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  • 99
    Thermo Fisher transfection transfection
    (A) Seed sequences of miR-148a in the wild- and mutant-type 3′-UTR of S1PR1. (B) Luciferase reporter assay data showed that <t>co-transfection</t> of HepG2 cells with miR-148a and wild-type S1PR1 3′-UTR led to a marked decrease in luciferase activity; however, co-transfection with miR-148a and mutant S1PR1 3′-UTR had no effect on luciferase activity, and co-transfection with NC miRNA and wild-type S1PR1 3′-UTR or mutant S1PR1 3′-UTR also showed no difference. ** P
    Transfection Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore transfection transfection
    Increased mir-155 promotes monocyte survival . (A) Healthy donor monocytes were transfected with a negative control miRNA mimic conjugated to a fluorescent molecule Dy547. Following incubation for 40 h, Dy547 positive cells were assessed by flow cytometry. (B) Monocytes were transfected with mir-155 mimic or negative control mimic (Neg Ctrl), or with <t>transfection</t> reagent only (mock) and incubated for 40 h. Mature mir-155 levels were measured using a TaqMan microRNA assay. (C, D) Representative plots (C) and cumulative data (D, n = 22) showing healthy donor CD14+ monocyte survival 40 h after transfection with negative control mimic (Neg Ctrl) or mir-155 mimic. (E) Healthy donor monocytes were transfected (n = 7) with Neg Ctrl or mir-155 mimic for 24 h followed by overnight culture with an agonistic anti-Fas antibody (aFas) or the isotype control (IgM) at 200 ng/mL and the percentage of live cells assessed. D and E tested by Wilcoxon matched-pairs test.
    Transfection Transfection, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 71 article reviews
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    99
    Qiagen transfection transfection
    miR-125b induces proliferation and apoptosis resistance in GBM cells. ( a ) Population growth of U87 and LN-18 cells overexpressing miR-125b or anti-miR-125b relative to the control using the resazurin assay ( n =3). ( b ) Cell cycle analysis of nocodazole-treated cells by flow cytometry ( n =3). ( c ) Apoptosis. Cells were transfected with pre-miR-125b or pre-control and treated with 10 ng/ml TNF α or 250 ng/ml TRAIL for 48 h beginning 24 h post <t>transfection.</t> Apoptosis was assessed using the ApoTox-Glo Triplex assay ( n =3) ( d ) LN-18 cells overexpressing anti-miR-125b or anti-control were induced with 10 ng/ml TNF α for 24 h and subjected to the ApoTox-Glo Triplex assay ( n =3)
    Transfection Transfection, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher transfection
    miR-125b induces proliferation and apoptosis resistance in GBM cells. ( a ) Population growth of U87 and LN-18 cells overexpressing miR-125b or anti-miR-125b relative to the control using the resazurin assay ( n =3). ( b ) Cell cycle analysis of nocodazole-treated cells by flow cytometry ( n =3). ( c ) Apoptosis. Cells were transfected with pre-miR-125b or pre-control and treated with 10 ng/ml TNF α or 250 ng/ml TRAIL for 48 h beginning 24 h post <t>transfection.</t> Apoptosis was assessed using the ApoTox-Glo Triplex assay ( n =3) ( d ) LN-18 cells overexpressing anti-miR-125b or anti-control were induced with 10 ng/ml TNF α for 24 h and subjected to the ApoTox-Glo Triplex assay ( n =3)
    Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega transfections
    miR-125b induces proliferation and apoptosis resistance in GBM cells. ( a ) Population growth of U87 and LN-18 cells overexpressing miR-125b or anti-miR-125b relative to the control using the resazurin assay ( n =3). ( b ) Cell cycle analysis of nocodazole-treated cells by flow cytometry ( n =3). ( c ) Apoptosis. Cells were transfected with pre-miR-125b or pre-control and treated with 10 ng/ml TNF α or 250 ng/ml TRAIL for 48 h beginning 24 h post <t>transfection.</t> Apoptosis was assessed using the ApoTox-Glo Triplex assay ( n =3) ( d ) LN-18 cells overexpressing anti-miR-125b or anti-control were induced with 10 ng/ml TNF α for 24 h and subjected to the ApoTox-Glo Triplex assay ( n =3)
    Transfections, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 810 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa transfections
    miR-125b induces proliferation and apoptosis resistance in GBM cells. ( a ) Population growth of U87 and LN-18 cells overexpressing miR-125b or anti-miR-125b relative to the control using the resazurin assay ( n =3). ( b ) Cell cycle analysis of nocodazole-treated cells by flow cytometry ( n =3). ( c ) Apoptosis. Cells were transfected with pre-miR-125b or pre-control and treated with 10 ng/ml TNF α or 250 ng/ml TRAIL for 48 h beginning 24 h post <t>transfection.</t> Apoptosis was assessed using the ApoTox-Glo Triplex assay ( n =3) ( d ) LN-18 cells overexpressing anti-miR-125b or anti-control were induced with 10 ng/ml TNF α for 24 h and subjected to the ApoTox-Glo Triplex assay ( n =3)
    Transfections, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen attractene transfection
    ( a ) Exogenous stimulation of S. gordonii lipoproteins augment TLR2 mRNA expressions in hDPCs, and were maintained after calcium hydroxide (CH) or HBD3-C15 treatment. ( b ) S. gordonii lipoprotein-stimulated NF-κB are mediated by TLR2. HEK-TLR2 cells (2.5 × 10 5 cells/mL) were transfected with an NF-κB luciferase reporter plasmid using <t>Attractene</t> <t>transfection</t> reagent. After 16 h, the cells were stimulated with lipoprotein purified from S. gordonii , and then treated with either of CH or HBD3-C15. After the cells were lysed, a luciferease assay was conducted.
    Attractene Transfection, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 33 article reviews
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    99
    Qiagen transfection agent
    Depletion of βIII spectrin impairs the anterograde transport of the VSV-G and the post-Golgi secretion of 35 S-labeled proteins. A , representative images of HeLa cells constitutively expressing VSV-G-GFP mutant form ts045 show the viral protein in the ER at 40 °C, or in the Golgi (30 min) or the plasma membrane ( PM ; 90 min) when cells were shifted to 32 °C. Bar , 10 μm. B , representative images of VSV-G-GFP expressing HeLa cells silenced with non-targeting or βIII spectrin siRNAs after 15 and 60 min after being shifted to 32 °C. C , quantitative analysis of results partially shown in B . The graph shows the percentage of the cells in which VSV-G-GFP is mainly visualized to the ER, to the Golgi, or to the plasma membrane. Data are shown as the mean ± S.E. of three independent experiments (100 cells each). D , biochemical transport assay for VSV-G-GFP using Endo H. HeLa cells constitutively expressing VSV-G-GFP were transfected for 96 h with siRNA control or against βIII spectrin and incubated at 40 °C for the last 24 h. Then, cells were shifted at 32 °C to induce the transport of VSV-G from the ER, lysed at indicated times, and subjected to Endo H treatment. R and S indicate Endo H-resistant and -sensitive forms, respectively. The ratio of the amount of Endo H-resistant form to that of total amount is plotted. Values are represented as the mean ± S.E. of three independent experiments. Statistical significance is shown, according to two-way analysis of variance using Bonferroni's post-tests (*, p ≤ 0.05). E , RPE1 cells were transfected with control or with βIII spectrin siRNA. Ninety-six h after the <t>transfection,</t> cells were pulse labeled with [ 35 S]Met/Cys, incubated at 19 °C for 3 h and then shifted to 37 °C. At the indicated times, proteins in the culture supernatants and the cell lysates were precipitated and quantified by scintillation counting. As additional controls, the secretion assay was performed at 4 °C or in cells treated with BFA. Results are the mean ± S.E. from at least three independent experiments. Statistical significance according to two-way analysis of variance using Bonferroni's post-tests (**, p ≤ 0.01 and ***, p ≤ 0.001).
    Transfection Agent, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 15 article reviews
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    99
    Thermo Fisher transfection system
    3D NEP Design Delivers High Dose Plasmid DNA at Single Cell Resolution with Lower Biological Variability (A) Photolithography-based adaptation of Transwell insert into a 3D NEP platform. (B) SEM of the modified insert surface showing a patterned microwell array (left, scale bar = 50 µm) over the nanochanneled membrane. Only exposed nanochannels (right, scale bar = 1 µm) can NEP-transfect the cells loaded on the apical side of the membrane. (C) Schematic diagram of how the <t>transfection</t> is conducted using the modified Transwell inserts. (D) Circuit diagram for the in-parallel array of nanochannels illustrating the relationship between the resistance across the membrane and the number of actively nanoporating channels. (E) Schematic illustration of a cell sitting directly on top of a nanoporating channel. (F, G) Finite element modeling of the electric field distribution during nanoporation. The electric field is maximized within the nanochannel which leads to enhanced and highly localized transmembrane potentials on the cells. (H–J) Bicistronic constructs for Ascl1 , Brn2 , and Myt1l (color coded green, red, and blue) were introduced into MEFs by NEP. Cells were allowed to recover for 24 hours as indicated on the schematic, and epifluorescent photomicrographs were captured. NEP cells are compared to cells that underwent BEP in parallel experiments. Arrowheads demarcate the same cell in the different panels. Mean fluorescent intensity was obtained from ImageJ and plotted in the respective channels. In addition to showing a higher cellular mean fluorescent intensity, the NEP’ed cells showed a much smaller interquartile range in fluorescent intensities, indicating a more uniform delivery across cells that underwent the NEP. Asterisk indicates statistical significance ( p
    Transfection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 84 article reviews
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    99
    Qiagen polyfect transfection
    3D NEP Design Delivers High Dose Plasmid DNA at Single Cell Resolution with Lower Biological Variability (A) Photolithography-based adaptation of Transwell insert into a 3D NEP platform. (B) SEM of the modified insert surface showing a patterned microwell array (left, scale bar = 50 µm) over the nanochanneled membrane. Only exposed nanochannels (right, scale bar = 1 µm) can NEP-transfect the cells loaded on the apical side of the membrane. (C) Schematic diagram of how the <t>transfection</t> is conducted using the modified Transwell inserts. (D) Circuit diagram for the in-parallel array of nanochannels illustrating the relationship between the resistance across the membrane and the number of actively nanoporating channels. (E) Schematic illustration of a cell sitting directly on top of a nanoporating channel. (F, G) Finite element modeling of the electric field distribution during nanoporation. The electric field is maximized within the nanochannel which leads to enhanced and highly localized transmembrane potentials on the cells. (H–J) Bicistronic constructs for Ascl1 , Brn2 , and Myt1l (color coded green, red, and blue) were introduced into MEFs by NEP. Cells were allowed to recover for 24 hours as indicated on the schematic, and epifluorescent photomicrographs were captured. NEP cells are compared to cells that underwent BEP in parallel experiments. Arrowheads demarcate the same cell in the different panels. Mean fluorescent intensity was obtained from ImageJ and plotted in the respective channels. In addition to showing a higher cellular mean fluorescent intensity, the NEP’ed cells showed a much smaller interquartile range in fluorescent intensities, indicating a more uniform delivery across cells that underwent the NEP. Asterisk indicates statistical significance ( p
    Polyfect Transfection, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyfect transfection/product/Qiagen
    Average 99 stars, based on 79 article reviews
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    polyfect transfection - by Bioz Stars, 2020-07
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    Image Search Results


