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
    Applications:
    Cell Culture|RNAi|RNAi Transfection|RNAi, Epigenetics & Non-Coding RNA Research|Stem Cell & Primary Cell Transfections|Synthetic siRNA Transfection|siRNA|Transfection
    Category:
    Cell Culture Transfection Reagents
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    Structured Review

    Thermo Fisher lipofectamine rnaimax
    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 <t>Lipofectamine</t> <t>RNAimax</t> ( 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.
    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
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    Images

    1) 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

    2) 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

    3) Product Images from "Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists"

    Article Title: Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists

    Journal: Journal of Virology

    doi: 10.1128/JVI.00845-15

    The RIG-I agonist sequence affects antiviral activity. (A) Schematic representation of modifications to the M8 5′pppRNA. Sequence changes were made to the poly(A·U) base pair stretch (M8A and M8C), the WT-derived blunt end (M8D), and the entire sequence (M8B) while keeping the structure intact. (B) A549 cells were transfected with 5′pppRNA (1 ng/ml) using Lipofectamine RNAiMax. After 24 h, cells were harvested and total mRNA was isolated. Antiviral and inflammatory gene expression was determined by qPCR. Data are from one experiment performed in triplicate and represent the means and SEM. (C) HEK293T cells were cotransfected ISRE or IFN-β promoter reporter plasmid (200 ng) along with 5′pppRNA (10 ng/ml). Luciferase activity was analyzed 24 h posttransfection by the dual-luciferase reporter assay. Relative luciferase activity was measured as fold induction relative to the basal level of the reporter gene. Data are from one experiment performed in triplicate and represent the means and SEM. (D) A549 cells were transfected with 5′pppRNA (1 ng/ml) for 24 h. Whole-cell extracts were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting for ISG56, STAT1, pIRF3 S396, IRF3, RIG-I, and β-actin. One representative Western blot from one experiment is shown. (E) A549 cells were transfected with 5′pppRNA (0.001 to 10 ng/ml) using Lipofectamine RNAiMax for 24 h and then challenged with dengue virus (MOI, 0.1) for 24 h. Percentage of infected cells was determined by intracellular staining of DenV E protein expression. Data are from one experiment performed in triplicate and represent the means.
    Figure Legend Snippet: The RIG-I agonist sequence affects antiviral activity. (A) Schematic representation of modifications to the M8 5′pppRNA. Sequence changes were made to the poly(A·U) base pair stretch (M8A and M8C), the WT-derived blunt end (M8D), and the entire sequence (M8B) while keeping the structure intact. (B) A549 cells were transfected with 5′pppRNA (1 ng/ml) using Lipofectamine RNAiMax. After 24 h, cells were harvested and total mRNA was isolated. Antiviral and inflammatory gene expression was determined by qPCR. Data are from one experiment performed in triplicate and represent the means and SEM. (C) HEK293T cells were cotransfected ISRE or IFN-β promoter reporter plasmid (200 ng) along with 5′pppRNA (10 ng/ml). Luciferase activity was analyzed 24 h posttransfection by the dual-luciferase reporter assay. Relative luciferase activity was measured as fold induction relative to the basal level of the reporter gene. Data are from one experiment performed in triplicate and represent the means and SEM. (D) A549 cells were transfected with 5′pppRNA (1 ng/ml) for 24 h. Whole-cell extracts were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting for ISG56, STAT1, pIRF3 S396, IRF3, RIG-I, and β-actin. One representative Western blot from one experiment is shown. (E) A549 cells were transfected with 5′pppRNA (0.001 to 10 ng/ml) using Lipofectamine RNAiMax for 24 h and then challenged with dengue virus (MOI, 0.1) for 24 h. Percentage of infected cells was determined by intracellular staining of DenV E protein expression. Data are from one experiment performed in triplicate and represent the means.

    Techniques Used: Sequencing, Activity Assay, Derivative Assay, Transfection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Luciferase, Reporter Assay, SDS Page, Western Blot, Infection, Staining

    Characterization of novel RIG-I agonists. (A) Schematic representation of 5′pppRNA sequences that include variations of the wild-type (WT) VSV-derived 5′pppRNA (M1 to M8), SELEX-selected RIG-I aptamers, and poly(I·C). (B) In vitro -transcribed 5′pppRNA was DNase treated, purified, and then run on a denaturing TBE-urea gel. (C) A549 cells were transfected with WT, M5, M8, CL9 aptamer, or poly(I·C) (2 fmol) using lipofectamine RNAiMax. After 24 h, cells were harvested and total mRNA was isolated. Antiviral and inflammatory gene expression was determined by qPCR. Data are from two independent experiments performed in triplicate and represent the means and standard errors of the means (SEM). (D) HEK293T cells were cotransfected ISRE or IFN-β promoter reporter plasmid (200 ng) along with WT, M5, or M8 5′pppRNA (10 ng/ml). Luciferase activity was analyzed 24 h posttransfection by the dual-luciferase reporter assay. Relative luciferase activity was measured as fold induction relative to the basal level of reporter gene. Data are from two independent experiments performed in triplicate and represent the means and SEM. (E) A549 cells were transfected with 5′pppRNA (0.1, 1, or 10 ng/ml), and whole-cell extracts were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting for IRF3 pSer396, IRF3, ISG56, STAT1, RIG-I, and β-actin 24 h later. One representative Western blot out of three independent triplicates is shown. (F) A549 cells were transfected with 5′pppRNA or CL9 aptamer (0.01, 0.1, 1, or 10 ng/ml) for 24 h and then infected with influenza (MOI, 0.2) for 24 h. Whole-cell extracts were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting for influenza viral protein NS1 and β-actin. One representative Western blot from one experiment is shown. (G) A549 cells were transfected with WT, M5, or M8 5′pppRNA, poly(I·C), or CL9 aptamer (1 ng/ml) for 24 h and then challenged with dengue virus (MOI, 0.5). The percentage of infected cells was determined 24 h postinfection by intracellular staining of DenV E protein expression. Data are from two independent experiments performed in triplicate and represent the means and SEM. *, P
    Figure Legend Snippet: Characterization of novel RIG-I agonists. (A) Schematic representation of 5′pppRNA sequences that include variations of the wild-type (WT) VSV-derived 5′pppRNA (M1 to M8), SELEX-selected RIG-I aptamers, and poly(I·C). (B) In vitro -transcribed 5′pppRNA was DNase treated, purified, and then run on a denaturing TBE-urea gel. (C) A549 cells were transfected with WT, M5, M8, CL9 aptamer, or poly(I·C) (2 fmol) using lipofectamine RNAiMax. After 24 h, cells were harvested and total mRNA was isolated. Antiviral and inflammatory gene expression was determined by qPCR. Data are from two independent experiments performed in triplicate and represent the means and standard errors of the means (SEM). (D) HEK293T cells were cotransfected ISRE or IFN-β promoter reporter plasmid (200 ng) along with WT, M5, or M8 5′pppRNA (10 ng/ml). Luciferase activity was analyzed 24 h posttransfection by the dual-luciferase reporter assay. Relative luciferase activity was measured as fold induction relative to the basal level of reporter gene. Data are from two independent experiments performed in triplicate and represent the means and SEM. (E) A549 cells were transfected with 5′pppRNA (0.1, 1, or 10 ng/ml), and whole-cell extracts were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting for IRF3 pSer396, IRF3, ISG56, STAT1, RIG-I, and β-actin 24 h later. One representative Western blot out of three independent triplicates is shown. (F) A549 cells were transfected with 5′pppRNA or CL9 aptamer (0.01, 0.1, 1, or 10 ng/ml) for 24 h and then infected with influenza (MOI, 0.2) for 24 h. Whole-cell extracts were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting for influenza viral protein NS1 and β-actin. One representative Western blot from one experiment is shown. (G) A549 cells were transfected with WT, M5, or M8 5′pppRNA, poly(I·C), or CL9 aptamer (1 ng/ml) for 24 h and then challenged with dengue virus (MOI, 0.5). The percentage of infected cells was determined 24 h postinfection by intracellular staining of DenV E protein expression. Data are from two independent experiments performed in triplicate and represent the means and SEM. *, P

    Techniques Used: Derivative Assay, In Vitro, Purification, Transfection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, SDS Page, Western Blot, Infection, Staining

    4) Product Images from "The differential statin effect on cytokine production of monocytes or macrophages is mediated by differential geranylgeranylation-dependent Rac1 activation"

    Article Title: The differential statin effect on cytokine production of monocytes or macrophages is mediated by differential geranylgeranylation-dependent Rac1 activation

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-2109-9

    microRNAs 146a, 146b, and 155 are down-regulated in statin-treated Mac. a microRNA array. Total RNA was isolated from macrophages differentiated in the absence or presence of statin (25 cm 2 flasks, 100,000 cells/cm 2 ) with the “RNeasy Plus Mini Kit”. Deep sequencing was performed by the “Core Unit DNA”, Universität Leipzig, using the “TruSeq™Small RNA sample prepkit v2” (illumina, San Diego, USA). The mean of the normalized data of macrophages pretreated without statin and macrophages pretreated with statin was calculated and data of samples with a mean > 100 counts (192 samples; compare Supplementary Table 2 ) were included into the analysis and blotted against each other. The orange line indicates unchanged expression. The red dots mark three selected miRs, which were down-regulated in statin-pretreated macrophages, as compared to Mac prepared in the absence of statin (compare Supplementary Table 2 ). A second array showed a similar result. b Blockade of miR-146a and miR-155 reverses the hypo-responsiveness in macrophages only to some degree. Macrophages were incubated as described in Fig. 1a (6-well plate; 100,000 cells/cm 2 ). The respective “miRCURY LNA™” anti-miR (Exiqon, Qiagen, Vedbaek, Denmark) were prepared in “Lipofectamine RNAiMax” and 250 µl of this solution was added to 2750 µl of culture medium in the absence (−) or presence (+) of statin. After 24 h LPS was added. After further 24 h, the supernatants were harvested and analyzed in ELISA. Four experiments with similar results were performed. Data analysis and color code as in Fig. 1c (“Ctrl” vs. “anti-miR”).
    Figure Legend Snippet: microRNAs 146a, 146b, and 155 are down-regulated in statin-treated Mac. a microRNA array. Total RNA was isolated from macrophages differentiated in the absence or presence of statin (25 cm 2 flasks, 100,000 cells/cm 2 ) with the “RNeasy Plus Mini Kit”. Deep sequencing was performed by the “Core Unit DNA”, Universität Leipzig, using the “TruSeq™Small RNA sample prepkit v2” (illumina, San Diego, USA). The mean of the normalized data of macrophages pretreated without statin and macrophages pretreated with statin was calculated and data of samples with a mean > 100 counts (192 samples; compare Supplementary Table 2 ) were included into the analysis and blotted against each other. The orange line indicates unchanged expression. The red dots mark three selected miRs, which were down-regulated in statin-pretreated macrophages, as compared to Mac prepared in the absence of statin (compare Supplementary Table 2 ). A second array showed a similar result. b Blockade of miR-146a and miR-155 reverses the hypo-responsiveness in macrophages only to some degree. Macrophages were incubated as described in Fig. 1a (6-well plate; 100,000 cells/cm 2 ). The respective “miRCURY LNA™” anti-miR (Exiqon, Qiagen, Vedbaek, Denmark) were prepared in “Lipofectamine RNAiMax” and 250 µl of this solution was added to 2750 µl of culture medium in the absence (−) or presence (+) of statin. After 24 h LPS was added. After further 24 h, the supernatants were harvested and analyzed in ELISA. Four experiments with similar results were performed. Data analysis and color code as in Fig. 1c (“Ctrl” vs. “anti-miR”).

