lipofectamine rnaimax  (Thermo Fisher)


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    Name:
    Lipofectamine RNAiMAX Transfection Reagent
    Description:
    Lipofectamine RNAiMAX Transfection Reagent provides the highest transfection efficiencies on the widest variety of cell types for siRNA mediated gene knockdown experiments Lipofectamine RNAiMAX is a proprietary RNAi specific cationic lipid formulation designed specifically for the delivery of siRNA and miRNA into all cell types With Lipofectamine RNAiMAX Transfection Reagent you will get • Superior transfection efficiency requiring lower RNAi concentrations leading to more effective gene knockdown with minimal nonspecific effects• Easy optimization due to minimal cytotoxicity across a 10 fold concentration range of transfection reagent• Superior transfection efficiencies for miRNA antagonists and mimics• Compatibility with a broad range of cell types providing the most versatile approach to all of your gene silencing experiments• A simple and rapid protocol for consistent and reproducible resultsHigh knockdown in a wide range of cellsLipofectamine RNAiMAX Transfection Reagent transfects a wide range of cell types see figure For gene silencing Lipofectamine RNAiMAX Transfection Reagent s high efficiency transfections lead to the high levels of gene knockdown needed to achieve convincing results Simple high throughput ready transfectionsSimply mix Lipofectamine RNAiMAX Transfection Reagent with siRNA add to your cells incubate and measure gene knockdown The simplicity and speed combined with high transfection efficiency make Lipofectamine RNAiMAX Transfection Reagent ideal for high throughput siRNA transfections Transfection conditions can be easily established for automated or robotic systems used in such applications Find an optimized Lipofectamine RNAiMAX transfection protocol for your cell line
    Catalog Number:
    13778030
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|RNAi|RNAi Transfection|RNAi, Epigenetics & Non-Coding RNA Research|Stem Cell & Primary Cell Transfections|Synthetic siRNA Transfection|siRNA|Transfection
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    Structured Review

    Thermo Fisher lipofectamine rnaimax
    Cellular sensitivity to oxidative stress is controlled by TRIM26 through NTH1 regulation. (A to C) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with <t>Lipofectamine</t> <t>RNAiMax</t> transfection reagent (10 μl) in the presence of 800 pmol NT siRNA or TRIM26 siRNA for 72 h. Cells were also treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 500 ng mammalian expression plasmid for NTH1 (NTH1 O/E) for 24 h. (A) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (B) Cells were treated with hydrogen peroxide (12.5 μM), and DNA single-strand breaks and alkali-labile sites were measured at various time points postincubation by the alkaline comet assay. Shown are the percentages of tail DNA with standard deviations from the results of at least three independent experiments. *, P
    Lipofectamine RNAiMAX Transfection Reagent provides the highest transfection efficiencies on the widest variety of cell types for siRNA mediated gene knockdown experiments Lipofectamine RNAiMAX is a proprietary RNAi specific cationic lipid formulation designed specifically for the delivery of siRNA and miRNA into all cell types With Lipofectamine RNAiMAX Transfection Reagent you will get • Superior transfection efficiency requiring lower RNAi concentrations leading to more effective gene knockdown with minimal nonspecific effects• Easy optimization due to minimal cytotoxicity across a 10 fold concentration range of transfection reagent• Superior transfection efficiencies for miRNA antagonists and mimics• Compatibility with a broad range of cell types providing the most versatile approach to all of your gene silencing experiments• A simple and rapid protocol for consistent and reproducible resultsHigh knockdown in a wide range of cellsLipofectamine RNAiMAX Transfection Reagent transfects a wide range of cell types see figure For gene silencing Lipofectamine RNAiMAX Transfection Reagent s high efficiency transfections lead to the high levels of gene knockdown needed to achieve convincing results Simple high throughput ready transfectionsSimply mix Lipofectamine RNAiMAX Transfection Reagent with siRNA add to your cells incubate and measure gene knockdown The simplicity and speed combined with high transfection efficiency make Lipofectamine RNAiMAX Transfection Reagent ideal for high throughput siRNA transfections Transfection conditions can be easily established for automated or robotic systems used in such applications Find an optimized Lipofectamine RNAiMAX transfection protocol for your cell line
    https://www.bioz.com/result/lipofectamine rnaimax/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lipofectamine rnaimax - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress"

    Article Title: NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00616-17

    Cellular sensitivity to oxidative stress is controlled by TRIM26 through NTH1 regulation. (A to C) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA or TRIM26 siRNA for 72 h. Cells were also treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 500 ng mammalian expression plasmid for NTH1 (NTH1 O/E) for 24 h. (A) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (B) Cells were treated with hydrogen peroxide (12.5 μM), and DNA single-strand breaks and alkali-labile sites were measured at various time points postincubation by the alkaline comet assay. Shown are the percentages of tail DNA with standard deviations from the results of at least three independent experiments. *, P
    Figure Legend Snippet: Cellular sensitivity to oxidative stress is controlled by TRIM26 through NTH1 regulation. (A to C) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA or TRIM26 siRNA for 72 h. Cells were also treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 500 ng mammalian expression plasmid for NTH1 (NTH1 O/E) for 24 h. (A) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (B) Cells were treated with hydrogen peroxide (12.5 μM), and DNA single-strand breaks and alkali-labile sites were measured at various time points postincubation by the alkaline comet assay. Shown are the percentages of tail DNA with standard deviations from the results of at least three independent experiments. *, P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, SDS Page, Alkaline Single Cell Gel Electrophoresis

    Cellular NTH1 protein levels are induced in response to oxidative stress controlled by TRIM26. (A and B) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA (A) or TRIM26 siRNA (Β) for 72 h. (C) HCT116 cells were also grown in 10-cm dishes for 24 h to ∼90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of a mammalian expression plasmid for TRIM26 (1 μg) for 24 h. Cells were either left untreated (lane C) or treated with hydrogen peroxide (150 μM for 15 min) and harvested at various time points following incubation. Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (D) Levels of NTH1 protein relative to tubulin were quantified from the results of at least three independent experiments. Shown is the mean NTH1/tubulin ratio with standard error normalized to that of the untreated control, which was set to 1.0. *, P
    Figure Legend Snippet: Cellular NTH1 protein levels are induced in response to oxidative stress controlled by TRIM26. (A and B) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA (A) or TRIM26 siRNA (Β) for 72 h. (C) HCT116 cells were also grown in 10-cm dishes for 24 h to ∼90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of a mammalian expression plasmid for TRIM26 (1 μg) for 24 h. Cells were either left untreated (lane C) or treated with hydrogen peroxide (150 μM for 15 min) and harvested at various time points following incubation. Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (D) Levels of NTH1 protein relative to tubulin were quantified from the results of at least three independent experiments. Shown is the mean NTH1/tubulin ratio with standard error normalized to that of the untreated control, which was set to 1.0. *, P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Incubation, SDS Page

    NTH1 protein accumulates on chromatin in response to oxidative stress, which is controlled by TRIM26. (A and B) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA (A) or TRIM26 siRNA (Β) for 72 h. Cells were either left untreated (lane C) or treated with hydrogen peroxide (150 μM for 15 min) and harvested at various time points following incubation, and proteins were separated by biochemical fractionation. The soluble (S) and chromatin-bound (CB) fractions were analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (C) Levels of NTH1 protein relative to fibrillarin in the chromatin-bound fraction were quantified from the results of at least three independent experiments; shown is the mean NTH1/fibrillarin ratio with standard deviation normalized to that of the NT siRNA-treated control, which was set to 1.0. *, P
    Figure Legend Snippet: NTH1 protein accumulates on chromatin in response to oxidative stress, which is controlled by TRIM26. (A and B) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA (A) or TRIM26 siRNA (Β) for 72 h. Cells were either left untreated (lane C) or treated with hydrogen peroxide (150 μM for 15 min) and harvested at various time points following incubation, and proteins were separated by biochemical fractionation. The soluble (S) and chromatin-bound (CB) fractions were analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (C) Levels of NTH1 protein relative to fibrillarin in the chromatin-bound fraction were quantified from the results of at least three independent experiments; shown is the mean NTH1/fibrillarin ratio with standard deviation normalized to that of the NT siRNA-treated control, which was set to 1.0. *, P

    Techniques Used: Transfection, Incubation, Fractionation, SDS Page, Standard Deviation

    Cellular NTH1 protein levels are regulated by ubiquitylation by TRIM26. (A) HCT116 cells were grown in 10-cm dishes for 24 h to 90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of mammalian expression plasmids for HA-tagged ubiquitin (1 μg), Flag-tagged NTH1 (500 ng), and HA-tagged TRIM26 (1 μg) for 24 h. The cells were then treated with MG-132 (10 μM) for 8 h, and whole-cell extracts were prepared and Flag-NTH1 purified, using anti-Flag magnetic beads, from extracts containing equal amounts of total protein. Proteins bound to the beads were analyzed by 10% SDS-PAGE and immunoblotting (IB) with HA antibodies to detect ubiquitylated NTH1. IP, immunoprecipitation. Molecular mass (kilodalton) markers are indicated on the left. (B to D) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT or TRIM26 siRNA for 72 h. (B) RNA and subsequently cDNA were prepared from cells, and quantitative PCRs using primer pairs for trim26 and actin were performed. Fold changes in the levels of trim26 mRNA relative to actin are shown. (C) Proteins were separated by biochemical fractionation, and the soluble (S) and chromatin-bound (CB) fractions were analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (D) Levels of NTH1 protein relative to lamin A in the chromatin-bound fraction were quantified from the results of at least three independent experiments, and the mean NTH1/lamin A ratio with standard deviation normalized to the NT siRNA-treated control, which was set to 1.0, is shown. (E and F) HCT116 cells were grown in 10-cm dishes for 24 h to ∼90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 250 ng mammalian expression plasmids for Flag-tagged WT or an NTH1 mutant (K67R) for 24 h. (E) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (F) Levels of Flag-tagged NTH1 proteins relative to tubulin were quantified from the results of at least three independent experiments. Shown is the mean Flag-NTH1/tubulin ratio with standard deviation normalized to that of the WT-NTH1-transfected cells, which was set to 1.0. *, P
    Figure Legend Snippet: Cellular NTH1 protein levels are regulated by ubiquitylation by TRIM26. (A) HCT116 cells were grown in 10-cm dishes for 24 h to 90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of mammalian expression plasmids for HA-tagged ubiquitin (1 μg), Flag-tagged NTH1 (500 ng), and HA-tagged TRIM26 (1 μg) for 24 h. The cells were then treated with MG-132 (10 μM) for 8 h, and whole-cell extracts were prepared and Flag-NTH1 purified, using anti-Flag magnetic beads, from extracts containing equal amounts of total protein. Proteins bound to the beads were analyzed by 10% SDS-PAGE and immunoblotting (IB) with HA antibodies to detect ubiquitylated NTH1. IP, immunoprecipitation. Molecular mass (kilodalton) markers are indicated on the left. (B to D) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT or TRIM26 siRNA for 72 h. (B) RNA and subsequently cDNA were prepared from cells, and quantitative PCRs using primer pairs for trim26 and actin were performed. Fold changes in the levels of trim26 mRNA relative to actin are shown. (C) Proteins were separated by biochemical fractionation, and the soluble (S) and chromatin-bound (CB) fractions were analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (D) Levels of NTH1 protein relative to lamin A in the chromatin-bound fraction were quantified from the results of at least three independent experiments, and the mean NTH1/lamin A ratio with standard deviation normalized to the NT siRNA-treated control, which was set to 1.0, is shown. (E and F) HCT116 cells were grown in 10-cm dishes for 24 h to ∼90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 250 ng mammalian expression plasmids for Flag-tagged WT or an NTH1 mutant (K67R) for 24 h. (E) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (F) Levels of Flag-tagged NTH1 proteins relative to tubulin were quantified from the results of at least three independent experiments. Shown is the mean Flag-NTH1/tubulin ratio with standard deviation normalized to that of the WT-NTH1-transfected cells, which was set to 1.0. *, P

    Techniques Used: Transfection, Expressing, Purification, Magnetic Beads, SDS Page, Immunoprecipitation, Fractionation, Standard Deviation, Mutagenesis

    2) Product Images from "Down-regulation of the tumor suppressor miR-34a contributes to head and neck cancer by up-regulating the MET oncogene and modulating tumor immune evasion"

    Article Title: Down-regulation of the tumor suppressor miR-34a contributes to head and neck cancer by up-regulating the MET oncogene and modulating tumor immune evasion

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-021-01865-2

    miR-34a over-expression induces early apoptosis, prevents tumor proliferation, and inhibits tumor growth in vivo. a Representative flow cytometry plot of annexin V (FITC) versus propidium iodide in control mimic treated cells or miR-34a-5p treated HTB-42 cells (25 nM, 48 h). Early apoptotic cells were defined as propidium iodide low and Annexin-V high. b , c The early apoptosis detection assay determined the percentage of early apoptotic cells in 2 different head and neck squamous cell lines (HTB-43 and CAL27 cells), and MET expression was confirmed by western blot. d The level of miR-34a-5p expression was determined by a TaqMan microRNA assay, 12 h post electroporation of miR-34a mimic or control mimic (25 nM). e miR-34a-5p mimic (25 nM or 50 nM) was administered to cells at day 0, and proliferation was measured using MTT reagent to assess the effect of miR-34a on the proliferation of CAL27 cells. f and g Anti-LNA-miR-34a (miR-34a-5p inhibitor) was administered to CAL27 cells and a TaqMan microRNA Assay was used to quantify levels of miR-34a-5p. MET siRNA (20 nM) or scramble siRNA (20 nM) were introduced to CAL27 cells which received miR-34a-5p inhibitor by Lipofectamine RNAiMAX and cell proliferation was quantified by MTT assay. All experiments performed at least in triplicate. Data represent results of at least three independent experiments. Data is presented as mean ± SD.* indicates p
    Figure Legend Snippet: miR-34a over-expression induces early apoptosis, prevents tumor proliferation, and inhibits tumor growth in vivo. a Representative flow cytometry plot of annexin V (FITC) versus propidium iodide in control mimic treated cells or miR-34a-5p treated HTB-42 cells (25 nM, 48 h). Early apoptotic cells were defined as propidium iodide low and Annexin-V high. b , c The early apoptosis detection assay determined the percentage of early apoptotic cells in 2 different head and neck squamous cell lines (HTB-43 and CAL27 cells), and MET expression was confirmed by western blot. d The level of miR-34a-5p expression was determined by a TaqMan microRNA assay, 12 h post electroporation of miR-34a mimic or control mimic (25 nM). e miR-34a-5p mimic (25 nM or 50 nM) was administered to cells at day 0, and proliferation was measured using MTT reagent to assess the effect of miR-34a on the proliferation of CAL27 cells. f and g Anti-LNA-miR-34a (miR-34a-5p inhibitor) was administered to CAL27 cells and a TaqMan microRNA Assay was used to quantify levels of miR-34a-5p. MET siRNA (20 nM) or scramble siRNA (20 nM) were introduced to CAL27 cells which received miR-34a-5p inhibitor by Lipofectamine RNAiMAX and cell proliferation was quantified by MTT assay. All experiments performed at least in triplicate. Data represent results of at least three independent experiments. Data is presented as mean ± SD.* indicates p

    Techniques Used: Over Expression, In Vivo, Flow Cytometry, Detection Assay, Expressing, Western Blot, TaqMan microRNA Assay, Electroporation, MTT Assay

    The anti-tumorigenic effect of miR-34a is dependent on MET, and miR-34a-5p inhibits epithelial mesenchymal transition (EMT). a MET vector or control vector (pBABE) were introduced to CAL27 cells and levels of MET mRNA expression were quantified by RT-qPCR. b miR-34a-5p (25 nM) mimic was administrated to the MET overexpressing CAL27 cells and cell proliferation was measured after 24 h using MTT assay. MET signaling was stimulated with HGF (20 ng/ml). c Percentage of early apoptotic cells were determined by Annexin V and PI in MET overexpressing CAL27 cells with and without introduction of miR-34a-5p mimic. MET signaling was stimulated with HGF (20 ng/ml). d Effect of miR-34a-5p on STAT3 protein phosphorylation (shaded line: control; open line: HGF (20 ng/ml); gray line: HGF (20 ng/ml) + miR34a-5p mimic). e , f Relative mRNA expression of epithelial marker (CDH1) and mesenchymal marker (Vimentin) were quantified 48 h after introduction of the miR control mimic or miR-34a-5p mimic by Lipofectamine RNAiMAX in CAL27 cells. RT- qPCR data was normalized using 18 s. g Protein expression of CDH1 and Vimentin were determined after 48 h administration of miR-34a-5p mimic or control mimic in CAL27 cells using western blot. Data represent results of at least three independent experiments. Data is presented as mean ± SD. * indicates p
    Figure Legend Snippet: The anti-tumorigenic effect of miR-34a is dependent on MET, and miR-34a-5p inhibits epithelial mesenchymal transition (EMT). a MET vector or control vector (pBABE) were introduced to CAL27 cells and levels of MET mRNA expression were quantified by RT-qPCR. b miR-34a-5p (25 nM) mimic was administrated to the MET overexpressing CAL27 cells and cell proliferation was measured after 24 h using MTT assay. MET signaling was stimulated with HGF (20 ng/ml). c Percentage of early apoptotic cells were determined by Annexin V and PI in MET overexpressing CAL27 cells with and without introduction of miR-34a-5p mimic. MET signaling was stimulated with HGF (20 ng/ml). d Effect of miR-34a-5p on STAT3 protein phosphorylation (shaded line: control; open line: HGF (20 ng/ml); gray line: HGF (20 ng/ml) + miR34a-5p mimic). e , f Relative mRNA expression of epithelial marker (CDH1) and mesenchymal marker (Vimentin) were quantified 48 h after introduction of the miR control mimic or miR-34a-5p mimic by Lipofectamine RNAiMAX in CAL27 cells. RT- qPCR data was normalized using 18 s. g Protein expression of CDH1 and Vimentin were determined after 48 h administration of miR-34a-5p mimic or control mimic in CAL27 cells using western blot. Data represent results of at least three independent experiments. Data is presented as mean ± SD. * indicates p

    Techniques Used: Plasmid Preparation, Expressing, Quantitative RT-PCR, MTT Assay, Marker, Western Blot

    3) Product Images from "Normal human cell proteins that interact with the adenovirus type 5 E1B 55 kDa protein"

    Article Title: Normal human cell proteins that interact with the adenovirus type 5 E1B 55 kDa protein

    Journal: Virology

    doi: 10.1016/j.virol.2017.01.013

    Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using lipofectamine RNAiMAX (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.
    Figure Legend Snippet: Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using lipofectamine RNAiMAX (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.

    Techniques Used: Expressing, Aqueous Normal-phase Chromatography, Infection

    4) Product Images from "Enhanced siRNA Delivery and Selective Apoptosis Induction in H1299 Cancer Cells by Layer-by-Layer-Assembled Se Nanocomplexes: Toward More Efficient Cancer Therapy"

    Article Title: Enhanced siRNA Delivery and Selective Apoptosis Induction in H1299 Cancer Cells by Layer-by-Layer-Assembled Se Nanocomplexes: Toward More Efficient Cancer Therapy

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2021.639184

    In vitro knockdown efficiency of LBL-Se-NCs. (A) GFP expression (%) of H1299 cells treated with LBL-Se-NCs as a function of siRNA concentration. (B) Comparison of the GFP expression level on H1299 cells after treatment with LBL-Se-NCs, Lipofectamine, jetPEI, CS-NCs, and naked siRNA, all at 8 nM effective siRNA concentration, for both freshly synthesized and 7-day-old nanocomplexes . (C) Confocal images of intracellular GFP knockdown of LBL-Se-NCs and control groups on H1299 cells. The scale bar corresponds to 50 μm. Data are presented as mean values ± SD ( n = 3, * p
    Figure Legend Snippet: In vitro knockdown efficiency of LBL-Se-NCs. (A) GFP expression (%) of H1299 cells treated with LBL-Se-NCs as a function of siRNA concentration. (B) Comparison of the GFP expression level on H1299 cells after treatment with LBL-Se-NCs, Lipofectamine, jetPEI, CS-NCs, and naked siRNA, all at 8 nM effective siRNA concentration, for both freshly synthesized and 7-day-old nanocomplexes . (C) Confocal images of intracellular GFP knockdown of LBL-Se-NCs and control groups on H1299 cells. The scale bar corresponds to 50 μm. Data are presented as mean values ± SD ( n = 3, * p

    Techniques Used: In Vitro, Expressing, Concentration Assay, Synthesized

    5) Product Images from "Role of MicroRNA 1207-5P and Its Host Gene, the Long Non-Coding RNA Pvt1, as Mediators of Extracellular Matrix Accumulation in the Kidney: Implications for Diabetic Nephropathy"

    Article Title: Role of MicroRNA 1207-5P and Its Host Gene, the Long Non-Coding RNA Pvt1, as Mediators of Extracellular Matrix Accumulation in the Kidney: Implications for Diabetic Nephropathy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077468

    Effect of PVT1 on TGF-β1, PAI-1 and FN1. Relative quantification of TGF-β1, PAI-1, and FN1 mRNA ( A ) or secreted protein ( B ) in MC with PVT1 over-expression. Cells were transfected with plasmid pCMV-PVT1 or empty vector pCMV (NC, negative control) by electroporation using the Neon System. ( C ) Changes in expression of FN1 mRNA in MC treated with 30 nM PVT1 or NC siRNA, in the presence or absence of the TGF-β signaling inhibitor SB431542. NC siRNA comprised of sequence not found in the human genome. MC were transfected using 5 µl Lipofectamine RNAiMax (Life Technologies) per ml of MsBM media. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significantly different from all the others. * P
    Figure Legend Snippet: Effect of PVT1 on TGF-β1, PAI-1 and FN1. Relative quantification of TGF-β1, PAI-1, and FN1 mRNA ( A ) or secreted protein ( B ) in MC with PVT1 over-expression. Cells were transfected with plasmid pCMV-PVT1 or empty vector pCMV (NC, negative control) by electroporation using the Neon System. ( C ) Changes in expression of FN1 mRNA in MC treated with 30 nM PVT1 or NC siRNA, in the presence or absence of the TGF-β signaling inhibitor SB431542. NC siRNA comprised of sequence not found in the human genome. MC were transfected using 5 µl Lipofectamine RNAiMax (Life Technologies) per ml of MsBM media. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significantly different from all the others. * P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Negative Control, Electroporation, Expressing, Sequencing

