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  • 99
    Thermo Fisher sirna transfection sirna transfection
    Knockdown of p73 with <t>siRNA</t> restores PARP cleavage . Western blot analysis after <t>transfection</t> with p73 siRNA in DB-1 cell lines A) at basal level and B) after TMZ treatment and C) Quantification of p73 to GAPDH and Cleaved PARP relative to uncleaved PARP. D) Transient transfection of tyrosinase DNA in DB-1 cell lines and western blot analysis of p53 and 73 expression.
    Sirna Transfection Sirna Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sirna transfection
    Knock-down of BC induced G1/S phase arrest and thus inhibited cell proliferation in NIH/3T3 cells. ( A ) Representative fluorescent images of EdU incorporation assay. Blue nuclei represent total cells visualized by UV excitation, while red nuclei represent EdU-positive cells visualized by green light excitation. ( B ) Quantitative analysis of EdU-positive cells (shown in A). A total of > 4000 cells were counted for each group. Data were mean ± s.d. of three independent experiments. ( C ) Flow cytometric analysis of cell cycle in NIH/3T3 cells transfected with nonsense or BC siRNAs at 48 h after <t>siRNA</t> <t>transfection.</t> 2n = cells in G0/G1 phase, and 4n = cells in G2/M phase. ( D ) Quantitative analysis of cell cycle phase distribution (shown in C). Data were mean ± s.d. of at least three independent experiments performed in triplicate. Values shown on top of bars are P values vs nonsense.
    Sirna Transfection, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1464 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    SABiosciences shrna transfection
    PARP3 mRNA expression and protein levels in Saos-2 cells after <t>transfection.</t> (A) Analysis of PARP3 expression levels by qRT-PCR, after <t>shRNA</t> transfection (data are the average of triplicate experiments, media ± standard error). (B) Western-blot assay for testing PARP3 protein levels in Saos-2 cell line (bars are the average of three experiments, media ± standard error).
    Shrna Transfection, supplied by SABiosciences, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Horizon Discovery sirna transfection
    Knockdown of Jag1 and Rbpj in cultured granulosa cells results in the retention of proliferative potential. Apoptosis and proliferation assays were done 3 days following <t>siRNA</t> <t>transfection,</t> with a total culture period of 7 days. (A, B) As measured by Annexin V staining and PI exclusion assay, Jag1 or Rbpj knockdown did not result in increased cell death. Instead, Jag1 and Rbpj knockdown led to decreased cellular apoptosis. (C, D) An increased rate of cell proliferation was observed following Jag1 and Rbpj knockdown. n = 3; * P
    Sirna Transfection, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 94/100, based on 1568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bio-Rad sirna transfection reagent transfection
    Knockdown of Jag1 and Rbpj in cultured granulosa cells results in the retention of proliferative potential. Apoptosis and proliferation assays were done 3 days following <t>siRNA</t> <t>transfection,</t> with a total culture period of 7 days. (A, B) As measured by Annexin V staining and PI exclusion assay, Jag1 or Rbpj knockdown did not result in increased cell death. Instead, Jag1 and Rbpj knockdown led to decreased cellular apoptosis. (C, D) An increased rate of cell proliferation was observed following Jag1 and Rbpj knockdown. n = 3; * P
    Sirna Transfection Reagent Transfection, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Genlantis sirna transfection