    Techniques Used: Isolation, Sequencing, Expressing, Incubation, Enzyme-linked Immunosorbent Assay

    5) Product Images from "The arginine methyltransferase PRMT6 regulates cell proliferation and senescence through transcriptional repression of tumor suppressor genes"

    Article Title: The arginine methyltransferase PRMT6 regulates cell proliferation and senescence through transcriptional repression of tumor suppressor genes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks767

    PRMT6 regulates p21 gene expression in U2OS cells and is recruited to the p21 gene locus accompanied by enhanced H3 R2me2a level. ( A ) U2OS cells were transfected with siNon-targeting (siNon), siScramble (siScr) or five alternative siRNAs against PRMT6 (siPRMT6_1, _2, _3, _4, _5). Additional controls were untransfected cells (−) and Lipofectamine RNAiMAX (Lipo)-treated cells. Seventy-two hours post transfection cells were harvested and 30 µg total protein of each sample were analyzed by western blot with the indicated antibodies. ( B–E ) U2OS cells were treated with siRNAs or control conditions as in (A) for 72 h. Subsequently, cells were harvested and total RNA was prepared and analyzed by RT–qPCR for transcript levels of (B) PRMT6, (C) p21, (D) p27 and p57, (E) cyclin A2 and D1, respectively, normalized to Ubiquitin. ( F and G ) U2OS cells were harvested after 72 h and subjected to ChIP analysis using antibodies against (F) PRMT6 (gray bars) and corresponding control antibody (black bars, IgG rabbit) or (G) H3 R2me2a and corresponding control antibody (H3). Immunoprecipitated DNA was analyzed in triplicates by qPCR with primers spanning the indicated regions of the p21 gene locus. In (F), mean values were expressed as percent input of chromatin or as fold IgG, which was equated 1. In (G ) , mean values were expressed as relative enrichment compared to histone H3.
    Figure Legend Snippet: PRMT6 regulates p21 gene expression in U2OS cells and is recruited to the p21 gene locus accompanied by enhanced H3 R2me2a level. ( A ) U2OS cells were transfected with siNon-targeting (siNon), siScramble (siScr) or five alternative siRNAs against PRMT6 (siPRMT6_1, _2, _3, _4, _5). Additional controls were untransfected cells (−) and Lipofectamine RNAiMAX (Lipo)-treated cells. Seventy-two hours post transfection cells were harvested and 30 µg total protein of each sample were analyzed by western blot with the indicated antibodies. ( B–E ) U2OS cells were treated with siRNAs or control conditions as in (A) for 72 h. Subsequently, cells were harvested and total RNA was prepared and analyzed by RT–qPCR for transcript levels of (B) PRMT6, (C) p21, (D) p27 and p57, (E) cyclin A2 and D1, respectively, normalized to Ubiquitin. ( F and G ) U2OS cells were harvested after 72 h and subjected to ChIP analysis using antibodies against (F) PRMT6 (gray bars) and corresponding control antibody (black bars, IgG rabbit) or (G) H3 R2me2a and corresponding control antibody (H3). Immunoprecipitated DNA was analyzed in triplicates by qPCR with primers spanning the indicated regions of the p21 gene locus. In (F), mean values were expressed as percent input of chromatin or as fold IgG, which was equated 1. In (G ) , mean values were expressed as relative enrichment compared to histone H3.

    Techniques Used: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    6) Product Images from "Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism"

    Article Title: Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17060800

    Sca-1+/CD31− CSCs hTERT CM protects HL-1 cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Sca-1+/CD31− CSCs hTERT were cultured in six-well plates at a density of 5 × 10 4 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes or NC siRNA duplexes using Lipofectamine RNAiMAX. After a 48 h transfection, MCP-1 mRNA expression was assessed by real-time PCR ( A ); The average value of MCP-1 mRNA was normalized to that of GAPDH for each sample. The data represent the mean ± SD from three experiments. Sca-1+/CD31− CSCs hTERT CM ( B ) after transfection of NC siRNA or MCP-1 siRNA were subjected to densitometry and are presented as fold changes for MCP-1 (indicated by number 6) and IL-6 (indicated by number 15) ( C ), taking MCP-1 and IL-6 levels in NC siRNA-transfected Sca-1+/CD31− CSCs hTERT as a one-fold value, each in triplicate, * p
    Figure Legend Snippet: Sca-1+/CD31− CSCs hTERT CM protects HL-1 cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Sca-1+/CD31− CSCs hTERT were cultured in six-well plates at a density of 5 × 10 4 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes or NC siRNA duplexes using Lipofectamine RNAiMAX. After a 48 h transfection, MCP-1 mRNA expression was assessed by real-time PCR ( A ); The average value of MCP-1 mRNA was normalized to that of GAPDH for each sample. The data represent the mean ± SD from three experiments. Sca-1+/CD31− CSCs hTERT CM ( B ) after transfection of NC siRNA or MCP-1 siRNA were subjected to densitometry and are presented as fold changes for MCP-1 (indicated by number 6) and IL-6 (indicated by number 15) ( C ), taking MCP-1 and IL-6 levels in NC siRNA-transfected Sca-1+/CD31− CSCs hTERT as a one-fold value, each in triplicate, * p

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

    7) Product Images from "A hMTR4‐PDIA3P1‐miR‐125/124‐TRAF6 Regulatory Axis and Its Function in NF kappa B Signaling and Chemoresistance"

    Article Title: A hMTR4‐PDIA3P1‐miR‐125/124‐TRAF6 Regulatory Axis and Its Function in NF kappa B Signaling and Chemoresistance

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.30931

    PDIA3P1 relieves the repression of miR‐125a/b/miR‐124 on TRAF6. (A) miR‐125a/b and miR‐124 suppressed the activity of firefly luciferase containing the miR‐125a/b or miR‐124 binding sequences of TRAF6‐3′ UTR. The short vertical lines in the schematic diagram indicate the target sites of miR‐125a/b and miR‐124 in TRAF6‐3′ UTR (upper panel). SK‐Hep‐1 cells were co‐transfected with the indicated RNA duplex, pRL‐PGK, and pGL3cm‐TRAF6‐3′ UTR for 48 hours before luciferase activity analysis. (B) Ectopic expression of miR‐125a/b or miR‐124 decreased the protein level of TRAF6. SK‐Hep‐1 cells were transfected with the indicated RNA duplexes for 48 hours before immunoblotting. RNAiMAX indicates cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. (C) PDIA3P1 knockdown suppressed the TRAF6‐3′ UTR reporter activity. SK‐Hep‐1 cells were transfected with NC or siPDIA3P1 for 24 hours, followed by co‐transfection with pRL‐PGK and pGL3cm‐TRAF6‐3′ UTR for 48 hours before luciferase activity analysis. (D) PDIA3P1 overexpression attenuated the function of miR‐125a/b and miR‐124 in decreasing the TRAF6‐3′ UTR reporter activity. SK‐PDIA3P1 and its control line SK‐Ctrl were co‐transfected with the indicated RNA duplexes, pRL‐PGK, and pGL3cm‐TRAF6‐3′ UTR for 48 hours before luciferase activity analysis. (E) Antagonism of cellular miR‐125a/b or miR‐124 rescued the siPDIA3P1‐induced reduction in the TRAF6 level. SK‐Hep‐1 cells were co‐transfected with the indicated siRNA duplexes and miRNA inhibitors for 48 hours before immunoblotting. GAPDH was used as an internal control. For (A), (C), and (D), data are expressed as the mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: PDIA3P1 relieves the repression of miR‐125a/b/miR‐124 on TRAF6. (A) miR‐125a/b and miR‐124 suppressed the activity of firefly luciferase containing the miR‐125a/b or miR‐124 binding sequences of TRAF6‐3′ UTR. The short vertical lines in the schematic diagram indicate the target sites of miR‐125a/b and miR‐124 in TRAF6‐3′ UTR (upper panel). SK‐Hep‐1 cells were co‐transfected with the indicated RNA duplex, pRL‐PGK, and pGL3cm‐TRAF6‐3′ UTR for 48 hours before luciferase activity analysis. (B) Ectopic expression of miR‐125a/b or miR‐124 decreased the protein level of TRAF6. SK‐Hep‐1 cells were transfected with the indicated RNA duplexes for 48 hours before immunoblotting. RNAiMAX indicates cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. (C) PDIA3P1 knockdown suppressed the TRAF6‐3′ UTR reporter activity. SK‐Hep‐1 cells were transfected with NC or siPDIA3P1 for 24 hours, followed by co‐transfection with pRL‐PGK and pGL3cm‐TRAF6‐3′ UTR for 48 hours before luciferase activity analysis. (D) PDIA3P1 overexpression attenuated the function of miR‐125a/b and miR‐124 in decreasing the TRAF6‐3′ UTR reporter activity. SK‐PDIA3P1 and its control line SK‐Ctrl were co‐transfected with the indicated RNA duplexes, pRL‐PGK, and pGL3cm‐TRAF6‐3′ UTR for 48 hours before luciferase activity analysis. (E) Antagonism of cellular miR‐125a/b or miR‐124 rescued the siPDIA3P1‐induced reduction in the TRAF6 level. SK‐Hep‐1 cells were co‐transfected with the indicated siRNA duplexes and miRNA inhibitors for 48 hours before immunoblotting. GAPDH was used as an internal control. For (A), (C), and (D), data are expressed as the mean ± SEM of at least three independent experiments. * P

    Techniques Used: Activity Assay, Luciferase, Binding Assay, Transfection, Expressing, Cotransfection, Over Expression

    PDIA3P1 inhibits apoptosis by enhancing TRAF6 expression. (A) PDIA3P1 knockdown reduced the protein level of TRAF6. SK‐Hep‐1 or QGY‐7703 cells were transfected with the indicated RNA duplexes for 48 hours before immunoblotting. RNAiMAX indicates cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. (B) Ectopic expression of PDIA3P1 increased the protein level of TRAF6. SK‐Hep‐1 or QGY‐7703 cells were transfected with pc3‐gab‐PDIA3P1 (PDIA3P1) or its control pc3‐gab (Ctrl) plasmid for 48 hours before western blotting. (C) The xenografts of shPDIA3P1 transfectants displayed lower levels of TRAF6. The intensity of TRAF6 immunohistochemical staining was analyzed. (D) Overexpression of TRAF6 abrogated the promotive effect of siPDIA3P1 on Dox‐induced apoptosis. SK‐TRAF6 and its control cell line SK‐Ctrl were transfected with NC or siPDIA3P1 for 24 hours and then treated with 0.25 μM Dox for 36 hours before DAPI staining or for 30 hours before immunoblotting. (E) Silencing of TRAF6 diminished the inhibitory role of PDIA3P1 on Dox‐induced apoptosis. SK‐PDIA3P1 and its control cell line SK‐Ctrl were transfected with NC or siTRAF6 for 24 hours and then treated with 1 μM Dox for 48 hours before DAPI staining. + or − indicates cells with (+) or without (−) the indicated treatment. For (A), (B), and (D), GAPDH was used as an internal control. For (C) and (D), data are expressed as the mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: PDIA3P1 inhibits apoptosis by enhancing TRAF6 expression. (A) PDIA3P1 knockdown reduced the protein level of TRAF6. SK‐Hep‐1 or QGY‐7703 cells were transfected with the indicated RNA duplexes for 48 hours before immunoblotting. RNAiMAX indicates cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. (B) Ectopic expression of PDIA3P1 increased the protein level of TRAF6. SK‐Hep‐1 or QGY‐7703 cells were transfected with pc3‐gab‐PDIA3P1 (PDIA3P1) or its control pc3‐gab (Ctrl) plasmid for 48 hours before western blotting. (C) The xenografts of shPDIA3P1 transfectants displayed lower levels of TRAF6. The intensity of TRAF6 immunohistochemical staining was analyzed. (D) Overexpression of TRAF6 abrogated the promotive effect of siPDIA3P1 on Dox‐induced apoptosis. SK‐TRAF6 and its control cell line SK‐Ctrl were transfected with NC or siPDIA3P1 for 24 hours and then treated with 0.25 μM Dox for 36 hours before DAPI staining or for 30 hours before immunoblotting. (E) Silencing of TRAF6 diminished the inhibitory role of PDIA3P1 on Dox‐induced apoptosis. SK‐PDIA3P1 and its control cell line SK‐Ctrl were transfected with NC or siTRAF6 for 24 hours and then treated with 1 μM Dox for 48 hours before DAPI staining. + or − indicates cells with (+) or without (−) the indicated treatment. For (A), (B), and (D), GAPDH was used as an internal control. For (C) and (D), data are expressed as the mean ± SEM of at least three independent experiments. * P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Immunohistochemistry, Staining, Over Expression

