Structured Review

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
Lipid Mediated Transfection, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells"

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

Journal: ACS synthetic biology

doi: 10.1021/acssynbio.5b00299

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
Figure Legend 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

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

2) Product Images from "The impact of chromatin dynamics on Cas9-mediated genome editing in human cells"

Article Title: The impact of chromatin dynamics on Cas9-mediated genome editing in human cells

Journal: bioRxiv

doi: 10.1101/071464

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
Figure Legend 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

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

Dynamic regulatory states impact Cas9-mediated editing at the luciferase transgene. (a) Illustration of the luciferase transgene in the basal expression state and in different, artificially-regulated states. (b) Background-subtracted Luciferase expression levels per cell were measured 96 hours after dox treatment (GAL4EED +dox), immediately before transfection with Gal4-p65 plasmid DNA (+p65), or mock-transfection (vehicle only). Luciferase expression was measured in siRNA-treated cells 336 hours after transfection. a.u.: arbitrary units. (c) Editing efficiency for Cas9/sg034 was determined by SURVEYOR assays. Editing was reduced in the hyperactive expression state (Luc14 +p65) compared to the Luc14 basal state (*p = 0.018) and the GAL4EED partially repressed state (*p = 0.004). Reversal of the closed state via siRNA treatment (GAL4EED +dox +siRNA) was accompanied by an increase in luciferase expression and editing efficiency (p = 0.045) compared to the fully repressed state. Editing efficiencies for Luc14, GAL4EED, and GAL4EED +dox shown here are the same data shown in Figure 3c .
Figure Legend Snippet: Dynamic regulatory states impact Cas9-mediated editing at the luciferase transgene. (a) Illustration of the luciferase transgene in the basal expression state and in different, artificially-regulated states. (b) Background-subtracted Luciferase expression levels per cell were measured 96 hours after dox treatment (GAL4EED +dox), immediately before transfection with Gal4-p65 plasmid DNA (+p65), or mock-transfection (vehicle only). Luciferase expression was measured in siRNA-treated cells 336 hours after transfection. a.u.: arbitrary units. (c) Editing efficiency for Cas9/sg034 was determined by SURVEYOR assays. Editing was reduced in the hyperactive expression state (Luc14 +p65) compared to the Luc14 basal state (*p = 0.018) and the GAL4EED partially repressed state (*p = 0.004). Reversal of the closed state via siRNA treatment (GAL4EED +dox +siRNA) was accompanied by an increase in luciferase expression and editing efficiency (p = 0.045) compared to the fully repressed state. Editing efficiencies for Luc14, GAL4EED, and GAL4EED +dox shown here are the same data shown in Figure 3c .

Techniques Used: Luciferase, Expressing, Transfection, Plasmid Preparation

Related Articles

Sequencing:

Article Title: The impact of chromatin dynamics on Cas9-mediated genome editing in human cells
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 : 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′. ..

Article Title: The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells
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′. .. Mock transfected cells (water used in place of siRNA duplex) were used as a control.

Transfection:

Article Title: The impact of chromatin dynamics on Cas9-mediated genome editing in human cells
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 : 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′. ..

Article Title: The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells
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′. .. Mock transfected cells (water used in place of siRNA duplex) were used as a control.