    Knockdown of Jag1 and Rbpj in cultured granulosa cells results in the retention of proliferative potential. Apoptosis and proliferation assays were done 3 days following <t>siRNA</t> <t>transfection,</t> with a total culture period of 7 days. (A, B) As measured by Annexin V staining and PI exclusion assay, Jag1 or Rbpj knockdown did not result in increased cell death. Instead, Jag1 and Rbpj knockdown led to decreased cellular apoptosis. (C, D) An increased rate of cell proliferation was observed following Jag1 and Rbpj knockdown. n = 3; * P
    Sirna Transfection, supplied by Genlantis, used in various techniques. Bioz Stars score: 92/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen sirna transfection sirna transfection
    Knockdown of Jag1 and Rbpj in cultured granulosa cells results in the retention of proliferative potential. Apoptosis and proliferation assays were done 3 days following <t>siRNA</t> <t>transfection,</t> with a total culture period of 7 days. (A, B) As measured by Annexin V staining and PI exclusion assay, Jag1 or Rbpj knockdown did not result in increased cell death. Instead, Jag1 and Rbpj knockdown led to decreased cellular apoptosis. (C, D) An increased rate of cell proliferation was observed following Jag1 and Rbpj knockdown. n = 3; * P
    Sirna Transfection Sirna Transfection, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Polypus Transfection sirna transfections
    DLC2 is required for junction maintenance and mitotic spindle stability. ( a ) Control and DLC2-depleted HCE cells stained for the adherens junction proteins E-cadherin (red) and β-catenin (green) and DNA (blue). ( b ) Immunoblot for DLC2 of control and DLC2 <t>siRNA-transfected</t> HCE cells; α-tubulin was used as a loading control. ( c ) Control and DLC2-depleted HCE cells stained for α-tubulin (green), α-catenin (red) and DNA (blue). ( d ) Quantification of cells with bipolar spindles with aligned and misaligned metaphase plates after <t>transfection</t> of control and DLC2 siRNAs (shown are means±1 s.d.; ** P
    Sirna Transfections, supplied by Polypus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology sirna transfection
    Depletion of V-ATPase subunit D1 inhibits HPV16 infectivity. (a) HeLa cells were transfected for 48 h with different siRNAs targeting the V-ATPase subunit D1 or a control <t>siRNA</t> and subsequently infected for 24 h with HPV16 pseudovirions. Depicted are the means ± SD relative to the control siRNA ( n = three independent experiments). (b) At 48 h after siRNA <t>transfection,</t> HeLa cells were analyzed by Western blotting for the levels of V-ATPase D1 and, as a loading control, tubulin.
    Sirna Transfection, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sirna
    Down-regulation of <t>CRLR</t> expression in HO8910 cells inhibited influence of exogenous AM on cell migration . Reduced CRLR mRNA expression (A) and protein expression (B) were determined by real-time PCR analysis or western blot in CRLR <t>siRNA</t> transfected cells, compared with scrambled siRNA transfected cells. After cells were transfected by CRLR siRNA, the effect of AM on cells migration was decreased consequently (C).
    Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 93491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology sirna transfection reagent
    Effect of HMGI-C <t>siRNA</t> on the MDA-MB-468 cells. 48 h after <t>transfection</t> with HMGI-C siRNA (40, 60, 80 pmol), cytotoxicity of treatments was determined by MTT assay. The results were expressed as mean ± SD (n = 3); **P
    Sirna Transfection Reagent, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1761 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polyplus Transfection sirna transfection