    8) Product Images from "Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin)"

    Article Title: Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0235563

    Verification of the band specific to endogenous Fpn1 protein. A) Caco-2 cells were transfected with Control- or Fpn1-siRNA using Lipofectamine RNAiMax for 18hr, followed by treatment with FAC at the final 250 and 500uM for additional 8hr in the presence of siRNA. Unheated Caco-2 WCLs in RIPA buffer along with unheated WCL from HEK293 cells transfected with pCMV or pCVMFpn1 were analyzed by western blotting for Fpn1 (top) followed by incubation with anti-GAPDH antibody. B) Caco-2 cells were untreated or treated with 500nM hepcidin (3 plates each) for 22h. 20ug of three-independent hepcidin (-) and hepcidin (+) WCLs in RIPA buffer were mixed with 2X SDSPAGE sample loading buffer, unheated, and subjected to Western blotting with the anti-Fpn1 antibody. Unheated WCL from HEK293 cells transfected with pCMV or pCVMFpn1 was loaded as a control. The membrane was incubated with the anti-GAPDH antibody to assess an equal amount of sample loading. The experiments were repeated five times and the representative western blots are shown.
    Figure Legend Snippet: Verification of the band specific to endogenous Fpn1 protein. A) Caco-2 cells were transfected with Control- or Fpn1-siRNA using Lipofectamine RNAiMax for 18hr, followed by treatment with FAC at the final 250 and 500uM for additional 8hr in the presence of siRNA. Unheated Caco-2 WCLs in RIPA buffer along with unheated WCL from HEK293 cells transfected with pCMV or pCVMFpn1 were analyzed by western blotting for Fpn1 (top) followed by incubation with anti-GAPDH antibody. B) Caco-2 cells were untreated or treated with 500nM hepcidin (3 plates each) for 22h. 20ug of three-independent hepcidin (-) and hepcidin (+) WCLs in RIPA buffer were mixed with 2X SDSPAGE sample loading buffer, unheated, and subjected to Western blotting with the anti-Fpn1 antibody. Unheated WCL from HEK293 cells transfected with pCMV or pCVMFpn1 was loaded as a control. The membrane was incubated with the anti-GAPDH antibody to assess an equal amount of sample loading. The experiments were repeated five times and the representative western blots are shown.

    Techniques Used: Transfection, Western Blot, Incubation

    9) 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

    10) 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

    11) 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

    12) 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

    13) Product Images from "Expression of MicroRNA miR-122 Facilitates an Efficient Replication in Nonhepatic Cells upon Infection with Hepatitis C Virus"

    Article Title: Expression of MicroRNA miR-122 Facilitates an Efficient Replication in Nonhepatic Cells upon Infection with Hepatitis C Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.00567-12

    Expression of miR-122 is essential for the enhancement of HCV replication in the Hec1B cells. (A) Huh7.5.1 and Hec1B cells were transduced with lentiviral vectors expressing miR-122 in a dose-dependent manner and infected with HCVcc at an MOI of 1. Intracellular miR-122 and HCV RNA were determined at 24 h postinfection by qRT-PCR. (B) Huh7.5.1 and Hec1B/miR-122 cells were infected with HCVcc at an MOI of 0.5 or 3 and subjected to immunoblotting and immunofluorescence analyses using anti-NS5A antibodies at 48 h postinfection. The asterisk indicates nonspecific bands. (C) LNAs specific to miR-122 at a final concentration of 5 nM, 20 nM, or 50 nM and control (LNA alone at 50 nM) were introduced into Huh7/Cont, Huh7/miR-122, Hec1B/miR-122, and Hec1B/Cont cells by using Lipofectamine RNAiMAX transfection reagent and infected with HCVcc at an MOI of 1 at 6 h posttransfection. Intracellular HCV RNA levels were determined by qRT-PCR at 12, 24, and 36 h postinfection. Asterisks indicate significant differences (*, P
    Figure Legend Snippet: Expression of miR-122 is essential for the enhancement of HCV replication in the Hec1B cells. (A) Huh7.5.1 and Hec1B cells were transduced with lentiviral vectors expressing miR-122 in a dose-dependent manner and infected with HCVcc at an MOI of 1. Intracellular miR-122 and HCV RNA were determined at 24 h postinfection by qRT-PCR. (B) Huh7.5.1 and Hec1B/miR-122 cells were infected with HCVcc at an MOI of 0.5 or 3 and subjected to immunoblotting and immunofluorescence analyses using anti-NS5A antibodies at 48 h postinfection. The asterisk indicates nonspecific bands. (C) LNAs specific to miR-122 at a final concentration of 5 nM, 20 nM, or 50 nM and control (LNA alone at 50 nM) were introduced into Huh7/Cont, Huh7/miR-122, Hec1B/miR-122, and Hec1B/Cont cells by using Lipofectamine RNAiMAX transfection reagent and infected with HCVcc at an MOI of 1 at 6 h posttransfection. Intracellular HCV RNA levels were determined by qRT-PCR at 12, 24, and 36 h postinfection. Asterisks indicate significant differences (*, P

    Techniques Used: Expressing, Transduction, Infection, Quantitative RT-PCR, Immunofluorescence, Concentration Assay, Transfection

    14) Product Images from "Tumor-Penetrating Delivery of siRNA against TNFα to Human Vestibular Schwannomas"

    Article Title: Tumor-Penetrating Delivery of siRNA against TNFα to Human Vestibular Schwannomas

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13032-9

    iRGD nanoparticles mediate TNF-α silencing by RNAi in vitro . ( A ) Nanoparticle delivery of TNFα siRNA to HEI-193 vestibular schwannoma cells. Relative TNFα gene expression quantified by qRT-PCR in cells treated with various formulations, in the absence of lipopolysaccharide (LPS) stimulation. Medium, untreated controls. siTNFa / scr, control nanoparticles carrying TNFα siRNA. si GFP / iRGD, iRGD nanoparticles carrying GFP siRNA. si TNFα / Lipofect, Lipofectamine RNAiMAX® complexed with TNFα siRNA as a positive transfection control. si TNF α / iRGD, iRGD nanoparticles carrying TNFα siRNA. Error bars represent standard deviation. N = 6 biological replicates with independently prepared nanoparticles. *** P
    Figure Legend Snippet: iRGD nanoparticles mediate TNF-α silencing by RNAi in vitro . ( A ) Nanoparticle delivery of TNFα siRNA to HEI-193 vestibular schwannoma cells. Relative TNFα gene expression quantified by qRT-PCR in cells treated with various formulations, in the absence of lipopolysaccharide (LPS) stimulation. Medium, untreated controls. siTNFa / scr, control nanoparticles carrying TNFα siRNA. si GFP / iRGD, iRGD nanoparticles carrying GFP siRNA. si TNFα / Lipofect, Lipofectamine RNAiMAX® complexed with TNFα siRNA as a positive transfection control. si TNF α / iRGD, iRGD nanoparticles carrying TNFα siRNA. Error bars represent standard deviation. N = 6 biological replicates with independently prepared nanoparticles. *** P

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, Standard Deviation

    15) Product Images from "Collateral Chemoresistance to Anti-Microtubule Agents in a Lung Cancer Cell Line with Acquired Resistance to Erlotinib"

    Article Title: Collateral Chemoresistance to Anti-Microtubule Agents in a Lung Cancer Cell Line with Acquired Resistance to Erlotinib

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123901

    Effect of siRNA-mediated knockdown for CDH1 in HCC4006ER cells. (A) Total cell lysates were harvested 72 hours after reverse-transfection of negative control siRNA (siNC) or validated siRNAs for CDH1 which encodes E-cadherin mixed with Lipofectamine RNAiMAX. (B-D) Tumor cells were reverse-transfected at the same time plating into 96-wells and then incubated for 24 hours. They were incubated with various concentrations of docetaxel, paclitaxel and vinorelbine for additional 72 hours. Percent growth relative to DMSO-treated controls was evaluated by Cell Counting Kit-8 assay.
    Figure Legend Snippet: Effect of siRNA-mediated knockdown for CDH1 in HCC4006ER cells. (A) Total cell lysates were harvested 72 hours after reverse-transfection of negative control siRNA (siNC) or validated siRNAs for CDH1 which encodes E-cadherin mixed with Lipofectamine RNAiMAX. (B-D) Tumor cells were reverse-transfected at the same time plating into 96-wells and then incubated for 24 hours. They were incubated with various concentrations of docetaxel, paclitaxel and vinorelbine for additional 72 hours. Percent growth relative to DMSO-treated controls was evaluated by Cell Counting Kit-8 assay.

    Techniques Used: Transfection, Negative Control, Incubation, Cell Counting

    Effect of siRNA-mediated knockdown for ABCB1 in HCC4006ER cells. (A) Total cell lysates were harvested 72 hours after reverse-transfection of negative control siRNA (siNC) or validated two siRNAs for ATP-binding cassette subfamily B, member 1 (ABCB1) mixed with Lipofectamine RNAiMAX. (B-E) Tumor cells were reverse-transfected at the same time plating into 96-wells and then incubated for 24 hours. They were incubated with various concentrations of docetaxel, paclitaxel, vinorelbine and erlotinib for additional 72 hours. Percent growth relative to DMSO-treated controls was evaluated by Cell Counting Kit-8 assay.
    Figure Legend Snippet: Effect of siRNA-mediated knockdown for ABCB1 in HCC4006ER cells. (A) Total cell lysates were harvested 72 hours after reverse-transfection of negative control siRNA (siNC) or validated two siRNAs for ATP-binding cassette subfamily B, member 1 (ABCB1) mixed with Lipofectamine RNAiMAX. (B-E) Tumor cells were reverse-transfected at the same time plating into 96-wells and then incubated for 24 hours. They were incubated with various concentrations of docetaxel, paclitaxel, vinorelbine and erlotinib for additional 72 hours. Percent growth relative to DMSO-treated controls was evaluated by Cell Counting Kit-8 assay.

    Techniques Used: Transfection, Negative Control, Binding Assay, Incubation, Cell Counting

    16) Product Images from "Polymeric nanoparticle-based delivery of microRNA-199a-3p inhibits proliferation and growth of osteosarcoma cells"

    Article Title: Polymeric nanoparticle-based delivery of microRNA-199a-3p inhibits proliferation and growth of osteosarcoma cells

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S79143

    Protein expression levels in osteosarcoma cells transfected with microRNAs delivered using dextran nanoparticles. Notes: Protein expression in miR-199a-3p dextran nanoparticle transfected osteosarcoma cells. U-2OS ( A ) and KHOS ( B ) cells were transfected with miR-199a-3p encapsulated with dextran-based nanoparticles, or transfected with Lipofectamine ® RNAiMAX. The expression of mTOR and Met were determined by Western blots, and the data were analyzed by densitometry ( C and D ). Abbreviation: β-actin, beta-actin.
    Figure Legend Snippet: Protein expression levels in osteosarcoma cells transfected with microRNAs delivered using dextran nanoparticles. Notes: Protein expression in miR-199a-3p dextran nanoparticle transfected osteosarcoma cells. U-2OS ( A ) and KHOS ( B ) cells were transfected with miR-199a-3p encapsulated with dextran-based nanoparticles, or transfected with Lipofectamine ® RNAiMAX. The expression of mTOR and Met were determined by Western blots, and the data were analyzed by densitometry ( C and D ). Abbreviation: β-actin, beta-actin.