    (A) Seed sequences of miR-148a in the wild- and mutant-type 3′-UTR of S1PR1. (B) Luciferase reporter assay data showed that co-transfection of HepG2 cells with miR-148a and wild-type S1PR1 3′-UTR led to a marked decrease in luciferase activity; however, co-transfection with miR-148a and mutant S1PR1 3′-UTR had no effect on luciferase activity, and co-transfection with NC miRNA and wild-type S1PR1 3′-UTR or mutant S1PR1 3′-UTR also showed no difference. ** P

    Journal: Experimental and Therapeutic Medicine

    Article Title: microRNA-148a inhibits hepatocellular carcinoma cell invasion by targeting sphingosine-1-phosphate receptor 1

    doi: 10.3892/etm.2014.2137

    Figure Lengend Snippet: (A) Seed sequences of miR-148a in the wild- and mutant-type 3′-UTR of S1PR1. (B) Luciferase reporter assay data showed that co-transfection of HepG2 cells with miR-148a and wild-type S1PR1 3′-UTR led to a marked decrease in luciferase activity; however, co-transfection with miR-148a and mutant S1PR1 3′-UTR had no effect on luciferase activity, and co-transfection with NC miRNA and wild-type S1PR1 3′-UTR or mutant S1PR1 3′-UTR also showed no difference. ** P

    Article Snippet: Transfection Transfection was performed using Lipofectamine® 2000 (Invitrogen Life Technologies), in accordance with the manufacturer’s instructions.

    Techniques: Mutagenesis, Luciferase, Reporter Assay, Cotransfection, Activity Assay

    Inhibition of gp64-initiated syncytium formation by lowering the concentration of accessible PtdIns(4,5) P 2 in the plasma membrane ( A ) Butan-1-ol application to Sf9 Op1D cells immediately after the end of low pH application inhibited syncytium formation (grey bars), but had no effect on lipid mixing (black bars). In control experiments, the cells were treated with t -butanol that, in contrast with butan-1-ol, does not induce PtdIns(4,5) P 2 depletion. ( B ) Expression of the PLCδ1PH–GFP construct in Sf9 Op1D cells inhibited cell fusion that was triggered 24 h post-transfection. Syncytium formation for the cells expressing PLCδ1PH–GFP was normalized to that observed for the cells expressing the PLCδ1PH–GFP mutant R40L. ( C ) Inhibition of syncytium formation by Sf9 Op1D cells by PBP10 (10 or 25 μM, bars 2 and 3) applied immediately after the end of low pH application. (1) Control with no reagents applied. For ( A ), ( B ) and ( C ), fusion between Sf9 Op1D cells was triggered by a 1 min application of pH 4.9 medium and assayed 20 min later. Results are means+S.E.M.

    Journal: Biochemical Journal

    Article Title: Intracellular curvature-generating proteins in cell-to-cell fusion

    doi: 10.1042/BJ20111243

    Figure Lengend Snippet: Inhibition of gp64-initiated syncytium formation by lowering the concentration of accessible PtdIns(4,5) P 2 in the plasma membrane ( A ) Butan-1-ol application to Sf9 Op1D cells immediately after the end of low pH application inhibited syncytium formation (grey bars), but had no effect on lipid mixing (black bars). In control experiments, the cells were treated with t -butanol that, in contrast with butan-1-ol, does not induce PtdIns(4,5) P 2 depletion. ( B ) Expression of the PLCδ1PH–GFP construct in Sf9 Op1D cells inhibited cell fusion that was triggered 24 h post-transfection. Syncytium formation for the cells expressing PLCδ1PH–GFP was normalized to that observed for the cells expressing the PLCδ1PH–GFP mutant R40L. ( C ) Inhibition of syncytium formation by Sf9 Op1D cells by PBP10 (10 or 25 μM, bars 2 and 3) applied immediately after the end of low pH application. (1) Control with no reagents applied. For ( A ), ( B ) and ( C ), fusion between Sf9 Op1D cells was triggered by a 1 min application of pH 4.9 medium and assayed 20 min later. Results are means+S.E.M.

    Article Snippet: Transfection Transfections were performed using Lipofectamine™ 2000 (Invitrogen) using the protocol suggested by the manufacturer.

    Techniques: Inhibition, Concentration Assay, Expressing, Construct, Transfection, Mutagenesis

    miR-223 promotes myoblast differentiation. ( a ) Relative miR-223 expression during CPM differentiation. ( b ) Relative miR-223 expression during qm-7 differentiation. ( c ) Relative expression of muscle differentiation marker genes after CPM transfected with miR-223 and NC. ( d ) Relative expression of muscle differentiation marker genes after qm-7 transfected with miR-223 and NC. ( e ) Relative expression of muscle differentiation marker genes after CPM transfected with miR-223 inhibitor and NC inhibitor. ( f ) Relative expression of muscle differentiation marker genes after qm-7 transfected with miR-223 inhibitor and NC inhibitor. ( g ) MyHC staining of CPM at 72 h after transfection of miR-223 or NC mimic. ( h ) Myotube area (%) at 72 h after transfection of miR-223 or NC mimic. Results are shown as the mean±S.E.M. of three independent experiments. Independent sample t- test was used to analysis the statistical differences between groups. * P

    Journal: Cell Death & Disease

    Article Title: miRNA-223 upregulated by MYOD inhibits myoblast proliferation by repressing IGF2 and facilitates myoblast differentiation by inhibiting ZEB1

    doi: 10.1038/cddis.2017.479

    Figure Lengend Snippet: miR-223 promotes myoblast differentiation. ( a ) Relative miR-223 expression during CPM differentiation. ( b ) Relative miR-223 expression during qm-7 differentiation. ( c ) Relative expression of muscle differentiation marker genes after CPM transfected with miR-223 and NC. ( d ) Relative expression of muscle differentiation marker genes after qm-7 transfected with miR-223 and NC. ( e ) Relative expression of muscle differentiation marker genes after CPM transfected with miR-223 inhibitor and NC inhibitor. ( f ) Relative expression of muscle differentiation marker genes after qm-7 transfected with miR-223 inhibitor and NC inhibitor. ( g ) MyHC staining of CPM at 72 h after transfection of miR-223 or NC mimic. ( h ) Myotube area (%) at 72 h after transfection of miR-223 or NC mimic. Results are shown as the mean±S.E.M. of three independent experiments. Independent sample t- test was used to analysis the statistical differences between groups. * P

    Article Snippet: Transfections Transfections were performed with Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s direction.

    Techniques: Expressing, Marker, Transfection, Staining

    miR-223 inhibits myoblast proliferation. ( a ) CCK-8 assay indicates miR-223 overexpression inhibited chicken primary myoblast (CPM) proliferation. ( b ) CCK-8 assay indicates miR-223 inhibition promoted CPM proliferation. ( c ) CPM was overexpressed with miR-223 and the negative control (NC) mimic, and the cell cycle phase was then analyzed. ( d ) Relative mRNA expression of the cell cycle-related genes after transfection of miR-223 and NC mimic. ( e ) CPM was transfected with miR-223 inhibitor and the NC inhibitor, and the cell cycle phase was then analyzed. ( f ) Relative mRNA expression of the cell cycle-related genes after transfection of miR-223 inhibitor and NC inhibitor. ( g ) EdU staining after transfection of miR-223 mimic and miR-223 inhibitor. Bar, 50 μ m. ( h ) The proliferation rate of myoblasts transfected with miR-223 mimic and miR-223 inhibitor. ( i ) CCK-8 assay showed that miR-223 overexpression inhibited qm-7 proliferation. ( j ) CCK-8 assay showed that miR-223 inhibition promoted qm-7 proliferation. ( k ) qm-7 was overexpressed with miR-223 and the NC mimic, and the cell cycle phase was then analyzed. ( l ) Relative mRNA expression of the cell cycle-related genes after transfection of miR-223 and NC mimic. ( m ) qm-7 was transfected with miR-223 inhibitor and the NC inhibitor, and the cell cycle phase was then analyzed. ( n ) Relative mRNA expression of the cell cycle-related genes after transfection of miR-223 inhibitor and NC inhibitor. ( o ) EdU staining after transfection of miR-223 mimic and miR-223 inhibitor. Bar, 50 μ m. ( p ) The proliferation rate of qm-7 cells transfected with miR-223 mimic and miR-223 inhibitor. Results are shown as the mean±S.E.M. of three independent experiments. Independent sample t -test was used to analysis the statistical differences between groups. * P

    Journal: Cell Death & Disease

    Article Title: miRNA-223 upregulated by MYOD inhibits myoblast proliferation by repressing IGF2 and facilitates myoblast differentiation by inhibiting ZEB1

    doi: 10.1038/cddis.2017.479

    Figure Lengend Snippet: miR-223 inhibits myoblast proliferation. ( a ) CCK-8 assay indicates miR-223 overexpression inhibited chicken primary myoblast (CPM) proliferation. ( b ) CCK-8 assay indicates miR-223 inhibition promoted CPM proliferation. ( c ) CPM was overexpressed with miR-223 and the negative control (NC) mimic, and the cell cycle phase was then analyzed. ( d ) Relative mRNA expression of the cell cycle-related genes after transfection of miR-223 and NC mimic. ( e ) CPM was transfected with miR-223 inhibitor and the NC inhibitor, and the cell cycle phase was then analyzed. ( f ) Relative mRNA expression of the cell cycle-related genes after transfection of miR-223 inhibitor and NC inhibitor. ( g ) EdU staining after transfection of miR-223 mimic and miR-223 inhibitor. Bar, 50 μ m. ( h ) The proliferation rate of myoblasts transfected with miR-223 mimic and miR-223 inhibitor. ( i ) CCK-8 assay showed that miR-223 overexpression inhibited qm-7 proliferation. ( j ) CCK-8 assay showed that miR-223 inhibition promoted qm-7 proliferation. ( k ) qm-7 was overexpressed with miR-223 and the NC mimic, and the cell cycle phase was then analyzed. ( l ) Relative mRNA expression of the cell cycle-related genes after transfection of miR-223 and NC mimic. ( m ) qm-7 was transfected with miR-223 inhibitor and the NC inhibitor, and the cell cycle phase was then analyzed. ( n ) Relative mRNA expression of the cell cycle-related genes after transfection of miR-223 inhibitor and NC inhibitor. ( o ) EdU staining after transfection of miR-223 mimic and miR-223 inhibitor. Bar, 50 μ m. ( p ) The proliferation rate of qm-7 cells transfected with miR-223 mimic and miR-223 inhibitor. Results are shown as the mean±S.E.M. of three independent experiments. Independent sample t -test was used to analysis the statistical differences between groups. * P

    Article Snippet: Transfections Transfections were performed with Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s direction.

