif -mutant mice as described in Materials and Methods . Cells were stimulated with increasing doses of poly I:C (pI:C) RNA alone or complexed with the cationic lipid (CL) <t>Lipofectamine.</t> Supernatants were harvested after 24 hours and analyzed by ELISA for the following mediators: Il-6 ( A ), IFN-α ( B ), and IFN-β ( C ). Data are means ± SEM from three experiments each analyzed in duplicate and presented in a logarithmic scale. ∗ P
    Siport Lipid Transfection Agent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipofectamine rnaimax lipid 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.
    Lipofectamine Rnaimax Lipid Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher rnai max lipid 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.
    Rnai Max Lipid Transfection Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher block it fluorescent oligo for lipid 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.
    Block It Fluorescent Oligo For Lipid Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    SignaGen genjet lipid transfection reagents
    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.
    Genjet Lipid Transfection Reagents, supplied by SignaGen, used in various techniques. Bioz Stars score: 85/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad silentfect lipid 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.
    Silentfect Lipid Transfection Reagent, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega transfast lipid 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.
    Transfast Lipid Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Mirus Bio lipid based transfection reagent
    RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with <t>transfection</t> vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.
    Lipid Based Transfection Reagent, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    lipid based transfection reagent - by Bioz Stars, 2020-09
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    99
    Thermo Fisher oligofectamine lipid transfection reagent
    RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with <t>transfection</t> vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.
    Oligofectamine Lipid Transfection Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Viral dsRNA complexed with cationic lipids activates mesangial cells to produce high amounts of Il-6 and type I IFN via a Trif-independent pathway. Primary mesangial cells were isolated from wild-type C56BL/6 mice and Trif -mutant mice as described in Materials and Methods . Cells were stimulated with increasing doses of poly I:C (pI:C) RNA alone or complexed with the cationic lipid (CL) Lipofectamine. Supernatants were harvested after 24 hours and analyzed by ELISA for the following mediators: Il-6 ( A ), IFN-α ( B ), and IFN-β ( C ). Data are means ± SEM from three experiments each analyzed in duplicate and presented in a logarithmic scale. ∗ P

    Journal: The American Journal of Pathology

    Article Title: Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5

    doi: 10.2353/ajpath.2009.080585

    Figure Lengend Snippet: Viral dsRNA complexed with cationic lipids activates mesangial cells to produce high amounts of Il-6 and type I IFN via a Trif-independent pathway. Primary mesangial cells were isolated from wild-type C56BL/6 mice and Trif -mutant mice as described in Materials and Methods . Cells were stimulated with increasing doses of poly I:C (pI:C) RNA alone or complexed with the cationic lipid (CL) Lipofectamine. Supernatants were harvested after 24 hours and analyzed by ELISA for the following mediators: Il-6 ( A ), IFN-α ( B ), and IFN-β ( C ). Data are means ± SEM from three experiments each analyzed in duplicate and presented in a logarithmic scale. ∗ P

    Article Snippet: Primary mesangial cells were stimulated with endotoxin-free poly I:C RNA (InvivoGen, Toulouse, France), poly I:C RNA transfected with the cationic lipid Lipofectamine 2000 (Invitrogen, Carlsbad, CA) or ultrapure LPS (InvivoGen) for 24 hours in RPMI 1640 containing 5% FCS in the presence or absence of murine IFN-α (AbD Serotec, Oxford, UK), IFN-β (PBL, Piscataway, NJ), or IFN-γ (PeproTech, Rocky Hill, NJ).

    Techniques: Isolation, Mouse Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay

    Activation of FXN expression and comparison to wild-type cells. ( A ) Time course showing activation of FXN mRNA expression starting 48 h post-transfection in FRDA patient-derived NPCs F4259 (5 µM, n = 2, Optimization 4). ( B ) Expression levels of FXN mRNA in FRDA patient-derived NPCs F4259 and wild-type NPCs C7522 ( n = 4). ( C ) Activation in FRDA patient-derived NPCs F4259. ( D ) Effect of adding anti-AAG ASOs to wild-type NPCs C7522. Relative FXN mRNA levels were measured by RT-qPCR when transfected with oligonucleotides using Optimization 4 ( n = 4) post 72 h transfection. EP(−) is non-oligo treatment, no electroporation control. All data are presented as ±STDEV. (*) P