    Techniques Used: Expressing, Transfection, Western Blot

    Expression levels of microRNAs transfected by dextran nanoparticles. Notes: Expression of miR-199a-3p or let-7a in osteosarcoma cells transfected by dextran nanoparticles. U-2OS cells were transfected with miR199a-3p ( A ) or 100 nM let-7a ( B ) encapsulated with dextran nanoparticle, or transfected with with Lipofectamine™ RNAiMax. The total RNA (including miRs) was extracted after 48 hours post transfection. Expressions of miR-199a-3p or let-7a were determined by TaqMan MicroRNA Assay kits. Abbreviation: RQ, relative quantity.
    Figure Legend Snippet: Expression levels of microRNAs transfected by dextran nanoparticles. Notes: Expression of miR-199a-3p or let-7a in osteosarcoma cells transfected by dextran nanoparticles. U-2OS cells were transfected with miR199a-3p ( A ) or 100 nM let-7a ( B ) encapsulated with dextran nanoparticle, or transfected with with Lipofectamine™ RNAiMax. The total RNA (including miRs) was extracted after 48 hours post transfection. Expressions of miR-199a-3p or let-7a were determined by TaqMan MicroRNA Assay kits. Abbreviation: RQ, relative quantity.

    Techniques Used: Expressing, Transfection, TaqMan microRNA Assay

    Osteosarcoma cell growth inhibition using microRNA encapsulated dextran nanoparticles. Notes: Effect on osteosarcoma cell growth and proliferation in dextran nanoparticle transfected cells. U-2OS ( A and B ) and KHOS ( C and D ) cells were transfected with miR-199a-3p ( A and C ) or let-7a ( B and D ) encapsulated with dextran-based nanoparticles, or transfected with Lipofectamine ® RNAiMAX. MTT assay was performed by measuring the absorbance at a wavelength of 490 nm. Data are represented as means ± SD and analyzed using Student’s t -test. *indicates P
    Figure Legend Snippet: Osteosarcoma cell growth inhibition using microRNA encapsulated dextran nanoparticles. Notes: Effect on osteosarcoma cell growth and proliferation in dextran nanoparticle transfected cells. U-2OS ( A and B ) and KHOS ( C and D ) cells were transfected with miR-199a-3p ( A and C ) or let-7a ( B and D ) encapsulated with dextran-based nanoparticles, or transfected with Lipofectamine ® RNAiMAX. MTT assay was performed by measuring the absorbance at a wavelength of 490 nm. Data are represented as means ± SD and analyzed using Student’s t -test. *indicates P

    Techniques Used: Inhibition, Transfection, MTT Assay

    17) 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

    18) Product Images from "Porcine Reproductive and Respiratory Syndrome Virus strains with Higher Virulence Cause Marked Protein Profile Changes in MARC-145 Cells"

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus strains with Higher Virulence Cause Marked Protein Profile Changes in MARC-145 Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32984-0

    Knockdown of Endogenous PSF Genes Decreases Replication of the PRRSV SX-1 Strain. ( A ) Endogenous PSF protein expression was down-regulated by PSF siRNA (SiPSF). SiRNA was transfected by Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Scrambled siRNA was used as a negative control. After transfected 48 h, cells were harvested, and western blotting was performed. ( B ) Graphical representation of t -test of the ratios of density between the PSF and β-actin bands. **** p
    Figure Legend Snippet: Knockdown of Endogenous PSF Genes Decreases Replication of the PRRSV SX-1 Strain. ( A ) Endogenous PSF protein expression was down-regulated by PSF siRNA (SiPSF). SiRNA was transfected by Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Scrambled siRNA was used as a negative control. After transfected 48 h, cells were harvested, and western blotting was performed. ( B ) Graphical representation of t -test of the ratios of density between the PSF and β-actin bands. **** p

    Techniques Used: Expressing, Transfection, Negative Control, Western Blot

    19) 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

    20) Product Images from "MicroRNA-143-3p contributes to the regulation of pain responses in collagen-induced arthritis"

    Article Title: MicroRNA-143-3p contributes to the regulation of pain responses in collagen-induced arthritis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.9322

    Effects of miR-143 on pain-associated target genes. Mouse DRG cells were transfected with Lipofectamine RNAiMAX transfection reagent and the miR-143-3p mimic NC-carboxyfluorescein. (A) Bright field (magnification, ×100). (B) Fluorescence microscopy (magnification, ×100). RNA was extracted from the DRG cells 48 h following transfection with an miR-143-3p inhibitor or mimic. (C) miR-143-3p expression following transfection with the mimic. (D) miR-143-3p expression following transfection with the inhibitor. (E) Ptgdr expression, (F) Mrgpre expression, (G) Ptgs2 expression and (H) Tnf expression. *P
    Figure Legend Snippet: Effects of miR-143 on pain-associated target genes. Mouse DRG cells were transfected with Lipofectamine RNAiMAX transfection reagent and the miR-143-3p mimic NC-carboxyfluorescein. (A) Bright field (magnification, ×100). (B) Fluorescence microscopy (magnification, ×100). RNA was extracted from the DRG cells 48 h following transfection with an miR-143-3p inhibitor or mimic. (C) miR-143-3p expression following transfection with the mimic. (D) miR-143-3p expression following transfection with the inhibitor. (E) Ptgdr expression, (F) Mrgpre expression, (G) Ptgs2 expression and (H) Tnf expression. *P

    Techniques Used: Transfection, Fluorescence, Microscopy, Expressing

    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 "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

    23) Product Images from "Crosstalk between p38 MAPK and caspase-9 regulates mitochondria-mediated apoptosis induced by tetra-α-(4-carboxyphenoxy) phthalocyanine zinc photodynamic therapy in LoVo cells"

    Article Title: Crosstalk between p38 MAPK and caspase-9 regulates mitochondria-mediated apoptosis induced by tetra-α-(4-carboxyphenoxy) phthalocyanine zinc photodynamic therapy in LoVo cells

    Journal: Oncology Reports

    doi: 10.3892/or.2017.6071

    Silencing of p38 MAPK in LoVo cells. LoVo cells were transfected with siRNA- p38 MAPK (12.5 nmol/l), the negative control siRNA and the GAPDH positive control siRNA using Lipofectamine RNAiMAX. In the presence of Block-iT Alexa Fluor Red Fluorescent Control for 48 h, the LoVo cells were exposed to red light irradiation (53.7 J/cm 2 ), incubated for 3 h and then, the transfected cells were observed using (A) an inverted microscope and (B) a fluorescence microscope. Following transfection, the expression levels of p38 MAPK mRNA and p38 MAPK protein were analyzed using (C and E) reverse transcription-PCR and (D and F) immunoblot assay, respectively. The LoVo cells were treated with Lipofectamine RNAiMAX only as the control treatment. GAPDH was used as an internal control. The values presented are representative of three independent experiments (mean ± standard deviation; **P
    Figure Legend Snippet: Silencing of p38 MAPK in LoVo cells. LoVo cells were transfected with siRNA- p38 MAPK (12.5 nmol/l), the negative control siRNA and the GAPDH positive control siRNA using Lipofectamine RNAiMAX. In the presence of Block-iT Alexa Fluor Red Fluorescent Control for 48 h, the LoVo cells were exposed to red light irradiation (53.7 J/cm 2 ), incubated for 3 h and then, the transfected cells were observed using (A) an inverted microscope and (B) a fluorescence microscope. Following transfection, the expression levels of p38 MAPK mRNA and p38 MAPK protein were analyzed using (C and E) reverse transcription-PCR and (D and F) immunoblot assay, respectively. The LoVo cells were treated with Lipofectamine RNAiMAX only as the control treatment. GAPDH was used as an internal control. The values presented are representative of three independent experiments (mean ± standard deviation; **P

    Techniques Used: Transfection, Negative Control, Positive Control, Blocking Assay, Irradiation, Incubation, Inverted Microscopy, Fluorescence, Microscopy, Expressing, Polymerase Chain Reaction, Standard Deviation

    24) Product Images from "Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide"

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033287

    A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).
    Figure Legend Snippet: A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Software, Isolation, Polymerase Chain Reaction, Luciferase, Negative Control, Incubation, Concentration Assay, Western Blot, Construct

    25) 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

    26) 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

    27) Product Images from "Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo"

    Article Title: Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo

    Journal: RNA

    doi: 10.1261/rna.031120.111

    In vitro activity and viability. ( A , B ) HeLa cells stably transfected with FVII ( A ) or parental HeLa cells ( B ) were reverse-transfected using Lipofectamine RNAiMax with 0.1 nM duplex. FVII protein ( A ) was quantified by chromogenic assay, while mRNA ( A–C
    Figure Legend Snippet: In vitro activity and viability. ( A , B ) HeLa cells stably transfected with FVII ( A ) or parental HeLa cells ( B ) were reverse-transfected using Lipofectamine RNAiMax with 0.1 nM duplex. FVII protein ( A ) was quantified by chromogenic assay, while mRNA ( A–C

    Techniques Used: In Vitro, Activity Assay, Stable Transfection, Transfection, Chromogenic Assay

    28) Product Images from "Pituitary Tumor-Transforming 1 Increases Cell Motility and Promotes Lymph Node Metastasis in Esophageal Squamous Cell Carcinoma"

    Article Title: Pituitary Tumor-Transforming 1 Increases Cell Motility and Promotes Lymph Node Metastasis in Esophageal Squamous Cell Carcinoma

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-07-3043

    Knockdown of PTTG1 expression in ESCC cells by siRNAs. HSA/c, KYSE140, and KYSE410 cells were transfected using the following conditions: LF−, untransfected control; LF+, transfection with Lipofectamine RNAiMAX alone; NTC, nontargeting control
    Figure Legend Snippet: Knockdown of PTTG1 expression in ESCC cells by siRNAs. HSA/c, KYSE140, and KYSE410 cells were transfected using the following conditions: LF−, untransfected control; LF+, transfection with Lipofectamine RNAiMAX alone; NTC, nontargeting control

    Techniques Used: Expressing, Transfection

    29) 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

    30) 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

    31) Product Images from "Towards genetic modification of plant-parasitic nematodes: delivery of macromolecules to adults and expression of exogenous mRNA in second stage juveniles"

    Article Title: Towards genetic modification of plant-parasitic nematodes: delivery of macromolecules to adults and expression of exogenous mRNA in second stage juveniles

    Journal: bioRxiv

    doi: 10.1101/2020.07.15.193052

    Expression of exogenous mRNAs in second stage juvenile H. schachtii . A) Quantification of fluorescence. Representative images of negative control and treated nematodes. Pixels above threshold marked (black) and counts are shown in the boxplot. Horizontal line in box plot represents median value, whiskers extend to data points that are less than 1.5 x IQR away from 1st/3rd quartile. Left, nematodes fixed post lipofection comparing nematodes soaked in empty liposomes (negative) with nematodes soaked in liposomes containing mRNA encoding GFP. Right, live nematodes comparing mRNA encoding GFP encapsulated in either CRISPRMAX or RNAiMAX lipofectamine. P-values are indicated for independent 2-group Mann–Whitney U test. B) Quantified bio-luminescence (arbitrary units plotted on log scale) of live nematodes soaked in mRNA encoding luciferase encapsulated in liposomes (red), or nematodes soaked in empty liposomes (turquoise), measured every 176 seconds for 48.84 hours. Inset, a zoom in of hours 0-20. Arrow indicated 10 hours. The half-life in treated nematodes is compared to the control using the independent 2-group Mann–Whitney U test. Error bars indicate standard deviation of the mean (n = 8) at each time point.
    Figure Legend Snippet: Expression of exogenous mRNAs in second stage juvenile H. schachtii . A) Quantification of fluorescence. Representative images of negative control and treated nematodes. Pixels above threshold marked (black) and counts are shown in the boxplot. Horizontal line in box plot represents median value, whiskers extend to data points that are less than 1.5 x IQR away from 1st/3rd quartile. Left, nematodes fixed post lipofection comparing nematodes soaked in empty liposomes (negative) with nematodes soaked in liposomes containing mRNA encoding GFP. Right, live nematodes comparing mRNA encoding GFP encapsulated in either CRISPRMAX or RNAiMAX lipofectamine. P-values are indicated for independent 2-group Mann–Whitney U test. B) Quantified bio-luminescence (arbitrary units plotted on log scale) of live nematodes soaked in mRNA encoding luciferase encapsulated in liposomes (red), or nematodes soaked in empty liposomes (turquoise), measured every 176 seconds for 48.84 hours. Inset, a zoom in of hours 0-20. Arrow indicated 10 hours. The half-life in treated nematodes is compared to the control using the independent 2-group Mann–Whitney U test. Error bars indicate standard deviation of the mean (n = 8) at each time point.