    Validation of putative miR-1207-5p target genes. miR-1207-5p target genes were identified by TargetScan software and four genes were selected using Pathway Studio software based on potential biological effects relevant to ECM accumulation. The four genes selected, G6PD, PMEPA1, PDPK1, and SMAD7 , were validated as putative miR-1207-5p target genes in MC using 25 nM miR-1207-5p or NC mimic ( A ), and 50 nM anti-miR-1207-5p or negative control ( B ). Results in A and B represent averages from three independent experiments. Data are means ± SD. About 20,000 human embryonic kidney 293 (HEK293) cells were seeded per well into a white 96-well plate and co-transfected with 100 ng of the indicated 3′UTR luciferase reporter vectors and 30 nM miR-1207-5p or negative control mimic (Dharmacon) using 0.2 µl per well of Lipofectamine 2000 ( C ). HEK293 cells were also co-transfected with reporter vectors and 50 nM miR-1207-5p inhibitor or negative control (Dharmacon) ( D ). Luciferase activity was measured 48 h after transfection using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to the corresponding renilla luciferase activity and plotted as a percentage of the control (HEK293 cells co-transfected with plasmid and control mimic or inhibitor). Approximately 70,000 MC/well were seeded in a 12-well plate and transfected with 3 µl Lipofectamine RNAiMAX (Life Technologies) mixed with 30 nM miR-1207-5p or negative control mimic, and 50 nM anti-miR-1207-5p or negative control. About 24 h after transfection, cells were stimulated with high glucose [30 mM] and incubated for another 24 h. Afterwards, cell culture media was removed, and cells were lysed and phosphorylathed Smad3 was quantified using phospho-SMAD3 (ser423/425) Instant One ELISA (eBiosciences), as per manufacturer's instructions ( E ). Experiments showed in C, D, and E were performed in quadruplicate. The significance is indicated only for samples that are significant different from all the others. * P
    Figure Legend Snippet: Validation of putative miR-1207-5p target genes. miR-1207-5p target genes were identified by TargetScan software and four genes were selected using Pathway Studio software based on potential biological effects relevant to ECM accumulation. The four genes selected, G6PD, PMEPA1, PDPK1, and SMAD7 , were validated as putative miR-1207-5p target genes in MC using 25 nM miR-1207-5p or NC mimic ( A ), and 50 nM anti-miR-1207-5p or negative control ( B ). Results in A and B represent averages from three independent experiments. Data are means ± SD. About 20,000 human embryonic kidney 293 (HEK293) cells were seeded per well into a white 96-well plate and co-transfected with 100 ng of the indicated 3′UTR luciferase reporter vectors and 30 nM miR-1207-5p or negative control mimic (Dharmacon) using 0.2 µl per well of Lipofectamine 2000 ( C ). HEK293 cells were also co-transfected with reporter vectors and 50 nM miR-1207-5p inhibitor or negative control (Dharmacon) ( D ). Luciferase activity was measured 48 h after transfection using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to the corresponding renilla luciferase activity and plotted as a percentage of the control (HEK293 cells co-transfected with plasmid and control mimic or inhibitor). Approximately 70,000 MC/well were seeded in a 12-well plate and transfected with 3 µl Lipofectamine RNAiMAX (Life Technologies) mixed with 30 nM miR-1207-5p or negative control mimic, and 50 nM anti-miR-1207-5p or negative control. About 24 h after transfection, cells were stimulated with high glucose [30 mM] and incubated for another 24 h. Afterwards, cell culture media was removed, and cells were lysed and phosphorylathed Smad3 was quantified using phospho-SMAD3 (ser423/425) Instant One ELISA (eBiosciences), as per manufacturer's instructions ( E ). Experiments showed in C, D, and E were performed in quadruplicate. The significance is indicated only for samples that are significant different from all the others. * P

    Techniques Used: Software, Negative Control, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effect of miR-1207-5p on TGF-β1, PAI-1 and FN1. Relative quantification of TGF-β1 ( A ) and PAI-1 ( B ) mRNA and secreted protein in MC over-expressing miR-1207-5p compared to control. ( C ) Relative TGF-β1 and PAI-1 protein secreted by MC transfected with miR-1207-5p inhibitor or NC inhibitor compared to control. ( D ) Relative quantification of secreted FN1 from MC with miR-1207-5p over-expression or knockdown compared to control. Cells were transfected with 3 µl Lipofectamine RNAiMAX mixed with 30 nM miR-1207-5p or negative control (NC) mimic, or 50 nM of miR-1207-5p inhibitor or NC inhibitor. Specific mRNAs were quantified by TaqMan qPCR using PPIA and UBC as endogenous controls. Secreted TGF-β1, PAI-1, and FN1 were determined by ELISA. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P
    Figure Legend Snippet: Effect of miR-1207-5p on TGF-β1, PAI-1 and FN1. Relative quantification of TGF-β1 ( A ) and PAI-1 ( B ) mRNA and secreted protein in MC over-expressing miR-1207-5p compared to control. ( C ) Relative TGF-β1 and PAI-1 protein secreted by MC transfected with miR-1207-5p inhibitor or NC inhibitor compared to control. ( D ) Relative quantification of secreted FN1 from MC with miR-1207-5p over-expression or knockdown compared to control. Cells were transfected with 3 µl Lipofectamine RNAiMAX mixed with 30 nM miR-1207-5p or negative control (NC) mimic, or 50 nM of miR-1207-5p inhibitor or NC inhibitor. Specific mRNAs were quantified by TaqMan qPCR using PPIA and UBC as endogenous controls. Secreted TGF-β1, PAI-1, and FN1 were determined by ELISA. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P

    Techniques Used: Expressing, Transfection, Over Expression, Negative Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Kdm4c is Recruited to Mitotic Chromosomes and Is Relevant for Chromosomal Stability, Cell Migration and Invasion of Triple Negative Breast Cancer Cells"

    Article Title: Kdm4c is Recruited to Mitotic Chromosomes and Is Relevant for Chromosomal Stability, Cell Migration and Invasion of Triple Negative Breast Cancer Cells

    Journal: Breast Cancer : Basic and Clinical Research

    doi: 10.1177/1178223418773075

    KDM4C depletion by siRNA treatment and its effect on cell proliferation. All images and data correspond to representative samples of at least two independent experiments performed by triplicate (A) IFAs with siRNA-treated or untreated HCC38 cells, determined as Methods section description. Note that KDM4C, but no KDM4A, is present on mitotic chromosomes during mitosis, and the significant reduction on fluorescence intensity for siRNA1 and siRNA2 samples with respect to the untreated cells, where KDM4C signal collocates with DAPI staining. (B) Western blot of total protein extracts from cells treated with each siRNA compared with total protein extracts from control cells treated with Lipofectamine RNAiMAX alone. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The intensity of the bands was analyzed by ImageJ software (NIH, USA) showing a reduction on KDM4C levels up to 85% (Data not shown), for the treatment with siRNAs 1 and 2, while a third tested siRNA 3C was not able to significantly reduce KDM4C expression and it wasn’t considered for further experiments. (C) Cell proliferation assays. Optical density at 5550 nm was evaluated at 24 hour and 48 hour by an MTT assay, with a significant reduction in the Optical density at 550 nm for siRNAi-treated cells (1 and 2) with respect to the control (C).
    Figure Legend Snippet: KDM4C depletion by siRNA treatment and its effect on cell proliferation. All images and data correspond to representative samples of at least two independent experiments performed by triplicate (A) IFAs with siRNA-treated or untreated HCC38 cells, determined as Methods section description. Note that KDM4C, but no KDM4A, is present on mitotic chromosomes during mitosis, and the significant reduction on fluorescence intensity for siRNA1 and siRNA2 samples with respect to the untreated cells, where KDM4C signal collocates with DAPI staining. (B) Western blot of total protein extracts from cells treated with each siRNA compared with total protein extracts from control cells treated with Lipofectamine RNAiMAX alone. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The intensity of the bands was analyzed by ImageJ software (NIH, USA) showing a reduction on KDM4C levels up to 85% (Data not shown), for the treatment with siRNAs 1 and 2, while a third tested siRNA 3C was not able to significantly reduce KDM4C expression and it wasn’t considered for further experiments. (C) Cell proliferation assays. Optical density at 5550 nm was evaluated at 24 hour and 48 hour by an MTT assay, with a significant reduction in the Optical density at 550 nm for siRNAi-treated cells (1 and 2) with respect to the control (C).

    Techniques Used: Fluorescence, Staining, Western Blot, Software, Expressing, MTT Assay

    7) Product Images from "Attenuation of Maladaptive Responses in Aortic Adventitial Fibroblasts through Stimuli-Triggered siRNA Release from Lipid-Polymer Nanocomplexes"

    Article Title: Attenuation of Maladaptive Responses in Aortic Adventitial Fibroblasts through Stimuli-Triggered siRNA Release from Lipid-Polymer Nanocomplexes

    Journal: Advanced biosystems

    doi: 10.1002/adbi.201700099

    Photo-controlled IL1β protein silencing with mPEG- b -P(APNBMA) polyplexes, Lipofectamine RNAiMAX lipoplexes, and hybrid nanocomplexes. AoAFs were treated with siRNA using the various carriers, irradiated with 365 nm light for either 0 min (black bars) or 10 min (gold bars), and lysed for western blot analysis at 48 h post-transfection. Data represent the IL1β protein expression levels relative to the levels of the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH), normalized to the protein levels in controls with no siRNA treatment. Results are shown as the mean ± standard deviation of data obtained from three independent experiments.
    Figure Legend Snippet: Photo-controlled IL1β protein silencing with mPEG- b -P(APNBMA) polyplexes, Lipofectamine RNAiMAX lipoplexes, and hybrid nanocomplexes. AoAFs were treated with siRNA using the various carriers, irradiated with 365 nm light for either 0 min (black bars) or 10 min (gold bars), and lysed for western blot analysis at 48 h post-transfection. Data represent the IL1β protein expression levels relative to the levels of the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH), normalized to the protein levels in controls with no siRNA treatment. Results are shown as the mean ± standard deviation of data obtained from three independent experiments.

    Techniques Used: Irradiation, Western Blot, Transfection, Expressing, Standard Deviation

    8) Product Images from "Lhx8 Mediated Wnt and TGFβ Pathways in Tooth Development and Regeneration"

    Article Title: Lhx8 Mediated Wnt and TGFβ Pathways in Tooth Development and Regeneration

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2015.06.004

    Lhx8 knockdown accelerates mesenchyme differentiation ( A ) Schematic representation of the Lhx8 intervention and dental epithelium and dental mesenchyme recombination procedures. Dental epithelium and mesenchyme of mandibular first molar at E16.5 are separated and then the dental mesenchyme is placed onto upper chamber of Transwell cell culture insert and further incubated with either RNAi or lentivirus for 1 hr at RT. Meanwhile the dental epithelium is kept in ice cold DPBS. Then the mesenchyme and epithelium are recombined and cultured for up to 7 days. ( B and C ) Representative data showing the location ( B ) and morphology ( C ) of the tooth germ of E16.5 mandibular first molar. ( D–F ) The top view of the recombined tooth germ right after recombination ( D ), 1 day culture ( E ), 3 days culture ( F ) and 7 days culture ( G, control group; K , siLhx8 group). ( H–N ) The recombined tooth germs from control ( G–J ) and siLhx8 (K–N) were harvested at day 7 and chemically stained with HE ( H , I , L and M ) and immunostained with Dspp ( J , N ) ( n=5) . ( O–Q ) Immunofluorescence of the isolated E16.5 dental mesenchyme transfected with Lipofectamine RNAiMAX coated Cy3 labeled RNAi duplex before recombination. Strong positive Cy3 dots indicate high transfection efficiency. ( R ) Knockdown efficiency of Lhx8. ( S , T ) Quantification data of dentin area ( S ) and thickness ( T ). ( U ) Tooth development related gene expression were examined by qPCR in the recombined tooth germs 4 days after in vitro culture from both control and siLhx8 groups. Data presented are a representative of 3 experiments. de, dental epithelium; dm, dental mesenchyme; d, dentin; od, odontoblast; am, ameloblast; e, enamel. Scale bars: 1mm ( B–F, Q and K, O–Q) ; 500 μm ( I and M ); 250 μm ( J and N ); Data represent mean ± SD. * P
    Figure Legend Snippet: Lhx8 knockdown accelerates mesenchyme differentiation ( A ) Schematic representation of the Lhx8 intervention and dental epithelium and dental mesenchyme recombination procedures. Dental epithelium and mesenchyme of mandibular first molar at E16.5 are separated and then the dental mesenchyme is placed onto upper chamber of Transwell cell culture insert and further incubated with either RNAi or lentivirus for 1 hr at RT. Meanwhile the dental epithelium is kept in ice cold DPBS. Then the mesenchyme and epithelium are recombined and cultured for up to 7 days. ( B and C ) Representative data showing the location ( B ) and morphology ( C ) of the tooth germ of E16.5 mandibular first molar. ( D–F ) The top view of the recombined tooth germ right after recombination ( D ), 1 day culture ( E ), 3 days culture ( F ) and 7 days culture ( G, control group; K , siLhx8 group). ( H–N ) The recombined tooth germs from control ( G–J ) and siLhx8 (K–N) were harvested at day 7 and chemically stained with HE ( H , I , L and M ) and immunostained with Dspp ( J , N ) ( n=5) . ( O–Q ) Immunofluorescence of the isolated E16.5 dental mesenchyme transfected with Lipofectamine RNAiMAX coated Cy3 labeled RNAi duplex before recombination. Strong positive Cy3 dots indicate high transfection efficiency. ( R ) Knockdown efficiency of Lhx8. ( S , T ) Quantification data of dentin area ( S ) and thickness ( T ). ( U ) Tooth development related gene expression were examined by qPCR in the recombined tooth germs 4 days after in vitro culture from both control and siLhx8 groups. Data presented are a representative of 3 experiments. de, dental epithelium; dm, dental mesenchyme; d, dentin; od, odontoblast; am, ameloblast; e, enamel. Scale bars: 1mm ( B–F, Q and K, O–Q) ; 500 μm ( I and M ); 250 μm ( J and N ); Data represent mean ± SD. * P

    Techniques Used: Cell Culture, Incubation, Staining, Immunofluorescence, Isolation, Transfection, Labeling, Expressing, Real-time Polymerase Chain Reaction, In Vitro

    9) Product Images from "Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells"

    Article Title: Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-18-65

    Effect of TLR3 knock-down on poly(I:C)-induced apoptosis in SK-N-AS cells . After transfection with siRNA (60 nM) for 24 h, the cells were incubated with poly(I:C) 50 μg/ml or vehicle for another 24 h and were then harvested for assay. C, control; LI, lipofectamine; SC. Scramble control siRNA; siTLR3, TLR3 siRNA. (A) Effect of TLR3 siRNA knockdown on poly(I:C)-induced caspase-3 activation. Arrows indicated the molecular weight of active caspase-3 at 17 and 19 kDa, respectively. Quantification results of caspase-3 activation in different treatment groups were expressed as mean ± SD from triplicate experiments (bottom panel). *: P
    Figure Legend Snippet: Effect of TLR3 knock-down on poly(I:C)-induced apoptosis in SK-N-AS cells . After transfection with siRNA (60 nM) for 24 h, the cells were incubated with poly(I:C) 50 μg/ml or vehicle for another 24 h and were then harvested for assay. C, control; LI, lipofectamine; SC. Scramble control siRNA; siTLR3, TLR3 siRNA. (A) Effect of TLR3 siRNA knockdown on poly(I:C)-induced caspase-3 activation. Arrows indicated the molecular weight of active caspase-3 at 17 and 19 kDa, respectively. Quantification results of caspase-3 activation in different treatment groups were expressed as mean ± SD from triplicate experiments (bottom panel). *: P

    Techniques Used: Transfection, Incubation, Activation Assay, Molecular Weight

    10) Product Images from "Heat shock proteins stimulate APOBEC-3–mediated cytidine deamination in the hepatitis B virus"

    Article Title: Heat shock proteins stimulate APOBEC-3–mediated cytidine deamination in the hepatitis B virus

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.760637

    Effect of Hsp knockdown through siRNA on HBV mutation frequency. Each Hsp siRNA was transfected into HepG2 cells by a reverse transfection method with Lipofectamine RNAiMax. After a 24-h siRNA treatment, the cells were transfected with an HBV viral genome-encoding plasmid with or without A3G. The HepG2 cells were harvested for RNA or HBV extraction 24 h after HBV transfection. A , Hsp mRNA levels after siRNA treatment by quantitative RT-PCR determination. The relative mRNA levels for each siRNA treatment were determined by quantitative RT-PCR, using GAPDH as an internal reference. The mRNA levels relative to control were calculated and are represented graphically as percentages with the control as 100%. Each bar represents the average of triplicates for each treatment. Blank , background control by a scrambled negative siRNA without A3G co-transfection. Control , another control by the scrambled negative siRNA with A3G co-transfection. B , Hsp protein expression level analyses after siRNA treatment. Total cellular proteins were extracted from HepG2 cells after siRNA treatment, and Hsp protein levels in the cell lysates were analyzed by Western blotting with antibodies against endogenous Hsp90β, Hsp90α, Hsp70, and Hsp40. GAPDH was analyzed as the protein loading reference. C , HBV DNA mutation analyses. HBV DNAs were extracted from the cell lysates after a 24-h HBV transfection, and the resultant HBV DNA mutations were determined by pe1453 using an 88 °C 3D-PCR. The data are presented graphically on the right . Each bar represents the average of triplicates for each treatment. Asterisks , statistically significant differences comparing treatments with their corresponding control: **, 0.01
    Figure Legend Snippet: Effect of Hsp knockdown through siRNA on HBV mutation frequency. Each Hsp siRNA was transfected into HepG2 cells by a reverse transfection method with Lipofectamine RNAiMax. After a 24-h siRNA treatment, the cells were transfected with an HBV viral genome-encoding plasmid with or without A3G. The HepG2 cells were harvested for RNA or HBV extraction 24 h after HBV transfection. A , Hsp mRNA levels after siRNA treatment by quantitative RT-PCR determination. The relative mRNA levels for each siRNA treatment were determined by quantitative RT-PCR, using GAPDH as an internal reference. The mRNA levels relative to control were calculated and are represented graphically as percentages with the control as 100%. Each bar represents the average of triplicates for each treatment. Blank , background control by a scrambled negative siRNA without A3G co-transfection. Control , another control by the scrambled negative siRNA with A3G co-transfection. B , Hsp protein expression level analyses after siRNA treatment. Total cellular proteins were extracted from HepG2 cells after siRNA treatment, and Hsp protein levels in the cell lysates were analyzed by Western blotting with antibodies against endogenous Hsp90β, Hsp90α, Hsp70, and Hsp40. GAPDH was analyzed as the protein loading reference. C , HBV DNA mutation analyses. HBV DNAs were extracted from the cell lysates after a 24-h HBV transfection, and the resultant HBV DNA mutations were determined by pe1453 using an 88 °C 3D-PCR. The data are presented graphically on the right . Each bar represents the average of triplicates for each treatment. Asterisks , statistically significant differences comparing treatments with their corresponding control: **, 0.01

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Quantitative RT-PCR, Cotransfection, Expressing, Western Blot, Polymerase Chain Reaction

    11) Product Images from "RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells"

    Article Title: RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells

    Journal: Scientific Reports

    doi: 10.1038/srep17184

    Chemical transfection of HSPCs with functional siRNAs shows no target protein knockdown but electroporation does. CD133 + HSPCs or indicated cell lines (Weri-RB1, K562) were transfected with siRNAs targeting CD133 ( a,c ), CD45 ( b,d ) or a non-targeting control siRNA (ctrl). Displayed is the median fluorescence intensity (MFI) of the respective surface marker as percentage relative to the control siRNA ( a,b ) Relative protein expression at the time of maximum knockdown (72 h after transfection, ( # ) 24 h after transfection; n = 2–10). ( c,d ) Kinetics of relative protein expression at the indicated time points after transfection. Open circles (О) represent cells transfected with non-targeting control siRNA (Neg-siRNA), closed triangles (▲) represent CD133- and CD45-siRNA in ( c,d ), respectively (mean of n = 3 at day 1–3, mean of n = 2 at day 6 and 8). LF: Lipofectamine 2000, RM: Lipofectamine RNAiMAX, HP: HiPerfect, Elpo: Electroporation, PEI: Polyethylenimine 2 K. Error bars represent SD. ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant, one-way ANOVA.
    Figure Legend Snippet: Chemical transfection of HSPCs with functional siRNAs shows no target protein knockdown but electroporation does. CD133 + HSPCs or indicated cell lines (Weri-RB1, K562) were transfected with siRNAs targeting CD133 ( a,c ), CD45 ( b,d ) or a non-targeting control siRNA (ctrl). Displayed is the median fluorescence intensity (MFI) of the respective surface marker as percentage relative to the control siRNA ( a,b ) Relative protein expression at the time of maximum knockdown (72 h after transfection, ( # ) 24 h after transfection; n = 2–10). ( c,d ) Kinetics of relative protein expression at the indicated time points after transfection. Open circles (О) represent cells transfected with non-targeting control siRNA (Neg-siRNA), closed triangles (▲) represent CD133- and CD45-siRNA in ( c,d ), respectively (mean of n = 3 at day 1–3, mean of n = 2 at day 6 and 8). LF: Lipofectamine 2000, RM: Lipofectamine RNAiMAX, HP: HiPerfect, Elpo: Electroporation, PEI: Polyethylenimine 2 K. Error bars represent SD. ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant, one-way ANOVA.