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    Horizon Discovery transfections hap1
    XKR8 activity is required for efficient EBOV VLP budding and particle PS levels. A) <t>HAP1</t> and ΔXKR8 cells were transfected with EBOV VLP components NP, GP, and VP40-nano-luciferase and budding activity was measured 24 hours following <t>transfection;</t> values were normalized to HAP1 budding efficiency. B) nLuc-VLPs were produced in HAP1 cells in the presence of DMSO or pan-caspase inhibitor Z-VAD-FMK (20μM). C) nLuc-VLPs were produced in HAP1 and ΔXKR8 cells with or without exogenous XKR8 FLAG , and in the presence of either DMSO or Z-VAD-FMK. D) nLuc-VLPs were produced in HAP1 and Vero cells with or without exogenous XKR8 FLAG . nLuc-VLP budding efficiencies were quantified. E) EBOV GP levels on VLPs produced in HAP1 and ΔXKR8 cells with or without exogenous XKR8 FLAG , VLPs were produced without GP for a negative control. Anti-EBOV-GP/anti-human AF647 fluorescence values for GFP+ VLPs are represented as histograms (left panel) and average MFIs (right panel). F) GFP-VLPs were also stained for surface PS using AnV. AnV-PE fluorescence values of GFP+ VLPs are represented as histograms (left panel) and average MFIs (right panel). Values shown are averages of at least three independent experiments ± SEM, * p
    Transfections Hap1, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery sirna transfections
    DNA methylation effects on alternative splcing in HeLa cells A, B, D ) Knock-down efficiencies of DNMTs in HeLa cells. <t>Transfection</t> of HeLa with the indicated <t>siRNA</t> targeting DNMT1 were used to extract total RNAs or proteins. A, D ) RNAs were extracted and subjected to RT-qPCR to quantify the RNA levels of DNMT genes. Relative RNA levels were expressed as fold change over the mock control. Non-targeting (NT) and GAPDH siRNA are used as negative controls. The right panel shows the relative levels of RNA corresponding to the imprinted H19 gene expressed as percent of RPLP0 reference gene. The two bars correspond to different primer pairs amplifying two separated regions of the transcripts. B) Prior to extraction, cells were treated with PMA for 2h and DNMT1 and histone H3 proteins were revealed by western blot. C) The proportion of CpG in the indicated loci ±200 bp surrounding the displayed qPCR amplicons in the MeDIP assays. CpG were counted on both DNA strands and expressed as percent of total dinucleotides. E) Relative levels of CD44 variant exons are decreased upon depletion of DNMT1 but not of DNMT3A or DNMT3B. Levels of each indicated exons were normalized by the average levels between mock and NT siRNA of corresponding exons. Data are average (± s.e.m.) of at least three independent experiments. Statistical comparison have been were evaluated using Student’s t-test (two-tailed), with p
    Sirna Transfections, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery nucleic acids all transfections
    Effect of Q17D57 and Q0IFK9 knock down on the exo-siRNA pathway. Aag2 cells were transfected with ( A , B ) dsRNA targeting eGFP (control), Ago2, Piwi4, Q17D57 or Q0IFK9, or ( C , D ) first infected with SFV4 at MOI 10 followed by dsRNA <t>transfection</t> 6 h later. After 48 h, cells were co-transfected with FFLuc (pIZ-Fluc) and Rluc (pAcIE1-Rluc) expressing reporter plasmids and ( A , C ) dsRNAs against FFLuc (dsFluc) or LacZ (dsLacZ) (as control) or alternatively, ( C , D ) small interfering RNAs (siRNAs) targeting FFLuc (siFluc) or Hygromycin B resistance gene (siHyg) (as control) to assess the silencing ability of siRNAs and bypass the necessity for Dcr2 processing. Cells were lysed at 24 h p.t., and luciferase activities determined. Relative luciferase levels are shown on the Y-axis (with FFLuc /Rluc ratio in dsLacZ or siHyg transfected cells set to 1). The mean values with standard error are shown for three independent experiment conducted in triplicate, * indicates significance, p
    Nucleic Acids All Transfections, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery dharmafect sirna transfection protocol
    AR coregulatory gene downregulation and decrease in PCa cancer cell viability are shRNA seed mediated. ( A ) Relative percent viability and TMEFF2 mRNA expression in LNCaP cells transfected with siTMEFF2-4 and control siRNAs (siNon-Target; Negative seed controls: siTMEFF2-4+3, siTMEFF2-4+5, 5p-siTMEFF2-4; Positive seed control: si634). 5p designates an ON-Target-Plus modification (Dharmacon) that blocks seed mediated gene downregulation. Cell viability was determined by trypan blue. mRNA expression was determined by RT qPCR using RPL8, RPL38, PSMA1 and PPP2CA housekeeping genes for normalization. Viability measurements, cell pictures and RNA extractions were done 72 hours after <t>siRNA</t> <t>transfections</t> N=4, error bars ±SD, * p
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    XKR8 activity is required for efficient EBOV VLP budding and particle PS levels. A) HAP1 and ΔXKR8 cells were transfected with EBOV VLP components NP, GP, and VP40-nano-luciferase and budding activity was measured 24 hours following transfection; values were normalized to HAP1 budding efficiency. B) nLuc-VLPs were produced in HAP1 cells in the presence of DMSO or pan-caspase inhibitor Z-VAD-FMK (20μM). C) nLuc-VLPs were produced in HAP1 and ΔXKR8 cells with or without exogenous XKR8 FLAG , and in the presence of either DMSO or Z-VAD-FMK. D) nLuc-VLPs were produced in HAP1 and Vero cells with or without exogenous XKR8 FLAG . nLuc-VLP budding efficiencies were quantified. E) EBOV GP levels on VLPs produced in HAP1 and ΔXKR8 cells with or without exogenous XKR8 FLAG , VLPs were produced without GP for a negative control. Anti-EBOV-GP/anti-human AF647 fluorescence values for GFP+ VLPs are represented as histograms (left panel) and average MFIs (right panel). F) GFP-VLPs were also stained for surface PS using AnV. AnV-PE fluorescence values of GFP+ VLPs are represented as histograms (left panel) and average MFIs (right panel). Values shown are averages of at least three independent experiments ± SEM, * p