    Techniques: CCK-8 Assay, Over Expression, Inhibition, Negative Control, Expressing, Transfection, Staining

    MYOD regulates miR-223 transcription by binding to the E-box 1 region. ( a ) The location of the two E-boxes in the 1932-kb upstream region of the gga-miR-223 gene TSS. ( b ) Identification of the core region in the gga-miR-223 gene promoter by luciferase reporter assays. ( c ) ChIP-qPCR analysis using anti-MYOG, anti-MYOD or chicken IgG antibodies, and the values showed that MYOD could bind to the R1 region of the chicken gga-miR-223 gene in myoblasts. A region from the GAPDH gene was amplified as a NC to verify the specificity of the enrichment. ( d ) MYOD overexpression promoted the relative luciferase activity of the pGL3-1932 reporter in DF-1 cell. ( e ) MYOD overexpression upregulated miR-223 expression in DF-1 cell. ( f) Relative expressions of miR-223 and its downstream genes after transfection of si-MYOD in chicken primary myoblast. ( g ) CCK-8 assay indicated that MYOG loss-of-function significantly reduced myoblast proliferation in chicken primary myoblast. Results are shown as the mean±S.E.M. of three independent experiments. Independent sample t -test was used to analysis the statistical differences between groups. * P

    Journal: Cell Death & Disease

    Article Title: miRNA-223 upregulated by MYOD inhibits myoblast proliferation by repressing IGF2 and facilitates myoblast differentiation by inhibiting ZEB1

    doi: 10.1038/cddis.2017.479

    Figure Lengend Snippet: MYOD regulates miR-223 transcription by binding to the E-box 1 region. ( a ) The location of the two E-boxes in the 1932-kb upstream region of the gga-miR-223 gene TSS. ( b ) Identification of the core region in the gga-miR-223 gene promoter by luciferase reporter assays. ( c ) ChIP-qPCR analysis using anti-MYOG, anti-MYOD or chicken IgG antibodies, and the values showed that MYOD could bind to the R1 region of the chicken gga-miR-223 gene in myoblasts. A region from the GAPDH gene was amplified as a NC to verify the specificity of the enrichment. ( d ) MYOD overexpression promoted the relative luciferase activity of the pGL3-1932 reporter in DF-1 cell. ( e ) MYOD overexpression upregulated miR-223 expression in DF-1 cell. ( f) Relative expressions of miR-223 and its downstream genes after transfection of si-MYOD in chicken primary myoblast. ( g ) CCK-8 assay indicated that MYOG loss-of-function significantly reduced myoblast proliferation in chicken primary myoblast. Results are shown as the mean±S.E.M. of three independent experiments. Independent sample t -test was used to analysis the statistical differences between groups. * P

    Article Snippet: Transfections Transfections were performed with Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s direction.

    Techniques: Binding Assay, Luciferase, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Over Expression, Activity Assay, Expressing, Transfection, CCK-8 Assay

    c-Jun protein interacts with TUB1A (Tubulin alpha chain) in melanoma cells and TUB1A affects AP-1 activity and stabilizes c-Jun protein. ( a , b ) Immunoprecipitation (IP) analyses of melanoma cell (Mel Juso, Mel Ju) lysates revealed co-precipitation of TUB1A with an ( a ) anti-c-Jun antibody and vice versa, ( b ) c-Jun with anti-TUB1A antibody. ( c ) Immunofluorescence analyses showed co-localization (white arrows) of c-Jun (red) and TUB1A (green) in the cytoplasm of melanoma cells. ( d ) Western blot analyses and densitometry of c-Jun and TUB1A in whole cell lysates of Mel Juso cells after TUB1A si-RNA (siTub1A) or control si-RNA (sictrl) transfection. GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was used as a loading control. The bar graph depicts the quantification of protein amounts (mean ± s.d.) of three independent experiments. ( e ) Analyses of c-Jun protein expression in TUB1A-suppressed (siTub1A) and control (sictrl) Mel Juso cells after cycloheximide (CHX) treatment showed a faster decline of c-Jun levels in siTub1A compared to control cells. The bar graph (mean ± s.d. of three western blot analyses) depicts c-Jun levels normalized to GAPDH. ( f ) Western blot analyses and densitometry of nuclear extracts of Mel Juso cells showed lower c-Jun protein levels in TUB1A-suppressed (siTub1A) compared to control (sictrl) cells. The bar graph depicts c-Jun levels of three western blot analyses relative to LAMIN (Lamin A/C), which was used as a loading control. ( g ) AP-1 luciferase reporter gene analyses showed reduced AP-1 activity in TUB1A-suppressed (siTub1A) Mel Juso cells compared to control (sictrl) cells. Bars show the means ± s.d. of three independent experiments; measurements were performed in replicates. (*: p

    Journal: Cancers

    Article Title: Complex Formation with Monomeric α-Tubulin and Importin 13 Fosters c-Jun Protein Stability and Is Required for c-Jun’s Nuclear Translocation and Activity

    doi: 10.3390/cancers11111806

    Figure Lengend Snippet: c-Jun protein interacts with TUB1A (Tubulin alpha chain) in melanoma cells and TUB1A affects AP-1 activity and stabilizes c-Jun protein. ( a , b ) Immunoprecipitation (IP) analyses of melanoma cell (Mel Juso, Mel Ju) lysates revealed co-precipitation of TUB1A with an ( a ) anti-c-Jun antibody and vice versa, ( b ) c-Jun with anti-TUB1A antibody. ( c ) Immunofluorescence analyses showed co-localization (white arrows) of c-Jun (red) and TUB1A (green) in the cytoplasm of melanoma cells. ( d ) Western blot analyses and densitometry of c-Jun and TUB1A in whole cell lysates of Mel Juso cells after TUB1A si-RNA (siTub1A) or control si-RNA (sictrl) transfection. GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was used as a loading control. The bar graph depicts the quantification of protein amounts (mean ± s.d.) of three independent experiments. ( e ) Analyses of c-Jun protein expression in TUB1A-suppressed (siTub1A) and control (sictrl) Mel Juso cells after cycloheximide (CHX) treatment showed a faster decline of c-Jun levels in siTub1A compared to control cells. The bar graph (mean ± s.d. of three western blot analyses) depicts c-Jun levels normalized to GAPDH. ( f ) Western blot analyses and densitometry of nuclear extracts of Mel Juso cells showed lower c-Jun protein levels in TUB1A-suppressed (siTub1A) compared to control (sictrl) cells. The bar graph depicts c-Jun levels of three western blot analyses relative to LAMIN (Lamin A/C), which was used as a loading control. ( g ) AP-1 luciferase reporter gene analyses showed reduced AP-1 activity in TUB1A-suppressed (siTub1A) Mel Juso cells compared to control (sictrl) cells. Bars show the means ± s.d. of three independent experiments; measurements were performed in replicates. (*: p

    Article Snippet: Gene Suppression Using siRNA siRNA transfection of Mel Juso cells was performed using the reverse transfection protocol of the Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

    Techniques: Activity Assay, Immunoprecipitation, Immunofluorescence, Western Blot, Transfection, Expressing, Luciferase

    Comparison of immune-stimulation in PBMCs after treatment with the linkage-modified and unmodified siRNA 728UU ( A ). IFN levels were measured 24 hours after siRNAs were transfected in duplicate using DOTAP. IFN-α levels ( B ) and IL-6 levels ( C ) in response to treatment with unmodified control siRNA (728UU ctrl) and its constituent single strands (RNA-S and RNA-AS); 2′-5′-modified siRNAs (728UU-25S and 728UU-25AS) and their constituent single strands (2′-5′S and 2′-5′-AS); mixmer siRNAs (728UU-MixS and 728UU-MixAS) and their constituent mixmer single strands (Mixmer S and Mixmer AS). Cells only was the negative control with no reagents. DOTAP only treatment was transfection without siRNA. Data were collected in duplicate for each of six donors. Bars indicate standard deviation.

    Journal: Nucleic Acids Research

    Article Title: Effect of 2′-5′/3′-5′ phosphodiester linkage heterogeneity on RNA interference

    doi: 10.1093/nar/gkaa222

    Figure Lengend Snippet: Comparison of immune-stimulation in PBMCs after treatment with the linkage-modified and unmodified siRNA 728UU ( A ). IFN levels were measured 24 hours after siRNAs were transfected in duplicate using DOTAP. IFN-α levels ( B ) and IL-6 levels ( C ) in response to treatment with unmodified control siRNA (728UU ctrl) and its constituent single strands (RNA-S and RNA-AS); 2′-5′-modified siRNAs (728UU-25S and 728UU-25AS) and their constituent single strands (2′-5′S and 2′-5′-AS); mixmer siRNAs (728UU-MixS and 728UU-MixAS) and their constituent mixmer single strands (Mixmer S and Mixmer AS). Cells only was the negative control with no reagents. DOTAP only treatment was transfection without siRNA. Data were collected in duplicate for each of six donors. Bars indicate standard deviation.

    Article Snippet: Subsequently, cells were washed once with serum-free DMEM media and then 80 μl of serum-free DMEM media was added. siRNA and control duplexes were diluted up to 20 μl with serum-free media and transfection reagent (Oligofectamine, Invitrogen) and added to the appropriate well (for a total of 100 μl) at increasing concentrations (0.16, 0.8, 4, 20 and 100 nM).

    Techniques: Modification, Transfection, Negative Control, Standard Deviation

    Increased mir-155 promotes monocyte survival . (A) Healthy donor monocytes were transfected with a negative control miRNA mimic conjugated to a fluorescent molecule Dy547. Following incubation for 40 h, Dy547 positive cells were assessed by flow cytometry. (B) Monocytes were transfected with mir-155 mimic or negative control mimic (Neg Ctrl), or with transfection reagent only (mock) and incubated for 40 h. Mature mir-155 levels were measured using a TaqMan microRNA assay. (C, D) Representative plots (C) and cumulative data (D, n = 22) showing healthy donor CD14+ monocyte survival 40 h after transfection with negative control mimic (Neg Ctrl) or mir-155 mimic. (E) Healthy donor monocytes were transfected (n = 7) with Neg Ctrl or mir-155 mimic for 24 h followed by overnight culture with an agonistic anti-Fas antibody (aFas) or the isotype control (IgM) at 200 ng/mL and the percentage of live cells assessed. D and E tested by Wilcoxon matched-pairs test.