    Techniques Used: Transfection, Functional Assay, Electroporation, Fluorescence, Marker, Expressing

    Transfection of fluorescent siRNA into CD133 + HSPCs with liposomal reagents and cationic polymers. Cells were analysed 19 to 24 h after transfection with different reagents and either a non-labelled, non-targeting negative control (siRNA-Neg) or a fluorescently labelled, non-targeting siRNA (siRNA-AF488). Cells were washed, stained with CD133-PE, and analysed via flow cytometry. ( a ) Representative dot plots after transfection with Lipofectamine 2000 (LF) and HiPerFect (HP), gated on viable cells. ( b ) Percentage of total AF488 + cells after optimisation of transfection conditions. Each dot represents one independent experiment (n = 2–8). RM: Lipofectamine RNAiMAX, DT: DOTAP, PEI: Polyethylenimine 2K, EG: ExGen 500.
    Figure Legend Snippet: Transfection of fluorescent siRNA into CD133 + HSPCs with liposomal reagents and cationic polymers. Cells were analysed 19 to 24 h after transfection with different reagents and either a non-labelled, non-targeting negative control (siRNA-Neg) or a fluorescently labelled, non-targeting siRNA (siRNA-AF488). Cells were washed, stained with CD133-PE, and analysed via flow cytometry. ( a ) Representative dot plots after transfection with Lipofectamine 2000 (LF) and HiPerFect (HP), gated on viable cells. ( b ) Percentage of total AF488 + cells after optimisation of transfection conditions. Each dot represents one independent experiment (n = 2–8). RM: Lipofectamine RNAiMAX, DT: DOTAP, PEI: Polyethylenimine 2K, EG: ExGen 500.

    Techniques Used: Transfection, Negative Control, Staining, Flow Cytometry, Cytometry

    12) Product Images from "Nucleotide Modifications Decrease Innate Immune Response Induced by Synthetic Analogs of snRNAs and snoRNAs"

    Article Title: Nucleotide Modifications Decrease Innate Immune Response Induced by Synthetic Analogs of snRNAs and snoRNAs

    Journal: Genes

    doi: 10.3390/genes9110531

    The viability of MCF-7 cells 24 h after transfection with snRNA and snoRNA analogs. Cells were transfected with 10 nM of non-modified ( NM ) and m 5 C- and Ψ-containing ( 20% m 5 C/30% Ψ , 50% m 5 C/50% Ψ , 100% m 5 C and 100% Ψ ) U25, U35a snoRNAs, and U12 snRNA analogs with Lipofectamine RNAiMAX. The asterisks (*) indicate analogs with additional trimethylated m 3 2,2,7 G cap. Control cells were incubated with Lipofectamine RNAiMAX only. Data are presented as the mean of at least three independent experiments. The error bars represent standard deviations. The difference between the “control” and transfected cell groups was statistically significant at p
    Figure Legend Snippet: The viability of MCF-7 cells 24 h after transfection with snRNA and snoRNA analogs. Cells were transfected with 10 nM of non-modified ( NM ) and m 5 C- and Ψ-containing ( 20% m 5 C/30% Ψ , 50% m 5 C/50% Ψ , 100% m 5 C and 100% Ψ ) U25, U35a snoRNAs, and U12 snRNA analogs with Lipofectamine RNAiMAX. The asterisks (*) indicate analogs with additional trimethylated m 3 2,2,7 G cap. Control cells were incubated with Lipofectamine RNAiMAX only. Data are presented as the mean of at least three independent experiments. The error bars represent standard deviations. The difference between the “control” and transfected cell groups was statistically significant at p

    Techniques Used: Transfection, Modification, Incubation

    13) Product Images from "Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleotide excision repair and cell-cycle checkpoints"

    Article Title: Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleotide excision repair and cell-cycle checkpoints

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17958-z

    Augmented synthetic lethality for Pt by co-targeting XPA and MK2 in vivo. a Schematic of dual-targeting peptide-based nanoplexes. b MK2 and XPA knockdown efficiency of nanoplex–siMK2/siXPA compared to lipofectamine RNAiMax–siMK2/siXPA measured by Western blotting for MK2 and XPA. Data representative of two independent experiments. c Representative bioluminescence images before and after indicated siRNA and cisplatin treatment on days 22, 29, 36, and 43. d Quantification of lung bioluminescence at 43 days after tumor implantation ( n = 3 animals were used for each condition; only two mice remained alive in the nanoplex–siControl + cisplatin at day 43, and they died by day 50). Error bars represent mean ± SEM. e Representative H E and Ki67 lung staining at the end of three rounds of the indicated treatments. Three animals were used for each condition. f Kaplan–Meier survival analysis of tumor-bearing mice treated with the indicated nanoplex–siRNA in combination with cisplatin treatment (nanoplex–siControl + cisplatin n = 6 animals, nanoplex–siMK2 + cisplatin n = 3 animals, nanoplex–siXPA + cisplatin n = 5 animals, and nanoplex–siMK2/siXPA + cisplatin n = 5 animals. * p ≤ 0.05 and ** p ≤ 0.01 calculated using the log-rank test). g Model illustrating crosstalk between the MK2 signaling pathway and nucleotide excision repair in p53-defective cells. Co-targeting these pathways in established tumors prolongs spontaneous survival and potently enhances the antitumor response to cisplatin treatment.
    Figure Legend Snippet: Augmented synthetic lethality for Pt by co-targeting XPA and MK2 in vivo. a Schematic of dual-targeting peptide-based nanoplexes. b MK2 and XPA knockdown efficiency of nanoplex–siMK2/siXPA compared to lipofectamine RNAiMax–siMK2/siXPA measured by Western blotting for MK2 and XPA. Data representative of two independent experiments. c Representative bioluminescence images before and after indicated siRNA and cisplatin treatment on days 22, 29, 36, and 43. d Quantification of lung bioluminescence at 43 days after tumor implantation ( n = 3 animals were used for each condition; only two mice remained alive in the nanoplex–siControl + cisplatin at day 43, and they died by day 50). Error bars represent mean ± SEM. e Representative H E and Ki67 lung staining at the end of three rounds of the indicated treatments. Three animals were used for each condition. f Kaplan–Meier survival analysis of tumor-bearing mice treated with the indicated nanoplex–siRNA in combination with cisplatin treatment (nanoplex–siControl + cisplatin n = 6 animals, nanoplex–siMK2 + cisplatin n = 3 animals, nanoplex–siXPA + cisplatin n = 5 animals, and nanoplex–siMK2/siXPA + cisplatin n = 5 animals. * p ≤ 0.05 and ** p ≤ 0.01 calculated using the log-rank test). g Model illustrating crosstalk between the MK2 signaling pathway and nucleotide excision repair in p53-defective cells. Co-targeting these pathways in established tumors prolongs spontaneous survival and potently enhances the antitumor response to cisplatin treatment.

    Techniques Used: In Vivo, Western Blot, Tumor Implantation, Mouse Assay, Staining

    14) Product Images from "Translationally controlled tumour protein TCTP is induced early in human colorectal tumours and contributes to the resistance of HCT116 colon cancer cells to 5-FU and oxaliplatin"

    Article Title: Translationally controlled tumour protein TCTP is induced early in human colorectal tumours and contributes to the resistance of HCT116 colon cancer cells to 5-FU and oxaliplatin

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-017-0164-3

    TCTP partially protects HCT116 colon cancer cells against toxicity induced by 5-FU or oxaliplatin. a Effect of TCTP knock-down on cellular sensitivity to 5-FU or oxaliplatin, assessed by the the xCELLigence RTCA System. HCT116 cells were either mock-transfected or transfected with TCTP siRNA or Luciferase siRNA (as a control) using Lipofectamine™ RNAiMAX transfection reagent for 24 h, and subsequently incubated in the presence of the indicated concentrations of either 5-FU or oxaliplatin. Cell growth was monitored in real-time using the xCELLigence RTCA System. Representative growth curves are shown in the left panels for control cells and for treatment with 12.5 μM 5-FU or oxaliplatin, as an example. Relative cell numbers are expressed as Cell Index (CI). Right panels compare the relative CI-values for Luc-siRNA, and TCTP-siRNA at the time point, when the control cells reached a CI value of 10 (5-FU treatment) or of 5 (oxaliplatin treatment). b Effect of TCTP knock-down on cellular sensitivity to 5-FU or oxaliplatin, measured by the MTS endpoint assay for cytotoxicity. TCTP-siRNA or GAPDH-siRNA (as a control) were transfected into HCT116 cells using Lipofectamine™ RNAiMAX transfection reagent and, after 24 h, incubated in the presence of the indicated concentrations of drugs for another 48 h. Cell viability was assessed using the MTS endpoint assay and was plotted against the concentration of 5-FU (left graph) and that of oxaliplatin (right graph). Statistical significance was ascertained using 2WAY ANOVA (**** P
    Figure Legend Snippet: TCTP partially protects HCT116 colon cancer cells against toxicity induced by 5-FU or oxaliplatin. a Effect of TCTP knock-down on cellular sensitivity to 5-FU or oxaliplatin, assessed by the the xCELLigence RTCA System. HCT116 cells were either mock-transfected or transfected with TCTP siRNA or Luciferase siRNA (as a control) using Lipofectamine™ RNAiMAX transfection reagent for 24 h, and subsequently incubated in the presence of the indicated concentrations of either 5-FU or oxaliplatin. Cell growth was monitored in real-time using the xCELLigence RTCA System. Representative growth curves are shown in the left panels for control cells and for treatment with 12.5 μM 5-FU or oxaliplatin, as an example. Relative cell numbers are expressed as Cell Index (CI). Right panels compare the relative CI-values for Luc-siRNA, and TCTP-siRNA at the time point, when the control cells reached a CI value of 10 (5-FU treatment) or of 5 (oxaliplatin treatment). b Effect of TCTP knock-down on cellular sensitivity to 5-FU or oxaliplatin, measured by the MTS endpoint assay for cytotoxicity. TCTP-siRNA or GAPDH-siRNA (as a control) were transfected into HCT116 cells using Lipofectamine™ RNAiMAX transfection reagent and, after 24 h, incubated in the presence of the indicated concentrations of drugs for another 48 h. Cell viability was assessed using the MTS endpoint assay and was plotted against the concentration of 5-FU (left graph) and that of oxaliplatin (right graph). Statistical significance was ascertained using 2WAY ANOVA (**** P

    Techniques Used: Transfection, Luciferase, Incubation, End Point Assay, Concentration Assay

    15) Product Images from "Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation"

    Article Title: Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00213-3

    NLRP3 regulates autophagy through an inflammasome-independent mechanism via direct binding to mTOR. a HT-29 cells were transfected with NLRP3-specific siRNA or negative control siRNA using Lipofectamine RNAiMAX. Forty-eight hours after transfection, total protein was isolated and western blot performed. Quantification of the ratio of LC3-II/LC3-I and the total amount of LC3 (LC3-I plus LC3-II) relative to β-actin is presented. Statistical analysis was performed using Student t -test. Results represent mean + s.e.m. of three independent experiments, * P
    Figure Legend Snippet: NLRP3 regulates autophagy through an inflammasome-independent mechanism via direct binding to mTOR. a HT-29 cells were transfected with NLRP3-specific siRNA or negative control siRNA using Lipofectamine RNAiMAX. Forty-eight hours after transfection, total protein was isolated and western blot performed. Quantification of the ratio of LC3-II/LC3-I and the total amount of LC3 (LC3-I plus LC3-II) relative to β-actin is presented. Statistical analysis was performed using Student t -test. Results represent mean + s.e.m. of three independent experiments, * P

    Techniques Used: Binding Assay, Transfection, Negative Control, Isolation, Western Blot

    16) Product Images from "Development of a 2?,4?-BNA/LNA-based siRNA for Dyslipidemia and Assessment of the Effects of Its Chemical Modifications In Vivo"

    Article Title: Development of a 2?,4?-BNA/LNA-based siRNA for Dyslipidemia and Assessment of the Effects of Its Chemical Modifications In Vivo

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2012.32

    RNAi effects on apoB mRNA of modified siRNAs based on siLNA-2 and the downstream effects on IFIT-1 mRNA levels. ( a ) All siRNAs were transfected into NMuLi cells at concentrations of 10 or 100 nmol/l using Lipofectamine RNAiMAX. ApoB mRNA expression was normalized to GAPDH expression, and relative values were calculated using the no-transfection group. ( b ) Expression of interferon-induced tetratricopeptide repeats 1 (IFIT-1) mRNA was measured as the inflammatory response of the cell; IFIT-1 expression was normalized to GAPDH expression. Error bars indicate SD.
    Figure Legend Snippet: RNAi effects on apoB mRNA of modified siRNAs based on siLNA-2 and the downstream effects on IFIT-1 mRNA levels. ( a ) All siRNAs were transfected into NMuLi cells at concentrations of 10 or 100 nmol/l using Lipofectamine RNAiMAX. ApoB mRNA expression was normalized to GAPDH expression, and relative values were calculated using the no-transfection group. ( b ) Expression of interferon-induced tetratricopeptide repeats 1 (IFIT-1) mRNA was measured as the inflammatory response of the cell; IFIT-1 expression was normalized to GAPDH expression. Error bars indicate SD.

    Techniques Used: Modification, Transfection, Expressing

    17) Product Images from "Epigenetic Suppression of the T-box Subfamily 2 (TBX2) in Human Non-Small Cell Lung Cancer"

    Article Title: Epigenetic Suppression of the T-box Subfamily 2 (TBX2) in Human Non-Small Cell Lung Cancer

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20051159

    Impact of simultaneous knockdown of all four members of TBX2 subfamily on anti-growth effects of Aza in H1299 cells. ( A ) H1299 cells were transfected with control (4× scrambled) siRNAs and siRNAs targeting all four members of the TBX2 subfamily simultaneously (combination of four target-specific siRNAs) using Lipofectamine RNAiMAX as described in Section 4 . The day following transfection, cells were re-seeded in six cm dishes. The next day cells were incubated overnight with reduced serum (1% FBS) medium prior to treatment with either DMSO control or 1 µM Aza for 72 h. Total RNA was extracted and RNA expression levels of TBX2, TBX3, TBX4 and TBX5 were analyzed using the 2 −∆∆ C t calculation method by normalization to GAPDH . H1299 cells were transfected in the same manner described above, except they were re-seeded the following day in 96-well plates for MTT assays ( B ) and trypan blue exclusion analysis ( C ). Cell numbers (% of control DMSO treatment) were determined using MTT assays ( B ) and percentages of cell growth inhibition in Aza-treated cells were obtained using the trypan blue exclusion method ( C ). Growth inhibition following the trypan blue exclusion analysis was calculated using the following formula: (100 − (treated/non-treated) × 100) and is depicted for the Aza-treated cells. Data depicted represent mean ± SD for MTT assays and mean ± SEM of triplicates for trypan blue exclusion analysis (* p
    Figure Legend Snippet: Impact of simultaneous knockdown of all four members of TBX2 subfamily on anti-growth effects of Aza in H1299 cells. ( A ) H1299 cells were transfected with control (4× scrambled) siRNAs and siRNAs targeting all four members of the TBX2 subfamily simultaneously (combination of four target-specific siRNAs) using Lipofectamine RNAiMAX as described in Section 4 . The day following transfection, cells were re-seeded in six cm dishes. The next day cells were incubated overnight with reduced serum (1% FBS) medium prior to treatment with either DMSO control or 1 µM Aza for 72 h. Total RNA was extracted and RNA expression levels of TBX2, TBX3, TBX4 and TBX5 were analyzed using the 2 −∆∆ C t calculation method by normalization to GAPDH . H1299 cells were transfected in the same manner described above, except they were re-seeded the following day in 96-well plates for MTT assays ( B ) and trypan blue exclusion analysis ( C ). Cell numbers (% of control DMSO treatment) were determined using MTT assays ( B ) and percentages of cell growth inhibition in Aza-treated cells were obtained using the trypan blue exclusion method ( C ). Growth inhibition following the trypan blue exclusion analysis was calculated using the following formula: (100 − (treated/non-treated) × 100) and is depicted for the Aza-treated cells. Data depicted represent mean ± SD for MTT assays and mean ± SEM of triplicates for trypan blue exclusion analysis (* p

    Techniques Used: Transfection, Incubation, RNA Expression, MTT Assay, Inhibition

    18) Product Images from "Electrophilic Lipid Mediator 15-Deoxy-Δ12,14-Prostaglandin J2 Modifies Glucocorticoid Signaling via Receptor SUMOylation"

    Article Title: Electrophilic Lipid Mediator 15-Deoxy-Δ12,14-Prostaglandin J2 Modifies Glucocorticoid Signaling via Receptor SUMOylation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00748-14

    SUMOylation modulates the inhibition of GR by 15d-PGJ 2 . HEK293 cells stably expressing wtGR- and GR3KR were transfected with 40 nM Dharmacon On-TARGETplus SMARTpool for human UBE2I (siUBC9) or Non-Targeting pool (siNON) by Lipofectamine RNAiMAX. After
    Figure Legend Snippet: SUMOylation modulates the inhibition of GR by 15d-PGJ 2 . HEK293 cells stably expressing wtGR- and GR3KR were transfected with 40 nM Dharmacon On-TARGETplus SMARTpool for human UBE2I (siUBC9) or Non-Targeting pool (siNON) by Lipofectamine RNAiMAX. After

    Techniques Used: Inhibition, Stable Transfection, Expressing, Transfection

    19) Product Images from "Possible Linkage of SP6 Transcriptional Activity with Amelogenesis by Protein Stabilization"

    Article Title: Possible Linkage of SP6 Transcriptional Activity with Amelogenesis by Protein Stabilization

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2011/320987

    Specificity of SP6 induction by MG132 treatment. (a) Subcellular localization of SP6 in C9 cells treated with MG132. SP6 localization was analysed by immunofluorescence signals. Red colour indicates SP6 detected by anti-HA antibody, blue colour shows nuclei stained with Hoechst 33342. (a–d): low magnification; (e–h): high magnification. Scale bar indicates 200 μ m. (b) Knockdown of SP6 in MG132-treated C9 cells by siRNA. Posi: positive control; N: no treatment; M: Lipofectamine RNAiMAX only; GC: negative control siRNA; si: siRNA against Sp6 . Upper panel: SP6 expression was detected by WB. Lower panel: relative SP6 levels estimated by densitometric analysis of WB results.
    Figure Legend Snippet: Specificity of SP6 induction by MG132 treatment. (a) Subcellular localization of SP6 in C9 cells treated with MG132. SP6 localization was analysed by immunofluorescence signals. Red colour indicates SP6 detected by anti-HA antibody, blue colour shows nuclei stained with Hoechst 33342. (a–d): low magnification; (e–h): high magnification. Scale bar indicates 200 μ m. (b) Knockdown of SP6 in MG132-treated C9 cells by siRNA. Posi: positive control; N: no treatment; M: Lipofectamine RNAiMAX only; GC: negative control siRNA; si: siRNA against Sp6 . Upper panel: SP6 expression was detected by WB. Lower panel: relative SP6 levels estimated by densitometric analysis of WB results.