    Journal: bioRxiv

    Article Title: Ebola virus requires phosphatidylserine scrambling activity for efficient budding and optimal infectivity

    doi: 10.1101/2020.03.16.994012

    Figure Lengend Snippet: XKR8 activity is required for efficient EBOV VLP budding and particle PS levels. A) HAP1 and ΔXKR8 cells were transfected with EBOV VLP components NP, GP, and VP40-nano-luciferase and budding activity was measured 24 hours following transfection; values were normalized to HAP1 budding efficiency. B) nLuc-VLPs were produced in HAP1 cells in the presence of DMSO or pan-caspase inhibitor Z-VAD-FMK (20μM). C) nLuc-VLPs were produced in HAP1 and ΔXKR8 cells with or without exogenous XKR8 FLAG , and in the presence of either DMSO or Z-VAD-FMK. D) nLuc-VLPs were produced in HAP1 and Vero cells with or without exogenous XKR8 FLAG . nLuc-VLP budding efficiencies were quantified. E) EBOV GP levels on VLPs produced in HAP1 and ΔXKR8 cells with or without exogenous XKR8 FLAG , VLPs were produced without GP for a negative control. Anti-EBOV-GP/anti-human AF647 fluorescence values for GFP+ VLPs are represented as histograms (left panel) and average MFIs (right panel). F) GFP-VLPs were also stained for surface PS using AnV. AnV-PE fluorescence values of GFP+ VLPs are represented as histograms (left panel) and average MFIs (right panel). Values shown are averages of at least three independent experiments ± SEM, * p

    Article Snippet: Cell lines and transfections HAP1, HAP1ΔXKR8 (HZGHC005916c007), and HAP1ΔTMEM16F (HZGHC002956c003) cells (Horizon Discovery) were maintained in Iscove’s media supplemented with 10% (vol/vol) fetal bovine serum (FBS).

    Techniques: Activity Assay, Transfection, Luciferase, Produced, Negative Control, Fluorescence, Staining