    Journal: Journal of Autoimmunity

    Article Title: MicroRNA-155 contributes to enhanced resistance to apoptosis in monocytes from patients with rheumatoid arthritis

    doi: 10.1016/j.jaut.2017.01.002

    Figure Lengend Snippet: Increased mir-155 promotes monocyte survival . (A) Healthy donor monocytes were transfected with a negative control miRNA mimic conjugated to a fluorescent molecule Dy547. Following incubation for 40 h, Dy547 positive cells were assessed by flow cytometry. (B) Monocytes were transfected with mir-155 mimic or negative control mimic (Neg Ctrl), or with transfection reagent only (mock) and incubated for 40 h. Mature mir-155 levels were measured using a TaqMan microRNA assay. (C, D) Representative plots (C) and cumulative data (D, n = 22) showing healthy donor CD14+ monocyte survival 40 h after transfection with negative control mimic (Neg Ctrl) or mir-155 mimic. (E) Healthy donor monocytes were transfected (n = 7) with Neg Ctrl or mir-155 mimic for 24 h followed by overnight culture with an agonistic anti-Fas antibody (aFas) or the isotype control (IgM) at 200 ng/mL and the percentage of live cells assessed. D and E tested by Wilcoxon matched-pairs test.

    Article Snippet: 2.6 Transfection Transfection of CD14+ cells was performed using the NTER nanoparticle transfection reagent (Sigma, St Louis, MO, USA) following manufacturer's instructions.

    Techniques: Transfection, Negative Control, Incubation, Flow Cytometry, Cytometry, TaqMan microRNA Assay

    Targeting of βarr2 to the centrosome does not depend on microtubules. (A) HeLa cells expressing βarr2-GFP fusion were untreated (control) or treated with nocodazole or taxol (10 µM) to depolymerize or stabilize microtubule network respectively. Cells were fixed and stained for microtubules (α-tubulin) and for the centrosome (pericentrin). Insets show higher magnifications of regions around centrosomes. Scale bar represents 5 µm. (B) Centrosome-associated fluorescence intensity for GFP was normalized to the cytoplasmic signal in the same cells for each condition. Values are the means (+/− SD) of at least 15 cells from three independent experiments. (C) Live HeLa cells transiently expressing βarr2-GFP fusion were treated for one hour at 37°C with nocodazole (10 µM) then used for FRAP experiments. A small region containing the centrosome was bleached twice with 100% of laser intensity (one second per bleach). Images of a representative cell before (bb), just after bleaching (pb) and 90 seconds after bleaching (+90s) are shown. Insets show higher magnifications of the bleached region. Fluorescence intensity of βarr2-GFP at the centrosome was normalized to cytoplasmic staining within an identical region in the cytoplasm. The FRAP experiments were done in at least 5 cells from two independent transfections.

    Journal: PLoS ONE

    Article Title: Targeting of ?-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

    doi: 10.1371/journal.pone.0003728

    Figure Lengend Snippet: Targeting of βarr2 to the centrosome does not depend on microtubules. (A) HeLa cells expressing βarr2-GFP fusion were untreated (control) or treated with nocodazole or taxol (10 µM) to depolymerize or stabilize microtubule network respectively. Cells were fixed and stained for microtubules (α-tubulin) and for the centrosome (pericentrin). Insets show higher magnifications of regions around centrosomes. Scale bar represents 5 µm. (B) Centrosome-associated fluorescence intensity for GFP was normalized to the cytoplasmic signal in the same cells for each condition. Values are the means (+/− SD) of at least 15 cells from three independent experiments. (C) Live HeLa cells transiently expressing βarr2-GFP fusion were treated for one hour at 37°C with nocodazole (10 µM) then used for FRAP experiments. A small region containing the centrosome was bleached twice with 100% of laser intensity (one second per bleach). Images of a representative cell before (bb), just after bleaching (pb) and 90 seconds after bleaching (+90s) are shown. Insets show higher magnifications of the bleached region. Fluorescence intensity of βarr2-GFP at the centrosome was normalized to cytoplasmic staining within an identical region in the cytoplasm. The FRAP experiments were done in at least 5 cells from two independent transfections.

    Article Snippet: Transfections Transfections were done following the recommended procedure of the Genejuice (Novagen) or of the FuGENE HD (Roche) transfection reagents.

    Techniques: Expressing, Staining, Fluorescence, Transfection

    βarr2 is found in the axoneme and basal body of primary cilia. (A) Confluent RPE1 cells were serum-starved for 24 hours, then fixed and stained for acetylated-tubulin (AT), which is highly enriched in primary cilia (axoneme) and for endogenous βarr2 using rARR antibody. (B) Z-stacks images of representative cells were deconvoluated as in Figure 2 and a 3D reconstruction of a representative cilium is shown. (C and D) RPE1 cells transfected with plasmids encoding for the βarr2-Cherry fusion (βarr2-Ch), were serum-starved for 24 hours after transfection, then fixed and stained for AT (C) or for the basal body marker pericentrin (D). In coloured images, βarr2 staining is in red, centrosome or cilia markers in green and nuclei stained with DAPI are in blue. (E) RPE1 cells transfected with plasmids encoding for Flag-tagged active form of smoothened (smo*) and the βarr2-Ch fusion were serum-starved for 24h after transfection, then fixed and stained for AT and smo*, using a rabbit polyclonal anti-Flag antibody. In coloured image, βarr2 staining is in red, AT in blue and smo* in green. Insets show higher magnifications of representative areas. Arrows stress basal bodies. Scale bars represent 5 µm. (F) Tissue sections from adult mouse kidney were stained for AT and for βarr2 using the rARR antibody. In coloured image, βarr2 staining is in red, AT in green and nuclei stained with DAPI in blue. The lumen of a representative tubule is indicated (Lu). Insets show higher magnifications of a representative ciliated tubular epithelial cell. Arrows stress AT positive structures.

    Journal: PLoS ONE

    Article Title: Targeting of ?-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

    doi: 10.1371/journal.pone.0003728

    Figure Lengend Snippet: βarr2 is found in the axoneme and basal body of primary cilia. (A) Confluent RPE1 cells were serum-starved for 24 hours, then fixed and stained for acetylated-tubulin (AT), which is highly enriched in primary cilia (axoneme) and for endogenous βarr2 using rARR antibody. (B) Z-stacks images of representative cells were deconvoluated as in Figure 2 and a 3D reconstruction of a representative cilium is shown. (C and D) RPE1 cells transfected with plasmids encoding for the βarr2-Cherry fusion (βarr2-Ch), were serum-starved for 24 hours after transfection, then fixed and stained for AT (C) or for the basal body marker pericentrin (D). In coloured images, βarr2 staining is in red, centrosome or cilia markers in green and nuclei stained with DAPI are in blue. (E) RPE1 cells transfected with plasmids encoding for Flag-tagged active form of smoothened (smo*) and the βarr2-Ch fusion were serum-starved for 24h after transfection, then fixed and stained for AT and smo*, using a rabbit polyclonal anti-Flag antibody. In coloured image, βarr2 staining is in red, AT in blue and smo* in green. Insets show higher magnifications of representative areas. Arrows stress basal bodies. Scale bars represent 5 µm. (F) Tissue sections from adult mouse kidney were stained for AT and for βarr2 using the rARR antibody. In coloured image, βarr2 staining is in red, AT in green and nuclei stained with DAPI in blue. The lumen of a representative tubule is indicated (Lu). Insets show higher magnifications of a representative ciliated tubular epithelial cell. Arrows stress AT positive structures.

    Article Snippet: Transfections Transfections were done following the recommended procedure of the Genejuice (Novagen) or of the FuGENE HD (Roche) transfection reagents.

    Techniques: Staining, Transfection, Marker

    Extracellular DNase I does not influence DNA transfection efficiency. ( A ) TKPTS cells were treated with different concentration of G-actin in culture medium, and endonuclease activity was measured with PIA. G-actin inhibited DNase I in culture medium ( n = 4 per concentration point, * p = 0.018, ** p = 0.0052, *** p = 0.006). ( B ) The efficiency of TKPTS cells transfection with pECFP plasmid in the presence of 1 mg/mL G-actin was not different from the one measured in the presence of 1 mg/mL albumin (15 ± 5 vs. 15 ± 8% of CFP-positive cells, n = 6).

    Journal: DNA and Cell Biology

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases

    doi: 10.1089/dna.2008.0850

    Figure Lengend Snippet: Extracellular DNase I does not influence DNA transfection efficiency. ( A ) TKPTS cells were treated with different concentration of G-actin in culture medium, and endonuclease activity was measured with PIA. G-actin inhibited DNase I in culture medium ( n = 4 per concentration point, * p = 0.018, ** p = 0.0052, *** p = 0.006). ( B ) The efficiency of TKPTS cells transfection with pECFP plasmid in the presence of 1 mg/mL G-actin was not different from the one measured in the presence of 1 mg/mL albumin (15 ± 5 vs. 15 ± 8% of CFP-positive cells, n = 6).

    Article Snippet: In brief, 4 μL transfection reagent was diluted to 50 μL with serum-free DMEM/Ham's F-12 medium (Sigma-Aldrich) incubated for 15 min, and then 15 μL (1 μM) siRNA was added and incubated for further 20 min at room temperature.

    Techniques: Transfection, Concentration Assay, Activity Assay, Plasmid Preparation

    Efficiency of RNA transfection of PTE cells with active or inactive EndoG. Primary EndoG KO or WT mouse tubular epithelial cells were transfected with fluorescent siRNA as described in the Materials and Methods section. About 48 h later RNA transfection was detected using a fluorescent microscope. Blue color of DAPI was used to stain the nuclei. EndoG KO cells show significantly higher rate of siRNA transfection than WT cells (14 ± 2 vs. 29 ± 4 arbitrary fluorescence units per cell, n = 3, * p

    Journal: DNA and Cell Biology

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases

    doi: 10.1089/dna.2008.0850

    Figure Lengend Snippet: Efficiency of RNA transfection of PTE cells with active or inactive EndoG. Primary EndoG KO or WT mouse tubular epithelial cells were transfected with fluorescent siRNA as described in the Materials and Methods section. About 48 h later RNA transfection was detected using a fluorescent microscope. Blue color of DAPI was used to stain the nuclei. EndoG KO cells show significantly higher rate of siRNA transfection than WT cells (14 ± 2 vs. 29 ± 4 arbitrary fluorescence units per cell, n = 3, * p

    Article Snippet: In brief, 4 μL transfection reagent was diluted to 50 μL with serum-free DMEM/Ham's F-12 medium (Sigma-Aldrich) incubated for 15 min, and then 15 μL (1 μM) siRNA was added and incubated for further 20 min at room temperature.