    Journal: RNA

    Article Title: Efficient electroporation of neuronal cells using synthetic oligonucleotides: identifying duplex RNA and antisense oligonucleotide activators of human frataxin expression

    doi: 10.1261/rna.071290.119

    Figure Lengend Snippet: Activation of FXN expression and comparison to wild-type cells. ( A ) Time course showing activation of FXN mRNA expression starting 48 h post-transfection in FRDA patient-derived NPCs F4259 (5 µM, n = 2, Optimization 4). ( B ) Expression levels of FXN mRNA in FRDA patient-derived NPCs F4259 and wild-type NPCs C7522 ( n = 4). ( C ) Activation in FRDA patient-derived NPCs F4259. ( D ) Effect of adding anti-AAG ASOs to wild-type NPCs C7522. Relative FXN mRNA levels were measured by RT-qPCR when transfected with oligonucleotides using Optimization 4 ( n = 4) post 72 h transfection. EP(−) is non-oligo treatment, no electroporation control. All data are presented as ±STDEV. (*) P

    Article Snippet: Lipid transfections were performed using Lipofectamine stem transfection reagent (Invitrogen) following reported protocols ( ; ).

    Techniques: Activation Assay, Expressing, Transfection, Derivative Assay, Quantitative RT-PCR, Electroporation

    Identifying an electroporation protocol using iXCells human motor neurons (HMNs). ( A, left ) Relative MALAT-1 RNA levels measured by q RT-PCR when transfected with control gapmer ctrl-gap (1 µM) and anti-MALAT1 gapmer anti-MALAT1 using Optimization 4 and 6 ( n = 2) post 24 h transfection. ( Right ) Cell viability post 24 h transfection measured by trypan blue staining ( n = 2) using 1× trypsin as detachment reagent. ( B, left ) Relative MALAT1 RNA levels measured by q RT-PCR when transfected with control gapmer ctrl-gap (1 µM) and anti-MALAT1 gapmer anti-MALAT1 using Optimization 4 ( n = 2) compared with Lipofectamine stem transfection reagent or gymnotic delivery post 24 h transfection. ( Right ) Cell viability post 24 h transfection measured by trypan blue staining ( n = 2) using accutase + 10 µM Y27632. EP(−) is non-oligo treatment, no electroporation control. All data are presented as ±STDEV.

    Journal: RNA

    Article Title: Efficient electroporation of neuronal cells using synthetic oligonucleotides: identifying duplex RNA and antisense oligonucleotide activators of human frataxin expression

    doi: 10.1261/rna.071290.119

    Figure Lengend Snippet: Identifying an electroporation protocol using iXCells human motor neurons (HMNs). ( A, left ) Relative MALAT-1 RNA levels measured by q RT-PCR when transfected with control gapmer ctrl-gap (1 µM) and anti-MALAT1 gapmer anti-MALAT1 using Optimization 4 and 6 ( n = 2) post 24 h transfection. ( Right ) Cell viability post 24 h transfection measured by trypan blue staining ( n = 2) using 1× trypsin as detachment reagent. ( B, left ) Relative MALAT1 RNA levels measured by q RT-PCR when transfected with control gapmer ctrl-gap (1 µM) and anti-MALAT1 gapmer anti-MALAT1 using Optimization 4 ( n = 2) compared with Lipofectamine stem transfection reagent or gymnotic delivery post 24 h transfection. ( Right ) Cell viability post 24 h transfection measured by trypan blue staining ( n = 2) using accutase + 10 µM Y27632. EP(−) is non-oligo treatment, no electroporation control. All data are presented as ±STDEV.

    Article Snippet: Lipid transfections were performed using Lipofectamine stem transfection reagent (Invitrogen) following reported protocols ( ; ).