    DNA methylation effects on alternative splcing in HeLa cells A, B, D ) Knock-down efficiencies of DNMTs in HeLa cells. Transfection of HeLa with the indicated siRNA targeting DNMT1 were used to extract total RNAs or proteins. A, D ) RNAs were extracted and subjected to RT-qPCR to quantify the RNA levels of DNMT genes. Relative RNA levels were expressed as fold change over the mock control. Non-targeting (NT) and GAPDH siRNA are used as negative controls. The right panel shows the relative levels of RNA corresponding to the imprinted H19 gene expressed as percent of RPLP0 reference gene. The two bars correspond to different primer pairs amplifying two separated regions of the transcripts. B) Prior to extraction, cells were treated with PMA for 2h and DNMT1 and histone H3 proteins were revealed by western blot. C) The proportion of CpG in the indicated loci ±200 bp surrounding the displayed qPCR amplicons in the MeDIP assays. CpG were counted on both DNA strands and expressed as percent of total dinucleotides. E) Relative levels of CD44 variant exons are decreased upon depletion of DNMT1 but not of DNMT3A or DNMT3B. Levels of each indicated exons were normalized by the average levels between mock and NT siRNA of corresponding exons. Data are average (± s.e.m.) of at least three independent experiments. Statistical comparison have been were evaluated using Student’s t-test (two-tailed), with p

    Journal: bioRxiv

    Article Title: CD44 alternative splicing is a sensor of intragenic DNA methylation in tumors

    doi: 10.1101/685651

    Figure Lengend Snippet: DNA methylation effects on alternative splcing in HeLa cells A, B, D ) Knock-down efficiencies of DNMTs in HeLa cells. Transfection of HeLa with the indicated siRNA targeting DNMT1 were used to extract total RNAs or proteins. A, D ) RNAs were extracted and subjected to RT-qPCR to quantify the RNA levels of DNMT genes. Relative RNA levels were expressed as fold change over the mock control. Non-targeting (NT) and GAPDH siRNA are used as negative controls. The right panel shows the relative levels of RNA corresponding to the imprinted H19 gene expressed as percent of RPLP0 reference gene. The two bars correspond to different primer pairs amplifying two separated regions of the transcripts. B) Prior to extraction, cells were treated with PMA for 2h and DNMT1 and histone H3 proteins were revealed by western blot. C) The proportion of CpG in the indicated loci ±200 bp surrounding the displayed qPCR amplicons in the MeDIP assays. CpG were counted on both DNA strands and expressed as percent of total dinucleotides. E) Relative levels of CD44 variant exons are decreased upon depletion of DNMT1 but not of DNMT3A or DNMT3B. Levels of each indicated exons were normalized by the average levels between mock and NT siRNA of corresponding exons. Data are average (± s.e.m.) of at least three independent experiments. Statistical comparison have been were evaluated using Student’s t-test (two-tailed), with p

    Article Snippet: Cell culture, siRNA transfections, and PMA treatmentColorectal carcinoma cell line HCT116 (CCL-247) and double knock-out (DKO) cells for DNMT1 and DNMT3b have been purchased from Horizon Discovery.

    Techniques: DNA Methylation Assay, Transfection, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Methylated DNA Immunoprecipitation, Variant Assay, Two Tailed Test

    Effect of Q17D57 and Q0IFK9 knock down on the exo-siRNA pathway. Aag2 cells were transfected with ( A , B ) dsRNA targeting eGFP (control), Ago2, Piwi4, Q17D57 or Q0IFK9, or ( C , D ) first infected with SFV4 at MOI 10 followed by dsRNA transfection 6 h later. After 48 h, cells were co-transfected with FFLuc (pIZ-Fluc) and Rluc (pAcIE1-Rluc) expressing reporter plasmids and ( A , C ) dsRNAs against FFLuc (dsFluc) or LacZ (dsLacZ) (as control) or alternatively, ( C , D ) small interfering RNAs (siRNAs) targeting FFLuc (siFluc) or Hygromycin B resistance gene (siHyg) (as control) to assess the silencing ability of siRNAs and bypass the necessity for Dcr2 processing. Cells were lysed at 24 h p.t., and luciferase activities determined. Relative luciferase levels are shown on the Y-axis (with FFLuc /Rluc ratio in dsLacZ or siHyg transfected cells set to 1). The mean values with standard error are shown for three independent experiment conducted in triplicate, * indicates significance, p