    Techniques: Transfection, Microscopy, Staining, Fluorescence

    Efficiency of plasmid transfection of PTE cells with active or inactive endonucleases. ( A ) Expression of CFP after pECFP-N1 plasmid transfection in DNase I KO PTE cells is higher than in WT cells. KO versus WT (21 ± 5% vs. 8 ± 5% transfected cells, n = 3, * p

    Journal: DNA and Cell Biology

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases

    doi: 10.1089/dna.2008.0850

    Figure Lengend Snippet: Efficiency of plasmid transfection of PTE cells with active or inactive endonucleases. ( A ) Expression of CFP after pECFP-N1 plasmid transfection in DNase I KO PTE cells is higher than in WT cells. KO versus WT (21 ± 5% vs. 8 ± 5% transfected cells, n = 3, * p

    Article Snippet: In brief, 4 μL transfection reagent was diluted to 50 μL with serum-free DMEM/Ham's F-12 medium (Sigma-Aldrich) incubated for 15 min, and then 15 μL (1 μM) siRNA was added and incubated for further 20 min at room temperature.

    Techniques: Plasmid Preparation, Transfection, Expressing

    Efficiency of pECFP-N1 plasmid transfection in TKPTS cells after EG siRNA, DNase I siRNA, and EG+DNase I siRNA treatment. TKPTS cells were treated with EndoG, DNase I, and EndoG+DNase I siRNA using TransIT-TKO transfection reagent (TKO), controlled with siCONTROL Non-Targeting siRNA (control siRNA) and transfected with pECFP-N1 cyan plasmid. SiRNA-silenced cells (EG, DNase I and both) show significantly higher plasmid transfection efficiency (8.8% vs. 2.4% of pECFP-N1 transfected cells, * p = 0.001). siRNA, small interfering RNA.

    Journal: DNA and Cell Biology

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases

    doi: 10.1089/dna.2008.0850

    Figure Lengend Snippet: Efficiency of pECFP-N1 plasmid transfection in TKPTS cells after EG siRNA, DNase I siRNA, and EG+DNase I siRNA treatment. TKPTS cells were treated with EndoG, DNase I, and EndoG+DNase I siRNA using TransIT-TKO transfection reagent (TKO), controlled with siCONTROL Non-Targeting siRNA (control siRNA) and transfected with pECFP-N1 cyan plasmid. SiRNA-silenced cells (EG, DNase I and both) show significantly higher plasmid transfection efficiency (8.8% vs. 2.4% of pECFP-N1 transfected cells, * p = 0.001). siRNA, small interfering RNA.

    Article Snippet: In brief, 4 μL transfection reagent was diluted to 50 μL with serum-free DMEM/Ham's F-12 medium (Sigma-Aldrich) incubated for 15 min, and then 15 μL (1 μM) siRNA was added and incubated for further 20 min at room temperature.

    Techniques: Plasmid Preparation, Transfection, Small Interfering RNA

    miR-125b induces proliferation and apoptosis resistance in GBM cells. ( a ) Population growth of U87 and LN-18 cells overexpressing miR-125b or anti-miR-125b relative to the control using the resazurin assay ( n =3). ( b ) Cell cycle analysis of nocodazole-treated cells by flow cytometry ( n =3). ( c ) Apoptosis. Cells were transfected with pre-miR-125b or pre-control and treated with 10 ng/ml TNF α or 250 ng/ml TRAIL for 48 h beginning 24 h post transfection. Apoptosis was assessed using the ApoTox-Glo Triplex assay ( n =3) ( d ) LN-18 cells overexpressing anti-miR-125b or anti-control were induced with 10 ng/ml TNF α for 24 h and subjected to the ApoTox-Glo Triplex assay ( n =3)

    Journal: Cell Death & Disease

    Article Title: miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas

    doi: 10.1038/cddis.2014.245

    Figure Lengend Snippet: miR-125b induces proliferation and apoptosis resistance in GBM cells. ( a ) Population growth of U87 and LN-18 cells overexpressing miR-125b or anti-miR-125b relative to the control using the resazurin assay ( n =3). ( b ) Cell cycle analysis of nocodazole-treated cells by flow cytometry ( n =3). ( c ) Apoptosis. Cells were transfected with pre-miR-125b or pre-control and treated with 10 ng/ml TNF α or 250 ng/ml TRAIL for 48 h beginning 24 h post transfection. Apoptosis was assessed using the ApoTox-Glo Triplex assay ( n =3) ( d ) LN-18 cells overexpressing anti-miR-125b or anti-control were induced with 10 ng/ml TNF α for 24 h and subjected to the ApoTox-Glo Triplex assay ( n =3)

    Article Snippet: Transfection Transfection was performed using Effectene, HiPerFect or Attractene reagents (Qiagen, Hombrechtikon, Switzerland) essentially as described except that 37.5 nM miRNA precursor hsa-miR-125a-5p and hsa-miR-125b-5p, pre-miR negative control #1 (Ambion, Carisbad, CA, USA) was used for transfection.

    Techniques: Resazurin Assay, Cell Cycle Assay, Flow Cytometry, Cytometry, Transfection

    miR-125b confers resistance to apoptosis and TMZ by targeting TNFAIP3 and NKIRAS2 . ( a ) Retrovirally transduced LN-18 cells were transfected with pre-miR-125b or pre-control and induced with 10 ng/ml TNF α for 24 h ( n =3). Apoptosis was assessed using the ApoTox-Glo Triplex assay. ( b ) Cell viability of GBM cells overexpressing miR-125b in the presence of 200 μ M TMZ. Cell viability was assessed 3 days after treatment with TMZ using resazurin assay ( n =3). ( c ) Cell viability of GBM cells overexpressing anti-miR-125b or anti-control in the presence of TMZ ( n =6). ( d ) Cell viability of LN-18 cells retrovirally transduced with miR-125b-refractory TNFAIP3, NKIRAS2 or pBABE control following transfection with pre-miR-125b or pre-control in the presence of TMZ ( n =6)

    Journal: Cell Death & Disease

    Article Title: miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas

    doi: 10.1038/cddis.2014.245

    Figure Lengend Snippet: miR-125b confers resistance to apoptosis and TMZ by targeting TNFAIP3 and NKIRAS2 . ( a ) Retrovirally transduced LN-18 cells were transfected with pre-miR-125b or pre-control and induced with 10 ng/ml TNF α for 24 h ( n =3). Apoptosis was assessed using the ApoTox-Glo Triplex assay. ( b ) Cell viability of GBM cells overexpressing miR-125b in the presence of 200 μ M TMZ. Cell viability was assessed 3 days after treatment with TMZ using resazurin assay ( n =3). ( c ) Cell viability of GBM cells overexpressing anti-miR-125b or anti-control in the presence of TMZ ( n =6). ( d ) Cell viability of LN-18 cells retrovirally transduced with miR-125b-refractory TNFAIP3, NKIRAS2 or pBABE control following transfection with pre-miR-125b or pre-control in the presence of TMZ ( n =6)

    Article Snippet: Transfection Transfection was performed using Effectene, HiPerFect or Attractene reagents (Qiagen, Hombrechtikon, Switzerland) essentially as described except that 37.5 nM miRNA precursor hsa-miR-125a-5p and hsa-miR-125b-5p, pre-miR negative control #1 (Ambion, Carisbad, CA, USA) was used for transfection.

    Techniques: Transfection, Resazurin Assay, Transduction

    miR-125b induces NF- κ B activity. ( a ) NF- κ B reporter activity of GBM cells overexpressing miR-125b or anti-miR-125b relative to control cells ( n =6). ( b ) NF- κ B reporter activity of LN-18 cells overexpressing anti-miR-125b. Cells were induced with 10 ng/ml TNF α for 4 h beginning 20 h post transfection ( n =6). Expression of ( c ) nuclear p65 and ( d ) cytoplasmic p65, I κ B α and phospho I κ B α in LN-18 cells transiently transfected with pre-miR-125b or pre-control by Western blotting. Cells were induced with 10 ng/ml TNF α beginning at 48 h post transfection. The ratio of pI κ B α to I κ B α protein levels is presented below the corresponding Western blot

    Journal: Cell Death & Disease

    Article Title: miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas

    doi: 10.1038/cddis.2014.245

    Figure Lengend Snippet: miR-125b induces NF- κ B activity. ( a ) NF- κ B reporter activity of GBM cells overexpressing miR-125b or anti-miR-125b relative to control cells ( n =6). ( b ) NF- κ B reporter activity of LN-18 cells overexpressing anti-miR-125b. Cells were induced with 10 ng/ml TNF α for 4 h beginning 20 h post transfection ( n =6). Expression of ( c ) nuclear p65 and ( d ) cytoplasmic p65, I κ B α and phospho I κ B α in LN-18 cells transiently transfected with pre-miR-125b or pre-control by Western blotting. Cells were induced with 10 ng/ml TNF α beginning at 48 h post transfection. The ratio of pI κ B α to I κ B α protein levels is presented below the corresponding Western blot

    Article Snippet: Transfection Transfection was performed using Effectene, HiPerFect or Attractene reagents (Qiagen, Hombrechtikon, Switzerland) essentially as described except that 37.5 nM miRNA precursor hsa-miR-125a-5p and hsa-miR-125b-5p, pre-miR negative control #1 (Ambion, Carisbad, CA, USA) was used for transfection.

    Techniques: Activity Assay, Transfection, Expressing, Western Blot

    No synergism on cell death . (A) Bcl2 expression. A549 or H2009 cells were transfected with 20 nM precursors and harvested 42 h post-transfection. Western blot analysis was performed using a monoclonal antibody against Bcl2. Protein levels were normalized to α-tubulin and presented relative to the level obtained for the control. (B) Time-course of propidium iodide (PI)-positive A549 and H2009 cells by flow cytometry. Cells were transfected with concentrations of precursors as indicated in the legend to Fig. 4B (n = 3). Cells were gated as shown in Additional file 1A . (C) Cleaved caspase-3-positive cells. H2009 cells were analysed for the presence of cleaved caspase 3 by flow cytometry 72 h post-transfection (n = 3). Values are relative to the level obtained for the control transfected with precursor control. As a positive control, cells were treated with UV.

    Journal: Molecular Cancer

    Article Title: miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

    doi: 10.1186/1476-4598-10-55

    Figure Lengend Snippet: No synergism on cell death . (A) Bcl2 expression. A549 or H2009 cells were transfected with 20 nM precursors and harvested 42 h post-transfection. Western blot analysis was performed using a monoclonal antibody against Bcl2. Protein levels were normalized to α-tubulin and presented relative to the level obtained for the control. (B) Time-course of propidium iodide (PI)-positive A549 and H2009 cells by flow cytometry. Cells were transfected with concentrations of precursors as indicated in the legend to Fig. 4B (n = 3). Cells were gated as shown in Additional file 1A . (C) Cleaved caspase-3-positive cells. H2009 cells were analysed for the presence of cleaved caspase 3 by flow cytometry 72 h post-transfection (n = 3). Values are relative to the level obtained for the control transfected with precursor control. As a positive control, cells were treated with UV.