    Techniques: Electroporation, Reverse Transcription Polymerase Chain Reaction, Transfection, Staining

    Activation of FXN protein expression, western analysis. ( A ) FXN protein in FRDA patient-derived NPCs F4259 and wild-type NPCs C7522 ( n = 3) with different confluency. ( B ) FXN protein expression in FRDA patient-derived NPCs F4259 when transfected with oligonucleotides (5 µM) using Optimization 4 ( n = 3) post 96 h transfection. All data are presented as ±STDEV. (*) P

    Journal: RNA

    Article Title: Efficient electroporation of neuronal cells using synthetic oligonucleotides: identifying duplex RNA and antisense oligonucleotide activators of human frataxin expression

    doi: 10.1261/rna.071290.119

    Figure Lengend Snippet: Activation of FXN protein expression, western analysis. ( A ) FXN protein in FRDA patient-derived NPCs F4259 and wild-type NPCs C7522 ( n = 3) with different confluency. ( B ) FXN protein expression in FRDA patient-derived NPCs F4259 when transfected with oligonucleotides (5 µM) using Optimization 4 ( n = 3) post 96 h transfection. All data are presented as ±STDEV. (*) P

    Article Snippet: Lipid transfections were performed using Lipofectamine stem transfection reagent (Invitrogen) following reported protocols ( ; ).

    Techniques: Activation Assay, Expressing, Western Blot, Derivative Assay, Transfection

    Identifying an electroporation protocol for FRDA patient-derived F4259 iPSC-NPCs. ( A, left ) Relative MALAT1 RNA levels measured by RT-qPCR when transfected with control gapmer ctrl-gap and anti-MALAT1 gapmer anti-MALAT1 using Optimization 4 ( n = 2) compared with Lipofectamine stem transfection reagent and gymnotic delivery post 24 h transfection. ( Right ) Cell viability post 24 h transfection measured by trypan blue staining ( n = 2). ( B, left ) Relative MALAT1 RNA levels measured by qRT-PCR when transfected with control gapmer ctrl-gap and anti-MALAT1 gapmer anti-MALAT1 using Optimization 4 ( n = 4) post 48 h transfection. ( Right ) Cell viability post 48 h transfection measured by trypan blue staining ( n = 4). ( C ) Time course experiments showing inhibition of MALAT1 RNA expression (1 µM, n = 2, Optimization 4). EP(−) is non-oligo treatment, no electroporation control. All data are presented as ± STDEV.

    Journal: RNA

    Article Title: Efficient electroporation of neuronal cells using synthetic oligonucleotides: identifying duplex RNA and antisense oligonucleotide activators of human frataxin expression

    doi: 10.1261/rna.071290.119

    Figure Lengend Snippet: Identifying an electroporation protocol for FRDA patient-derived F4259 iPSC-NPCs. ( A, left ) Relative MALAT1 RNA levels measured by RT-qPCR when transfected with control gapmer ctrl-gap and anti-MALAT1 gapmer anti-MALAT1 using Optimization 4 ( n = 2) compared with Lipofectamine stem transfection reagent and gymnotic delivery post 24 h transfection. ( Right ) Cell viability post 24 h transfection measured by trypan blue staining ( n = 2). ( B, left ) Relative MALAT1 RNA levels measured by qRT-PCR when transfected with control gapmer ctrl-gap and anti-MALAT1 gapmer anti-MALAT1 using Optimization 4 ( n = 4) post 48 h transfection. ( Right ) Cell viability post 48 h transfection measured by trypan blue staining ( n = 4). ( C ) Time course experiments showing inhibition of MALAT1 RNA expression (1 µM, n = 2, Optimization 4). EP(−) is non-oligo treatment, no electroporation control. All data are presented as ± STDEV.

    Article Snippet: Lipid transfections were performed using Lipofectamine stem transfection reagent (Invitrogen) following reported protocols ( ; ).