    Techniques Used: Expressing, Fluorescence, Negative Control, MANN-WHITNEY, Luciferase, Standard Deviation

    32) 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

    33) Product Images from "siRNA enhances DNA-mediated interferon lambda-1 response through crosstalk between RIG-I and IFI16 signalling pathway"

    Article Title: siRNA enhances DNA-mediated interferon lambda-1 response through crosstalk between RIG-I and IFI16 signalling pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt844

    Characterization of siRNA-induced IFN-λ1 production. ( A ) HeLa cells were transfected with a transfection lipid reagent Lipofectamine RNAiMAX, naked siRNA or siRNA with the lipid, followed by IFN-α treatment and DNA transfection on day 2 and day 3, respectively. ( B ) HeLa cells were transfected with double-stranded siRNA, sense or antisense single-stranded siRNA at 10 nM, followed by IFN-α treatment and DNA transfection. ( C ) HeLa cells were transfected with various concentrations of Non-human Ctrl siRNA (0.0001–100 nM), followed by 1000 U/ml IFN-α treatment and 1 µg of DNA transfection as described above. For experiments (A), (B) and (C), total RNA was extracted for relative mRNA expression measurements using real-time RT-PCR. The gene expression level was compared with the cells treated with IFN-α and DNA but not siRNA. ( D ) HeLa cells were transfected with or without Non-human Ctrl siRNA, followed by IFN-α treatment and DNA transfection. Total RNA was extracted at different time points after plasmid DNA transfection. Gene expression was compared with untreated cells at time zero. ** P
    Figure Legend Snippet: Characterization of siRNA-induced IFN-λ1 production. ( A ) HeLa cells were transfected with a transfection lipid reagent Lipofectamine RNAiMAX, naked siRNA or siRNA with the lipid, followed by IFN-α treatment and DNA transfection on day 2 and day 3, respectively. ( B ) HeLa cells were transfected with double-stranded siRNA, sense or antisense single-stranded siRNA at 10 nM, followed by IFN-α treatment and DNA transfection. ( C ) HeLa cells were transfected with various concentrations of Non-human Ctrl siRNA (0.0001–100 nM), followed by 1000 U/ml IFN-α treatment and 1 µg of DNA transfection as described above. For experiments (A), (B) and (C), total RNA was extracted for relative mRNA expression measurements using real-time RT-PCR. The gene expression level was compared with the cells treated with IFN-α and DNA but not siRNA. ( D ) HeLa cells were transfected with or without Non-human Ctrl siRNA, followed by IFN-α treatment and DNA transfection. Total RNA was extracted at different time points after plasmid DNA transfection. Gene expression was compared with untreated cells at time zero. ** P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Plasmid Preparation

    34) 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

    35) Product Images from "MiR-27b regulates podocyte survival through targeting adenosine receptor 2B in podocytes from non-human primate"

    Article Title: MiR-27b regulates podocyte survival through targeting adenosine receptor 2B in podocytes from non-human primate

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1178-5

    Stimulating Adenosine signaling through Adora2b reversed PAN-induced injury in NHP podocytes. a Adora2b was efficiently knocked-down by siRNAs in NHP podocytes. A total of 50 nM siRNAs were transfected into the cells using Lipofectamine RNAiMax (Life Technologies). Total RNAs were isolated for RT-qPCR at 48 h post transfection. Data were normalized to GAPDH. Error bar represents data from two replicates (Left panel). Protein expression of Adora2b was also significantly decreased in siA2B transfected cells (Right panel). Total proteins were isolated for western blotting at 48 h post transfection. Tubulin was used as the loading control. b Knockdown of Adora2b promoted PAN-induced injury in NHP podocytes. Cells were transfected with siControl, miR-27b mimic, and siA2B at the final concentration of 50 nM. PAN treatment (10 µg/ml) was initiated 1 day after transfection. Cells were subject to viability test using CellTiter-96 (Promega) at day 5 post PAN treatment. Relative cell viability was calculated by normalizing absorbance readings from PAN-treated samples to non-treated controls. Error bar represents data from two independent experiments with eight replicates. * p
    Figure Legend Snippet: Stimulating Adenosine signaling through Adora2b reversed PAN-induced injury in NHP podocytes. a Adora2b was efficiently knocked-down by siRNAs in NHP podocytes. A total of 50 nM siRNAs were transfected into the cells using Lipofectamine RNAiMax (Life Technologies). Total RNAs were isolated for RT-qPCR at 48 h post transfection. Data were normalized to GAPDH. Error bar represents data from two replicates (Left panel). Protein expression of Adora2b was also significantly decreased in siA2B transfected cells (Right panel). Total proteins were isolated for western blotting at 48 h post transfection. Tubulin was used as the loading control. b Knockdown of Adora2b promoted PAN-induced injury in NHP podocytes. Cells were transfected with siControl, miR-27b mimic, and siA2B at the final concentration of 50 nM. PAN treatment (10 µg/ml) was initiated 1 day after transfection. Cells were subject to viability test using CellTiter-96 (Promega) at day 5 post PAN treatment. Relative cell viability was calculated by normalizing absorbance readings from PAN-treated samples to non-treated controls. Error bar represents data from two independent experiments with eight replicates. * p

    Techniques Used: Transfection, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Concentration Assay

    MiR-27b regulated PAN-induced apoptosis in NHP podocytes. a miR-27b was significantly suppressed upon PAN treatment. Cells were exposed to 10 µg/ml PAN for 3–5 days before collected for RT-qPCR. Relative miR expression was calculated using U6 as normalization control. Error bar represents data from two independent experiments. b PAN-induced miR-27 change was also observed in primary rat podocytes. Cells were treated with PAN for 3 and 5 days before collected for miR-qPCR analysis. Error bar represents data from two independent experiments. c miR-27b was efficiently transfected into NHP podocytes. 50 nM miRNA mimic and siControl were transfected into NHP podocytes using Lipofectamine RNAiMax. Cells were collected at day 3 and day 5 post transfection and miRNA expression was analysis using miR-X kit (Clontech). Error bar represents data from two independent experiments. Data were normalized to U6. d Endogenous miR-27b was efficiently knocked-down by transfecting miR-27b hairpin inhibitor into NHP podocytes. miR inhibitor control and miR-27b inhibitor were transfected into NHP podocytes at the final concentration of 50 nM. Cells were collected for miRNA qPCR at day 3 and day 5 post transfection. Data were normalized to U6. Error bar represents data from two independent experiments. e miR-27b overexpression enhanced PAN-induced cell death while miRNA inhibition led to increased cell viability. Cells were transfected with 50 nM miRNA mimic or inhibitor and treated with 10 µg/ml PAN for 5 days before collected for cell viability test using CellTiter 96 kit (Promega). Relative cell viability was calculated by normalizing absorbance readings from PAN-treated samples to non-treated controls. Error bar represents data from three independent experiments with eight replicates. ** p
    Figure Legend Snippet: MiR-27b regulated PAN-induced apoptosis in NHP podocytes. a miR-27b was significantly suppressed upon PAN treatment. Cells were exposed to 10 µg/ml PAN for 3–5 days before collected for RT-qPCR. Relative miR expression was calculated using U6 as normalization control. Error bar represents data from two independent experiments. b PAN-induced miR-27 change was also observed in primary rat podocytes. Cells were treated with PAN for 3 and 5 days before collected for miR-qPCR analysis. Error bar represents data from two independent experiments. c miR-27b was efficiently transfected into NHP podocytes. 50 nM miRNA mimic and siControl were transfected into NHP podocytes using Lipofectamine RNAiMax. Cells were collected at day 3 and day 5 post transfection and miRNA expression was analysis using miR-X kit (Clontech). Error bar represents data from two independent experiments. Data were normalized to U6. d Endogenous miR-27b was efficiently knocked-down by transfecting miR-27b hairpin inhibitor into NHP podocytes. miR inhibitor control and miR-27b inhibitor were transfected into NHP podocytes at the final concentration of 50 nM. Cells were collected for miRNA qPCR at day 3 and day 5 post transfection. Data were normalized to U6. Error bar represents data from two independent experiments. e miR-27b overexpression enhanced PAN-induced cell death while miRNA inhibition led to increased cell viability. Cells were transfected with 50 nM miRNA mimic or inhibitor and treated with 10 µg/ml PAN for 5 days before collected for cell viability test using CellTiter 96 kit (Promega). Relative cell viability was calculated by normalizing absorbance readings from PAN-treated samples to non-treated controls. Error bar represents data from three independent experiments with eight replicates. ** p

    Techniques Used: Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Transfection, Concentration Assay, Over Expression, Inhibition

    36) Product Images from "Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate"

    Article Title: Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2476-2

    Either BAK or BAX knockdown decreases TNFα+CHX or navitoclax-induced pyroptosis. a Efficiency of BAK and/or BAX knockdown was detected by immunoblotting. b–e After BAK siRNA, BAX siRNA, BAK siRNA+BAX siRNA, or negative control siRNA were transfected into HCT116 cells by Lipofectamine RNAiMAX, cells were reseeded into 24-well plate followed by treatment of TNFα+CHX ( b , d ) or navitoclax ( c , e ) for 24 h. Culture supernatants were collected to measure the percentage of LDH release ( b , c ) and phase contrast images were taken ( d , e ) at the indicated time points.
    Figure Legend Snippet: Either BAK or BAX knockdown decreases TNFα+CHX or navitoclax-induced pyroptosis. a Efficiency of BAK and/or BAX knockdown was detected by immunoblotting. b–e After BAK siRNA, BAX siRNA, BAK siRNA+BAX siRNA, or negative control siRNA were transfected into HCT116 cells by Lipofectamine RNAiMAX, cells were reseeded into 24-well plate followed by treatment of TNFα+CHX ( b , d ) or navitoclax ( c , e ) for 24 h. Culture supernatants were collected to measure the percentage of LDH release ( b , c ) and phase contrast images were taken ( d , e ) at the indicated time points.