    Journal: Viruses

    Article Title: aBravo Is a Novel Aedes aegypti Antiviral Protein That Interacts with, but Acts Independently of, the Exogenous siRNA Pathway Effector Dicer 2

    doi: 10.3390/v12070748

    Figure Lengend Snippet: Effect of Q17D57 and Q0IFK9 knock down on the exo-siRNA pathway. Aag2 cells were transfected with ( A , B ) dsRNA targeting eGFP (control), Ago2, Piwi4, Q17D57 or Q0IFK9, or ( C , D ) first infected with SFV4 at MOI 10 followed by dsRNA transfection 6 h later. After 48 h, cells were co-transfected with FFLuc (pIZ-Fluc) and Rluc (pAcIE1-Rluc) expressing reporter plasmids and ( A , C ) dsRNAs against FFLuc (dsFluc) or LacZ (dsLacZ) (as control) or alternatively, ( C , D ) small interfering RNAs (siRNAs) targeting FFLuc (siFluc) or Hygromycin B resistance gene (siHyg) (as control) to assess the silencing ability of siRNAs and bypass the necessity for Dcr2 processing. Cells were lysed at 24 h p.t., and luciferase activities determined. Relative luciferase levels are shown on the Y-axis (with FFLuc /Rluc ratio in dsLacZ or siHyg transfected cells set to 1). The mean values with standard error are shown for three independent experiment conducted in triplicate, * indicates significance, p

    Article Snippet: Transfection of Nucleic Acids All transfections were carried out using Dharmafect 2 reagent (Horizon Discovery, Cambridge, UK).

    Techniques: Transfection, Infection, Expressing, Luciferase

    AR coregulatory gene downregulation and decrease in PCa cancer cell viability are shRNA seed mediated. ( A ) Relative percent viability and TMEFF2 mRNA expression in LNCaP cells transfected with siTMEFF2-4 and control siRNAs (siNon-Target; Negative seed controls: siTMEFF2-4+3, siTMEFF2-4+5, 5p-siTMEFF2-4; Positive seed control: si634). 5p designates an ON-Target-Plus modification (Dharmacon) that blocks seed mediated gene downregulation. Cell viability was determined by trypan blue. mRNA expression was determined by RT qPCR using RPL8, RPL38, PSMA1 and PPP2CA housekeeping genes for normalization. Viability measurements, cell pictures and RNA extractions were done 72 hours after siRNA transfections N=4, error bars ±SD, * p

    Journal: bioRxiv

    Article Title: SEED-MEDIATED RNA INTERFERENCE OF ANDROGEN SIGNALING AND SURVIVAL NETWORKS INDUCES CELL DEATH IN PROSTATE CANCER CELLS

    doi: 10.1101/2020.07.17.209379

    Figure Lengend Snippet: AR coregulatory gene downregulation and decrease in PCa cancer cell viability are shRNA seed mediated. ( A ) Relative percent viability and TMEFF2 mRNA expression in LNCaP cells transfected with siTMEFF2-4 and control siRNAs (siNon-Target; Negative seed controls: siTMEFF2-4+3, siTMEFF2-4+5, 5p-siTMEFF2-4; Positive seed control: si634). 5p designates an ON-Target-Plus modification (Dharmacon) that blocks seed mediated gene downregulation. Cell viability was determined by trypan blue. mRNA expression was determined by RT qPCR using RPL8, RPL38, PSMA1 and PPP2CA housekeeping genes for normalization. Viability measurements, cell pictures and RNA extractions were done 72 hours after siRNA transfections N=4, error bars ±SD, * p

    Article Snippet: Dharmafect siRNA transfection protocol was used for transfections, ( https://horizondiscovery.com/-/media/Files/Horizon/resources/Protocols/basic-dharmafect-protocol.pdf).Dharmafect reagent #1 (0.2%) was used for RWPE1, BHPre1 and NHPre1 cell lines, and 0.2% Dharmafect reagent #3 was used for LNCaP, C4-2B and 22Rv1 cell lines.