    Techniques: Electroporation, Derivative Assay, Quantitative RT-PCR, Transfection, Staining, Inhibition, RNA Expression

    Huntingtin is required for normal cell heat shock stress response and mutant huntingtin affects the rate of the nuclear cofilin rod stress response. Temporal imaging in live STHdh Q7/Q7 or STHdh Q111/Q111 cells stably expressing mCerulean–cofilin fusion protein. ( A ) mCerulean–cofilin imaged over time showing nuclear cofilin rod formation in both wild-type and mutant cell lines at 10 and 32 min, respectively (b versus g), following panels show length of times rods exist and time of clearance during maintained heat shock at 42°C (c–e and h–j). ( B ) Comparison graph of average time to nuclear rod formation for stable mCerulean–cofilin wild-type ( N = 15) and mutant ( N = 9) STHdh cells during live cell imaging experiments. * P -value of 0.007. ( C ) Temporal imaging in live STHdh Q7/Q7 cells stably expressing mCerulean–cofilin. Cells were co-transfected with control siRNA or huntingtin-specific siRNA and Block-iT™ Alexa Fluor® red for 72 h prior to experiments. Single-cell visualization of control (a–b) or huntingtin siRNA (h–i) transfection with labeled Block-iT™Alexa Fluor® red. Visualization of STHdh Q7/Q7 cells with mCerulean–cofilin during heat shock, treated with control siRNA (c–g). STHdh Q7/Q7 cells with cofilin–mCerulean during heat shock, treated with siRNA to huntingtin (j–n). ( D ) Comparison graph of average time to cell death in mCerulean–cofilin stable STHdh Q7/Q7 ( N = 13) or STHdh Q111/Q111 ( N = 24) cells imaged live and STHdh Q7/Q7 mCerulean–cofilin cells treated with control siRNA ( N = 5) or huntingtin siRNA ( N = 6) imaged live during heat shock. * P -value of 0.003. Scale bars are 10 µm.

    Journal: Human Molecular Genetics

    Article Title: Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

    doi: 10.1093/hmg/ddr075

    Figure Lengend Snippet: Huntingtin is required for normal cell heat shock stress response and mutant huntingtin affects the rate of the nuclear cofilin rod stress response. Temporal imaging in live STHdh Q7/Q7 or STHdh Q111/Q111 cells stably expressing mCerulean–cofilin fusion protein. ( A ) mCerulean–cofilin imaged over time showing nuclear cofilin rod formation in both wild-type and mutant cell lines at 10 and 32 min, respectively (b versus g), following panels show length of times rods exist and time of clearance during maintained heat shock at 42°C (c–e and h–j). ( B ) Comparison graph of average time to nuclear rod formation for stable mCerulean–cofilin wild-type ( N = 15) and mutant ( N = 9) STHdh cells during live cell imaging experiments. * P -value of 0.007. ( C ) Temporal imaging in live STHdh Q7/Q7 cells stably expressing mCerulean–cofilin. Cells were co-transfected with control siRNA or huntingtin-specific siRNA and Block-iT™ Alexa Fluor® red for 72 h prior to experiments. Single-cell visualization of control (a–b) or huntingtin siRNA (h–i) transfection with labeled Block-iT™Alexa Fluor® red. Visualization of STHdh Q7/Q7 cells with mCerulean–cofilin during heat shock, treated with control siRNA (c–g). STHdh Q7/Q7 cells with cofilin–mCerulean during heat shock, treated with siRNA to huntingtin (j–n). ( D ) Comparison graph of average time to cell death in mCerulean–cofilin stable STHdh Q7/Q7 ( N = 13) or STHdh Q111/Q111 ( N = 24) cells imaged live and STHdh Q7/Q7 mCerulean–cofilin cells treated with control siRNA ( N = 5) or huntingtin siRNA ( N = 6) imaged live during heat shock. * P -value of 0.003. Scale bars are 10 µm.

    Article Snippet: 120 pmol of RNAi was transfected into 25 mm dishes plated at 50% confluency using the lipid-based transfection reagent, Lipofectamine™ 2000 (Invitrogen) according to the manufacturers protocols.