    Techniques Used: Negative Control, Transfection

    37) Product Images from "Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium"

    Article Title: Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium

    Journal: Journal of Virology

    doi: 10.1128/JVI.03005-12

    Matriptase is involved in multicycle replication of H1N1 influenza virus in human bronchial epithelial cells. (A) Calu-3 cells grown for 10 days at the air-liquid interface were infected with A/Puerto Rico/8/34 (PR8) H1N1 at an MOI of 0.0001 and incubated in the absence of trypsin. At various time points, the virus titer was determined by plaque assay. (B) Calu-3 cells grown under air-liquid conditions or submerged were infected at an MOI of 0.0001 and incubated in the absence of trypsin. The virus titer was determined at 24, 48, and 72 h postinfection by plaque assay. (C) Submerged Calu-3 cells were transfected with 50 nM matriptase siRNA or scrambled siRNA using Lipofectamine RNAiMax (Invitrogen). Knockdown of matriptase was confirmed at 48 h posttransfection by Western blotting and quantified by densitometric analysis. β-Actin confirmed equal loading. (D) siRNA-transfected submerged Calu-3 cells were infected at 48 h posttransfection at an MOI of 0.0001. Supernatants were collected at 92 h postinfection and titrated by standard plaque assays for the presence of infectious virus. Data represent the means ± standard errors of the means of duplicate samples. Experiments were performed at least twice. A Student paired t test was used to analyze the data. P values are relative to scrambled siRNA-transfected cells. **, P ≤ 0.01;***, P ≤ 0.001.
    Figure Legend Snippet: Matriptase is involved in multicycle replication of H1N1 influenza virus in human bronchial epithelial cells. (A) Calu-3 cells grown for 10 days at the air-liquid interface were infected with A/Puerto Rico/8/34 (PR8) H1N1 at an MOI of 0.0001 and incubated in the absence of trypsin. At various time points, the virus titer was determined by plaque assay. (B) Calu-3 cells grown under air-liquid conditions or submerged were infected at an MOI of 0.0001 and incubated in the absence of trypsin. The virus titer was determined at 24, 48, and 72 h postinfection by plaque assay. (C) Submerged Calu-3 cells were transfected with 50 nM matriptase siRNA or scrambled siRNA using Lipofectamine RNAiMax (Invitrogen). Knockdown of matriptase was confirmed at 48 h posttransfection by Western blotting and quantified by densitometric analysis. β-Actin confirmed equal loading. (D) siRNA-transfected submerged Calu-3 cells were infected at 48 h posttransfection at an MOI of 0.0001. Supernatants were collected at 92 h postinfection and titrated by standard plaque assays for the presence of infectious virus. Data represent the means ± standard errors of the means of duplicate samples. Experiments were performed at least twice. A Student paired t test was used to analyze the data. P values are relative to scrambled siRNA-transfected cells. **, P ≤ 0.01;***, P ≤ 0.001.

    Techniques Used: Infection, Incubation, Plaque Assay, Transfection, Western Blot

    38) Product Images from "Dynamic Actin Reorganization and Vav/Cdc42-dependent Actin Polymerization Promote Macrophage Aggregated LDL Uptake and Catabolism"

    Article Title: Dynamic Actin Reorganization and Vav/Cdc42-dependent Actin Polymerization Promote Macrophage Aggregated LDL Uptake and Catabolism

    Journal: Arteriosclerosis, thrombosis, and vascular biology

    doi: 10.1161/ATVBAHA.118.312087

    Cdc42 regulates actin polymerization and compartment formation in response to agLDL. (A-C) J774 cells were transfected with scrambled (Scr), Rac1 or Cdc42 siRNA using Lipofectamine RNAiMAX reagent, and cultured for 48 h. (A) Cell lysates were taken and used to confirm Rac1 and Cdc42 protein levels were reduced by immunoblot analysis. (B) J774 cells treated under the same conditions were plated onto coverslip dishes and treated with Alexa546-agLDL for 1 h, fixed, stained for F-actin using Alexa488-phalloidin and analysed by confocal microscopy. (C) Confocal images were used to quantify F-actin colocalized with agLDL per cell for at least 10 fields containing > 100 cells. Median and interquartile range are shown. (D-F) WT BMMs were transfected with scrambled (Scr) and Cdc42 siRNA and cultured for 72 h. (D) Cell lysates were taken and used to confirm Cdc42 protein levels were reduced by immunoblot analysis. (E) BMMs treated under the same conditions were plated onto coverslip dishes and treated with Alexa546-agLDL for 1 h, fixed, stained for F-actin using Alexa488-phalloidin and analysed by confocal microscopy. (F) Confocal images were used to quantify F-actin colocalized with agLDL per cell for at least 10 fields containing > 100 cells. Median and interquartile range are shown. (G-H) BMMs were treated with DMSO (control) or 50 µM ZCL 278 (Cdc42 inhibitor) for 30 min prior to treatment with Alexa546-agLDL for 1 h in the presence of inhibitor. Cells were analysed by confocal microscopy (G) and images used to quantify F-actin colocalized with agLDL per cell for at least 10 fields containing > 100 cells. Median and interquartile range are shown. * p
    Figure Legend Snippet: Cdc42 regulates actin polymerization and compartment formation in response to agLDL. (A-C) J774 cells were transfected with scrambled (Scr), Rac1 or Cdc42 siRNA using Lipofectamine RNAiMAX reagent, and cultured for 48 h. (A) Cell lysates were taken and used to confirm Rac1 and Cdc42 protein levels were reduced by immunoblot analysis. (B) J774 cells treated under the same conditions were plated onto coverslip dishes and treated with Alexa546-agLDL for 1 h, fixed, stained for F-actin using Alexa488-phalloidin and analysed by confocal microscopy. (C) Confocal images were used to quantify F-actin colocalized with agLDL per cell for at least 10 fields containing > 100 cells. Median and interquartile range are shown. (D-F) WT BMMs were transfected with scrambled (Scr) and Cdc42 siRNA and cultured for 72 h. (D) Cell lysates were taken and used to confirm Cdc42 protein levels were reduced by immunoblot analysis. (E) BMMs treated under the same conditions were plated onto coverslip dishes and treated with Alexa546-agLDL for 1 h, fixed, stained for F-actin using Alexa488-phalloidin and analysed by confocal microscopy. (F) Confocal images were used to quantify F-actin colocalized with agLDL per cell for at least 10 fields containing > 100 cells. Median and interquartile range are shown. (G-H) BMMs were treated with DMSO (control) or 50 µM ZCL 278 (Cdc42 inhibitor) for 30 min prior to treatment with Alexa546-agLDL for 1 h in the presence of inhibitor. Cells were analysed by confocal microscopy (G) and images used to quantify F-actin colocalized with agLDL per cell for at least 10 fields containing > 100 cells. Median and interquartile range are shown. * p

    Techniques Used: Transfection, Cell Culture, Staining, Confocal Microscopy

    Cortical F-actin depolymerization occurs in parallel to actin polymerization at the compartment and regulates macrophage responsiveness to agLDL. (A-B) RAW 264.7 macrophages were transiently transfected with LifeAct-GFP, treated with Alexa546-agLDL and imaged live cell (A). Arrows shows F-actin associated with Alexa546-agLDL and arrowheads denote cortical F-actin adjacent to sites of contact with agLDL. (B) Images from (A) were used to quantify total LifeAct-GFP (total), LifeAct-GFP colocalized with the LS (LS), and LifeAct-GFP not localized at the LS (non-LS) for n = 12 cells. Pairwise statistical comparisons between total and non-LS, and LS vs LS 0’ are shown. ( C-F) J774 cells were transfected with scrambled (Scr) or Cofilin-1 siRNA (Cofilin-1KD) using Lipofectamine RNAiMAX reagent, and cultured for 48 h. (C) Cells were lysed and Cofilin-1 knockdown was confirmed by immunoblot analysis. (D) Scr and (E) Cofilin-1KD cells were treated with Alexa546-agLDL for 1 h, fixed, stained for F-actin using Alexa488-phalloidin and analysed by confocal microscopy. (F) Confocal images were used to quantify F-actin colocalized with agLDL per cell for at least 10 fields containing > 100 cells. Median and interquartile range are shown. (G-K) Scr (G-H) and (I-J) Cofilin-1KD cells were treated with agLDL for 4 h prior to fixation, neutral lipid staining using LipidTOX green, and analysis by confocal microscopy. (K) Confocal images were used to quantify LipidTOX green per cell for at least 10 fields containing > 100 cells. * p ≤ 0.05, ** p ≤ 0.01,**** p ≤ 0.0001. # p = 0.0556. Error bars s.e.m (B and K).
    Figure Legend Snippet: Cortical F-actin depolymerization occurs in parallel to actin polymerization at the compartment and regulates macrophage responsiveness to agLDL. (A-B) RAW 264.7 macrophages were transiently transfected with LifeAct-GFP, treated with Alexa546-agLDL and imaged live cell (A). Arrows shows F-actin associated with Alexa546-agLDL and arrowheads denote cortical F-actin adjacent to sites of contact with agLDL. (B) Images from (A) were used to quantify total LifeAct-GFP (total), LifeAct-GFP colocalized with the LS (LS), and LifeAct-GFP not localized at the LS (non-LS) for n = 12 cells. Pairwise statistical comparisons between total and non-LS, and LS vs LS 0’ are shown. ( C-F) J774 cells were transfected with scrambled (Scr) or Cofilin-1 siRNA (Cofilin-1KD) using Lipofectamine RNAiMAX reagent, and cultured for 48 h. (C) Cells were lysed and Cofilin-1 knockdown was confirmed by immunoblot analysis. (D) Scr and (E) Cofilin-1KD cells were treated with Alexa546-agLDL for 1 h, fixed, stained for F-actin using Alexa488-phalloidin and analysed by confocal microscopy. (F) Confocal images were used to quantify F-actin colocalized with agLDL per cell for at least 10 fields containing > 100 cells. Median and interquartile range are shown. (G-K) Scr (G-H) and (I-J) Cofilin-1KD cells were treated with agLDL for 4 h prior to fixation, neutral lipid staining using LipidTOX green, and analysis by confocal microscopy. (K) Confocal images were used to quantify LipidTOX green per cell for at least 10 fields containing > 100 cells. * p ≤ 0.05, ** p ≤ 0.01,**** p ≤ 0.0001. # p = 0.0556. Error bars s.e.m (B and K).

    Techniques Used: Transfection, Cell Culture, Staining, Confocal Microscopy

    39) Product Images from "MicroRNA-143-3p contributes to the regulation of pain responses in collagen-induced arthritis"

    Article Title: MicroRNA-143-3p contributes to the regulation of pain responses in collagen-induced arthritis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.9322

    Effects of miR-143 on pain-associated target genes. Mouse DRG cells were transfected with Lipofectamine RNAiMAX transfection reagent and the miR-143-3p mimic NC-carboxyfluorescein. (A) Bright field (magnification, ×100). (B) Fluorescence microscopy (magnification, ×100). RNA was extracted from the DRG cells 48 h following transfection with an miR-143-3p inhibitor or mimic. (C) miR-143-3p expression following transfection with the mimic. (D) miR-143-3p expression following transfection with the inhibitor. (E) Ptgdr expression, (F) Mrgpre expression, (G) Ptgs2 expression and (H) Tnf expression. *P
    Figure Legend Snippet: Effects of miR-143 on pain-associated target genes. Mouse DRG cells were transfected with Lipofectamine RNAiMAX transfection reagent and the miR-143-3p mimic NC-carboxyfluorescein. (A) Bright field (magnification, ×100). (B) Fluorescence microscopy (magnification, ×100). RNA was extracted from the DRG cells 48 h following transfection with an miR-143-3p inhibitor or mimic. (C) miR-143-3p expression following transfection with the mimic. (D) miR-143-3p expression following transfection with the inhibitor. (E) Ptgdr expression, (F) Mrgpre expression, (G) Ptgs2 expression and (H) Tnf expression. *P

    Techniques Used: Transfection, Fluorescence, Microscopy, Expressing

    40) Product Images from "Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA"