    Low-dose pemetrexed plus cisplatin synergistically up-regulates NOXA expression and down-regulates Mcl-1 expression in choroidal melanoma cells. (A) To analyze the synergistic effect of the co-treatment, the indicated cells were treated with the indicated concentrations of the chemotherapeutic drugs for 24 hours and then harvested for western blotting. NOXA and Mcl-1 expression was quantified using Image J software and analyzed with GraphPad Prism 5.0 software. OCM1 and M619 cells were seeded in 6-well plates and transfected with control or NOXA <t>siRNA</t> on the second day. (B, C, D)At 48 hours after <t>transfection,</t> the cells were treated with 1.25 μmol/L pemetrexed combined with 5 μmol/L cisplatin for another 24 hours and then harvested for western blotting and apoptosis analysis. CF: cleaved form. All data are presented as the mean ± S.D.
    Sirna Transfection, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare sirna transfection
    Embryo like cells decreased after CCAAT/enhancer binding protein-α (C/EBPα) was downregulation. (A) The C/EBPα downregulation in endometrial polyp stem cells after <t>siRNA</t> <t>transfection</t> (p
    Sirna Transfection, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene sirna transfection
    Embryo like cells decreased after CCAAT/enhancer binding protein-α (C/EBPα) was downregulation. (A) The C/EBPα downregulation in endometrial polyp stem cells after <t>siRNA</t> <t>transfection</t> (p
    Sirna Transfection, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amaxa sirna transfection
    PDZ-RhoGEF/LARG is associated with the increase of ABCA1 protein levels. A , knockdown of RhoGEF expression by <t>siRNA</t> suppressed the apoA-I-mediated ABCA1 stabilization in primary human fibroblast. The cells were transfected with either a nontargeting pool of siRNA duplexes (negative control), siRNA duplex targeting PDZ-RhoGEF, or LARG, or a mixture of siRNA duplexes targeting both PDZ-RhoGEF and LARG. After 48 h <t>transfection,</t> the cells were incubated with apoA-I for 1 h. The level of ABCA1 protein was quantified using a LAS-3000 imager. B , PDZ-RhoGEF expression increased ABCA1 protein level in cells expressing wild-type ( WT ) ABCA1 but in cells expressing the ABCA1-Δ4 mutant, which disrupts the ABCA1 PDZ-binding motif. 293 cells were transfected with wild-type or Δ4 mutant of ABCA1 cDNA either alone or together with PDZ-RhoGEF cDNA. The results are representatives of two or more experiments.
    Sirna Transfection, supplied by Amaxa, used in various techniques. Bioz Stars score: 93/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins sirna transfection sirna
    PDZ-RhoGEF/LARG is associated with the increase of ABCA1 protein levels. A , knockdown of RhoGEF expression by <t>siRNA</t> suppressed the apoA-I-mediated ABCA1 stabilization in primary human fibroblast. The cells were transfected with either a nontargeting pool of siRNA duplexes (negative control), siRNA duplex targeting PDZ-RhoGEF, or LARG, or a mixture of siRNA duplexes targeting both PDZ-RhoGEF and LARG. After 48 h <t>transfection,</t> the cells were incubated with apoA-I for 1 h. The level of ABCA1 protein was quantified using a LAS-3000 imager. B , PDZ-RhoGEF expression increased ABCA1 protein level in cells expressing wild-type ( WT ) ABCA1 but in cells expressing the ABCA1-Δ4 mutant, which disrupts the ABCA1 PDZ-binding motif. 293 cells were transfected with wild-type or Δ4 mutant of ABCA1 cDNA either alone or together with PDZ-RhoGEF cDNA. The results are representatives of two or more experiments.
    Sirna Transfection Sirna, supplied by Eurofins, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Knockdown of p73 with siRNA restores PARP cleavage . Western blot analysis after transfection with p73 siRNA in DB-1 cell lines A) at basal level and B) after TMZ treatment and C) Quantification of p73 to GAPDH and Cleaved PARP relative to uncleaved PARP. D) Transient transfection of tyrosinase DNA in DB-1 cell lines and western blot analysis of p53 and 73 expression.