    Techniques: shRNA, Expressing, Transfection, Modification, Quantitative RT-PCR

    AN-DISE siRNAs do not reduce the viability of normal prostate epithelial cell lines. Relative percent viability of normal prostate (RWPE1, BHPre1, NHPre1) and PCa cell lines (LNCaP, C4-2B and 22Rv1) transfected with Cy5 labeled siNon-Target, siTMEFF2-4 or siTMEFF2-9 siRNAs. Viability was determined by trypan blue 72 hours after siRNA transfection. N=3, error bars ±SD, * p

    Journal: bioRxiv

    Article Title: SEED-MEDIATED RNA INTERFERENCE OF ANDROGEN SIGNALING AND SURVIVAL NETWORKS INDUCES CELL DEATH IN PROSTATE CANCER CELLS

    doi: 10.1101/2020.07.17.209379

    Figure Lengend Snippet: AN-DISE siRNAs do not reduce the viability of normal prostate epithelial cell lines. Relative percent viability of normal prostate (RWPE1, BHPre1, NHPre1) and PCa cell lines (LNCaP, C4-2B and 22Rv1) transfected with Cy5 labeled siNon-Target, siTMEFF2-4 or siTMEFF2-9 siRNAs. Viability was determined by trypan blue 72 hours after siRNA transfection. N=3, error bars ±SD, * p

    Article Snippet: Dharmafect siRNA transfection protocol was used for transfections, ( https://horizondiscovery.com/-/media/Files/Horizon/resources/Protocols/basic-dharmafect-protocol.pdf).Dharmafect reagent #1 (0.2%) was used for RWPE1, BHPre1 and NHPre1 cell lines, and 0.2% Dharmafect reagent #3 was used for LNCaP, C4-2B and 22Rv1 cell lines.

    Techniques: Transfection, Labeling

    Reduction in PCa cancer cell viability is seed mediated. ( A/B ) Relative percent viability and TMEFF2 mRNA expression in LNCaP cells transfected with siTMEFF2-9 ( A ) or siTMEFF2-3 ( B ) and control siRNAs (siNon-Target; Negative seed control: 5p-siTMEFF2). 5p designates an ON-Target-Plus modification (Dharmacon) that blocks seed mediated gene downregulation. Cell viability was determined by trypan blue. Viability measurements, cell pictures and RNA extractions were done 72 hours after siRNA transfections. mRNA expression was determined by RT qPCR using RPL8, RPL38, PSMA1 and PPP2CA housekeeping genes for normalization. N=4, error bars ±SD, * p

    Journal: bioRxiv

    Article Title: SEED-MEDIATED RNA INTERFERENCE OF ANDROGEN SIGNALING AND SURVIVAL NETWORKS INDUCES CELL DEATH IN PROSTATE CANCER CELLS

    doi: 10.1101/2020.07.17.209379

    Figure Lengend Snippet: Reduction in PCa cancer cell viability is seed mediated. ( A/B ) Relative percent viability and TMEFF2 mRNA expression in LNCaP cells transfected with siTMEFF2-9 ( A ) or siTMEFF2-3 ( B ) and control siRNAs (siNon-Target; Negative seed control: 5p-siTMEFF2). 5p designates an ON-Target-Plus modification (Dharmacon) that blocks seed mediated gene downregulation. Cell viability was determined by trypan blue. Viability measurements, cell pictures and RNA extractions were done 72 hours after siRNA transfections. mRNA expression was determined by RT qPCR using RPL8, RPL38, PSMA1 and PPP2CA housekeeping genes for normalization. N=4, error bars ±SD, * p

    Article Snippet: Dharmafect siRNA transfection protocol was used for transfections, ( https://horizondiscovery.com/-/media/Files/Horizon/resources/Protocols/basic-dharmafect-protocol.pdf).Dharmafect reagent #1 (0.2%) was used for RWPE1, BHPre1 and NHPre1 cell lines, and 0.2% Dharmafect reagent #3 was used for LNCaP, C4-2B and 22Rv1 cell lines.

    Techniques: Expressing, Transfection, Modification, Quantitative RT-PCR