    Article Title: Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA

    Journal: Nature materials

    doi: 10.1038/nmat2444

    In vitro cytotoxicity and bioactivity of siRNA nanoparticles Polymer PLGA nanoparticles encapsulating siRNA were evaluated for cytotoxicity using cultured (a) HepG2 hepatocytes and HeLa cervical carcinoma cells. The toxicity of free spermidine (green diamonds), spermidine-loaded PLGA nanoparticles (black squares), dsDNA/spermidine- (red circles), and siRNA/spermidine-loaded (purple stars) nanoparticles were evaluated over a range of concentrations from 0-10 mg/mL. CellTiter Blue® fluorescence (Promega) was used to measure cell viability compared to untreated cells (blue line/triangles). Neither the PLGA nanoparticles singularly or in combination with spermidine, the mimic (dsDNA), or siRNA showed cytotoxicity in the cell types and over this range of concentrations. (b) Dose-response curves comparing the bioactivity of siRNA delivered with a transfection agent (red circles) or using PLGA nanoparticles (open squares). An siRNA targeted against the luciferase gene (siLUC) was delivered to cultured HEK-293T cells stably expressing luciferase. Luciferase activity was measured with the Bright-Glo™ (Promega) reagent and separate treated wells were used to measure cell viability using CellTiter Blue®. Luciferase activity normalized to viable cell number and plotted against the amount of delivered siLUC. PLGA nanoparticles show equal or better activity compared to a commercial transfection agent (Lipofectamine RNAiMax).
    Figure Legend Snippet: In vitro cytotoxicity and bioactivity of siRNA nanoparticles Polymer PLGA nanoparticles encapsulating siRNA were evaluated for cytotoxicity using cultured (a) HepG2 hepatocytes and HeLa cervical carcinoma cells. The toxicity of free spermidine (green diamonds), spermidine-loaded PLGA nanoparticles (black squares), dsDNA/spermidine- (red circles), and siRNA/spermidine-loaded (purple stars) nanoparticles were evaluated over a range of concentrations from 0-10 mg/mL. CellTiter Blue® fluorescence (Promega) was used to measure cell viability compared to untreated cells (blue line/triangles). Neither the PLGA nanoparticles singularly or in combination with spermidine, the mimic (dsDNA), or siRNA showed cytotoxicity in the cell types and over this range of concentrations. (b) Dose-response curves comparing the bioactivity of siRNA delivered with a transfection agent (red circles) or using PLGA nanoparticles (open squares). An siRNA targeted against the luciferase gene (siLUC) was delivered to cultured HEK-293T cells stably expressing luciferase. Luciferase activity was measured with the Bright-Glo™ (Promega) reagent and separate treated wells were used to measure cell viability using CellTiter Blue®. Luciferase activity normalized to viable cell number and plotted against the amount of delivered siLUC. PLGA nanoparticles show equal or better activity compared to a commercial transfection agent (Lipofectamine RNAiMax).

    Techniques Used: In Vitro, Cell Culture, Fluorescence, Transfection, Luciferase, Stable Transfection, Expressing, Activity Assay

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    Article Snippet: .. Transfection with Small Interfering RNAs Transfection of non-treated and Aza-treated H1299 cells with control (scrambled) small interfering RNAs (siRNAs) or siRNAs specific to the TBX genes was performed using Lipofectamine RNAiMAX from Invitrogen according to the manufacturer’s instructions. ..

    Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness
    Article Snippet: .. Silencing of TLR3 and TRIF SW620 or HT29 cells were transfected for 24–48 h with 10–20 nm siRNA against TLR3 (TLR3_5, Qiagen, SI02630768; TLR3_8, Qiagen, SI02655156) or non-silencing control siRNA (Qiagen, SI03650325) using Lipofectamine RNAiMAX (Invitrogen) for TLR3 knockdown or with siRNA against TICAM-1/TRIF (Ambion, s45115) for TICAM/TRIF knockdown using Lipofectamine RNAiMAX. siRNA and transfection reagent (ratio, 1:2) were preincubated for 15 min in RPMI medium before being added to newly seeded cells. .. Knockdown of targets were confirmed in cell lysates by qPCR.

    Article Title: Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism
    Article Snippet: .. Apoptosis Assay of HL-1 Cardiomyocytes Sca-1+/CD31− CSCshTERT were cultured on six-well plates at a density of 5 × 104 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes (5′-CACAACCACCTCAAGCACT-3′) or NC siRNA (all from Bioneer) using Lipofectamine RNAiMAX (Invitrogen) for 48 h as suggested by the manufacturer. .. For CoCl2 - (Sigma-Aldrich) or H2 O2 -induced hypoxia, HL-1 cells, a cardiomyocyte cell line that continuously divides and spontaneously contracts while maintaining a differentiated cardiac phenotype [ ] was used.

    Article Title: miR-548aq-3p is a novel target of Far infrared radiation which predicts coronary artery disease endothelial colony forming cell responsiveness
    Article Snippet: .. To knockdown miR-548aq-3p in ECFCs, a commercial synthetic miRIDIAN microRNA Hairpin Inhibitor (hsa-miR-548aq-3p, IH-302531-01-0005) (Dharmacon, Lafayette, CO, USA) was added to the culture medium at a final concentration of 20 nM at 70~80% cell confluence using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA). .. The expression level of miR-548aq-3p was measured by RT-qPCR after 48 hours.

    Article Title: Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists
    Article Snippet: .. Transfection of RNA and small interfering RNA (siRNA) in A549 cells was performed with Lipofectamine RNAiMax (Invitrogen) for 18 to 24 h and 48 h, respectively. .. Transfections of RNA and siRNA in monocyte-derived dendritic cells (Mo-DCs) were performed with HiPerFect transfection reagent (Qiagen).

    Cell Culture:

    Article Title: Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism
    Article Snippet: .. Apoptosis Assay of HL-1 Cardiomyocytes Sca-1+/CD31− CSCshTERT were cultured on six-well plates at a density of 5 × 104 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes (5′-CACAACCACCTCAAGCACT-3′) or NC siRNA (all from Bioneer) using Lipofectamine RNAiMAX (Invitrogen) for 48 h as suggested by the manufacturer. .. For CoCl2 - (Sigma-Aldrich) or H2 O2 -induced hypoxia, HL-1 cells, a cardiomyocyte cell line that continuously divides and spontaneously contracts while maintaining a differentiated cardiac phenotype [ ] was used.

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  • 99
    Thermo Fisher sirna
    Netrin-1 receptors on RBMVECs and their function. (A) <t>UNC5H2</t> receptor, not DCC receptor, was expressed on RBMVECs. Arrows indicates positive green signal by immunofluorescence. (B) Western blot analysis of UNC5H2 receptor. OGD-induced UNC5H2 was efficiently knocked down by UNC5H2 <t>siRNA</t> transfection. (C) Cell viability shown by CCK-8 and Annexin V-FITC kit. (D) Western blot analysis of autophagy-related protein p62 and LC3. UNC5H2 siRNA transfection did not change cell viability or autophagy activity after OGD, but suppressed the strengthened effects of netrin-1 (50 ng/mL) on cell viability and autophagy activity. * P
    Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher mock transfected
    Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of <t>transfected</t> SCs. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Mitochondrial staining in SCs. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p
    Mock Transfected, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Netrin-1 receptors on RBMVECs and their function. (A) UNC5H2 receptor, not DCC receptor, was expressed on RBMVECs. Arrows indicates positive green signal by immunofluorescence. (B) Western blot analysis of UNC5H2 receptor. OGD-induced UNC5H2 was efficiently knocked down by UNC5H2 siRNA transfection. (C) Cell viability shown by CCK-8 and Annexin V-FITC kit. (D) Western blot analysis of autophagy-related protein p62 and LC3. UNC5H2 siRNA transfection did not change cell viability or autophagy activity after OGD, but suppressed the strengthened effects of netrin-1 (50 ng/mL) on cell viability and autophagy activity. * P

    Journal: Frontiers in Neuroscience

    Article Title: Netrin-1 Ameliorates Blood-Brain Barrier Impairment Secondary to Ischemic Stroke via the Activation of PI3K Pathway

    doi: 10.3389/fnins.2017.00700

    Figure Lengend Snippet: Netrin-1 receptors on RBMVECs and their function. (A) UNC5H2 receptor, not DCC receptor, was expressed on RBMVECs. Arrows indicates positive green signal by immunofluorescence. (B) Western blot analysis of UNC5H2 receptor. OGD-induced UNC5H2 was efficiently knocked down by UNC5H2 siRNA transfection. (C) Cell viability shown by CCK-8 and Annexin V-FITC kit. (D) Western blot analysis of autophagy-related protein p62 and LC3. UNC5H2 siRNA transfection did not change cell viability or autophagy activity after OGD, but suppressed the strengthened effects of netrin-1 (50 ng/mL) on cell viability and autophagy activity. * P

    Article Snippet: UNC5H2 siRNA-lipid or negative control siRNA-lipid complexes were made by adding siRNA to Lipofectamine RNAiMAX Reagent (cat. No. 13778; Thermo Fisher Scientific) diluted in Opti-MEM I reduced Serum medium (cat. No. 31985070; Thermo Fisher Scientific) to achieve a final concentration of 100 nmol/L.

    Techniques: Droplet Countercurrent Chromatography, Immunofluorescence, Western Blot, Transfection, CCK-8 Assay, Activity Assay

    AMFR and Erlin-2 regulation and cortisol metabolism with Enz treatment. ( A ) Erlin-2 but not AMFR is consistently up-regulated with Enz treatment of LAPC4 cells. ( B ) Erlin-2 is up-regulated in 8 of 11 human prostate tissues. Immunoblots were performed as described previously. ( C ) Erlin-2 overexpression (OE) suppresses expression of 11β-HSD2 protein in LAPC4 cells and Erlin-2 knockdown by siRNA increases 11β-HSD2 expression and activity in the long-term Enz-treated LAPC4 cells. ( D ) AMFR silencing does not regulate HSD11B2 transcript. qPCR was performed in triplicate and expression is normalized to shControl-expressing cells and RPLP0 . ( E ) Reversal of the metabolic phenotype that sustains cortisol with Enz treatment by AMFR knockdown is reversed again by 11β-HSD2 knockdown (compare cortisol at 24 hr in shAMFR groups between siCTRL and siHSD11B2). The specificity of siHSD11B2 is shown by qPCR and immunoblot. LAPC4 cells stably expressing stably shCTRL or an shAMFR construct were treated with Enz as described for Figure 5 , transiently transfected with siHSD11B2 or siCTRL and treated with [ 3 H]-cortisol (100 nM). Experiments were performed in duplicate. DOI: http://dx.doi.org/10.7554/eLife.20183.011

    Journal: eLife

    Article Title: Aberrant corticosteroid metabolism in tumor cells enables GR takeover in enzalutamide resistant prostate cancer

    doi: 10.7554/eLife.20183

    Figure Lengend Snippet: AMFR and Erlin-2 regulation and cortisol metabolism with Enz treatment. ( A ) Erlin-2 but not AMFR is consistently up-regulated with Enz treatment of LAPC4 cells. ( B ) Erlin-2 is up-regulated in 8 of 11 human prostate tissues. Immunoblots were performed as described previously. ( C ) Erlin-2 overexpression (OE) suppresses expression of 11β-HSD2 protein in LAPC4 cells and Erlin-2 knockdown by siRNA increases 11β-HSD2 expression and activity in the long-term Enz-treated LAPC4 cells. ( D ) AMFR silencing does not regulate HSD11B2 transcript. qPCR was performed in triplicate and expression is normalized to shControl-expressing cells and RPLP0 . ( E ) Reversal of the metabolic phenotype that sustains cortisol with Enz treatment by AMFR knockdown is reversed again by 11β-HSD2 knockdown (compare cortisol at 24 hr in shAMFR groups between siCTRL and siHSD11B2). The specificity of siHSD11B2 is shown by qPCR and immunoblot. LAPC4 cells stably expressing stably shCTRL or an shAMFR construct were treated with Enz as described for Figure 5 , transiently transfected with siHSD11B2 or siCTRL and treated with [ 3 H]-cortisol (100 nM). Experiments were performed in duplicate. DOI: http://dx.doi.org/10.7554/eLife.20183.011

    Article Snippet: After incubation overnight, the cells were transfected with siRNA following the Lipofectamine RNAiMAX Reagent (ThermoFisher, Waltham, MA) protocol provided by the manufacturer for 48 hr.