    Techniques Used: Immunofluorescence, Staining, Positive Control, Negative Control, Expressing, Western Blot

    20) Product Images from "Downregulation of microRNA-100 enhances the ICMT-Rac1 signaling and promotes metastasis of hepatocellular carcinoma cells"

    Article Title: Downregulation of microRNA-100 enhances the ICMT-Rac1 signaling and promotes metastasis of hepatocellular carcinoma cells

    Journal: Oncotarget

    doi:

    Rac1 and ICMT are direct targets of miR-100 (A) miR-100 and its putative binding sequences in the 3′-UTR of Rac1 and ICMT. Mutations were generated in the complementary site that binds to the seed region of miR-100. (B) miR-100 overexpression suppressed the activity of renilla luciferase that carried the wild-type but not mutant 3′-UTR of Rac1 and ICMT. QGY-7703 cells were co-transfected with the indicated RNA duplex and psiCHECK2 luciferase reporter plasmid containing wild-type or mutant 3′-UTR (indicated as WT or MUT on the X axis) of putative target genes. The values for the luciferase activity assays were from three independent experiments that were performed in duplicate. (C) Reintroduction of miR-100 reduced the endogenous level of Rac1 and ICMT proteins in HCC cell lines. Left and middle panels, QGY-7703 and SMMC-7721 cells without treatment (lane 1), treated with Lipofectamine RNAiMax (lane 2), or transfected with the indicated RNA duplex (lanes 3-4). Right panel, Hepa1-6 stable subclones. (D) Inhibition of miR-100 increased the protein levels of Rac1 and ICMT. Forty-eight hours after transfection with anti-miR-C or anti-miR-100, SMMC-7721 cells were analyzed by immunoblotting. For (C and D), the results were reproducible in three independent experiments. β-actin, internal control. (E and F) Mouse orthotopic xenografts of Hepa-miR-100 cells showed much lower Rac1 and ICMT expression than those of Hepa-Ctrl cells. (G) The level of miR-100 was inversely correlated with Rac1 expression in human HCC tissues. Rac1 expression was quantified based on immunohistochemical staining and miR-100 levels were detected by qPCR. Brown signal was considered as positive staining. Scale bar, 50 μm. * P
    Figure Legend Snippet: Rac1 and ICMT are direct targets of miR-100 (A) miR-100 and its putative binding sequences in the 3′-UTR of Rac1 and ICMT. Mutations were generated in the complementary site that binds to the seed region of miR-100. (B) miR-100 overexpression suppressed the activity of renilla luciferase that carried the wild-type but not mutant 3′-UTR of Rac1 and ICMT. QGY-7703 cells were co-transfected with the indicated RNA duplex and psiCHECK2 luciferase reporter plasmid containing wild-type or mutant 3′-UTR (indicated as WT or MUT on the X axis) of putative target genes. The values for the luciferase activity assays were from three independent experiments that were performed in duplicate. (C) Reintroduction of miR-100 reduced the endogenous level of Rac1 and ICMT proteins in HCC cell lines. Left and middle panels, QGY-7703 and SMMC-7721 cells without treatment (lane 1), treated with Lipofectamine RNAiMax (lane 2), or transfected with the indicated RNA duplex (lanes 3-4). Right panel, Hepa1-6 stable subclones. (D) Inhibition of miR-100 increased the protein levels of Rac1 and ICMT. Forty-eight hours after transfection with anti-miR-C or anti-miR-100, SMMC-7721 cells were analyzed by immunoblotting. For (C and D), the results were reproducible in three independent experiments. β-actin, internal control. (E and F) Mouse orthotopic xenografts of Hepa-miR-100 cells showed much lower Rac1 and ICMT expression than those of Hepa-Ctrl cells. (G) The level of miR-100 was inversely correlated with Rac1 expression in human HCC tissues. Rac1 expression was quantified based on immunohistochemical staining and miR-100 levels were detected by qPCR. Brown signal was considered as positive staining. Scale bar, 50 μm. * P

    Techniques Used: Binding Assay, Generated, Over Expression, Activity Assay, Luciferase, Mutagenesis, Transfection, Plasmid Preparation, Inhibition, Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

    21) Product Images from "Kdm4c is Recruited to Mitotic Chromosomes and Is Relevant for Chromosomal Stability, Cell Migration and Invasion of Triple Negative Breast Cancer Cells"

    Article Title: Kdm4c is Recruited to Mitotic Chromosomes and Is Relevant for Chromosomal Stability, Cell Migration and Invasion of Triple Negative Breast Cancer Cells

    Journal: Breast Cancer : Basic and Clinical Research

    doi: 10.1177/1178223418773075

    KDM4C depletion by siRNA treatment and its effect on cell proliferation. All images and data correspond to representative samples of at least two independent experiments performed by triplicate (A) IFAs with siRNA-treated or untreated HCC38 cells, determined as Methods section description. Note that KDM4C, but no KDM4A, is present on mitotic chromosomes during mitosis, and the significant reduction on fluorescence intensity for siRNA1 and siRNA2 samples with respect to the untreated cells, where KDM4C signal collocates with DAPI staining. (B) Western blot of total protein extracts from cells treated with each siRNA compared with total protein extracts from control cells treated with Lipofectamine RNAiMAX alone. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The intensity of the bands was analyzed by ImageJ software (NIH, USA) showing a reduction on KDM4C levels up to 85% (Data not shown), for the treatment with siRNAs 1 and 2, while a third tested siRNA 3C was not able to significantly reduce KDM4C expression and it wasn’t considered for further experiments. (C) Cell proliferation assays. Optical density at 5550 nm was evaluated at 24 hour and 48 hour by an MTT assay, with a significant reduction in the Optical density at 550 nm for siRNAi-treated cells (1 and 2) with respect to the control (C).
    Figure Legend Snippet: KDM4C depletion by siRNA treatment and its effect on cell proliferation. All images and data correspond to representative samples of at least two independent experiments performed by triplicate (A) IFAs with siRNA-treated or untreated HCC38 cells, determined as Methods section description. Note that KDM4C, but no KDM4A, is present on mitotic chromosomes during mitosis, and the significant reduction on fluorescence intensity for siRNA1 and siRNA2 samples with respect to the untreated cells, where KDM4C signal collocates with DAPI staining. (B) Western blot of total protein extracts from cells treated with each siRNA compared with total protein extracts from control cells treated with Lipofectamine RNAiMAX alone. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The intensity of the bands was analyzed by ImageJ software (NIH, USA) showing a reduction on KDM4C levels up to 85% (Data not shown), for the treatment with siRNAs 1 and 2, while a third tested siRNA 3C was not able to significantly reduce KDM4C expression and it wasn’t considered for further experiments. (C) Cell proliferation assays. Optical density at 5550 nm was evaluated at 24 hour and 48 hour by an MTT assay, with a significant reduction in the Optical density at 550 nm for siRNAi-treated cells (1 and 2) with respect to the control (C).

    Techniques Used: Fluorescence, Staining, Western Blot, Software, Expressing, MTT Assay

    22) Product Images from "Switching the intracellular pathway and enhancing the therapeutic efficacy of small interfering RNA by auroliposome"

    Article Title: Switching the intracellular pathway and enhancing the therapeutic efficacy of small interfering RNA by auroliposome

    Journal: Science Advances

    doi: 10.1126/sciadv.aba5379

    Effect of nanoparticle size, shape, and core material in gene silencing efficacy AuroLPs. ( A to C ) WB demonstrating effect on MICU1 protein expression by treatment with nanoparticles of different sizes, shapes, and core materials containing MICU1-siRNA entrapped in traditional liposome or along with inorganic nanoparticles. (C) MICU1-siRNA-20 nm Fe 3 O 4 LPs effect on MICU1 protein expression as observed by WB. ( D ) Analysis of MICU1 silencing at the mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR) via the treatment of cells with CTL-siRNA-AuroLPs, 20-nm AuNP, CTL-siRNA-HF, MICU1-siRNA-AuroLPs, MICU1-siRNA-cLPs, complex (1) (25 nM) or complex (2) (100 nM), and MICU1-siRNA-20 nm AuNP. ( E ) WB analysis demonstrating silencing efficacy of MICU1-siRNA in CTL-siRNA-HF complex (100 nM), complex (1) and complex-2 or MICU1-siRNA-cLPs, MICU1-siRNA-AuroLPs, MICU1-siRNA-Lipofectamine 3000, and MICU1-siRNA-RNAiMax complex (25 nM) by treating the cells for 72 hours. ( F ) Stability of MICU1-siRNA, MICU1-siRNA-cLPs, MICU1-siRNA-AuroLPs, MICU1-siRNA-HF complex, and MICU1-siRNA-20 nm AuNP for 96 hours by electrophoresis. ( G ) Inhibition of clonal growth by treatment with CTL-siRNA-AuroLPs, 20-nm AuNP, CTL-siRNA complex, MICU1-siRNA-AuroLPs, MICU1-siRNA-cLPs, complex (1) or complex (2), and conjugate (25 nM) for 12-day through crystal violet staining. Data were expressed as means ± SD. ImageJ was used for quantitation of MICU1 protein, normalized by GAPDH as loading control.
    Figure Legend Snippet: Effect of nanoparticle size, shape, and core material in gene silencing efficacy AuroLPs. ( A to C ) WB demonstrating effect on MICU1 protein expression by treatment with nanoparticles of different sizes, shapes, and core materials containing MICU1-siRNA entrapped in traditional liposome or along with inorganic nanoparticles. (C) MICU1-siRNA-20 nm Fe 3 O 4 LPs effect on MICU1 protein expression as observed by WB. ( D ) Analysis of MICU1 silencing at the mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR) via the treatment of cells with CTL-siRNA-AuroLPs, 20-nm AuNP, CTL-siRNA-HF, MICU1-siRNA-AuroLPs, MICU1-siRNA-cLPs, complex (1) (25 nM) or complex (2) (100 nM), and MICU1-siRNA-20 nm AuNP. ( E ) WB analysis demonstrating silencing efficacy of MICU1-siRNA in CTL-siRNA-HF complex (100 nM), complex (1) and complex-2 or MICU1-siRNA-cLPs, MICU1-siRNA-AuroLPs, MICU1-siRNA-Lipofectamine 3000, and MICU1-siRNA-RNAiMax complex (25 nM) by treating the cells for 72 hours. ( F ) Stability of MICU1-siRNA, MICU1-siRNA-cLPs, MICU1-siRNA-AuroLPs, MICU1-siRNA-HF complex, and MICU1-siRNA-20 nm AuNP for 96 hours by electrophoresis. ( G ) Inhibition of clonal growth by treatment with CTL-siRNA-AuroLPs, 20-nm AuNP, CTL-siRNA complex, MICU1-siRNA-AuroLPs, MICU1-siRNA-cLPs, complex (1) or complex (2), and conjugate (25 nM) for 12-day through crystal violet staining. Data were expressed as means ± SD. ImageJ was used for quantitation of MICU1 protein, normalized by GAPDH as loading control.

    Techniques Used: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Electrophoresis, Inhibition, Staining, Quantitation Assay

    23) Product Images from "Efficient cell penetration and delivery of peptide nucleic acids by an argininocalix[4]arene"

    Article Title: Efficient cell penetration and delivery of peptide nucleic acids by an argininocalix[4]arene

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39211-4

    Effects of calixarene 1 on viability, cell proliferation and morphology of U251 cells. ( A ) Viability profile in U251 cells was reported for three incremental concentrations of 1 : 1.25 µM (white bars), 2.5 µM (grey bars) and 10 µM (black bars). Three different time points: 24, 48 and 72 hours after the transfection were considered. ( B – G ) Comparison between the effects of calixarene 1 ( B , E ) and Lipofectamine RNAiMAX ( C , F , G ) on U251 cell growth ( B , C ) and morphology ( D – G ), determined after 72 hours cell culture. D = control untreated U251 cells. Appearance of vacuolization and methuosis-like patterns are underlined by the arrowheads shown in panel F (representative example in the panel G).
    Figure Legend Snippet: Effects of calixarene 1 on viability, cell proliferation and morphology of U251 cells. ( A ) Viability profile in U251 cells was reported for three incremental concentrations of 1 : 1.25 µM (white bars), 2.5 µM (grey bars) and 10 µM (black bars). Three different time points: 24, 48 and 72 hours after the transfection were considered. ( B – G ) Comparison between the effects of calixarene 1 ( B , E ) and Lipofectamine RNAiMAX ( C , F , G ) on U251 cell growth ( B , C ) and morphology ( D – G ), determined after 72 hours cell culture. D = control untreated U251 cells. Appearance of vacuolization and methuosis-like patterns are underlined by the arrowheads shown in panel F (representative example in the panel G).

    Techniques Used: Transfection, Cell Culture

    24) Product Images from "Intracellular Progesterone Receptor and cSrc Protein Working Together to Regulate the Activity of Proteins Involved in Migration and Invasion of Human Glioblastoma Cells"

    Article Title: Intracellular Progesterone Receptor and cSrc Protein Working Together to Regulate the Activity of Proteins Involved in Migration and Invasion of Human Glioblastoma Cells

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2021.640298

    P4 induces the activation of Fak and Pax through cSrc in glioblastoma cells. (A, B) U251 and U87 cells were treated with P4 (50 nM) or vehicle (V, DMSO 0.01%) for 20 min. (C) U251 cells were transfected with cSrc siRNA and a control siRNA (an aleatory RNA sequence) (100 nM) or were only treated with lipofectamine (Control). (D, E) Transfected cells with cSrc siRNA or control siRNA were treated with P4 (50 nM) or vehicle (V, DMSO 0.01%) for 20 min. Upper panels show the representative western blots for, cSrc, p-Fak, Fak, p-Pax, Pax, and α-tubulin. Lower panels show the densitometric analysis. Data were normalized respect to the vehicle or control. Results are expressed as the mean ± S.E.M. n = 4; *p
    Figure Legend Snippet: P4 induces the activation of Fak and Pax through cSrc in glioblastoma cells. (A, B) U251 and U87 cells were treated with P4 (50 nM) or vehicle (V, DMSO 0.01%) for 20 min. (C) U251 cells were transfected with cSrc siRNA and a control siRNA (an aleatory RNA sequence) (100 nM) or were only treated with lipofectamine (Control). (D, E) Transfected cells with cSrc siRNA or control siRNA were treated with P4 (50 nM) or vehicle (V, DMSO 0.01%) for 20 min. Upper panels show the representative western blots for, cSrc, p-Fak, Fak, p-Pax, Pax, and α-tubulin. Lower panels show the densitometric analysis. Data were normalized respect to the vehicle or control. Results are expressed as the mean ± S.E.M. n = 4; *p

    Techniques Used: Activation Assay, Transfection, Sequencing, Western Blot

    P4 induces the activation of cSrc through PR. (A, B) U251 and U87 cells were treated with P4 (10, 50 and 250 nM) and P4 (50 nM) respectively or vehicle (V, DMSO 0.01%) for 10 min. (C, D) U251 and U87 cells were treated with R5020 (10 nM) or vehicle (V, DMSO 0.01%) for 10 min. (E) U251 cells were transfected with PR siRNA and a control siRNA (an aleatory RNA sequence) (100 nM) or were only treated with lipofectamine (Control). (F) Transfected cells with PR siRNA or control siRNA were treated with P4 (50 nM) or vehicle (V, DMSO 0.01%) for 10 min. Upper panels show the representative western blots for p-cSrc, cSrc, and α-tubulin or representative RT-PCR bands for PR and 18S mRNA. Lower panels show the densitometric analysis. (G) U251 cells were treated with P4 (50 nM) or vehicle (V, DMSO 0.01%) for 5 min and co-immunoprecipitated with PR. Data were normalized respect to the vehicle or control. Results are expressed as the mean ± S.E.M. (A–F) n = 4 (G) n = 3; *p
    Figure Legend Snippet: P4 induces the activation of cSrc through PR. (A, B) U251 and U87 cells were treated with P4 (10, 50 and 250 nM) and P4 (50 nM) respectively or vehicle (V, DMSO 0.01%) for 10 min. (C, D) U251 and U87 cells were treated with R5020 (10 nM) or vehicle (V, DMSO 0.01%) for 10 min. (E) U251 cells were transfected with PR siRNA and a control siRNA (an aleatory RNA sequence) (100 nM) or were only treated with lipofectamine (Control). (F) Transfected cells with PR siRNA or control siRNA were treated with P4 (50 nM) or vehicle (V, DMSO 0.01%) for 10 min. Upper panels show the representative western blots for p-cSrc, cSrc, and α-tubulin or representative RT-PCR bands for PR and 18S mRNA. Lower panels show the densitometric analysis. (G) U251 cells were treated with P4 (50 nM) or vehicle (V, DMSO 0.01%) for 5 min and co-immunoprecipitated with PR. Data were normalized respect to the vehicle or control. Results are expressed as the mean ± S.E.M. (A–F) n = 4 (G) n = 3; *p

    Techniques Used: Activation Assay, Transfection, Sequencing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation

    25) Product Images from "Hepatitis C Virus Induced miR200c Down Modulates FAP-1, a Negative Regulator of Src Signaling and Promotes Hepatic Fibrosis"

    Article Title: Hepatitis C Virus Induced miR200c Down Modulates FAP-1, a Negative Regulator of Src Signaling and Promotes Hepatic Fibrosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070744

    Increased miR200c decreases FAP-1 expression. Human liver fibroblasts were transfe cted with pre-miR200c miRNA or scrambled miRNA (200 nM) using Lipofectamine RNAiMax and stimulated with TGF-β (50 ng/mL). RNA was isolated using trizol reagent and expression levels of miR200c and FAP-1 were measured using pre-developed Taqman miRNA and mRNA assays respectively. The small RNA U6b (miRNA) and GAPDH (mRNA) was used as an endogenous control and relative levels was calculated by the ΔΔCt method. Bars represent the mean expression observed in 3 different experiments performed with 3 different fibroblasts. In order to further define the role of miR200c, we cotransfected fibroblasts with pre-miR200c and mir Vana® miRNA inhibitor for miR200c and analyzed for the expression levels of miR200c and FAP-1. Cotransfection with mir Vana® miRNA inhibitor (50 nM) resulted in restoration of the levels of miR200c and FAP-1 to the levels observed in the untreated fibroblasts. a) Relative expression levels of miR200c; b: Relative expression levels of FAP-1 at the mRNA level; c: Relative expression levels of FAP-1 at the protein level and d: Representative western blot analysis of FAP-1. Lanes 1) Fibroblasts; 2) Fibroblast + TGF-β; 3) Fibroblast + TGF-β+pre-miR-miR200c; 4) Fibroblast + TGF-β+ scrambled pre-miR; 5) Fibroblast + TGF-β+ pre-miR –miR200c+ mir Vana® miRNA inhibitor control; and 6) Fibroblast + TGF-β+ pre-miR –miR200c+ mir Vana® miRNA inhibitor miR200c.
    Figure Legend Snippet: Increased miR200c decreases FAP-1 expression. Human liver fibroblasts were transfe cted with pre-miR200c miRNA or scrambled miRNA (200 nM) using Lipofectamine RNAiMax and stimulated with TGF-β (50 ng/mL). RNA was isolated using trizol reagent and expression levels of miR200c and FAP-1 were measured using pre-developed Taqman miRNA and mRNA assays respectively. The small RNA U6b (miRNA) and GAPDH (mRNA) was used as an endogenous control and relative levels was calculated by the ΔΔCt method. Bars represent the mean expression observed in 3 different experiments performed with 3 different fibroblasts. In order to further define the role of miR200c, we cotransfected fibroblasts with pre-miR200c and mir Vana® miRNA inhibitor for miR200c and analyzed for the expression levels of miR200c and FAP-1. Cotransfection with mir Vana® miRNA inhibitor (50 nM) resulted in restoration of the levels of miR200c and FAP-1 to the levels observed in the untreated fibroblasts. a) Relative expression levels of miR200c; b: Relative expression levels of FAP-1 at the mRNA level; c: Relative expression levels of FAP-1 at the protein level and d: Representative western blot analysis of FAP-1. Lanes 1) Fibroblasts; 2) Fibroblast + TGF-β; 3) Fibroblast + TGF-β+pre-miR-miR200c; 4) Fibroblast + TGF-β+ scrambled pre-miR; 5) Fibroblast + TGF-β+ pre-miR –miR200c+ mir Vana® miRNA inhibitor control; and 6) Fibroblast + TGF-β+ pre-miR –miR200c+ mir Vana® miRNA inhibitor miR200c.

    Techniques Used: Expressing, Isolation, Cotransfection, Western Blot

    26) Product Images from "Inhibition of Tunneling Nanotubes between Cancer Cell and the Endothelium Alters the Metastatic Phenotype"

    Article Title: Inhibition of Tunneling Nanotubes between Cancer Cell and the Endothelium Alters the Metastatic Phenotype

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22116161

    Involvement of exocyst proteins in TNT formation. ( a ) Schematic representation of the nanotube connection between a cancer cell and an endothelial cell and the involvement of actin remodeling pathway. The involvement of the exocyst complex and small GTPases in actin polymerization and TNT formation is shown schematically. The exocyst complex, is a combination of eight proteins (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that interact with small Ras GTPases (like, Cdc42, Rac1, RalA/B) to fulfill the energy requirement for the actin polymerization. ( b ) Schematic representation of the experimental design of the Sec3 knockdown in the cancer cells and the co-culture setup with preformed endothelium. ( c ) Graph showing the reduction of CFSE transfer upon knockdown of Sec3 (one of the major exocyst proteins) in cancer cells. siRNA-mediated knockdown of Sec3 was performed in cancer cells by lipofectamine-based transfection. Post knockdown, cancer cells were stained with CFSE and employed in co-culture with Dil-Ac-LDL stained HUVECs. After 24 h the amount of CFSE transfer was checked by FACS analysis and compared with the control (without Sec3 knockdown) condition. ( d ) Representative image of co-culture showing a reduced amount of TNT formation in the case of Sec3 knockdown of cancer cells. Cancer cells were stained with CellTrace red and co-cultured with unstained HUVECs. After 24 h, the co-culture was fixed, stained with phalloidin green and DAPI, and images were captured with an inverted fluorescence microscope. ( e ) Graph representing a quantification of the number of heterotypic (cancer cell–endothelial cell) and homotypic (cancer cell–cancer cell) nanoscale connections in the control and the Sec3 knockdown condition. The number of homotypic and heterotypic TNTs was counted from at least eight images in each control and knockdown group. The number of cancer cell–endothelial cell (heterotypic) and cancer cell–cancer cell (homotypic) TNT connections in the knockdown group was significantly reduced in comparison to the control group. Data are represented as ±SEM, and an unpaired two-tailed t -test was performed with Welch’s correction * p = 0.0215 (for cancer–endothelial) and **** p
    Figure Legend Snippet: Involvement of exocyst proteins in TNT formation. ( a ) Schematic representation of the nanotube connection between a cancer cell and an endothelial cell and the involvement of actin remodeling pathway. The involvement of the exocyst complex and small GTPases in actin polymerization and TNT formation is shown schematically. The exocyst complex, is a combination of eight proteins (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that interact with small Ras GTPases (like, Cdc42, Rac1, RalA/B) to fulfill the energy requirement for the actin polymerization. ( b ) Schematic representation of the experimental design of the Sec3 knockdown in the cancer cells and the co-culture setup with preformed endothelium. ( c ) Graph showing the reduction of CFSE transfer upon knockdown of Sec3 (one of the major exocyst proteins) in cancer cells. siRNA-mediated knockdown of Sec3 was performed in cancer cells by lipofectamine-based transfection. Post knockdown, cancer cells were stained with CFSE and employed in co-culture with Dil-Ac-LDL stained HUVECs. After 24 h the amount of CFSE transfer was checked by FACS analysis and compared with the control (without Sec3 knockdown) condition. ( d ) Representative image of co-culture showing a reduced amount of TNT formation in the case of Sec3 knockdown of cancer cells. Cancer cells were stained with CellTrace red and co-cultured with unstained HUVECs. After 24 h, the co-culture was fixed, stained with phalloidin green and DAPI, and images were captured with an inverted fluorescence microscope. ( e ) Graph representing a quantification of the number of heterotypic (cancer cell–endothelial cell) and homotypic (cancer cell–cancer cell) nanoscale connections in the control and the Sec3 knockdown condition. The number of homotypic and heterotypic TNTs was counted from at least eight images in each control and knockdown group. The number of cancer cell–endothelial cell (heterotypic) and cancer cell–cancer cell (homotypic) TNT connections in the knockdown group was significantly reduced in comparison to the control group. Data are represented as ±SEM, and an unpaired two-tailed t -test was performed with Welch’s correction * p = 0.0215 (for cancer–endothelial) and **** p

    Techniques Used: Co-Culture Assay, Transfection, Staining, FACS, Cell Culture, Fluorescence, Microscopy, Two Tailed Test

    27) Product Images from "Role of PDZ-binding motif from West Nile virus NS5 protein on viral replication"

    Article Title: Role of PDZ-binding motif from West Nile virus NS5 protein on viral replication