    Article Snippet: Co-transfections with plasmid DNA were performed using Effectene reagent (Qiagen), all other transfections were performed using HiPerFect (Qiagen).

    Techniques: Expressing, Transfection, Western Blot, Flow Cytometry, Cytometry, Positive Control

    Synergistic action on cell cycle arrest is due to the down-regulation of unique mRNA targets . A549 (A) or H1299 cells (B) were co-transfected with 20 nM miRNA precursors and 7.8 nM siRNA against CCNE1 and subsequently treated for 18 h with nocodazole beginning 24 h post-transfection. Comparable results were also obtained 48 h post-transfection (data not shown).

    Journal: Molecular Cancer

    Article Title: miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

    doi: 10.1186/1476-4598-10-55

    Figure Lengend Snippet: Synergistic action on cell cycle arrest is due to the down-regulation of unique mRNA targets . A549 (A) or H1299 cells (B) were co-transfected with 20 nM miRNA precursors and 7.8 nM siRNA against CCNE1 and subsequently treated for 18 h with nocodazole beginning 24 h post-transfection. Comparable results were also obtained 48 h post-transfection (data not shown).

    Article Snippet: Co-transfections with plasmid DNA were performed using Effectene reagent (Qiagen), all other transfections were performed using HiPerFect (Qiagen).

    Techniques: Transfection

    H2009 cells are refractory to miR-34a -induced cell cycle arrest . (A) DNA content distribution of NSCLC cells transfected with precursor miRNA or precursor control. Cells were treated for 18 h with nocodazole beginning 24 h post-transfection. (B) Percent difference in G 1 -G 0 between cells transfected with pre-miR-34a and cells transfected with precursor control. H2009, A549, n = 3; H1299 and H358, n = 1. (C) mRNA levels of known miR-34a targets. H2009 cells were transfected with pre-miR-34a and harvested 42 h post-transfection (n = 3). Values are relative to the level obtained for the control transfected with precursor control.

    Journal: Molecular Cancer

    Article Title: miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

    doi: 10.1186/1476-4598-10-55

    Figure Lengend Snippet: H2009 cells are refractory to miR-34a -induced cell cycle arrest . (A) DNA content distribution of NSCLC cells transfected with precursor miRNA or precursor control. Cells were treated for 18 h with nocodazole beginning 24 h post-transfection. (B) Percent difference in G 1 -G 0 between cells transfected with pre-miR-34a and cells transfected with precursor control. H2009, A549, n = 3; H1299 and H358, n = 1. (C) mRNA levels of known miR-34a targets. H2009 cells were transfected with pre-miR-34a and harvested 42 h post-transfection (n = 3). Values are relative to the level obtained for the control transfected with precursor control.

    Article Snippet: Co-transfections with plasmid DNA were performed using Effectene reagent (Qiagen), all other transfections were performed using HiPerFect (Qiagen).

    Techniques: Transfection

    miR-34a -induced cell cycle arrest depends on the expression of Rb . (A, D), DNA content distribution by flow cytometry of H2009 (A) and A549 cells (D) treated for 18 h with nocodazole beginning 48 h post-transfection. The percent cells in G 1 -G 0 is shown in the upper panel (n = 3) and a representative histogram of the cell cycle profile is shown in the lower panel. (B, C), Western blot analysis of H2009 (B) and A549 cells (C) subjected to the same conditions as in (A) and (D) using antibodies directed against Rb or phospho-Rb. Protein levels were normalised to α-tubulin.

    Journal: Molecular Cancer

    Article Title: miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

    doi: 10.1186/1476-4598-10-55

    Figure Lengend Snippet: miR-34a -induced cell cycle arrest depends on the expression of Rb . (A, D), DNA content distribution by flow cytometry of H2009 (A) and A549 cells (D) treated for 18 h with nocodazole beginning 48 h post-transfection. The percent cells in G 1 -G 0 is shown in the upper panel (n = 3) and a representative histogram of the cell cycle profile is shown in the lower panel. (B, C), Western blot analysis of H2009 (B) and A549 cells (C) subjected to the same conditions as in (A) and (D) using antibodies directed against Rb or phospho-Rb. Protein levels were normalised to α-tubulin.

    Article Snippet: Co-transfections with plasmid DNA were performed using Effectene reagent (Qiagen), all other transfections were performed using HiPerFect (Qiagen).

    Techniques: Expressing, Flow Cytometry, Cytometry, Transfection, Western Blot

    miR-15a/16 and miR-34a act synergistically to induce cell cycle arrest . (A) Cell cycle analysis of A549 cells transfected with pre-miR-34a and/or pre-miR-15a/16 under non-saturating conditions. Precursors were supplemented with precursor control to yield a total concentration of 2.5 nM per transfection. *, transfection with 2.5 nM precursor control. Cells were treated for 18 h with nocodazole beginning 24 h post-transfection (n = 3). (B) Cell cycle analysis under saturating conditions. A549 cells were transfected with 20 nM precursor or precursor control or co-transfected with 10 nM pre-miR-34a and 10 nM pre-miR-15a/16 and treated for 18 h with nocodazole beginning 24 h (left panel) or 48 h (right panel) post-transfection (n = 3).

    Journal: Molecular Cancer

    Article Title: miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

    doi: 10.1186/1476-4598-10-55

    Figure Lengend Snippet: miR-15a/16 and miR-34a act synergistically to induce cell cycle arrest . (A) Cell cycle analysis of A549 cells transfected with pre-miR-34a and/or pre-miR-15a/16 under non-saturating conditions. Precursors were supplemented with precursor control to yield a total concentration of 2.5 nM per transfection. *, transfection with 2.5 nM precursor control. Cells were treated for 18 h with nocodazole beginning 24 h post-transfection (n = 3). (B) Cell cycle analysis under saturating conditions. A549 cells were transfected with 20 nM precursor or precursor control or co-transfected with 10 nM pre-miR-34a and 10 nM pre-miR-15a/16 and treated for 18 h with nocodazole beginning 24 h (left panel) or 48 h (right panel) post-transfection (n = 3).

    Article Snippet: Co-transfections with plasmid DNA were performed using Effectene reagent (Qiagen), all other transfections were performed using HiPerFect (Qiagen).

    Techniques: Activated Clotting Time Assay, Cell Cycle Assay, Transfection, Concentration Assay

    miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer . (A) miR-16 and miR-34a levels in adenocarcinomas and squamous cell carcinomas relative to matched normal tissues. (B) miR-21 levels in the same tumour samples, relative to matched normal tissue. (C) miR-34a and miR-15a/16 are not able to mutually regulate their expression. H2009 cells were transfected with pre-miR-34a (upper panel) or pre-miR-15a/16 (lower panel) and analysed for the expression of the miRNA counterpart or CDK6 mRNA 42 h post-transfection. Values are relative to the value obtained for the control transfected with precursor control (n = 3). *, P

    Journal: Molecular Cancer

    Article Title: miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

    doi: 10.1186/1476-4598-10-55

    Figure Lengend Snippet: miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer . (A) miR-16 and miR-34a levels in adenocarcinomas and squamous cell carcinomas relative to matched normal tissues. (B) miR-21 levels in the same tumour samples, relative to matched normal tissue. (C) miR-34a and miR-15a/16 are not able to mutually regulate their expression. H2009 cells were transfected with pre-miR-34a (upper panel) or pre-miR-15a/16 (lower panel) and analysed for the expression of the miRNA counterpart or CDK6 mRNA 42 h post-transfection. Values are relative to the value obtained for the control transfected with precursor control (n = 3). *, P

    Article Snippet: Co-transfections with plasmid DNA were performed using Effectene reagent (Qiagen), all other transfections were performed using HiPerFect (Qiagen).

    Techniques: Expressing, Transfection

    GAPDH in retinal endothelial cells. Retinal endothelial cells were transfected using 0.5 to 5.0 μg GAPDH expression plasmid for 8 hours. ( a ) GAPDH activity was measured spectrophotometrically to determine the degree of transfection. ( b , c ) BRECs were transfected with 2.5 μg GAPDH plasmid or no plasmid but with transfection reagent alone (R) for 8 hours and were incubated for 4 days in 5 mM or 20 mM glucose media. ( b ) GAPDH abundance was assessed by Western blot analysis, and ( c ) its glycolytic activity was assessed by measuring the production of NADH at 340 nm. Each parameter was measured in duplicate using four different cell preparations. Results are presented as mean ± SD of the values obtained from three different cell preparations, with each measurement made in duplicate. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected BRECs incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone, followed by incubation in 5 mM or 20 mM glucose, respectively. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy

    doi: 10.1167/iovs.09-4171

    Figure Lengend Snippet: GAPDH in retinal endothelial cells. Retinal endothelial cells were transfected using 0.5 to 5.0 μg GAPDH expression plasmid for 8 hours. ( a ) GAPDH activity was measured spectrophotometrically to determine the degree of transfection. ( b , c ) BRECs were transfected with 2.5 μg GAPDH plasmid or no plasmid but with transfection reagent alone (R) for 8 hours and were incubated for 4 days in 5 mM or 20 mM glucose media. ( b ) GAPDH abundance was assessed by Western blot analysis, and ( c ) its glycolytic activity was assessed by measuring the production of NADH at 340 nm. Each parameter was measured in duplicate using four different cell preparations. Results are presented as mean ± SD of the values obtained from three different cell preparations, with each measurement made in duplicate. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected BRECs incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone, followed by incubation in 5 mM or 20 mM glucose, respectively. * P

    Article Snippet: Transfection complexes were formed with transfection reagent (Effectene; Qiagen, Valencia, CA) and were incubated with BRECs for 8 hours before incubation in 5 mM or 20 mM glucose media for 4 days.