    Techniques: Mutagenesis, Imaging, Stable Transfection, Expressing, Live Cell Imaging, Transfection, Blocking Assay, Labeling

    Expression of ORAI homologs in human airway smooth muscle (HASM) and siRNA-targeted knockdown of ORAI homologs. ( A ) mRNA expression of ORAI1, 2, and 3 in HASM cells using reverse transcriptase–polymerase chain reaction (RT-PCR) (O1: ORAI1, O2: ORAI2, O3: ORAI3). PCR products were sequenced to confirm expression. No products were detected in the RT samples (i.e., RNA samples that have not been reverse transcribed) to confirm no genomic DNA contamination. ( B ) Western blot analysis of ORAI1 protein expression after ORAI1 siRNA transfection; expression of smooth muscle α-actin was assessed as a control for equal protein loading. ( C ) siRNA-mediated knockdown of ORAI1, 2, and 3 mRNA assessed by real-time, quantitative PCR; the effects of individual siRNAs on expression of all ORAI homologs were assessed. The level of ORAI mRNA expression in HASM cells transfected with siRNA was compared relative to an untreated control, which was set to 100%. Results are expressed as the mean ± SEM, cDNA samples were tested in triplicate and the graph represents data from three separate transfections.

    Journal: American Journal of Respiratory Cell and Molecular Biology

    Article Title: ORAI and Store-Operated Calcium Influx in Human Airway Smooth Muscle Cells

    doi: 10.1165/rcmb.2007-0395OC

    Figure Lengend Snippet: Expression of ORAI homologs in human airway smooth muscle (HASM) and siRNA-targeted knockdown of ORAI homologs. ( A ) mRNA expression of ORAI1, 2, and 3 in HASM cells using reverse transcriptase–polymerase chain reaction (RT-PCR) (O1: ORAI1, O2: ORAI2, O3: ORAI3). PCR products were sequenced to confirm expression. No products were detected in the RT samples (i.e., RNA samples that have not been reverse transcribed) to confirm no genomic DNA contamination. ( B ) Western blot analysis of ORAI1 protein expression after ORAI1 siRNA transfection; expression of smooth muscle α-actin was assessed as a control for equal protein loading. ( C ) siRNA-mediated knockdown of ORAI1, 2, and 3 mRNA assessed by real-time, quantitative PCR; the effects of individual siRNAs on expression of all ORAI homologs were assessed. The level of ORAI mRNA expression in HASM cells transfected with siRNA was compared relative to an untreated control, which was set to 100%. Results are expressed as the mean ± SEM, cDNA samples were tested in triplicate and the graph represents data from three separate transfections.

    Article Snippet: Delivery of siRNA to the cells was achieved through use of lipid-based transfection by Lipofectamine 2000 (Invitrogen).

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

    SPC25 knockdown inhibits the proliferation of PC-3 cells. (A) Expression of SPC25 mRNA following transfection with the indicated shRNAs in PC-3 cells. (B) An MTT assay demonstrated that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (C) The Celigo ® system revealed that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (D) Cell number of shCtrl or shSPC25 group in each day was calculated using the Celigo ® system. P

    Journal: Oncology Letters

    Article Title: Knockdown of spindle pole body component 25 homolog inhibits cell proliferation and cycle progression in prostate cancer

    doi: 10.3892/ol.2018.8003

    Figure Lengend Snippet: SPC25 knockdown inhibits the proliferation of PC-3 cells. (A) Expression of SPC25 mRNA following transfection with the indicated shRNAs in PC-3 cells. (B) An MTT assay demonstrated that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (C) The Celigo ® system revealed that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (D) Cell number of shCtrl or shSPC25 group in each day was calculated using the Celigo ® system. P

    Article Snippet: Opti-modified Eagle's medium (Opti-MEM) which was ideal for use during cationic lipid transfections especially Lipofectamine 2000 transfection reagents was purchased from Thermo Fisher Scientific, Inc (cat. no. 31985062).

    Techniques: Expressing, Transfection, MTT Assay

    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

    RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with transfection vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.

    Journal: Virology Journal

    Article Title: Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA

    doi: 10.1186/1743-422X-2-53

    Figure Lengend Snippet: RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with transfection vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.

    Article Snippet: Because the lipid-based transfection reagent targets nucleic acids to the cytoplasm (Mirus Corp, personal communication), the presence of additional membranes between the viral RNA and the cytoplasm could prevent the siRNA from reaching the actively replicating WNV RNA complex.

    Techniques: Infection, Transfection, Flow Cytometry, Cytometry, Expressing, Inhibition