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-82751-x

    SiRNA knocked down ARHGAP21, CNKSR3, DLG1, HTRA1, MAGI1, PARD3, RIMS2, SHANK2, SNX27 and TJP1 expression. HEK-293T cells were transfected with two siRNAs against each protein: ARHGAP21, CNKSR3, DLG1, HTRA1, MAGI1, PARD3, RIMS2, SHANK2, SNX27 and TJP1 with 10 µM for each target siRNA in Lipofectamine RNAimax. After 48 h, the corresponding transcripts were measured by RTqPCR ( A ) and gene expression measured by Western blot assay (β-actin = loading control) ( B ). ( A ) Total RNA from transfected HEK-293T cells was extracted and used for real time quantitative PCR. Relative expression was normalised to the housekeeping gene GAPDH and is presented as the mean ± SD (*p
    Figure Legend Snippet: SiRNA knocked down ARHGAP21, CNKSR3, DLG1, HTRA1, MAGI1, PARD3, RIMS2, SHANK2, SNX27 and TJP1 expression. HEK-293T cells were transfected with two siRNAs against each protein: ARHGAP21, CNKSR3, DLG1, HTRA1, MAGI1, PARD3, RIMS2, SHANK2, SNX27 and TJP1 with 10 µM for each target siRNA in Lipofectamine RNAimax. After 48 h, the corresponding transcripts were measured by RTqPCR ( A ) and gene expression measured by Western blot assay (β-actin = loading control) ( B ). ( A ) Total RNA from transfected HEK-293T cells was extracted and used for real time quantitative PCR. Relative expression was normalised to the housekeeping gene GAPDH and is presented as the mean ± SD (*p

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

    28) Product Images from "Proteinase-activated receptor 2 promotes TGF-β-dependent cell motility in pancreatic cancer cells by sustaining expression of the TGF-β type I receptor ALK5"

    Article Title: Proteinase-activated receptor 2 promotes TGF-β-dependent cell motility in pancreatic cancer cells by sustaining expression of the TGF-β type I receptor ALK5

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9600

    Effects of PAR2 depletion on the TGF-β1 response of invasion associated genes A. Panc-1, Colo357, and HaCaT cells were transfected twice on two consecutive days with 50 nM of control (Co), PAR2, PAR1 or ALK5 siRNA, as indicated, using Lipofectamine RNAiMAX. Twenty-four h after the second round of transfection, cells were stimulated, or not, with 5 ng/ml TGF-β1 for another 24 h (Panc-1, Colo357) or 48 h (HaCaT) followed by RNA isolation and qPCR for the indicated genes. B. Panc-1 cells were transfected as described in A using the indicated siRNAs. Twenty-four h after the second round of transfection, cells were stimulated, or not, with 5 ng/ml TGF-β1 for 1 h followed by RNA isolation and qPCR for GADD45β and Smad7. In A and B, bars represent mean values ± SD of three wells normalised to β-actin and TBP. Successful knockdown of PAR2, ALK5 and PAR1 expression was verified by qPCR (not shown). One representative experiment out of three experiments performed in total is shown. P
    Figure Legend Snippet: Effects of PAR2 depletion on the TGF-β1 response of invasion associated genes A. Panc-1, Colo357, and HaCaT cells were transfected twice on two consecutive days with 50 nM of control (Co), PAR2, PAR1 or ALK5 siRNA, as indicated, using Lipofectamine RNAiMAX. Twenty-four h after the second round of transfection, cells were stimulated, or not, with 5 ng/ml TGF-β1 for another 24 h (Panc-1, Colo357) or 48 h (HaCaT) followed by RNA isolation and qPCR for the indicated genes. B. Panc-1 cells were transfected as described in A using the indicated siRNAs. Twenty-four h after the second round of transfection, cells were stimulated, or not, with 5 ng/ml TGF-β1 for 1 h followed by RNA isolation and qPCR for GADD45β and Smad7. In A and B, bars represent mean values ± SD of three wells normalised to β-actin and TBP. Successful knockdown of PAR2, ALK5 and PAR1 expression was verified by qPCR (not shown). One representative experiment out of three experiments performed in total is shown. P

    Techniques Used: Transfection, Isolation, Real-time Polymerase Chain Reaction, Expressing

    Depletion of PAR2 decreases the sensitivity of TGF-β/Smad responsive reporters to TGF-β1 stimulation A. Panc-1 and Colo357 cells were transfected on day 1 with RNAiMAX along with negative control siRNA (Co), PAR2 siRNA or ALK5 siRNA. On day 2, cells received the same siRNAs along with either p6SBE-Luc ( upper two graphs) or p(CAGA) 12 MLP-Luc ( lower two graphs), and the Renilla luciferase encoding vector pRL-TK-Luc using Lipofectamine 2000. Forty-eight h after the start of the first transfection, cells were stimulated with TGF-β1 for another 24 h followed by dual luciferase measurements. Data are the mean ± SD from six parallel wells. Asterisks indicate significance vs. TGF-β1-treated Co. B. , C. Ectopic expression of PAR2 cells increases the sensitivity of Smad responsive reporter genes to TGF-β1. B . Panc-1 cells were transiently transfected with either empty pcDNA3 vector (vector) or PAR2 encoding vector (PAR2-HA) along with either p6SBE-Luc ( upper graph) or p(CAGA) 12 MLP-Luc ( lower graph), and pRL-TK-Luc. Two days later, cells were treated with 5 ng/ml TGF-β1 for 24 h followed by lysis and dual luciferase assay. Data represent the normalised mean ± SD of six wells. C . HEK293T cells were cotransfected with p3TP-Lux, pRL-TK-Luc, and either empty vector or PAR2 encoding vector. Forty-eight h after the start of transfection, cells were stimulated with TGF-β1 for another 24 h followed by dual luciferase measurements. Data represent the mean ± SD from six wells. Data in A-C are representative of at least four independent experiments.
    Figure Legend Snippet: Depletion of PAR2 decreases the sensitivity of TGF-β/Smad responsive reporters to TGF-β1 stimulation A. Panc-1 and Colo357 cells were transfected on day 1 with RNAiMAX along with negative control siRNA (Co), PAR2 siRNA or ALK5 siRNA. On day 2, cells received the same siRNAs along with either p6SBE-Luc ( upper two graphs) or p(CAGA) 12 MLP-Luc ( lower two graphs), and the Renilla luciferase encoding vector pRL-TK-Luc using Lipofectamine 2000. Forty-eight h after the start of the first transfection, cells were stimulated with TGF-β1 for another 24 h followed by dual luciferase measurements. Data are the mean ± SD from six parallel wells. Asterisks indicate significance vs. TGF-β1-treated Co. B. , C. Ectopic expression of PAR2 cells increases the sensitivity of Smad responsive reporter genes to TGF-β1. B . Panc-1 cells were transiently transfected with either empty pcDNA3 vector (vector) or PAR2 encoding vector (PAR2-HA) along with either p6SBE-Luc ( upper graph) or p(CAGA) 12 MLP-Luc ( lower graph), and pRL-TK-Luc. Two days later, cells were treated with 5 ng/ml TGF-β1 for 24 h followed by lysis and dual luciferase assay. Data represent the normalised mean ± SD of six wells. C . HEK293T cells were cotransfected with p3TP-Lux, pRL-TK-Luc, and either empty vector or PAR2 encoding vector. Forty-eight h after the start of transfection, cells were stimulated with TGF-β1 for another 24 h followed by dual luciferase measurements. Data represent the mean ± SD from six wells. Data in A-C are representative of at least four independent experiments.

    Techniques Used: Transfection, Negative Control, Luciferase, Plasmid Preparation, Expressing, Lysis

    29) Product Images from "Normal human cell proteins that interact with the adenovirus type 5 E1B 55 kDa protein"

    Article Title: Normal human cell proteins that interact with the adenovirus type 5 E1B 55 kDa protein

    Journal: Virology

    doi: 10.1016/j.virol.2017.01.013

    Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using lipofectamine RNAiMAX (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.
    Figure Legend Snippet: Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using lipofectamine RNAiMAX (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.

    Techniques Used: Expressing, Aqueous Normal-phase Chromatography, Infection

    Impact of ANP32A knockdown on viral DNA synthesis in type I IFN-treated cells. A. SiRNAs specific for ANP32A or for EGFP (control, C) were introduced into proliferating HFFs using lipofectamine as described in Materials and Methods, or cells were mock-treated (M). Whole cells lysates were prepared 4 days thereafter and ANP32A and cellular β-actin examined by immunoblotting. B. Proliferating HFFs were treated in siRNAs specific for ANP32A, SIN3A or EGFP (Control) or mock treated as described in Materials and methods, and incubated for 48 h. β-interferon or solvent only was then added to the medium and incubation continued for a further 24 h., when cells were infected with 50 pfu/cell AdEasyE1 or AdEasy E1Δ2347. Total DNA was isolated 24 h. after infection and the concentrations of viral DNA and cellular GAPDH DNA determined by qPCR as described in Materials and Methods. Shown are the ratios of the relative viral DNA concentrations in cells treated with IFN and cells not so treated. C. Viral DNA concentrations were measured as described for panel B in HFFs treated with lipofectamine for 48 h (+LF) or mock treated (−LF) prior to exposure to 250 U/ml type I IFN or solvent for 24 h. followed by infection with 50 pfu/cell AdEasyE1 for 24 h.
    Figure Legend Snippet: Impact of ANP32A knockdown on viral DNA synthesis in type I IFN-treated cells. A. SiRNAs specific for ANP32A or for EGFP (control, C) were introduced into proliferating HFFs using lipofectamine as described in Materials and Methods, or cells were mock-treated (M). Whole cells lysates were prepared 4 days thereafter and ANP32A and cellular β-actin examined by immunoblotting. B. Proliferating HFFs were treated in siRNAs specific for ANP32A, SIN3A or EGFP (Control) or mock treated as described in Materials and methods, and incubated for 48 h. β-interferon or solvent only was then added to the medium and incubation continued for a further 24 h., when cells were infected with 50 pfu/cell AdEasyE1 or AdEasy E1Δ2347. Total DNA was isolated 24 h. after infection and the concentrations of viral DNA and cellular GAPDH DNA determined by qPCR as described in Materials and Methods. Shown are the ratios of the relative viral DNA concentrations in cells treated with IFN and cells not so treated. C. Viral DNA concentrations were measured as described for panel B in HFFs treated with lipofectamine for 48 h (+LF) or mock treated (−LF) prior to exposure to 250 U/ml type I IFN or solvent for 24 h. followed by infection with 50 pfu/cell AdEasyE1 for 24 h.

    Techniques Used: DNA Synthesis, Incubation, Infection, Isolation, Real-time Polymerase Chain Reaction

    30) Product Images from "Heat shock proteins stimulate APOBEC-3–mediated cytidine deamination in the hepatitis B virus"

    Article Title: Heat shock proteins stimulate APOBEC-3–mediated cytidine deamination in the hepatitis B virus

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.760637

    Effect of Hsp knockdown through siRNA on HBV mutation frequency. Each Hsp siRNA was transfected into HepG2 cells by a reverse transfection method with Lipofectamine RNAiMax. After a 24-h siRNA treatment, the cells were transfected with an HBV viral genome-encoding plasmid with or without A3G. The HepG2 cells were harvested for RNA or HBV extraction 24 h after HBV transfection. A , Hsp mRNA levels after siRNA treatment by quantitative RT-PCR determination. The relative mRNA levels for each siRNA treatment were determined by quantitative RT-PCR, using GAPDH as an internal reference. The mRNA levels relative to control were calculated and are represented graphically as percentages with the control as 100%. Each bar represents the average of triplicates for each treatment. Blank , background control by a scrambled negative siRNA without A3G co-transfection. Control , another control by the scrambled negative siRNA with A3G co-transfection. B , Hsp protein expression level analyses after siRNA treatment. Total cellular proteins were extracted from HepG2 cells after siRNA treatment, and Hsp protein levels in the cell lysates were analyzed by Western blotting with antibodies against endogenous Hsp90β, Hsp90α, Hsp70, and Hsp40. GAPDH was analyzed as the protein loading reference. C , HBV DNA mutation analyses. HBV DNAs were extracted from the cell lysates after a 24-h HBV transfection, and the resultant HBV DNA mutations were determined by pe1453 using an 88 °C 3D-PCR. The data are presented graphically on the right . Each bar represents the average of triplicates for each treatment. Asterisks , statistically significant differences comparing treatments with their corresponding control: **, 0.01
    Figure Legend Snippet: Effect of Hsp knockdown through siRNA on HBV mutation frequency. Each Hsp siRNA was transfected into HepG2 cells by a reverse transfection method with Lipofectamine RNAiMax. After a 24-h siRNA treatment, the cells were transfected with an HBV viral genome-encoding plasmid with or without A3G. The HepG2 cells were harvested for RNA or HBV extraction 24 h after HBV transfection. A , Hsp mRNA levels after siRNA treatment by quantitative RT-PCR determination. The relative mRNA levels for each siRNA treatment were determined by quantitative RT-PCR, using GAPDH as an internal reference. The mRNA levels relative to control were calculated and are represented graphically as percentages with the control as 100%. Each bar represents the average of triplicates for each treatment. Blank , background control by a scrambled negative siRNA without A3G co-transfection. Control , another control by the scrambled negative siRNA with A3G co-transfection. B , Hsp protein expression level analyses after siRNA treatment. Total cellular proteins were extracted from HepG2 cells after siRNA treatment, and Hsp protein levels in the cell lysates were analyzed by Western blotting with antibodies against endogenous Hsp90β, Hsp90α, Hsp70, and Hsp40. GAPDH was analyzed as the protein loading reference. C , HBV DNA mutation analyses. HBV DNAs were extracted from the cell lysates after a 24-h HBV transfection, and the resultant HBV DNA mutations were determined by pe1453 using an 88 °C 3D-PCR. The data are presented graphically on the right . Each bar represents the average of triplicates for each treatment. Asterisks , statistically significant differences comparing treatments with their corresponding control: **, 0.01

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Quantitative RT-PCR, Cotransfection, Expressing, Western Blot, Polymerase Chain Reaction

    31) Product Images from "Use of small interfering ribonucleic acids to inhibit the adipogenic effect of alcohol on human bone marrow-derived mesenchymal cells"

    Article Title: Use of small interfering ribonucleic acids to inhibit the adipogenic effect of alcohol on human bone marrow-derived mesenchymal cells

    Journal:

    doi: 10.1007/s00264-009-0914-y

    Alizarin red staining identification of mineralisation in cell cultures for 28 days. a Cell control. b Osteogenic control. c Adipogenic control. d Lipofectamine™ RNAiMAX control. e Negative siRNA control. f PPARγ-siRNA (magnification
    Figure Legend Snippet: Alizarin red staining identification of mineralisation in cell cultures for 28 days. a Cell control. b Osteogenic control. c Adipogenic control. d Lipofectamine™ RNAiMAX control. e Negative siRNA control. f PPARγ-siRNA (magnification

    Techniques Used: Staining

    Oil red O staining of hBMSCs transfected with PPRAγ-siRNA or controls followed by treatment for 24 days. a Cell control. b Osteogenic control. c Adipogenic control. d Lipofectamine™ RNAiMAX control. e Negative siRNA control. f
    Figure Legend Snippet: Oil red O staining of hBMSCs transfected with PPRAγ-siRNA or controls followed by treatment for 24 days. a Cell control. b Osteogenic control. c Adipogenic control. d Lipofectamine™ RNAiMAX control. e Negative siRNA control. f

    Techniques Used: Staining, Transfection

    32) Product Images from "The Effects of Propionate and Valerate on Insulin Responsiveness for Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myotubes via G Protein-Coupled Receptor 41"

    Article Title: The Effects of Propionate and Valerate on Insulin Responsiveness for Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myotubes via G Protein-Coupled Receptor 41

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095268

    Effects of siRNA for GPR41 on SCFA-induced rise in insulin-stimulated glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes. After confirming the expression of GPR41 protein expression by transfecting with siRNA for GPR41 (siGPR41,100 nM) using Lipofectamine RNAiMAX for 48 h in 3T3-L1 adipocytes (A) or C2C12 myotubes (C), cells were treated with 300 µM propionic acid or 500 µM valeric acid for 30 min in the absence or presence of insulin (100 nM) in KRPH buffer. Glucose uptake was measured in the lysates of 3T3-L1 adipocytes (B) or C2C12 myotubes (D) as described in the Methods . Results are the means ± SEM of three similar independent experiments, each performed in quadruplicate. ** P
    Figure Legend Snippet: Effects of siRNA for GPR41 on SCFA-induced rise in insulin-stimulated glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes. After confirming the expression of GPR41 protein expression by transfecting with siRNA for GPR41 (siGPR41,100 nM) using Lipofectamine RNAiMAX for 48 h in 3T3-L1 adipocytes (A) or C2C12 myotubes (C), cells were treated with 300 µM propionic acid or 500 µM valeric acid for 30 min in the absence or presence of insulin (100 nM) in KRPH buffer. Glucose uptake was measured in the lysates of 3T3-L1 adipocytes (B) or C2C12 myotubes (D) as described in the Methods . Results are the means ± SEM of three similar independent experiments, each performed in quadruplicate. ** P

    Techniques Used: Expressing

    33) Product Images from "Divergent androgen regulation of unfolded protein response pathways drives prostate cancer"

    Article Title: Divergent androgen regulation of unfolded protein response pathways drives prostate cancer

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201404509

    IRE1α and XBP-1 are proliferative factors in PCa cells both in vitro and in vivo A Knockdown of IRE1α or XBP-1 leads to a decrease in cell survival. LNCaP cells were transfected with siRNA targeting either IRE1α or XBP-1 (5 nM) and starved in 2% CT-FCS medium for 3 days before cell viability was measured using the CCK-8 assay. The graph is representative of one experiment in triplicate and was repeated three times with similar results. Error bars represent SD with * P = 6.6 × 10 −5 and 4.5 × 10 −5 for comparison between Ctrl and siRNA against IRE1α and XBP-1, respectively, using paired Student's t -test. B XBP-1 rescues the growth defect of siIRE1α-transfected LNCaP cells. LNCaP cells were transfected with 5 nM of indicated siRNA using Lipofectamine RNAiMax reagent. One day after siRNA transfection, the cells were transfected with either vector control (Empty) or Flag-XBP-1S (XBP-1S). Three days after transfection, cells were harvested for Western analysis or cultured for three more days before being applied to cell proliferation assay using the CCK-8 reagent. The data are representative of two experiments in triplicate. Error bars represent SE. * P = 0.02, ** P = 8.54 × 10 −7 using paired Student's t -test. C, D IRE1α and XBP-1 knockdown inhibits clonogenic capacity of LNCaP cells. Control LN-Scr (Scr), LN-shIRE1 (shIRE1), or LN-shXBP1 (shXBP-1) cells were cultured for 3 weeks. The colonies formed were stained with crystal violet and photographed. The extent of IRE1α and XBP-1 knockdown was determined by Western blot analysis. The area covered by colonies was quantified using the Gene Tools software (SynGene). The data are representative of three experiments in triplicate. Error bars represent SEM. * P = 4.38 × 10 −7 and ** P = 3.39 × 10 −20 using paired Student's t -test. E Growth analysis of xenografted LNCaP tumors in nude mice. LNCaP cells expressing shRNA against IRE1α (LN-shIRE1), XBP1 (LN-shXBP-1), or control shRNA (LN-Scr) were subcutaneously implanted into both flanks of male nude mice (6 mice per group). Tumor size was measured at the indicated time points. Representative pictures of the tumors at harvest are shown. Error bars indicate SEM. * P = 0.03 for shIRE1 at week 7, P = 0.02 for shXBP-1 at week 7, ** P = 0.01 for both shIRE1 and shXBP-1 at week 8 using unpaired Student's t -test. F PCNA immunostaining in tumors from animals bearing LN-shIRE1, LN-shXBP-1, or LN-Scr tumors. Scale bars: 100 μm.
    Figure Legend Snippet: IRE1α and XBP-1 are proliferative factors in PCa cells both in vitro and in vivo A Knockdown of IRE1α or XBP-1 leads to a decrease in cell survival. LNCaP cells were transfected with siRNA targeting either IRE1α or XBP-1 (5 nM) and starved in 2% CT-FCS medium for 3 days before cell viability was measured using the CCK-8 assay. The graph is representative of one experiment in triplicate and was repeated three times with similar results. Error bars represent SD with * P = 6.6 × 10 −5 and 4.5 × 10 −5 for comparison between Ctrl and siRNA against IRE1α and XBP-1, respectively, using paired Student's t -test. B XBP-1 rescues the growth defect of siIRE1α-transfected LNCaP cells. LNCaP cells were transfected with 5 nM of indicated siRNA using Lipofectamine RNAiMax reagent. One day after siRNA transfection, the cells were transfected with either vector control (Empty) or Flag-XBP-1S (XBP-1S). Three days after transfection, cells were harvested for Western analysis or cultured for three more days before being applied to cell proliferation assay using the CCK-8 reagent. The data are representative of two experiments in triplicate. Error bars represent SE. * P = 0.02, ** P = 8.54 × 10 −7 using paired Student's t -test. C, D IRE1α and XBP-1 knockdown inhibits clonogenic capacity of LNCaP cells. Control LN-Scr (Scr), LN-shIRE1 (shIRE1), or LN-shXBP1 (shXBP-1) cells were cultured for 3 weeks. The colonies formed were stained with crystal violet and photographed. The extent of IRE1α and XBP-1 knockdown was determined by Western blot analysis. The area covered by colonies was quantified using the Gene Tools software (SynGene). The data are representative of three experiments in triplicate. Error bars represent SEM. * P = 4.38 × 10 −7 and ** P = 3.39 × 10 −20 using paired Student's t -test. E Growth analysis of xenografted LNCaP tumors in nude mice. LNCaP cells expressing shRNA against IRE1α (LN-shIRE1), XBP1 (LN-shXBP-1), or control shRNA (LN-Scr) were subcutaneously implanted into both flanks of male nude mice (6 mice per group). Tumor size was measured at the indicated time points. Representative pictures of the tumors at harvest are shown. Error bars indicate SEM. * P = 0.03 for shIRE1 at week 7, P = 0.02 for shXBP-1 at week 7, ** P = 0.01 for both shIRE1 and shXBP-1 at week 8 using unpaired Student's t -test. F PCNA immunostaining in tumors from animals bearing LN-shIRE1, LN-shXBP-1, or LN-Scr tumors. Scale bars: 100 μm.