    Techniques: Transfection, Expressing, Plasmid Preparation, Activity Assay, Incubation, Western Blot

    Effect of GAPDH overexpression on endothelial cell death: GAPDH-transfected BRECs (using 2.5 μg plasmid) or untransfected BRECs were incubated in 5 mM or 20 mM glucose for 4 days. ( a ) Apoptosis was determined by cytoplasmic histone-associated DNA fragments using ELISA. The graph shows the mean ± SD adjusted to the total DNA in each sample. ( b ) Activation of apoptosis execution enzyme caspase-3 was determined in the cells by measuring the cleavage of the substrate Ac-DEVD-pNA. Each experiment was repeated with at least three different BREC preparations, and measurements were made in duplicate. ( c ) The appearance of a 85-kDa band of PARP was determined by Western blot technique, and β-actin was used as a loading standard. Western blot is representative of four different experiments. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 20+Fe, 20 mM glucose and 2.5 μM FeTPPS; 20+PJ, 20 mM glucose and 1.0 μM PJ34; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone before incubation with 5 mM or 20 mM glucose, respectively. The values, represented as mean ± SD, obtained with 5 mM glucose were considered 100%. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy

    doi: 10.1167/iovs.09-4171

    Figure Lengend Snippet: Effect of GAPDH overexpression on endothelial cell death: GAPDH-transfected BRECs (using 2.5 μg plasmid) or untransfected BRECs were incubated in 5 mM or 20 mM glucose for 4 days. ( a ) Apoptosis was determined by cytoplasmic histone-associated DNA fragments using ELISA. The graph shows the mean ± SD adjusted to the total DNA in each sample. ( b ) Activation of apoptosis execution enzyme caspase-3 was determined in the cells by measuring the cleavage of the substrate Ac-DEVD-pNA. Each experiment was repeated with at least three different BREC preparations, and measurements were made in duplicate. ( c ) The appearance of a 85-kDa band of PARP was determined by Western blot technique, and β-actin was used as a loading standard. Western blot is representative of four different experiments. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 20+Fe, 20 mM glucose and 2.5 μM FeTPPS; 20+PJ, 20 mM glucose and 1.0 μM PJ34; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone before incubation with 5 mM or 20 mM glucose, respectively. The values, represented as mean ± SD, obtained with 5 mM glucose were considered 100%. * P

    Article Snippet: Transfection complexes were formed with transfection reagent (Effectene; Qiagen, Valencia, CA) and were incubated with BRECs for 8 hours before incubation in 5 mM or 20 mM glucose media for 4 days.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot

    Subcellular distribution of GAPDH. BRECs either transfected with 2.5 μg GAPDH plasmid or left untransfected were incubated for 4 days in 5 mM or 20 mM glucose media. The nuclear fraction was prepared by centrifugation, and the expression of GAPDH was determined by Western blot analysis using histone 2B as a loading control. Western blot represents results obtained from three or four different experiments, with each measurement made in duplicate. Histogram represents mean ± SD. GAPDH band intensity adjusted to H2B band intensity, and the values obtained from BRECs incubated in 5 mM glucose are considered 100%. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 20+R, BRECs treated with the transfection reagent alone before incubation with 20 mM glucose. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy

    doi: 10.1167/iovs.09-4171

    Figure Lengend Snippet: Subcellular distribution of GAPDH. BRECs either transfected with 2.5 μg GAPDH plasmid or left untransfected were incubated for 4 days in 5 mM or 20 mM glucose media. The nuclear fraction was prepared by centrifugation, and the expression of GAPDH was determined by Western blot analysis using histone 2B as a loading control. Western blot represents results obtained from three or four different experiments, with each measurement made in duplicate. Histogram represents mean ± SD. GAPDH band intensity adjusted to H2B band intensity, and the values obtained from BRECs incubated in 5 mM glucose are considered 100%. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 20+R, BRECs treated with the transfection reagent alone before incubation with 20 mM glucose. * P

    Article Snippet: Transfection complexes were formed with transfection reagent (Effectene; Qiagen, Valencia, CA) and were incubated with BRECs for 8 hours before incubation in 5 mM or 20 mM glucose media for 4 days.

    Techniques: Transfection, Plasmid Preparation, Incubation, Centrifugation, Expressing, Western Blot

    PACA as a covalent activator for PPAR-γ. ( A ) Docking of OXO-PACA to PPAR-γ LBD. OXO-PACA was docked to PPAR-γ LBD (PDB:) by Autodock vina. ( B ) Amino acid residues for recognizing OXO-PACA. Hydrogen bonds are shown in green, whereas pi-alkyl interactions are shown in pink. ( C ) Luciferase assay for PPAR-γ activation. RAW264.7 macrophages were transiently transfected with PPRE-X3-TK luciferase and pRL control using Effectene transfection reagent from Qiagen. Transfected macrophages were treated with PACA for 24 h. Luciferase activities were measured using the Dual-Luciferase reporter assay system. Data were expressed as mean ± SD (n = 5). * p

    Journal: Aging (Albany NY)

    Article Title: N-Propargyl caffeate amide (PACA) prevents cardiac fibrosis in experimental myocardial infarction by promoting pro-resolving macrophage polarization

    doi: 10.18632/aging.102959

    Figure Lengend Snippet: PACA as a covalent activator for PPAR-γ. ( A ) Docking of OXO-PACA to PPAR-γ LBD. OXO-PACA was docked to PPAR-γ LBD (PDB:) by Autodock vina. ( B ) Amino acid residues for recognizing OXO-PACA. Hydrogen bonds are shown in green, whereas pi-alkyl interactions are shown in pink. ( C ) Luciferase assay for PPAR-γ activation. RAW264.7 macrophages were transiently transfected with PPRE-X3-TK luciferase and pRL control using Effectene transfection reagent from Qiagen. Transfected macrophages were treated with PACA for 24 h. Luciferase activities were measured using the Dual-Luciferase reporter assay system. Data were expressed as mean ± SD (n = 5). * p

    Article Snippet: Luciferase reporter assays RAW264.7 macrophages in 12-well plated were co-transfected with 300 ng of PPRE-X3-TK controlled Firefly luciferase plasmid from Addgene (Cambridge, MA, USA) and 30 ng of pRL-TK-renilla luciferase plasmid using Effectene transfection reagent from Qiagen (Hilden, Germany).

    Techniques: Luciferase, Activation Assay, Transfection, Reporter Assay

    ( a ) Exogenous stimulation of S. gordonii lipoproteins augment TLR2 mRNA expressions in hDPCs, and were maintained after calcium hydroxide (CH) or HBD3-C15 treatment. ( b ) S. gordonii lipoprotein-stimulated NF-κB are mediated by TLR2. HEK-TLR2 cells (2.5 × 10 5 cells/mL) were transfected with an NF-κB luciferase reporter plasmid using Attractene transfection reagent. After 16 h, the cells were stimulated with lipoprotein purified from S. gordonii , and then treated with either of CH or HBD3-C15. After the cells were lysed, a luciferease assay was conducted.

    Journal: International Journal of Molecular Sciences

    Article Title: Synthetic Human β Defensin-3-C15 Peptide in Endodontics: Potential Therapeutic Agent in Streptococcus gordonii Lipoprotein-Stimulated Human Dental Pulp-Derived Cells

    doi: 10.3390/ijms21010071

    Figure Lengend Snippet: ( a ) Exogenous stimulation of S. gordonii lipoproteins augment TLR2 mRNA expressions in hDPCs, and were maintained after calcium hydroxide (CH) or HBD3-C15 treatment. ( b ) S. gordonii lipoprotein-stimulated NF-κB are mediated by TLR2. HEK-TLR2 cells (2.5 × 10 5 cells/mL) were transfected with an NF-κB luciferase reporter plasmid using Attractene transfection reagent. After 16 h, the cells were stimulated with lipoprotein purified from S. gordonii , and then treated with either of CH or HBD3-C15. After the cells were lysed, a luciferease assay was conducted.

    Article Snippet: Transfections were performed in Opti-MEM using Attractene transfection reagent for 16 h (Qiagen, Germantown, MD, USA).

    Techniques: Transfection, Luciferase, Plasmid Preparation, Purification

    Depletion of βIII spectrin impairs the anterograde transport of the VSV-G and the post-Golgi secretion of 35 S-labeled proteins. A , representative images of HeLa cells constitutively expressing VSV-G-GFP mutant form ts045 show the viral protein in the ER at 40 °C, or in the Golgi (30 min) or the plasma membrane ( PM ; 90 min) when cells were shifted to 32 °C. Bar , 10 μm. B , representative images of VSV-G-GFP expressing HeLa cells silenced with non-targeting or βIII spectrin siRNAs after 15 and 60 min after being shifted to 32 °C. C , quantitative analysis of results partially shown in B . The graph shows the percentage of the cells in which VSV-G-GFP is mainly visualized to the ER, to the Golgi, or to the plasma membrane. Data are shown as the mean ± S.E. of three independent experiments (100 cells each). D , biochemical transport assay for VSV-G-GFP using Endo H. HeLa cells constitutively expressing VSV-G-GFP were transfected for 96 h with siRNA control or against βIII spectrin and incubated at 40 °C for the last 24 h. Then, cells were shifted at 32 °C to induce the transport of VSV-G from the ER, lysed at indicated times, and subjected to Endo H treatment. R and S indicate Endo H-resistant and -sensitive forms, respectively. The ratio of the amount of Endo H-resistant form to that of total amount is plotted. Values are represented as the mean ± S.E. of three independent experiments. Statistical significance is shown, according to two-way analysis of variance using Bonferroni's post-tests (*, p ≤ 0.05). E , RPE1 cells were transfected with control or with βIII spectrin siRNA. Ninety-six h after the transfection, cells were pulse labeled with [ 35 S]Met/Cys, incubated at 19 °C for 3 h and then shifted to 37 °C. At the indicated times, proteins in the culture supernatants and the cell lysates were precipitated and quantified by scintillation counting. As additional controls, the secretion assay was performed at 4 °C or in cells treated with BFA. Results are the mean ± S.E. from at least three independent experiments. Statistical significance according to two-way analysis of variance using Bonferroni's post-tests (**, p ≤ 0.01 and ***, p ≤ 0.001).

    Journal: The Journal of Biological Chemistry

    Article Title: ?III Spectrin Regulates the Structural Integrity and the Secretory Protein Transport of the Golgi Complex *

    doi: 10.1074/jbc.M112.406462

    Figure Lengend Snippet: Depletion of βIII spectrin impairs the anterograde transport of the VSV-G and the post-Golgi secretion of 35 S-labeled proteins. A , representative images of HeLa cells constitutively expressing VSV-G-GFP mutant form ts045 show the viral protein in the ER at 40 °C, or in the Golgi (30 min) or the plasma membrane ( PM ; 90 min) when cells were shifted to 32 °C. Bar , 10 μm. B , representative images of VSV-G-GFP expressing HeLa cells silenced with non-targeting or βIII spectrin siRNAs after 15 and 60 min after being shifted to 32 °C. C , quantitative analysis of results partially shown in B . The graph shows the percentage of the cells in which VSV-G-GFP is mainly visualized to the ER, to the Golgi, or to the plasma membrane. Data are shown as the mean ± S.E. of three independent experiments (100 cells each). D , biochemical transport assay for VSV-G-GFP using Endo H. HeLa cells constitutively expressing VSV-G-GFP were transfected for 96 h with siRNA control or against βIII spectrin and incubated at 40 °C for the last 24 h. Then, cells were shifted at 32 °C to induce the transport of VSV-G from the ER, lysed at indicated times, and subjected to Endo H treatment. R and S indicate Endo H-resistant and -sensitive forms, respectively. The ratio of the amount of Endo H-resistant form to that of total amount is plotted. Values are represented as the mean ± S.E. of three independent experiments. Statistical significance is shown, according to two-way analysis of variance using Bonferroni's post-tests (*, p ≤ 0.05). E , RPE1 cells were transfected with control or with βIII spectrin siRNA. Ninety-six h after the transfection, cells were pulse labeled with [ 35 S]Met/Cys, incubated at 19 °C for 3 h and then shifted to 37 °C. At the indicated times, proteins in the culture supernatants and the cell lysates were precipitated and quantified by scintillation counting. As additional controls, the secretion assay was performed at 4 °C or in cells treated with BFA. Results are the mean ± S.E. from at least three independent experiments. Statistical significance according to two-way analysis of variance using Bonferroni's post-tests (**, p ≤ 0.01 and ***, p ≤ 0.001).