    Techniques: Western Blot, Over Expression, Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Construct, Transfection

    RNF12/RLIM E3 ubiquitin ligase is selectively phosphorylated by SRPKs at a SR-rich motif. (A) RNF12 knockout ( Rlim -/y ) mESCs were transfected with WT RNF12 or the indicated point mutants and SR-motif phosphorylation analysed by Phos-tag immunoblotting for RNF12. RNF12 4xSA = S212A/S214A/S227A/S229A. RNF12 and ERK1/2 levels are shown as a loading control. (B) RNF12 knockout ( Rlim -/y ) mESCs were transfected with the indicated RNF12 constructs and lysates treated with λ-phosphatase and analysed by Phos-tag immunoblotting for RNF12. Recombinant RNF12 is included as an un-phosphorylated control. (C) mESCs were treated with 10 µM of the of the following CMGC kinase inhibitors: AZ-191 (DYRK1B inhibitor), KH-CB19 (CLK-DYRK inhibitor), T3 (CLK inhibitor), SPHINX31 (SRPK1 inhibitor), CHIR-99021 (GSK3 inhibitor), PD-0325901 (MEK inhibitor), VX-745 (p38 inhibitor), JNK-IN-8 (JNK inhibitor), RO-3306 (CDK1 inhibitor) and Flavopiridol (CDK7/9 inhibitor) for 4 h and RNF12 phosphorylation analysed via Phos-tag immunoblotting for RNF12. RNF12 4xSA is included as an unphosphorylated control. RNF12 and ERK1/2 levels are shown as a control. (D) ESCs were treated with the indicated concentrations of SRPKIN-1 for 4 h and RNF12 phosphorylation analysed via Phos-tag immunoblotting for RNF12. (E) mESCs were treated with 10 µM SRPKIN-1 for 4 h and RNF12 phosphorylation analysed from HA-RNF12 immunoprecipitates via RNF12 phos-tag and phospho-Ser214 immunoblotting using multiplex infrared immunoblot. (F) Phosphorylated peptides detected via mass spectrometry analysis of RNF12 following in vitro phosphorylation by SRPK1. pS=phospho-Serine. (G) Autoradiography of RNF12 wild-type (WT) or S212A/S214A/S227A/S229A (4xSA) following a radioactive kinase reaction with SRPK1, SRPK2 or SRPK3. Coomassie staining of RNF12 protein is shown as a loading control. (H) Srpk1 -/- mESCs were transfected with control or SRPK2 siRNA and RNF12 phosphorylation was analysed via phos-tag immunoblotting. ERK1/2 levels are shown as a loading control.

    Journal: bioRxiv

    Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

    doi: 10.1101/2020.04.02.005041

    Figure Lengend Snippet: RNF12/RLIM E3 ubiquitin ligase is selectively phosphorylated by SRPKs at a SR-rich motif. (A) RNF12 knockout ( Rlim -/y ) mESCs were transfected with WT RNF12 or the indicated point mutants and SR-motif phosphorylation analysed by Phos-tag immunoblotting for RNF12. RNF12 4xSA = S212A/S214A/S227A/S229A. RNF12 and ERK1/2 levels are shown as a loading control. (B) RNF12 knockout ( Rlim -/y ) mESCs were transfected with the indicated RNF12 constructs and lysates treated with λ-phosphatase and analysed by Phos-tag immunoblotting for RNF12. Recombinant RNF12 is included as an un-phosphorylated control. (C) mESCs were treated with 10 µM of the of the following CMGC kinase inhibitors: AZ-191 (DYRK1B inhibitor), KH-CB19 (CLK-DYRK inhibitor), T3 (CLK inhibitor), SPHINX31 (SRPK1 inhibitor), CHIR-99021 (GSK3 inhibitor), PD-0325901 (MEK inhibitor), VX-745 (p38 inhibitor), JNK-IN-8 (JNK inhibitor), RO-3306 (CDK1 inhibitor) and Flavopiridol (CDK7/9 inhibitor) for 4 h and RNF12 phosphorylation analysed via Phos-tag immunoblotting for RNF12. RNF12 4xSA is included as an unphosphorylated control. RNF12 and ERK1/2 levels are shown as a control. (D) ESCs were treated with the indicated concentrations of SRPKIN-1 for 4 h and RNF12 phosphorylation analysed via Phos-tag immunoblotting for RNF12. (E) mESCs were treated with 10 µM SRPKIN-1 for 4 h and RNF12 phosphorylation analysed from HA-RNF12 immunoprecipitates via RNF12 phos-tag and phospho-Ser214 immunoblotting using multiplex infrared immunoblot. (F) Phosphorylated peptides detected via mass spectrometry analysis of RNF12 following in vitro phosphorylation by SRPK1. pS=phospho-Serine. (G) Autoradiography of RNF12 wild-type (WT) or S212A/S214A/S227A/S229A (4xSA) following a radioactive kinase reaction with SRPK1, SRPK2 or SRPK3. Coomassie staining of RNF12 protein is shown as a loading control. (H) Srpk1 -/- mESCs were transfected with control or SRPK2 siRNA and RNF12 phosphorylation was analysed via phos-tag immunoblotting. ERK1/2 levels are shown as a loading control.

    Article Snippet: ESCs were transfected with siRNA using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific). siRNA oligos used are listed in Table S6.

    Techniques: Knock-Out, Transfection, Construct, Recombinant, Multiplex Assay, Mass Spectrometry, In Vitro, Autoradiography, Staining

    Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of transfected SCs. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Mitochondrial staining in SCs. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of transfected SCs. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Mitochondrial staining in SCs. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: At 60–80% confluence, SCs were mock-transfected (Lipofectamine® RNAiMAX Regent only; Thermo Fisher Scientific) or transfected with complexes of Lipofectamine® RNAiMAX, miR-7450 agomir negative control (NC; 50 nM), miR-7450 antagomir NC (100 nM), miR-7450 agomir (chemically-modified double-stranded miRNA mimic; 50 nM) in the presence or absence of non-thermal plasma treatment at 22.0 kV for 120 s, and miR-7450 antagomir (chemically-modified single-stranded miRNA inhibitor; 100 nM) from GenePharma Co., Ltd. (Shanghai, China), according to the manufacturer’s protocol.

    Techniques: Activity Assay, Transfection, Staining, Fluorescence

    Chicken SC protein expression. ( A ) Representative western blot analysis of protein bands in SCs exposed to 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S5 . Relative protein levels of ( B ) NRF2, KEAP1, PRDX4, ( C ) ATP5A, ( D ) p-AMPKα/AMPKα, and ( E ) p-mTOR/mTOR in SCs exposed to plasma. ( F ) Representative western blot analysis of protein bands in SCs trasfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S6 . Relative protein levels of ( G ) ATP5A, ( H ) p-AMPKα/AMPKα, and ( I ) p-mTOR/mTOR in transfected SCs. One independent replicate on western blot analysis of protein bands in SCs is presented in Supplementary Figure S7 . Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: Chicken SC protein expression. ( A ) Representative western blot analysis of protein bands in SCs exposed to 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S5 . Relative protein levels of ( B ) NRF2, KEAP1, PRDX4, ( C ) ATP5A, ( D ) p-AMPKα/AMPKα, and ( E ) p-mTOR/mTOR in SCs exposed to plasma. ( F ) Representative western blot analysis of protein bands in SCs trasfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S6 . Relative protein levels of ( G ) ATP5A, ( H ) p-AMPKα/AMPKα, and ( I ) p-mTOR/mTOR in transfected SCs. One independent replicate on western blot analysis of protein bands in SCs is presented in Supplementary Figure S7 . Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: At 60–80% confluence, SCs were mock-transfected (Lipofectamine® RNAiMAX Regent only; Thermo Fisher Scientific) or transfected with complexes of Lipofectamine® RNAiMAX, miR-7450 agomir negative control (NC; 50 nM), miR-7450 antagomir NC (100 nM), miR-7450 agomir (chemically-modified double-stranded miRNA mimic; 50 nM) in the presence or absence of non-thermal plasma treatment at 22.0 kV for 120 s, and miR-7450 antagomir (chemically-modified single-stranded miRNA inhibitor; 100 nM) from GenePharma Co., Ltd. (Shanghai, China), according to the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Transfection

    Expression of miR-7450 and mRNA levels of AMPKα and mTOR in SCs. ( A ) miR-7450 relative level and ( B ) relative mRNA levels of AMPKα and mTOR in SCs exposed to 22.0 kV of plasma for 120 s. ( C ) miR-7450 relative level and ( D ) relative mRNA levels of AMPKα and mTOR in SCs transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. RT-PCR analysis of a non-target gene ( POU1F1 ) and an unrelated target gene ( PDE10A ) of miR-7450 in transfected SCs is presented in Supplementary Figure S4 . Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: Expression of miR-7450 and mRNA levels of AMPKα and mTOR in SCs. ( A ) miR-7450 relative level and ( B ) relative mRNA levels of AMPKα and mTOR in SCs exposed to 22.0 kV of plasma for 120 s. ( C ) miR-7450 relative level and ( D ) relative mRNA levels of AMPKα and mTOR in SCs transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. RT-PCR analysis of a non-target gene ( POU1F1 ) and an unrelated target gene ( PDE10A ) of miR-7450 in transfected SCs is presented in Supplementary Figure S4 . Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: At 60–80% confluence, SCs were mock-transfected (Lipofectamine® RNAiMAX Regent only; Thermo Fisher Scientific) or transfected with complexes of Lipofectamine® RNAiMAX, miR-7450 agomir negative control (NC; 50 nM), miR-7450 antagomir NC (100 nM), miR-7450 agomir (chemically-modified double-stranded miRNA mimic; 50 nM) in the presence or absence of non-thermal plasma treatment at 22.0 kV for 120 s, and miR-7450 antagomir (chemically-modified single-stranded miRNA inhibitor; 100 nM) from GenePharma Co., Ltd. (Shanghai, China), according to the manufacturer’s protocol.

    Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction

    SC viability and apoptosis after miRNA transfection. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Relative viability of SCs. ( B ) Flow cytometric analysis of SC cell apoptosis. ( C ) JC-1 staining of SCs. Scale bar: 50 μm. ( D ) Relative green/red fluorescence intensity for JC-1 staining. Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: SC viability and apoptosis after miRNA transfection. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Relative viability of SCs. ( B ) Flow cytometric analysis of SC cell apoptosis. ( C ) JC-1 staining of SCs. Scale bar: 50 μm. ( D ) Relative green/red fluorescence intensity for JC-1 staining. Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: At 60–80% confluence, SCs were mock-transfected (Lipofectamine® RNAiMAX Regent only; Thermo Fisher Scientific) or transfected with complexes of Lipofectamine® RNAiMAX, miR-7450 agomir negative control (NC; 50 nM), miR-7450 antagomir NC (100 nM), miR-7450 agomir (chemically-modified double-stranded miRNA mimic; 50 nM) in the presence or absence of non-thermal plasma treatment at 22.0 kV for 120 s, and miR-7450 antagomir (chemically-modified single-stranded miRNA inhibitor; 100 nM) from GenePharma Co., Ltd. (Shanghai, China), according to the manufacturer’s protocol.

    Techniques: Transfection, Flow Cytometry, Staining, Fluorescence