    Techniques Used: In Vitro, In Vivo, Transfection, CCK-8 Assay, Plasmid Preparation, Western Blot, Cell Culture, Proliferation Assay, Staining, Software, Mouse Assay, Expressing, shRNA, Immunostaining

    34) Product Images from "Design, Synthesis and Biological Evaluation of novel Hedgehog Inhibitors for treating Pancreatic Cancer"

    Article Title: Design, Synthesis and Biological Evaluation of novel Hedgehog Inhibitors for treating Pancreatic Cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01942-7

    Effect of MDB5 on Hh pathway and target genes. ( a ) Western blot analysis for Gli-1, Shh, Bcl-2, Bax and GAPDH of MIA PaCa-2 cells treated with GDC-0449 and MDB5. ( b ) Real-time RT-PCR analysis of Gli-1, Gli-2, Ptch-1 and Shh of MIA PaCa-2 cells treated with GDC-0449 and MDB5. (c) Western blot analysis for Gli-1, Shh, Bcl-2, Bax and GAPDH of PANC-1 cells treated with GDC-0449 and MDB5. ( d ) Transfection of labeled oligonucleotide using Lipofectamine® RNAiMAX in MIA PaCa-2 cells. ( e ) Cell cytotoxicity assay of GDC-0449 and MDB5 in Smo knockdown MIA PaCa-2 cells. Percentage of viable MIA PaCa-2 cells was estimated by MTT assay. Data is shown as mean ± SD of three separate experiments (Student t-test; *p
    Figure Legend Snippet: Effect of MDB5 on Hh pathway and target genes. ( a ) Western blot analysis for Gli-1, Shh, Bcl-2, Bax and GAPDH of MIA PaCa-2 cells treated with GDC-0449 and MDB5. ( b ) Real-time RT-PCR analysis of Gli-1, Gli-2, Ptch-1 and Shh of MIA PaCa-2 cells treated with GDC-0449 and MDB5. (c) Western blot analysis for Gli-1, Shh, Bcl-2, Bax and GAPDH of PANC-1 cells treated with GDC-0449 and MDB5. ( d ) Transfection of labeled oligonucleotide using Lipofectamine® RNAiMAX in MIA PaCa-2 cells. ( e ) Cell cytotoxicity assay of GDC-0449 and MDB5 in Smo knockdown MIA PaCa-2 cells. Percentage of viable MIA PaCa-2 cells was estimated by MTT assay. Data is shown as mean ± SD of three separate experiments (Student t-test; *p

    Techniques Used: Western Blot, Quantitative RT-PCR, Transfection, Labeling, Cytotoxicity Assay, MTT Assay

    35) Product Images from "Novel Application of Radotinib for the Treatment of Solid Tumors via Natural Killer Cell Activation"

    Article Title: Novel Application of Radotinib for the Treatment of Solid Tumors via Natural Killer Cell Activation

    Journal: Journal of Immunology Research

    doi: 10.1155/2018/9580561

    Radotinib enhances cytolytic activity of NK cells against Fas-expressing A549 cells. (a) Primary NK cells were isolated from healthy donors to perform NK cell cytotoxicity assay. The purity of CD3 − CD56 + NK cells was 96.6%. (b) To determine the effect of radotinib on the cytolytic activity of NK cells against A549 cells, the cells were treated with various concentrations of radotinib (0, 12.5, 25, 50, 100, and 200 μ M) for 48 h and the cytotoxicity assay was performed (E : T ratio = 5 : 1). (c) To determine if the effect of radotinib on NK cytotoxicity was mediated by the Fas receptor, Fas expression was transiently downregulated by Fas siRNA transfection into A549 cells. At approximately 70% confluency, A549 cells were incubated with 50 pmole Fas-specific siRNA or negative control siRNA using Lipofectamine RNAiMAX. Surface expression of Fas on A549 cells was determined by staining with PE-conjugated Fas antibody (solid line). PE-conjugated mouse IgG antibody was used as an isotype control (dotted line). (d) The effect of radotinib on NK cytolytic activity against Fas siRNA-transfected A549 cells was determined by cytotoxicity assay. Radotinib-treated NK cells were used as effector cells, and Fas siRNA-transfected A549 cells or control cells were used as target cells (E : T ratio = 2 : 1). All values were normalized relative to the control (radotinib 0 μ M). The relative level was set to 1 for the control. (e) To further confirm the involvement of Fas-Fas ligand interaction in the radotinib-enhanced NK cytotoxicity, recombinant human soluble Fas was used to block Fas ligand on NK cells. Various concentrations of soluble Fas were preincubated with resting NK cells or radotinib-treated NK cells for 1 h, and then cytotoxicity assays were performed (E : T ratio = 2 : 1). All values were normalized relative to the control (radotinib 0 μ M). The relative level was set to 1 for the control. Data are reported as mean ± SD. All values were analyzed by unpaired Student's t -tests using GraphPad Prism 5. ∗ P
    Figure Legend Snippet: Radotinib enhances cytolytic activity of NK cells against Fas-expressing A549 cells. (a) Primary NK cells were isolated from healthy donors to perform NK cell cytotoxicity assay. The purity of CD3 − CD56 + NK cells was 96.6%. (b) To determine the effect of radotinib on the cytolytic activity of NK cells against A549 cells, the cells were treated with various concentrations of radotinib (0, 12.5, 25, 50, 100, and 200 μ M) for 48 h and the cytotoxicity assay was performed (E : T ratio = 5 : 1). (c) To determine if the effect of radotinib on NK cytotoxicity was mediated by the Fas receptor, Fas expression was transiently downregulated by Fas siRNA transfection into A549 cells. At approximately 70% confluency, A549 cells were incubated with 50 pmole Fas-specific siRNA or negative control siRNA using Lipofectamine RNAiMAX. Surface expression of Fas on A549 cells was determined by staining with PE-conjugated Fas antibody (solid line). PE-conjugated mouse IgG antibody was used as an isotype control (dotted line). (d) The effect of radotinib on NK cytolytic activity against Fas siRNA-transfected A549 cells was determined by cytotoxicity assay. Radotinib-treated NK cells were used as effector cells, and Fas siRNA-transfected A549 cells or control cells were used as target cells (E : T ratio = 2 : 1). All values were normalized relative to the control (radotinib 0 μ M). The relative level was set to 1 for the control. (e) To further confirm the involvement of Fas-Fas ligand interaction in the radotinib-enhanced NK cytotoxicity, recombinant human soluble Fas was used to block Fas ligand on NK cells. Various concentrations of soluble Fas were preincubated with resting NK cells or radotinib-treated NK cells for 1 h, and then cytotoxicity assays were performed (E : T ratio = 2 : 1). All values were normalized relative to the control (radotinib 0 μ M). The relative level was set to 1 for the control. Data are reported as mean ± SD. All values were analyzed by unpaired Student's t -tests using GraphPad Prism 5. ∗ P

    Techniques Used: Activity Assay, Expressing, Isolation, Cytotoxicity Assay, Transfection, Incubation, Negative Control, Staining, Recombinant, Blocking Assay

    36) Product Images from "Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness"

    Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.784090

    IRF3 is phosphorylated, translocates to the nucleus, and binds the CXCL10 promoter in HT29 cells in response to addition of poly(I:C). A , Western blots of HT29 cells were stimulated with poly(I:C) alone ( Poly(I:C) , 2.5 μg/ml) or transfected with poly(I:C) using Lipofectamine RNAimax ( LF + Poly(I:C) , 0–1200 min) and stained with antibodies against phospho-IRF3 Ser-396 , total IRF3, phosphor-p65 Ser-536 , total p65, or GAPDH. The results are representative of two independent experiments. MW , molecular weight. B and C , nuclear accumulation of IRF3 ( B ) and IRF1 ( C ) in HT29 cells left untreated ( 0 ), stimulated with poly(I:C) (5–2 μg/ml), or transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml) for 3 h or overnight ( o/n ). Stimulated cells were fixed and immunostained for IRF3 or IRF1, and cell nuclei were stained with Hoechst 3342. Cells were visualized by automated imaging, and analysis was done using ScanR. The results show the percentage of cells with positive staining of IRF3 and IRF1 in the nucleus. The results show mean ± S.D. of triplicate samples with a minimum of 1300 cells assayed and are representative of three independent experiments. D , CXCL10 promotor occupancy by IRF3 in HT29 cells after poly(I:C) (2 μg/ml) stimulation for 3 h. IRF3 binding to the CXCL10 promoter was investigated by ChIP followed by qPCR of the CXCL10 promoter region. RNA polymerase II occupancy was measured as a control. E and F , CXCL10 production ( left panels ) and IRF mRNA expression ( right panels ) in HT29 cells left untreated ( No add ), treated with siRNA against IRF3 ( E ) or IRF7 ( F ) (10 n m ), NS RNA (10 n m ), or transfection reagent alone ( LF ) for 24 h. Cells were subsequently stimulated with poly(I:C) (2.5 μg/ml) for 6 h. CXCL10 release was assessed by ELISA, whereas silencing of IRF3 and 7 was confirmed by assessing mRNA expression by qPCR using GAPDH as a reference control. The results show mean ± S.D. of triplicate samples.
    Figure Legend Snippet: IRF3 is phosphorylated, translocates to the nucleus, and binds the CXCL10 promoter in HT29 cells in response to addition of poly(I:C). A , Western blots of HT29 cells were stimulated with poly(I:C) alone ( Poly(I:C) , 2.5 μg/ml) or transfected with poly(I:C) using Lipofectamine RNAimax ( LF + Poly(I:C) , 0–1200 min) and stained with antibodies against phospho-IRF3 Ser-396 , total IRF3, phosphor-p65 Ser-536 , total p65, or GAPDH. The results are representative of two independent experiments. MW , molecular weight. B and C , nuclear accumulation of IRF3 ( B ) and IRF1 ( C ) in HT29 cells left untreated ( 0 ), stimulated with poly(I:C) (5–2 μg/ml), or transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml) for 3 h or overnight ( o/n ). Stimulated cells were fixed and immunostained for IRF3 or IRF1, and cell nuclei were stained with Hoechst 3342. Cells were visualized by automated imaging, and analysis was done using ScanR. The results show the percentage of cells with positive staining of IRF3 and IRF1 in the nucleus. The results show mean ± S.D. of triplicate samples with a minimum of 1300 cells assayed and are representative of three independent experiments. D , CXCL10 promotor occupancy by IRF3 in HT29 cells after poly(I:C) (2 μg/ml) stimulation for 3 h. IRF3 binding to the CXCL10 promoter was investigated by ChIP followed by qPCR of the CXCL10 promoter region. RNA polymerase II occupancy was measured as a control. E and F , CXCL10 production ( left panels ) and IRF mRNA expression ( right panels ) in HT29 cells left untreated ( No add ), treated with siRNA against IRF3 ( E ) or IRF7 ( F ) (10 n m ), NS RNA (10 n m ), or transfection reagent alone ( LF ) for 24 h. Cells were subsequently stimulated with poly(I:C) (2.5 μg/ml) for 6 h. CXCL10 release was assessed by ELISA, whereas silencing of IRF3 and 7 was confirmed by assessing mRNA expression by qPCR using GAPDH as a reference control. The results show mean ± S.D. of triplicate samples.

    Techniques Used: Western Blot, Transfection, Staining, Molecular Weight, Imaging, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    IFNβ is induced in IECs in response to transfection with poly(I:C) but not in response to poly(I:C) addition. A , HT29 cells were left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before CXCL10 in the supernatant was assessed by ELISA. B , kinetics of CXCL10 release assessed by ELISA in supernatant from HT29 cells stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 5, 12, 20, and 24 h. The results are presented as mean ± S.D. of triplicates. C , IFNβ mRNA induction in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone ( Poly(I:C) ), transfected with poly(I:C) complexed with Lipofectamine RNAimax ( LF + Poly(I:C) , 2 μg/ml), or treated with only Lipofectamine RNAimax ( LF ) for 20 h. IFNβ mRNA induction was determined by qPCR. The results are presented as relative induction compared with medium-treated Caco-2 cells. GAPDH served as an internal control. Results show mean -fold induction ± S.D. of triplicates. D , IFNβ protein production in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone, transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 20 h. IFNβ in the supernatant was assessed by ELISA, and the results show mean ± S.D. of three samples. E , HT29 cells were stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 6, 12, 20, or 24 h before CXCL10 and IFNβ mRNA induction was determined by qPCR. The results show relative induction with a non-treated sample as reference. GAPDH served as an internal control. The results show mean -fold induction ± S.D. of triplicates. F , CXCL10 mRNA induction in HT29 cells pretreated with cycloheximide (0, 15, or 30 μg/ml) for 30 min prior to stimulation with poly(I:C) (2.5 μg/ml) for 8 h. CXCL10 mRNA was determined by qPCR (normalized to medium control and the endogenous control TBP). G , viability in HT29 cells left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before viability was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. H , viability in IECs left untreated ( 0 ), stimulated with poly(I:C) alone (2 μg/ml), transfected with poly(I:C) using Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 43 h before the viability of the cells was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. The results show mean ± S.D. of five samples. All results are representative of at least two independent experiments.
    Figure Legend Snippet: IFNβ is induced in IECs in response to transfection with poly(I:C) but not in response to poly(I:C) addition. A , HT29 cells were left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before CXCL10 in the supernatant was assessed by ELISA. B , kinetics of CXCL10 release assessed by ELISA in supernatant from HT29 cells stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 5, 12, 20, and 24 h. The results are presented as mean ± S.D. of triplicates. C , IFNβ mRNA induction in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone ( Poly(I:C) ), transfected with poly(I:C) complexed with Lipofectamine RNAimax ( LF + Poly(I:C) , 2 μg/ml), or treated with only Lipofectamine RNAimax ( LF ) for 20 h. IFNβ mRNA induction was determined by qPCR. The results are presented as relative induction compared with medium-treated Caco-2 cells. GAPDH served as an internal control. Results show mean -fold induction ± S.D. of triplicates. D , IFNβ protein production in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone, transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 20 h. IFNβ in the supernatant was assessed by ELISA, and the results show mean ± S.D. of three samples. E , HT29 cells were stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 6, 12, 20, or 24 h before CXCL10 and IFNβ mRNA induction was determined by qPCR. The results show relative induction with a non-treated sample as reference. GAPDH served as an internal control. The results show mean -fold induction ± S.D. of triplicates. F , CXCL10 mRNA induction in HT29 cells pretreated with cycloheximide (0, 15, or 30 μg/ml) for 30 min prior to stimulation with poly(I:C) (2.5 μg/ml) for 8 h. CXCL10 mRNA was determined by qPCR (normalized to medium control and the endogenous control TBP). G , viability in HT29 cells left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before viability was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. H , viability in IECs left untreated ( 0 ), stimulated with poly(I:C) alone (2 μg/ml), transfected with poly(I:C) using Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 43 h before the viability of the cells was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. The results show mean ± S.D. of five samples. All results are representative of at least two independent experiments.

    Techniques Used: Transfection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, MTT Assay

    37) Product Images from "AUTS2 confers gene activation to Polycomb group proteins in the CNS"

    Article Title: AUTS2 confers gene activation to Polycomb group proteins in the CNS

    Journal: Nature

    doi: 10.1038/nature13921

    Requirement of the integrity of the PRC1-AUTS2 complex for transcriptional activation. a Luciferase activity in screened stable cell clones expressing GAl4-PCGF5, 24 hr after induction by doxycycline at 100 µg/ml. b Fold change in luciferase activity in GAl4-AUTS2 cells upon knockdown of RING1B or PCGF5. Cells were transfected with Lipofectamine 2000 RNAiMAX and siRNAs against RING1B or PCGF5, or control siRNAs for two days and then 100 µg/ml doxycycline was added to induce GAL4-AUTS2 expression. After 24 hr after induction luciferase activity was measured. Each value is the mean of three independent measurements. Error bars represent standard error. c Immunoblotting of samples used for luciferase activity reporter assay as in b using the antibodies indicated. d Fold change in luciferase activity in GAl4-PCGF5 cells upon knockdown of AUTS2. Cells were treated as in b. e Immunoblotting of samples used for luciferase activity reporter assay as in d using the antibodies indicated.
    Figure Legend Snippet: Requirement of the integrity of the PRC1-AUTS2 complex for transcriptional activation. a Luciferase activity in screened stable cell clones expressing GAl4-PCGF5, 24 hr after induction by doxycycline at 100 µg/ml. b Fold change in luciferase activity in GAl4-AUTS2 cells upon knockdown of RING1B or PCGF5. Cells were transfected with Lipofectamine 2000 RNAiMAX and siRNAs against RING1B or PCGF5, or control siRNAs for two days and then 100 µg/ml doxycycline was added to induce GAL4-AUTS2 expression. After 24 hr after induction luciferase activity was measured. Each value is the mean of three independent measurements. Error bars represent standard error. c Immunoblotting of samples used for luciferase activity reporter assay as in b using the antibodies indicated. d Fold change in luciferase activity in GAl4-PCGF5 cells upon knockdown of AUTS2. Cells were treated as in b. e Immunoblotting of samples used for luciferase activity reporter assay as in d using the antibodies indicated.

    Techniques Used: Activation Assay, Luciferase, Activity Assay, Stable Transfection, Clone Assay, Expressing, Transfection, Reporter Assay

    38) Product Images from "Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity"

    Article Title: Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003298

    5′pppRNA stimulates an antiviral and inflammatory response in lung epithelial A549 cells. ( A ) Schematic representation of VSV-derived 5′pppRNA and gel analysis. The 5′ppp-containing 67-mer RNA oligonucleotide is derived from the untranslated regions (UTRs) of VSV and the product of in vitro transcription runs as a single product degraded by RNase I. ( B ) 5′pppRNA or a homologous control RNA lacking a 5′-triphosphate end was mixed with Lipofectamine RNAiMax and transfected at different RNA concentrations (0.1–500 ng/ml) into A549 cells. At 8 h post treatment, whole cell extracts (WCEs) were prepared, resolved by SDS-page and analyzed by immunoblotting for IRF3 pSer396, IRF3, ISG56, NOXA, cleaved caspase 3, PARP and β-actin. Results are from a representative experiment; all immunoblots are from the same samples. ( C ) A549 cells were transfected with 10 ng/ml 5′pppRNA and WCEs were prepared at different times after transfection (0–48 h), subjected to SDS-PAGE and probed with antibodies for IRF3 pSer-396, IRF3, IRF7, STAT1 pTyr-701, STAT1, ISG56, RIG-I, IκBα pSer-32, IkBα and β-actin; all immunoblots are from the same samples. To detect IRF3 dimerization, WCEs were resolved by native-PAGE and analyzed by immunoblotting for IRF3. ( D ) ELISA was performed on cell culture supernatants to quantify the release of IFNβ and IFNα over time. Error bars represent SEM from two independent samples.
    Figure Legend Snippet: 5′pppRNA stimulates an antiviral and inflammatory response in lung epithelial A549 cells. ( A ) Schematic representation of VSV-derived 5′pppRNA and gel analysis. The 5′ppp-containing 67-mer RNA oligonucleotide is derived from the untranslated regions (UTRs) of VSV and the product of in vitro transcription runs as a single product degraded by RNase I. ( B ) 5′pppRNA or a homologous control RNA lacking a 5′-triphosphate end was mixed with Lipofectamine RNAiMax and transfected at different RNA concentrations (0.1–500 ng/ml) into A549 cells. At 8 h post treatment, whole cell extracts (WCEs) were prepared, resolved by SDS-page and analyzed by immunoblotting for IRF3 pSer396, IRF3, ISG56, NOXA, cleaved caspase 3, PARP and β-actin. Results are from a representative experiment; all immunoblots are from the same samples. ( C ) A549 cells were transfected with 10 ng/ml 5′pppRNA and WCEs were prepared at different times after transfection (0–48 h), subjected to SDS-PAGE and probed with antibodies for IRF3 pSer-396, IRF3, IRF7, STAT1 pTyr-701, STAT1, ISG56, RIG-I, IκBα pSer-32, IkBα and β-actin; all immunoblots are from the same samples. To detect IRF3 dimerization, WCEs were resolved by native-PAGE and analyzed by immunoblotting for IRF3. ( D ) ELISA was performed on cell culture supernatants to quantify the release of IFNβ and IFNα over time. Error bars represent SEM from two independent samples.

    Techniques Used: Derivative Assay, In Vitro, Transfection, SDS Page, Western Blot, Clear Native PAGE, Enzyme-linked Immunosorbent Assay, Cell Culture

    Gene expression profiling of differentially expressed genes in response to 5′pppRNA and IFNα-2b. A549 cells were transfected with 10 ng/ml of 5′pppRNA using Lipofectamine RNAiMax or treated with IFNα-2b (100 IU/ml or 1000 IU/ml). Samples were collected at 6 h or 24 h post-treatment and were analyzed by Illumina gene expression array. Genes with a fold change ≥±2.0 and p -value ≤0.001 were considered differentially expressed. ( A ) Heatmap showing top DEG affected by 5′pppRNA and IFNα-2b treatments. Genes regulated by both 5′pppRNA and IFNα-2b in at least one condition are indicated in black (up-regulated) or blue (down-regulated). Genes uniquely induced by 5′pppRNA in at least one time point but not by IFNα-2b in any conditions are highlighted in red (up-regulated) or green (down-regulated). Top genes are listed in each instance. ( B ) Cell culture supernatant was collected at the time of treatment with IFNα-2b (0 h; input), or at 24 h following 5′pppRNA or IFNα-2b treatment and assayed by ELISA for multiple subunits of IFNα. Error bars represent SEM from two independent samples. ( C ) Surface proportional Venn diagram illustrating the magnitude of the response by 5′pppRNA and IFNα-2b at 6 h and 24 h. Number of DEG is indicated in each area. ( D ) Comparison of genes induced by each treatment - 5′pppRNA 6 h (dark blue); 5′pppRNA 24 h (light blue); IFNα-2b (1000 IU/ml) 6 h (red); IFNα-2b (1000 IU/ml) 24 h (pink) - based on functional classification by Ingenuity Pathway Analysis.
    Figure Legend Snippet: Gene expression profiling of differentially expressed genes in response to 5′pppRNA and IFNα-2b. A549 cells were transfected with 10 ng/ml of 5′pppRNA using Lipofectamine RNAiMax or treated with IFNα-2b (100 IU/ml or 1000 IU/ml). Samples were collected at 6 h or 24 h post-treatment and were analyzed by Illumina gene expression array. Genes with a fold change ≥±2.0 and p -value ≤0.001 were considered differentially expressed. ( A ) Heatmap showing top DEG affected by 5′pppRNA and IFNα-2b treatments. Genes regulated by both 5′pppRNA and IFNα-2b in at least one condition are indicated in black (up-regulated) or blue (down-regulated). Genes uniquely induced by 5′pppRNA in at least one time point but not by IFNα-2b in any conditions are highlighted in red (up-regulated) or green (down-regulated). Top genes are listed in each instance. ( B ) Cell culture supernatant was collected at the time of treatment with IFNα-2b (0 h; input), or at 24 h following 5′pppRNA or IFNα-2b treatment and assayed by ELISA for multiple subunits of IFNα. Error bars represent SEM from two independent samples. ( C ) Surface proportional Venn diagram illustrating the magnitude of the response by 5′pppRNA and IFNα-2b at 6 h and 24 h. Number of DEG is indicated in each area. ( D ) Comparison of genes induced by each treatment - 5′pppRNA 6 h (dark blue); 5′pppRNA 24 h (light blue); IFNα-2b (1000 IU/ml) 6 h (red); IFNα-2b (1000 IU/ml) 24 h (pink) - based on functional classification by Ingenuity Pathway Analysis.