    Article Snippet: Experiments to evaluate the effects of transfection were performed at least 16 h later. siRNAs for human βIII spectrin were purchased from Dharmacon (ON-TARGET plus siRNAs; Ref. L-012770-01-0005). siRNAs were employed in two cycles of siRNA treatment (20 n m each) with a delay between them of 24 h, and for a total of 96 h using Hiperfect® as transfection agent (Qiagen, Hilden, Germany).

    Techniques: Labeling, Expressing, Mutagenesis, Transport Assay, Transfection, Incubation

    Role of sesn2 in apoptosis and mitochondrial dysfunction in podocytes under HG conditions through AMPK. Cells were divided into 6 groups: i) the control (CTL), where cells were cultured in 5 mM glucose for 24 h; ii) scramble, where cells were transfected with scramble siRNA and HiPerFect transfection reagent under normal conditions for 24 h; iii) siRNA sesn2, where cells were transfected with 6 nM sesn2 siRNA and HiPerFect transfection reagent under normal conditions for 24 h; iv) HG, where cells were cultured in 35 mM glucose for 24 h; v) HG + pcDNA3.1, where cells were transfected with negative control with pcDNA3.1 and the X-tremeGENETM Transfection Reagent under the 35 mM glucose condition for 24 h; vi) HG + pcDNA3.1 sesn2, cells were transfected with sesn2 plasmid and the X-tremeGENETM Transfection Reagent under the 35 mM glucose condition for 24 h. (A and B) Western blots to measure the protein levels of sesn2, p-AMPK and AMPK in cultured podocytes transfected with siRNA sesn2, siRNA-scrambles, pcDNA3.1-sesn2 or pcDNA3.1 ( * P

    Journal: International Journal of Molecular Medicine

    Article Title: Sestrin-2 regulates podocyte mitochondrial dysfunction and apoptosis under high-glucose conditions via AMPK

    doi: 10.3892/ijmm.2020.4508

    Figure Lengend Snippet: Role of sesn2 in apoptosis and mitochondrial dysfunction in podocytes under HG conditions through AMPK. Cells were divided into 6 groups: i) the control (CTL), where cells were cultured in 5 mM glucose for 24 h; ii) scramble, where cells were transfected with scramble siRNA and HiPerFect transfection reagent under normal conditions for 24 h; iii) siRNA sesn2, where cells were transfected with 6 nM sesn2 siRNA and HiPerFect transfection reagent under normal conditions for 24 h; iv) HG, where cells were cultured in 35 mM glucose for 24 h; v) HG + pcDNA3.1, where cells were transfected with negative control with pcDNA3.1 and the X-tremeGENETM Transfection Reagent under the 35 mM glucose condition for 24 h; vi) HG + pcDNA3.1 sesn2, cells were transfected with sesn2 plasmid and the X-tremeGENETM Transfection Reagent under the 35 mM glucose condition for 24 h. (A and B) Western blots to measure the protein levels of sesn2, p-AMPK and AMPK in cultured podocytes transfected with siRNA sesn2, siRNA-scrambles, pcDNA3.1-sesn2 or pcDNA3.1 ( * P

    Article Snippet: Transfection sesn2-siRNA was purchased from Shanghai GenePharma Co., Ltd., and transfection was performed using the HiPerFect Transfection Reagent according to the manufacturer’s protocol (Qiagen GmbH).

    Techniques: Cell Culture, Transfection, Negative Control, Plasmid Preparation, Western Blot

    RCAD/Ufl1 and its binding partner C53/LZAP were involved in ufmylation of endogenous Ufm1 targets. A. The endogenous Ufm1 conjugates. HCT116 cells were transiently transfected with siRNAs, and the cell lysates were collected two days after transfection and subject to WB using Ufm1 antibody. Specific Ufm1 conjugates were marked by arrowheads, and Ufm1 knockdown efficiency was evaluated by Ufm1 immunoblotting. A “70 kD” non-specific band marked by “*”. B. Ufmylation of endogenous targets was reduced by shRNA-mediated knockdown of Ufm1, Uba5 and Ufc1. HCT116 cells were infected with lentiviral vectors expressing specific Ufm1, Uba5 and Ufc1 shRNAs. The cells were selected with puromycin and the cell lysates were collected after 4-day incubation. Knockdown of specific genes were confirmed by immunoblotting of specific antibodies, respectively. The Ufm1 conjugates were marked by arrowheads. C. RCAD/Ufl1 and its binding partner C53/LZAP were involved in ufmylation of endogenous Ufm1 targets. HCT116 cells were infected with specific lentiviral shRNAs as indicated, and knockdown of corresponding genes was confirmed by immunoblotting. Scramble shRNA was used as the negative control. After 4-day selection and incubation, the cells were treated with cycloheximide (CHX, 10 µg/ml) for 6 hours, and the cell lysates were collected and subject to immunoblotting.

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of the Ufm1 Conjugation System in Response to Disturbance of the Endoplasmic Reticulum Homeostasis and Inhibition of Vesicle Trafficking

    doi: 10.1371/journal.pone.0048587

    Figure Lengend Snippet: RCAD/Ufl1 and its binding partner C53/LZAP were involved in ufmylation of endogenous Ufm1 targets. A. The endogenous Ufm1 conjugates. HCT116 cells were transiently transfected with siRNAs, and the cell lysates were collected two days after transfection and subject to WB using Ufm1 antibody. Specific Ufm1 conjugates were marked by arrowheads, and Ufm1 knockdown efficiency was evaluated by Ufm1 immunoblotting. A “70 kD” non-specific band marked by “*”. B. Ufmylation of endogenous targets was reduced by shRNA-mediated knockdown of Ufm1, Uba5 and Ufc1. HCT116 cells were infected with lentiviral vectors expressing specific Ufm1, Uba5 and Ufc1 shRNAs. The cells were selected with puromycin and the cell lysates were collected after 4-day incubation. Knockdown of specific genes were confirmed by immunoblotting of specific antibodies, respectively. The Ufm1 conjugates were marked by arrowheads. C. RCAD/Ufl1 and its binding partner C53/LZAP were involved in ufmylation of endogenous Ufm1 targets. HCT116 cells were infected with specific lentiviral shRNAs as indicated, and knockdown of corresponding genes was confirmed by immunoblotting. Scramble shRNA was used as the negative control. After 4-day selection and incubation, the cells were treated with cycloheximide (CHX, 10 µg/ml) for 6 hours, and the cell lysates were collected and subject to immunoblotting.

    Article Snippet: Reverse transfections were performed using Hiperfect (Qiagen) following the manufacturer's instruction with minor modification.

    Techniques: Binding Assay, Transfection, Western Blot, shRNA, Infection, Expressing, Incubation, Negative Control, Selection

    3D NEP Design Delivers High Dose Plasmid DNA at Single Cell Resolution with Lower Biological Variability (A) Photolithography-based adaptation of Transwell insert into a 3D NEP platform. (B) SEM of the modified insert surface showing a patterned microwell array (left, scale bar = 50 µm) over the nanochanneled membrane. Only exposed nanochannels (right, scale bar = 1 µm) can NEP-transfect the cells loaded on the apical side of the membrane. (C) Schematic diagram of how the transfection is conducted using the modified Transwell inserts. (D) Circuit diagram for the in-parallel array of nanochannels illustrating the relationship between the resistance across the membrane and the number of actively nanoporating channels. (E) Schematic illustration of a cell sitting directly on top of a nanoporating channel. (F, G) Finite element modeling of the electric field distribution during nanoporation. The electric field is maximized within the nanochannel which leads to enhanced and highly localized transmembrane potentials on the cells. (H–J) Bicistronic constructs for Ascl1 , Brn2 , and Myt1l (color coded green, red, and blue) were introduced into MEFs by NEP. Cells were allowed to recover for 24 hours as indicated on the schematic, and epifluorescent photomicrographs were captured. NEP cells are compared to cells that underwent BEP in parallel experiments. Arrowheads demarcate the same cell in the different panels. Mean fluorescent intensity was obtained from ImageJ and plotted in the respective channels. In addition to showing a higher cellular mean fluorescent intensity, the NEP’ed cells showed a much smaller interquartile range in fluorescent intensities, indicating a more uniform delivery across cells that underwent the NEP. Asterisk indicates statistical significance ( p

    Journal: Nanomedicine : nanotechnology, biology, and medicine

    Article Title: Deterministic Transfection Drives Efficient Nonviral Reprogramming and Uncovers Reprogramming Barriers

    doi: 10.1016/j.nano.2015.11.015

    Figure Lengend Snippet: 3D NEP Design Delivers High Dose Plasmid DNA at Single Cell Resolution with Lower Biological Variability (A) Photolithography-based adaptation of Transwell insert into a 3D NEP platform. (B) SEM of the modified insert surface showing a patterned microwell array (left, scale bar = 50 µm) over the nanochanneled membrane. Only exposed nanochannels (right, scale bar = 1 µm) can NEP-transfect the cells loaded on the apical side of the membrane. (C) Schematic diagram of how the transfection is conducted using the modified Transwell inserts. (D) Circuit diagram for the in-parallel array of nanochannels illustrating the relationship between the resistance across the membrane and the number of actively nanoporating channels. (E) Schematic illustration of a cell sitting directly on top of a nanoporating channel. (F, G) Finite element modeling of the electric field distribution during nanoporation. The electric field is maximized within the nanochannel which leads to enhanced and highly localized transmembrane potentials on the cells. (H–J) Bicistronic constructs for Ascl1 , Brn2 , and Myt1l (color coded green, red, and blue) were introduced into MEFs by NEP. Cells were allowed to recover for 24 hours as indicated on the schematic, and epifluorescent photomicrographs were captured. NEP cells are compared to cells that underwent BEP in parallel experiments. Arrowheads demarcate the same cell in the different panels. Mean fluorescent intensity was obtained from ImageJ and plotted in the respective channels. In addition to showing a higher cellular mean fluorescent intensity, the NEP’ed cells showed a much smaller interquartile range in fluorescent intensities, indicating a more uniform delivery across cells that underwent the NEP. Asterisk indicates statistical significance ( p

    Article Snippet: A Neon (Life Technologies) transfection system was used for BEP transfection.

    Techniques: Plasmid Preparation, Modification, Transfection, Construct