    Journal: BMC Cancer

    Article Title: Quercetin abrogates chemoresistance in melanoma cells by modulating ?Np73

    doi: 10.1186/1471-2407-10-282

    Figure Lengend Snippet: Knockdown of p73 with siRNA restores PARP cleavage . Western blot analysis after transfection with p73 siRNA in DB-1 cell lines A) at basal level and B) after TMZ treatment and C) Quantification of p73 to GAPDH and Cleaved PARP relative to uncleaved PARP. D) Transient transfection of tyrosinase DNA in DB-1 cell lines and western blot analysis of p53 and 73 expression.

    Article Snippet: siRNA transfection siRNA transfection was carried out according to manufacturer's protocol (Invitrogen, CA, USA).

    Techniques: Western Blot, Transfection, Expressing

    Cell-cycle analysis after DHODH depletion ( A , B ) At 72 h after DHODH siRNA transfection, cells were fixed and stained with propidium iodide. Then, the DNA content was measured by flow cytometry. Three separate experiments were carried out, all of which exhibited similar trends. The results of one representative experiment are shown. The cell-cycle phase distribution in each condition is shown as the percentage of cells present in the G 1 -, S- or G 2 /M-phases.

    Journal: Bioscience Reports

    Article Title: Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction

    doi: 10.1042/BSR20120097

    Figure Lengend Snippet: Cell-cycle analysis after DHODH depletion ( A , B ) At 72 h after DHODH siRNA transfection, cells were fixed and stained with propidium iodide. Then, the DNA content was measured by flow cytometry. Three separate experiments were carried out, all of which exhibited similar trends. The results of one representative experiment are shown. The cell-cycle phase distribution in each condition is shown as the percentage of cells present in the G 1 -, S- or G 2 /M-phases.

    Article Snippet: Knockdown using siRNAs The following 25-bp double-stranded DHODH siRNAs were generated by Stealth Select RNAi (RNA interference) (Invitrogen): 5′-AUU UAU GGC CCA GAA CUC UCA CUU C-3′ and 5′-GAA GUG AGA GUU CUG GGC CAU AAA U-3′ as DHODH siRNA-1; 5′-AUA CCU GUU AAU GAC AGC UUG GUC C-3′ and 5′-GGA CCA AGC UGU CAU UAA CAG GUA U-3′ as DHODH siRNA-2. siRNA transfections were performed according to the manufacturer's instructions (Invitrogen).

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

    Effects of siRNA-mediated knockdown of DHODH ( A ) HeLa cells were transfected with control siRNA or two independent DHODH siRNAs, #1 and #2, using Oligofectamine™. At 3 days after the transfection, the cells were lysed and immunoblotted with antibodies against DHODH, NDUFA9 (complex I), SDHA (complex II), complex III subunit core I, complex V, p53 and β-actin. ( B ) Depletion of DHODH causes growth retardation. The proliferation rate of siRNA-transfected HeLa cells is shown. HeLa cells were transfected with control or two independent DHODH siRNAs on day 0. At the indicated time, the cells were harvested and counted using a cell counter. Left-hand panel, lines represent control siRNA, DHODH siRNA #1 and DHODH siRNA #2 respectively. Uridine (1 mM) was added on day 0 and day 2 to DHODH-depleted HeLa cells. The cell number was counted at the indicated time after seeding. Right-panel panel, proliferation rate of LFN-treated HeLa cells. HeLa cells were treated with 1 mM LFN on day 0. Uridine was added on day 0 and day 2.

    Journal: Bioscience Reports

    Article Title: Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction

    doi: 10.1042/BSR20120097

    Figure Lengend Snippet: Effects of siRNA-mediated knockdown of DHODH ( A ) HeLa cells were transfected with control siRNA or two independent DHODH siRNAs, #1 and #2, using Oligofectamine™. At 3 days after the transfection, the cells were lysed and immunoblotted with antibodies against DHODH, NDUFA9 (complex I), SDHA (complex II), complex III subunit core I, complex V, p53 and β-actin. ( B ) Depletion of DHODH causes growth retardation. The proliferation rate of siRNA-transfected HeLa cells is shown. HeLa cells were transfected with control or two independent DHODH siRNAs on day 0. At the indicated time, the cells were harvested and counted using a cell counter. Left-hand panel, lines represent control siRNA, DHODH siRNA #1 and DHODH siRNA #2 respectively. Uridine (1 mM) was added on day 0 and day 2 to DHODH-depleted HeLa cells. The cell number was counted at the indicated time after seeding. Right-panel panel, proliferation rate of LFN-treated HeLa cells. HeLa cells were treated with 1 mM LFN on day 0. Uridine was added on day 0 and day 2.