    Techniques Used: Expressing, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Functional Assay

    Transcriptome analysis of the host antiviral response to 5′pppRNA. A549 cells were transfected with 10 ng/ml of 5′pppRNA using Lipofectamine RNAiMax for designated periods of time. Samples were analyzed by Illumina gene expression array and DEG were identified based on fold change ≥±2 and p -value ≤0.001 ( A ) Number of up-regulated and down-regulated DEG at each time point. ( B ) Heatmap of all DEG sorted by fold change; top 30 genes are listed. Red, Up-regulated; blue, down-regulated. ( C ) Functional characterization of DEGs following 5′pppRNA treatment based on Ingenuity Pathway Analysis software. Bar height refers to the number of DEG in each pathway and the color refers to the contribution from up-regulated or down-regulated genes. ( D ) Genes among the top up-regulated genes were selected based on three different expression patterns: early, sustained, late.
    Figure Legend Snippet: Transcriptome analysis of the host antiviral response to 5′pppRNA. A549 cells were transfected with 10 ng/ml of 5′pppRNA using Lipofectamine RNAiMax for designated periods of time. Samples were analyzed by Illumina gene expression array and DEG were identified based on fold change ≥±2 and p -value ≤0.001 ( A ) Number of up-regulated and down-regulated DEG at each time point. ( B ) Heatmap of all DEG sorted by fold change; top 30 genes are listed. Red, Up-regulated; blue, down-regulated. ( C ) Functional characterization of DEGs following 5′pppRNA treatment based on Ingenuity Pathway Analysis software. Bar height refers to the number of DEG in each pathway and the color refers to the contribution from up-regulated or down-regulated genes. ( D ) Genes among the top up-regulated genes were selected based on three different expression patterns: early, sustained, late.

    Techniques Used: Transfection, Expressing, Functional Assay, Software

    39) Product Images from "Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation"

    Article Title: Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation

    Journal: Veterinary Immunology and Immunopathology

    doi: 10.1016/j.vetimm.2014.02.002

    Comparison of the efficacy of eleven commercially available reagents to transfect siRNA into bovine monocyte-derived macrophages (bMDM). bMDM were transfected with 50 nM FITC-labelled siRNA using three different concentrations of the transfection reagents HiPerFect (HPT), INTERFERin (INT), Lipofectamine RNAiMAX (RNA), Lipofectamine 2000 (L2K), siPORT Amine (SPA), X-tremeGENE (XTG), N-TER (NTR) and DharmaFECT 1 (DF1), DharmaFECT 2 (DF2), DharmaFECT 3 (DF3) and DharmaFECT 4 (DF4) following the manufacturer's instructions. In addition bMDM were cultured with 50 nM FITC-labelled siRNA in the absence of transfection reagents as a control (C). After 16 h the percentage of bMDM transfected with FITC-siRNA (grey bars) and the percentage of viable bMDM (black diamonds) were measured by flow cytometry. The result is representative of three repeat experiments using bMDM from different animals.
    Figure Legend Snippet: Comparison of the efficacy of eleven commercially available reagents to transfect siRNA into bovine monocyte-derived macrophages (bMDM). bMDM were transfected with 50 nM FITC-labelled siRNA using three different concentrations of the transfection reagents HiPerFect (HPT), INTERFERin (INT), Lipofectamine RNAiMAX (RNA), Lipofectamine 2000 (L2K), siPORT Amine (SPA), X-tremeGENE (XTG), N-TER (NTR) and DharmaFECT 1 (DF1), DharmaFECT 2 (DF2), DharmaFECT 3 (DF3) and DharmaFECT 4 (DF4) following the manufacturer's instructions. In addition bMDM were cultured with 50 nM FITC-labelled siRNA in the absence of transfection reagents as a control (C). After 16 h the percentage of bMDM transfected with FITC-siRNA (grey bars) and the percentage of viable bMDM (black diamonds) were measured by flow cytometry. The result is representative of three repeat experiments using bMDM from different animals.

    Techniques Used: Derivative Assay, Transfection, Cell Culture, Flow Cytometry, Cytometry

    Transient transfection reagents induce a type I interferon (IFN)-response in bovine monocyte-derived macrophages (bMDM) in the absence of siRNA. The siRNA uptake efficacy and off-target effects of five transfection reagents, INTERFERin (INT), Lipofectamine RNAiMAX (RNA), Lipofectamine 2000 (L2K), X-tremeGENE (XTG) and DharmaFECT 3 (DF3) were investigated. (A) bMDM were cultured with pre-determined concentrations of the transfection reagents in the absence of siRNA for 48 h (48 h incubation) or the medium was changed 24 h post transfection reagent treatment and cultured for a further 24 h (24 h incubation). The mRNA levels of the type I IFN-response gene interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were measured by qRT-PCR. The results are expressed as the IFIT1 mRNA fold difference compared to that measured in untreated cells (NC). Error bars denote the variation between three technical replicates. (B) bMDM were transfected with MEFV siRNA using the five transfection reagents for 24 h before the medium was replaced. MEFV mRNA levels in bMDM transfected with (NTC) and without (TC) non-target control siRNA using Lipofectam ine RNAiMAX were also investigated. After 48 h the bMDM were activated with 100 ng/ml LPS for 2 h. The results are expressed as the percentage MEFV mRNA detected compared to that measured in NC. Error bars denote the variation between three technical replicates. The results are representative of three repeat experiments using bMDM from different animals.
    Figure Legend Snippet: Transient transfection reagents induce a type I interferon (IFN)-response in bovine monocyte-derived macrophages (bMDM) in the absence of siRNA. The siRNA uptake efficacy and off-target effects of five transfection reagents, INTERFERin (INT), Lipofectamine RNAiMAX (RNA), Lipofectamine 2000 (L2K), X-tremeGENE (XTG) and DharmaFECT 3 (DF3) were investigated. (A) bMDM were cultured with pre-determined concentrations of the transfection reagents in the absence of siRNA for 48 h (48 h incubation) or the medium was changed 24 h post transfection reagent treatment and cultured for a further 24 h (24 h incubation). The mRNA levels of the type I IFN-response gene interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were measured by qRT-PCR. The results are expressed as the IFIT1 mRNA fold difference compared to that measured in untreated cells (NC). Error bars denote the variation between three technical replicates. (B) bMDM were transfected with MEFV siRNA using the five transfection reagents for 24 h before the medium was replaced. MEFV mRNA levels in bMDM transfected with (NTC) and without (TC) non-target control siRNA using Lipofectam ine RNAiMAX were also investigated. After 48 h the bMDM were activated with 100 ng/ml LPS for 2 h. The results are expressed as the percentage MEFV mRNA detected compared to that measured in NC. Error bars denote the variation between three technical replicates. The results are representative of three repeat experiments using bMDM from different animals.

    Techniques Used: Transfection, Derivative Assay, Cell Culture, Incubation, Quantitative RT-PCR

    40) Product Images from "Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts *"

    Article Title: Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.143693

    Collagen I-induced DDR1 and MMP-10 expression is mediated by JAK2 in NHLFs. A , C , and D , NHLFs were reverse transfected with JAK2-specific siRNA or negative control ( CTRL ) siRNA using Lipofectamine RNAiMAX. 48 h after transfection, NHLFs were serum-starved
    Figure Legend Snippet: Collagen I-induced DDR1 and MMP-10 expression is mediated by JAK2 in NHLFs. A , C , and D , NHLFs were reverse transfected with JAK2-specific siRNA or negative control ( CTRL ) siRNA using Lipofectamine RNAiMAX. 48 h after transfection, NHLFs were serum-starved

    Techniques Used: Expressing, Transfection, Negative Control

    Collagen I-induced DDR1 and MMP-10 expression is mediated by ERK1/2 in NHLFs. A , C , and D , NHLFs were reverse transfected with ERK1/2-specific siRNA or negative control siRNA using Lipofectamine RNAiMAX. 48 h after transfection, NHLFs were serum-starved
    Figure Legend Snippet: Collagen I-induced DDR1 and MMP-10 expression is mediated by ERK1/2 in NHLFs. A , C , and D , NHLFs were reverse transfected with ERK1/2-specific siRNA or negative control siRNA using Lipofectamine RNAiMAX. 48 h after transfection, NHLFs were serum-starved

    Techniques Used: Expressing, Transfection, Negative Control

    Collagen I induces DDR1 and MMP-10 expression through DDR2 in NHLFs. A , C , and D , NHLFs were reverse transfected with DDR2-specific siRNA or negative control ( CTRL ) siRNA using Lipofectamine RNAiMAX. 48 h after transfection, NHLFs were serum-starved for
    Figure Legend Snippet: Collagen I induces DDR1 and MMP-10 expression through DDR2 in NHLFs. A , C , and D , NHLFs were reverse transfected with DDR2-specific siRNA or negative control ( CTRL ) siRNA using Lipofectamine RNAiMAX. 48 h after transfection, NHLFs were serum-starved for

    Techniques Used: Expressing, Transfection, Negative Control

    Related Articles

    Transfection:

    Article Title: Rickettsia Sca4 reduces vinculin-mediated intercellular tension to promote spread
    Article Snippet: Stained samples were overlaid with 50% sterile glycerol in 1x PBS and imaged directly in the plates. .. For , the same procedure was followed as described above except 1.5×104 donor cells/well and 9×104 recipient cells per well were reverse transfected with 5 nM siRNA and Lipofectamine RNAiMAX (Thermo Fisher) 2 days before infection. .. In assays where host vinculin expression was reduced, the following Silencer Select siRNAs (Ambion) were used: siVCL #1 (s14763; target sequence: UUCGAAUUUUGAUUGAAGCag) and siVCL #2 (s14764; target sequence: UCCUAAGUAAGAUACGAGCag).

    Article Title: GADD34 suppresses lipopolysaccharide-induced sepsis and tissue injury through the regulation of macrophage activation
    Article Snippet: THP-1 cells were cultured in RPMI-1640 (Sigma) supplemented with 10% heat-inactivated FBS (Hyclone, Logan, UT, USA). .. THP-1 was transfected with 10 nM siRNA using Lipofectamine RNAiMax (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. siRNAs were obtained from Ambion (Waltham, MA, USA). .. The sequence of siRNA used to knockdown GADD34 is 5′-GGAUCAGCCCGAGGAUGAAA-3′.

    Article Title: The critical role of catalase in prooxidant and antioxidant function of p53
    Article Snippet: Control siRNA was purchased from Bioneer (Daejeon, Korea). .. Cells were transiently transfected with siRNA duplexes using Lipofectamine RNAiMAX (Invitrogen). .. Intracellular ROS production was assayed using the fluorochrome marker 5-(and-6)-chloromethyl-2′,7′-diclorodihydrofluoescein diacetate, acetyl ester (CM-H2 DCFDA) probe (Invitrogen, Molecular Probes).

    Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer
    Article Snippet: The oligos were annealed as per manufactures directions, digested with Bgl II and Hind III restriction enzymes and clones into pSUPER-neo-gfp vector digested with the same enzymes. .. Transfection of siRNA and shRNA was performed using the Lipofectamine RNAiMax transfection reagent (Invitrogen) following manufactures instructions, and assayed for silencing at 24 and 48 hours after transfection. .. Effect of SMC1 overexpression and suppression on colony propagation MDA-MB-231 cells (0.1×106 cells/500 µL) untreated and treated with control-liposome, SMC1-liposomes (each liposomes final purified SMC1 protein conc.

    Article Title: Evaluation of in vitro and in vivo therapeutic antitumor efficacy of transduction of polo-like kinase 1 and heat shock transcription factor 1 small interfering RNA
    Article Snippet: Cell growth inhibition MCF-7, MDA-MB-231 and HeLa cells were seeded in 96-well plates at a density of 2×104 cells per well 24 h prior to transfection. .. Cells at confluences of 50% in the well were transfected with 2.5, 5, 10, 20, 30, 40 and 50 nM Cont siRNA, PLK1 siRNA or HSF1 siRNA using Lipofectamine RNAiMax reagent and then incubated for 48 h at 37°C. .. In combined treatment with doxorubicin (DXR; LC Laboratories, Woburn, MA, USA), the cells were incubated for 24 h at 37°C after transfection at 50 nM Cont siRNA, PLK1 siRNA or HSF1 siRNA using Lipofectamine RNAiMax reagent, and then treated with various concentrations (0.016–0.5 µM) of DXR as reported previously ( ).

    Article Title: LIPG signaling promotes tumor initiation and metastasis of human basal-like triple-negative breast cancer
    Article Snippet: To establish stable gene knockdown cell lines, infected MDA-MB-468 cells with or without DTX3L overexpression were selected in the culture medium containing puromycin (0.8 μg/ml). .. The siRNA transfection was performed with 40 nM of siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to manufacturer’s instructions. siRNAs were obtained from Sigma-Aldrich. ..

    Infection:

    Article Title: Rickettsia Sca4 reduces vinculin-mediated intercellular tension to promote spread
    Article Snippet: Stained samples were overlaid with 50% sterile glycerol in 1x PBS and imaged directly in the plates. .. For , the same procedure was followed as described above except 1.5×104 donor cells/well and 9×104 recipient cells per well were reverse transfected with 5 nM siRNA and Lipofectamine RNAiMAX (Thermo Fisher) 2 days before infection. .. In assays where host vinculin expression was reduced, the following Silencer Select siRNAs (Ambion) were used: siVCL #1 (s14763; target sequence: UUCGAAUUUUGAUUGAAGCag) and siVCL #2 (s14764; target sequence: UCCUAAGUAAGAUACGAGCag).

    Over Expression:

    Article Title: NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress
    Article Snippet: After a final wash with PBS, the proteins were visualized and quantified using the Odyssey image analysis system (Li-Cor Biosciences, Cambridge, United Kingdom). .. HCT116p53+/+ cells grown in 35-mm dishes were treated in the absence and presence of TRIM26 siRNA (800 pmol) using Lipofectamine RNAiMax (Life Technologies, Paisley, United Kingdom) for 48 h. For NTH1 overexpression, cells were treated with 50 ng pCMV-Tag3a-NTH mammalian expression plasmid using Lipofectamine 2000 (Life Technologies, Paisley, United Kingdom) for 24 h. The cells were treated with hydrogen peroxide (0 to 300 μM) for 15 min, trypsinized, and counted, and a defined number were seeded in triplicate into 6-well plates and incubated at 37°C in 5% CO2 . ..

    Expressing:

    Article Title: NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress
    Article Snippet: After a final wash with PBS, the proteins were visualized and quantified using the Odyssey image analysis system (Li-Cor Biosciences, Cambridge, United Kingdom). .. HCT116p53+/+ cells grown in 35-mm dishes were treated in the absence and presence of TRIM26 siRNA (800 pmol) using Lipofectamine RNAiMax (Life Technologies, Paisley, United Kingdom) for 48 h. For NTH1 overexpression, cells were treated with 50 ng pCMV-Tag3a-NTH mammalian expression plasmid using Lipofectamine 2000 (Life Technologies, Paisley, United Kingdom) for 24 h. The cells were treated with hydrogen peroxide (0 to 300 μM) for 15 min, trypsinized, and counted, and a defined number were seeded in triplicate into 6-well plates and incubated at 37°C in 5% CO2 . ..

    Plasmid Preparation:

    Article Title: NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress
    Article Snippet: After a final wash with PBS, the proteins were visualized and quantified using the Odyssey image analysis system (Li-Cor Biosciences, Cambridge, United Kingdom). .. HCT116p53+/+ cells grown in 35-mm dishes were treated in the absence and presence of TRIM26 siRNA (800 pmol) using Lipofectamine RNAiMax (Life Technologies, Paisley, United Kingdom) for 48 h. For NTH1 overexpression, cells were treated with 50 ng pCMV-Tag3a-NTH mammalian expression plasmid using Lipofectamine 2000 (Life Technologies, Paisley, United Kingdom) for 24 h. The cells were treated with hydrogen peroxide (0 to 300 μM) for 15 min, trypsinized, and counted, and a defined number were seeded in triplicate into 6-well plates and incubated at 37°C in 5% CO2 . ..

    Incubation:

    Article Title: NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress
    Article Snippet: After a final wash with PBS, the proteins were visualized and quantified using the Odyssey image analysis system (Li-Cor Biosciences, Cambridge, United Kingdom). .. HCT116p53+/+ cells grown in 35-mm dishes were treated in the absence and presence of TRIM26 siRNA (800 pmol) using Lipofectamine RNAiMax (Life Technologies, Paisley, United Kingdom) for 48 h. For NTH1 overexpression, cells were treated with 50 ng pCMV-Tag3a-NTH mammalian expression plasmid using Lipofectamine 2000 (Life Technologies, Paisley, United Kingdom) for 24 h. The cells were treated with hydrogen peroxide (0 to 300 μM) for 15 min, trypsinized, and counted, and a defined number were seeded in triplicate into 6-well plates and incubated at 37°C in 5% CO2 . ..

    Article Title: Evaluation of in vitro and in vivo therapeutic antitumor efficacy of transduction of polo-like kinase 1 and heat shock transcription factor 1 small interfering RNA
    Article Snippet: Cell growth inhibition MCF-7, MDA-MB-231 and HeLa cells were seeded in 96-well plates at a density of 2×104 cells per well 24 h prior to transfection. .. Cells at confluences of 50% in the well were transfected with 2.5, 5, 10, 20, 30, 40 and 50 nM Cont siRNA, PLK1 siRNA or HSF1 siRNA using Lipofectamine RNAiMax reagent and then incubated for 48 h at 37°C. .. In combined treatment with doxorubicin (DXR; LC Laboratories, Woburn, MA, USA), the cells were incubated for 24 h at 37°C after transfection at 50 nM Cont siRNA, PLK1 siRNA or HSF1 siRNA using Lipofectamine RNAiMax reagent, and then treated with various concentrations (0.016–0.5 µM) of DXR as reported previously ( ).

    shRNA:

    Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer
    Article Snippet: The oligos were annealed as per manufactures directions, digested with Bgl II and Hind III restriction enzymes and clones into pSUPER-neo-gfp vector digested with the same enzymes. .. Transfection of siRNA and shRNA was performed using the Lipofectamine RNAiMax transfection reagent (Invitrogen) following manufactures instructions, and assayed for silencing at 24 and 48 hours after transfection. .. Effect of SMC1 overexpression and suppression on colony propagation MDA-MB-231 cells (0.1×106 cells/500 µL) untreated and treated with control-liposome, SMC1-liposomes (each liposomes final purified SMC1 protein conc.

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    Thermo Fisher lipofectamine rnaimax
    Cellular sensitivity to oxidative stress is controlled by TRIM26 through NTH1 regulation. (A to C) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with <t>Lipofectamine</t> <t>RNAiMax</t> transfection reagent (10 μl) in the presence of 800 pmol NT siRNA or TRIM26 siRNA for 72 h. Cells were also treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 500 ng mammalian expression plasmid for NTH1 (NTH1 O/E) for 24 h. (A) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (B) Cells were treated with hydrogen peroxide (12.5 μM), and DNA single-strand breaks and alkali-labile sites were measured at various time points postincubation by the alkaline comet assay. Shown are the percentages of tail DNA with standard deviations from the results of at least three independent experiments. *, P
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    Cellular sensitivity to oxidative stress is controlled by TRIM26 through NTH1 regulation. (A to C) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA or TRIM26 siRNA for 72 h. Cells were also treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 500 ng mammalian expression plasmid for NTH1 (NTH1 O/E) for 24 h. (A) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (B) Cells were treated with hydrogen peroxide (12.5 μM), and DNA single-strand breaks and alkali-labile sites were measured at various time points postincubation by the alkaline comet assay. Shown are the percentages of tail DNA with standard deviations from the results of at least three independent experiments. *, P

    Journal: Molecular and Cellular Biology

    Article Title: NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress

    doi: 10.1128/MCB.00616-17

    Figure Lengend Snippet: Cellular sensitivity to oxidative stress is controlled by TRIM26 through NTH1 regulation. (A to C) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA or TRIM26 siRNA for 72 h. Cells were also treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 500 ng mammalian expression plasmid for NTH1 (NTH1 O/E) for 24 h. (A) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (B) Cells were treated with hydrogen peroxide (12.5 μM), and DNA single-strand breaks and alkali-labile sites were measured at various time points postincubation by the alkaline comet assay. Shown are the percentages of tail DNA with standard deviations from the results of at least three independent experiments. *, P

    Article Snippet: HCT116p53+/+ cells grown in 35-mm dishes were treated in the absence and presence of TRIM26 siRNA (800 pmol) using Lipofectamine RNAiMax (Life Technologies, Paisley, United Kingdom) for 48 h. For NTH1 overexpression, cells were treated with 50 ng pCMV-Tag3a-NTH mammalian expression plasmid using Lipofectamine 2000 (Life Technologies, Paisley, United Kingdom) for 24 h. The cells were treated with hydrogen peroxide (0 to 300 μM) for 15 min, trypsinized, and counted, and a defined number were seeded in triplicate into 6-well plates and incubated at 37°C in 5% CO2 .

    Techniques: Transfection, Expressing, Plasmid Preparation, SDS Page, Alkaline Single Cell Gel Electrophoresis

    Cellular NTH1 protein levels are induced in response to oxidative stress controlled by TRIM26. (A and B) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA (A) or TRIM26 siRNA (Β) for 72 h. (C) HCT116 cells were also grown in 10-cm dishes for 24 h to ∼90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of a mammalian expression plasmid for TRIM26 (1 μg) for 24 h. Cells were either left untreated (lane C) or treated with hydrogen peroxide (150 μM for 15 min) and harvested at various time points following incubation. Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (D) Levels of NTH1 protein relative to tubulin were quantified from the results of at least three independent experiments. Shown is the mean NTH1/tubulin ratio with standard error normalized to that of the untreated control, which was set to 1.0. *, P

    Journal: Molecular and Cellular Biology

    Article Title: NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress

    doi: 10.1128/MCB.00616-17

    Figure Lengend Snippet: Cellular NTH1 protein levels are induced in response to oxidative stress controlled by TRIM26. (A and B) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA (A) or TRIM26 siRNA (Β) for 72 h. (C) HCT116 cells were also grown in 10-cm dishes for 24 h to ∼90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of a mammalian expression plasmid for TRIM26 (1 μg) for 24 h. Cells were either left untreated (lane C) or treated with hydrogen peroxide (150 μM for 15 min) and harvested at various time points following incubation. Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (D) Levels of NTH1 protein relative to tubulin were quantified from the results of at least three independent experiments. Shown is the mean NTH1/tubulin ratio with standard error normalized to that of the untreated control, which was set to 1.0. *, P

    Article Snippet: HCT116p53+/+ cells grown in 35-mm dishes were treated in the absence and presence of TRIM26 siRNA (800 pmol) using Lipofectamine RNAiMax (Life Technologies, Paisley, United Kingdom) for 48 h. For NTH1 overexpression, cells were treated with 50 ng pCMV-Tag3a-NTH mammalian expression plasmid using Lipofectamine 2000 (Life Technologies, Paisley, United Kingdom) for 24 h. The cells were treated with hydrogen peroxide (0 to 300 μM) for 15 min, trypsinized, and counted, and a defined number were seeded in triplicate into 6-well plates and incubated at 37°C in 5% CO2 .

    Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, SDS Page

    NTH1 protein accumulates on chromatin in response to oxidative stress, which is controlled by TRIM26. (A and B) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA (A) or TRIM26 siRNA (Β) for 72 h. Cells were either left untreated (lane C) or treated with hydrogen peroxide (150 μM for 15 min) and harvested at various time points following incubation, and proteins were separated by biochemical fractionation. The soluble (S) and chromatin-bound (CB) fractions were analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (C) Levels of NTH1 protein relative to fibrillarin in the chromatin-bound fraction were quantified from the results of at least three independent experiments; shown is the mean NTH1/fibrillarin ratio with standard deviation normalized to that of the NT siRNA-treated control, which was set to 1.0. *, P

    Journal: Molecular and Cellular Biology

    Article Title: NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress

    doi: 10.1128/MCB.00616-17

    Figure Lengend Snippet: NTH1 protein accumulates on chromatin in response to oxidative stress, which is controlled by TRIM26. (A and B) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT siRNA (A) or TRIM26 siRNA (Β) for 72 h. Cells were either left untreated (lane C) or treated with hydrogen peroxide (150 μM for 15 min) and harvested at various time points following incubation, and proteins were separated by biochemical fractionation. The soluble (S) and chromatin-bound (CB) fractions were analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (C) Levels of NTH1 protein relative to fibrillarin in the chromatin-bound fraction were quantified from the results of at least three independent experiments; shown is the mean NTH1/fibrillarin ratio with standard deviation normalized to that of the NT siRNA-treated control, which was set to 1.0. *, P

    Article Snippet: HCT116p53+/+ cells grown in 35-mm dishes were treated in the absence and presence of TRIM26 siRNA (800 pmol) using Lipofectamine RNAiMax (Life Technologies, Paisley, United Kingdom) for 48 h. For NTH1 overexpression, cells were treated with 50 ng pCMV-Tag3a-NTH mammalian expression plasmid using Lipofectamine 2000 (Life Technologies, Paisley, United Kingdom) for 24 h. The cells were treated with hydrogen peroxide (0 to 300 μM) for 15 min, trypsinized, and counted, and a defined number were seeded in triplicate into 6-well plates and incubated at 37°C in 5% CO2 .

    Techniques: Transfection, Incubation, Fractionation, SDS Page, Standard Deviation

    Cellular NTH1 protein levels are regulated by ubiquitylation by TRIM26. (A) HCT116 cells were grown in 10-cm dishes for 24 h to 90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of mammalian expression plasmids for HA-tagged ubiquitin (1 μg), Flag-tagged NTH1 (500 ng), and HA-tagged TRIM26 (1 μg) for 24 h. The cells were then treated with MG-132 (10 μM) for 8 h, and whole-cell extracts were prepared and Flag-NTH1 purified, using anti-Flag magnetic beads, from extracts containing equal amounts of total protein. Proteins bound to the beads were analyzed by 10% SDS-PAGE and immunoblotting (IB) with HA antibodies to detect ubiquitylated NTH1. IP, immunoprecipitation. Molecular mass (kilodalton) markers are indicated on the left. (B to D) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT or TRIM26 siRNA for 72 h. (B) RNA and subsequently cDNA were prepared from cells, and quantitative PCRs using primer pairs for trim26 and actin were performed. Fold changes in the levels of trim26 mRNA relative to actin are shown. (C) Proteins were separated by biochemical fractionation, and the soluble (S) and chromatin-bound (CB) fractions were analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (D) Levels of NTH1 protein relative to lamin A in the chromatin-bound fraction were quantified from the results of at least three independent experiments, and the mean NTH1/lamin A ratio with standard deviation normalized to the NT siRNA-treated control, which was set to 1.0, is shown. (E and F) HCT116 cells were grown in 10-cm dishes for 24 h to ∼90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 250 ng mammalian expression plasmids for Flag-tagged WT or an NTH1 mutant (K67R) for 24 h. (E) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (F) Levels of Flag-tagged NTH1 proteins relative to tubulin were quantified from the results of at least three independent experiments. Shown is the mean Flag-NTH1/tubulin ratio with standard deviation normalized to that of the WT-NTH1-transfected cells, which was set to 1.0. *, P

    Journal: Molecular and Cellular Biology

    Article Title: NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress

    doi: 10.1128/MCB.00616-17

    Figure Lengend Snippet: Cellular NTH1 protein levels are regulated by ubiquitylation by TRIM26. (A) HCT116 cells were grown in 10-cm dishes for 24 h to 90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of mammalian expression plasmids for HA-tagged ubiquitin (1 μg), Flag-tagged NTH1 (500 ng), and HA-tagged TRIM26 (1 μg) for 24 h. The cells were then treated with MG-132 (10 μM) for 8 h, and whole-cell extracts were prepared and Flag-NTH1 purified, using anti-Flag magnetic beads, from extracts containing equal amounts of total protein. Proteins bound to the beads were analyzed by 10% SDS-PAGE and immunoblotting (IB) with HA antibodies to detect ubiquitylated NTH1. IP, immunoprecipitation. Molecular mass (kilodalton) markers are indicated on the left. (B to D) HCT116 cells were grown in 10-cm dishes for 24 h to 30 to 50% confluence and then treated with Lipofectamine RNAiMax transfection reagent (10 μl) in the presence of 800 pmol NT or TRIM26 siRNA for 72 h. (B) RNA and subsequently cDNA were prepared from cells, and quantitative PCRs using primer pairs for trim26 and actin were performed. Fold changes in the levels of trim26 mRNA relative to actin are shown. (C) Proteins were separated by biochemical fractionation, and the soluble (S) and chromatin-bound (CB) fractions were analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (D) Levels of NTH1 protein relative to lamin A in the chromatin-bound fraction were quantified from the results of at least three independent experiments, and the mean NTH1/lamin A ratio with standard deviation normalized to the NT siRNA-treated control, which was set to 1.0, is shown. (E and F) HCT116 cells were grown in 10-cm dishes for 24 h to ∼90% confluence and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 250 ng mammalian expression plasmids for Flag-tagged WT or an NTH1 mutant (K67R) for 24 h. (E) Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (F) Levels of Flag-tagged NTH1 proteins relative to tubulin were quantified from the results of at least three independent experiments. Shown is the mean Flag-NTH1/tubulin ratio with standard deviation normalized to that of the WT-NTH1-transfected cells, which was set to 1.0. *, P

    Article Snippet: HCT116p53+/+ cells grown in 35-mm dishes were treated in the absence and presence of TRIM26 siRNA (800 pmol) using Lipofectamine RNAiMax (Life Technologies, Paisley, United Kingdom) for 48 h. For NTH1 overexpression, cells were treated with 50 ng pCMV-Tag3a-NTH mammalian expression plasmid using Lipofectamine 2000 (Life Technologies, Paisley, United Kingdom) for 24 h. The cells were treated with hydrogen peroxide (0 to 300 μM) for 15 min, trypsinized, and counted, and a defined number were seeded in triplicate into 6-well plates and incubated at 37°C in 5% CO2 .

    Techniques: Transfection, Expressing, Purification, Magnetic Beads, SDS Page, Immunoprecipitation, Fractionation, Standard Deviation, Mutagenesis

    Effects of combining a PARP-inhibitor with SMC1 siRNA in TNBC basal-like cell lines. IC 50 (half maximal inhibitory concentration) of the PARP-inhibitor, ABT-888 combined scrambled (scr) or SMC1 siRNA was tested in normal (HUVEC) and TNBC basal-like (MDA-MB-468, HCC1937) cell lines by MTT assay as detailed in methods section. Briefly, cells were transfected with scrambled siRNA or SMC1 siRNA (50 nM) using Lipofectamine RNAiMax following manufactures instructions and after 24 hours, transfected cells were treated with a range of ABT-888 (0–100 µM) and MTT assay was performed after 72 hours ( Panel A and C ). To further check the effect of SMC1 silencing on the efficacy of ABT-888, colony propagation assay was performed as described [37] , [38] . Briefly, MDA-MB-468 and HCC1937 were transfected with scrambled or SMC1 siRNA (0.1×10 6 cells/500 µl in triplicates). Aliquots of 50 and 100 µl (in triplicates) were taken in 60 mm size petri-dishes, separately, in a total volume of 4 ml with medium containing 20 µM ABT-888 (MDA-MB-468) and 5 µM (HCC1937). The medium was changed every 2 days and after 10 days, the cells were stained with 0.5% methylene blue and colonies were counted by Alpha Innotech Imager ( Panel B and D ). These results showed that SMC1 siRNA sensitized basal like TNBC cells irrespective of their BRCA1 mutation status.

    Journal: PLoS ONE

    Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

    doi: 10.1371/journal.pone.0064338

    Figure Lengend Snippet: Effects of combining a PARP-inhibitor with SMC1 siRNA in TNBC basal-like cell lines. IC 50 (half maximal inhibitory concentration) of the PARP-inhibitor, ABT-888 combined scrambled (scr) or SMC1 siRNA was tested in normal (HUVEC) and TNBC basal-like (MDA-MB-468, HCC1937) cell lines by MTT assay as detailed in methods section. Briefly, cells were transfected with scrambled siRNA or SMC1 siRNA (50 nM) using Lipofectamine RNAiMax following manufactures instructions and after 24 hours, transfected cells were treated with a range of ABT-888 (0–100 µM) and MTT assay was performed after 72 hours ( Panel A and C ). To further check the effect of SMC1 silencing on the efficacy of ABT-888, colony propagation assay was performed as described [37] , [38] . Briefly, MDA-MB-468 and HCC1937 were transfected with scrambled or SMC1 siRNA (0.1×10 6 cells/500 µl in triplicates). Aliquots of 50 and 100 µl (in triplicates) were taken in 60 mm size petri-dishes, separately, in a total volume of 4 ml with medium containing 20 µM ABT-888 (MDA-MB-468) and 5 µM (HCC1937). The medium was changed every 2 days and after 10 days, the cells were stained with 0.5% methylene blue and colonies were counted by Alpha Innotech Imager ( Panel B and D ). These results showed that SMC1 siRNA sensitized basal like TNBC cells irrespective of their BRCA1 mutation status.

    Article Snippet: Transfection of siRNA and shRNA was performed using the Lipofectamine RNAiMax transfection reagent (Invitrogen) following manufactures instructions, and assayed for silencing at 24 and 48 hours after transfection.

    Techniques: Concentration Assay, Multiple Displacement Amplification, MTT Assay, Transfection, Staining, Mutagenesis

    Effect of SMC1 on cellular Viability and Anchorage Independent Cell Growth. The MDA-MB-231 cells were transfected with the eukaryotic expression vector alone (pcDNA3.1) and SMC1/pcDNA3.1, using Lipofectamine 2000 transfection reagent (Invitrogen). Transfection of scrambled and SMC1 siRNA was performed with Lipofectamine RNAiMax kit (Invitrogen) following manufactures instructions. Total RNA was purified using Trizol reagent and quantified using a nano-drop spectrophotometer (Thermo Scientific). Expression of SMC1 mRNA was evaluated by RT-PCR analysis using gene specific primers [nt 307–326 bp (upstream primer) and nt 730–750 bp (downstream primer)] and β-actin was used as a control. Equal amount of DNA was loaded on 1% agarose gel; lane 1, pcDNA3.1 (control vector); lane 2, pcDNA3.1/SMC1; lane 3, scrambled siRNA; and lane 4 SMC1 siRNA to check for the overexpression and silencing of SMC1( Panel A ). For SMC1-liposomes, SMC1 was purified by DNP-SG affinity chromatography and reconstituted into liposomes using our established procedure [36] , [38] . Viability of colonies was determined by colony forming assay, performed in untreated, control-liposomes, SMC1-liposomes, scrambled and SMC1 siRNA, pcDNA3.1 and SMC1/pcDNA3.1 transfected MDA-MB-231 cells (0.1×10 6 cells/500 µl in triplicates). Aliquots of 50 and 100 µl (in triplicates) were taken in 60 mm size petri-dishes, separately, in a total volume of 4 ml with medium. After 10 days, cells were stained with 0.5% methylene blue for 30 min and colonies were counted using Innotech Alpha Imager. The results shown are normalized to control untreated cells. Values are means ± S.D. of three separate experiments ( Panel B ). The effect of SMC1 overexpression (SMC1 transfected) and suppression (SMC1 siRNA) was also checked on the cell apoptosis by using TUNEL apoptosis kit (Promega). Briefly, approximately 0.1×10 6 MDA-MB-231 cells were grown on the cover slips in 12 well plate and transfected with SMC1/pcDNA3.1 and SMC1 siRNA as described in method section. TUNEL apoptosis assay was performed using the Promega Apoptosis Detection Kit according to manufactures instructions. The slides were analyzed by fluorescence microscope (Olympus IX81 automated Inverted) using a standard fluorescein filter set to view the green fluorescence at 520 nm, and blue fluorescence at > 340 nm. Photographs taken at identical exposure at 20× magnification are presented. Apoptotic cells showed green fluorescence and characteristic cell shrinkage ( Panel C ). MDA-MB-231 cells transfected with scrambled shRNA and SMC1 shRNA were also tested for the anchorage-independent growth on soft agar as described in the methods section and colonies were counted after 21 days and plotted ( Panel D ).

    Journal: PLoS ONE

    Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

    doi: 10.1371/journal.pone.0064338

    Figure Lengend Snippet: Effect of SMC1 on cellular Viability and Anchorage Independent Cell Growth. The MDA-MB-231 cells were transfected with the eukaryotic expression vector alone (pcDNA3.1) and SMC1/pcDNA3.1, using Lipofectamine 2000 transfection reagent (Invitrogen). Transfection of scrambled and SMC1 siRNA was performed with Lipofectamine RNAiMax kit (Invitrogen) following manufactures instructions. Total RNA was purified using Trizol reagent and quantified using a nano-drop spectrophotometer (Thermo Scientific). Expression of SMC1 mRNA was evaluated by RT-PCR analysis using gene specific primers [nt 307–326 bp (upstream primer) and nt 730–750 bp (downstream primer)] and β-actin was used as a control. Equal amount of DNA was loaded on 1% agarose gel; lane 1, pcDNA3.1 (control vector); lane 2, pcDNA3.1/SMC1; lane 3, scrambled siRNA; and lane 4 SMC1 siRNA to check for the overexpression and silencing of SMC1( Panel A ). For SMC1-liposomes, SMC1 was purified by DNP-SG affinity chromatography and reconstituted into liposomes using our established procedure [36] , [38] . Viability of colonies was determined by colony forming assay, performed in untreated, control-liposomes, SMC1-liposomes, scrambled and SMC1 siRNA, pcDNA3.1 and SMC1/pcDNA3.1 transfected MDA-MB-231 cells (0.1×10 6 cells/500 µl in triplicates). Aliquots of 50 and 100 µl (in triplicates) were taken in 60 mm size petri-dishes, separately, in a total volume of 4 ml with medium. After 10 days, cells were stained with 0.5% methylene blue for 30 min and colonies were counted using Innotech Alpha Imager. The results shown are normalized to control untreated cells. Values are means ± S.D. of three separate experiments ( Panel B ). The effect of SMC1 overexpression (SMC1 transfected) and suppression (SMC1 siRNA) was also checked on the cell apoptosis by using TUNEL apoptosis kit (Promega). Briefly, approximately 0.1×10 6 MDA-MB-231 cells were grown on the cover slips in 12 well plate and transfected with SMC1/pcDNA3.1 and SMC1 siRNA as described in method section. TUNEL apoptosis assay was performed using the Promega Apoptosis Detection Kit according to manufactures instructions. The slides were analyzed by fluorescence microscope (Olympus IX81 automated Inverted) using a standard fluorescein filter set to view the green fluorescence at 520 nm, and blue fluorescence at > 340 nm. Photographs taken at identical exposure at 20× magnification are presented. Apoptotic cells showed green fluorescence and characteristic cell shrinkage ( Panel C ). MDA-MB-231 cells transfected with scrambled shRNA and SMC1 shRNA were also tested for the anchorage-independent growth on soft agar as described in the methods section and colonies were counted after 21 days and plotted ( Panel D ).

    Article Snippet: Transfection of siRNA and shRNA was performed using the Lipofectamine RNAiMax transfection reagent (Invitrogen) following manufactures instructions, and assayed for silencing at 24 and 48 hours after transfection.

    Techniques: Multiple Displacement Amplification, Transfection, Expressing, Plasmid Preparation, Purification, Spectrophotometry, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Over Expression, Affinity Chromatography, Staining, TUNEL Assay, Apoptosis Assay, Fluorescence, Microscopy, shRNA

    Expression of PLK1 and HSF1 mRNA in MCF-7, MDA-MB-231 and HeLa cells, and suppression of PLK1 or HSF1 mRNA expression by transfection with siRNA in MDA-MB-231 and HeLa cells. (A) The expression levels of HSF1 and PLK1 mRNA in MCF-7, MDA-MB-231 and HeLa cells were analyzed by RT-PCR. MDA-MB-231 and HeLa cells were transfected with 50 nM Cont, HSF1 or PLK1 siRNA using Lipofectamine RNAiMax reagent, and the expression levels of (B) PLK1 and (C) HSF1 mRNA in the cells were analyzed by RT-quantitative PCR. Data are presented as the mean + standard deviation (n=3). **P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Evaluation of in vitro and in vivo therapeutic antitumor efficacy of transduction of polo-like kinase 1 and heat shock transcription factor 1 small interfering RNA

    doi: 10.3892/etm.2017.5060

    Figure Lengend Snippet: Expression of PLK1 and HSF1 mRNA in MCF-7, MDA-MB-231 and HeLa cells, and suppression of PLK1 or HSF1 mRNA expression by transfection with siRNA in MDA-MB-231 and HeLa cells. (A) The expression levels of HSF1 and PLK1 mRNA in MCF-7, MDA-MB-231 and HeLa cells were analyzed by RT-PCR. MDA-MB-231 and HeLa cells were transfected with 50 nM Cont, HSF1 or PLK1 siRNA using Lipofectamine RNAiMax reagent, and the expression levels of (B) PLK1 and (C) HSF1 mRNA in the cells were analyzed by RT-quantitative PCR. Data are presented as the mean + standard deviation (n=3). **P

    Article Snippet: Cells at confluences of 50% in the well were transfected with 2.5, 5, 10, 20, 30, 40 and 50 nM Cont siRNA, PLK1 siRNA or HSF1 siRNA using Lipofectamine RNAiMax reagent and then incubated for 48 h at 37°C.

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Standard Deviation

    p53R2 interacts with catalase and is required for antioxidant function of p53. ( a and b ) U2OS, RKO, and HCT116 cells were transfected with control (white columns) or p53 (gray columns) siRNA, and ROS levels ( a ) and catalase activity ( b ) were measured

    Journal: Cell Death and Differentiation

    Article Title: The critical role of catalase in prooxidant and antioxidant function of p53

    doi: 10.1038/cdd.2012.102

    Figure Lengend Snippet: p53R2 interacts with catalase and is required for antioxidant function of p53. ( a and b ) U2OS, RKO, and HCT116 cells were transfected with control (white columns) or p53 (gray columns) siRNA, and ROS levels ( a ) and catalase activity ( b ) were measured

    Article Snippet: Cells were transiently transfected with siRNA duplexes using Lipofectamine RNAiMAX (Invitrogen).

    Techniques: Transfection, Activity Assay