    Article Snippet: Knockdown using siRNAs The following 25-bp double-stranded DHODH siRNAs were generated by Stealth Select RNAi (RNA interference) (Invitrogen): 5′-AUU UAU GGC CCA GAA CUC UCA CUU C-3′ and 5′-GAA GUG AGA GUU CUG GGC CAU AAA U-3′ as DHODH siRNA-1; 5′-AUA CCU GUU AAU GAC AGC UUG GUC C-3′ and 5′-GGA CCA AGC UGU CAU UAA CAG GUA U-3′ as DHODH siRNA-2. siRNA transfections were performed according to the manufacturer's instructions (Invitrogen).

    Techniques: Transfection

    Knockdown S6 by siRNA transfection reduces the expression of GLS and activity of GDH. HEY cells were transfected with RPS6 siRNA for 24 h. Western blotting showed the expression of phosphorylation of S6 was inhibited after siRNA transfection (A). HEY cells were treated with 2.0 mM glutamine for 24 h after siRNA transfection. Both of S6 siRNA and rapamycin reduced the expression levels of GLS and phosphorylation of S6 (B). S6 siRNA transfection decreased GDH activity induced by glutamine (C). HEY cells were treated with 2.0 mM glutamine for 48 h after siRNA transfection. Cell proliferation was determined by MTT assay (D). * P

    Journal: Endocrine-Related Cancer

    Article Title: Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway

    doi: 10.1530/ERC-15-0192

    Figure Lengend Snippet: Knockdown S6 by siRNA transfection reduces the expression of GLS and activity of GDH. HEY cells were transfected with RPS6 siRNA for 24 h. Western blotting showed the expression of phosphorylation of S6 was inhibited after siRNA transfection (A). HEY cells were treated with 2.0 mM glutamine for 24 h after siRNA transfection. Both of S6 siRNA and rapamycin reduced the expression levels of GLS and phosphorylation of S6 (B). S6 siRNA transfection decreased GDH activity induced by glutamine (C). HEY cells were treated with 2.0 mM glutamine for 48 h after siRNA transfection. Cell proliferation was determined by MTT assay (D). * P

    Article Snippet: siRNA transfection Transfection of siRNA was performed using Ambion RPS6 siRNA and Qiagen HiperFect Transfection Reagent.

    Techniques: Transfection, Expressing, Activity Assay, Western Blot, MTT Assay

    RhoA and acto-myosin regulate the activity of hSREBP1 and dSREBP. a Screening of low density lipoprotein receptor promoter-luciferase activity in MDA-MB-231 cells transfected with constructs expressing firefly (LDLR-Luc) and renilla (Rluc) luciferase and either control siRNA (siCTL) or siRNAs targeting genes encoding geranylgeranyl pyrophosphate transferase I (GGTase1) protein substrates. b Western blot analysis of MDA-MB-231 and Mahlavu cells 48 h after transfection with siCTL or RhoA targeting siRNAs (siR#1 and siR#2). Hsp90 was used as loading control. mSREBP indicates mature protein. c Western blot analyses of immunoprecipitated (IP) RhoA in MCF-10A cells treated DMSO (as control), 1 μM cerivastatin (STAT), 1 μM cerivastatin and 20 μM GGPP (STAT+GGPP), 5 μM GGTI-298 or 5 μM FTI-277. IgGs were used as IP control. d LDLR-Luc assay in MCF-10A cells 12 h after transfection with pcDNA3-GFP control plasmid, pcDNA3-GFP-RhoA G14V construct with a 6 h DMSO treatment, pcDNA3-GFP-RhoA G14V construct with a 6 h Latrunculin A (G14V + Lat.A) treatment, or pcDNA3-GFP-RhoA T19N construct. e LDLR-Luc assay in MCF-10A cells treated with either DMSO as control or C3 for 24 h. f LDLR-Luc assay in MCF-10A cells treated with either DMSO as control, Y-27632, or Blebbistatin (Blebbist.) for 24 h. g BODIPY 493/503 staining of lipid droplets (in red) in Mahlavu cells treated with either DMSO, C3 or Y-27632 for 24 h. Scale bar, 15 μm. h , i BODIPY 493/503 staining of lipid droplets (in red) and immunofluorescence analysis of dSREBP (in green) and phosphorylated myosin light chain (pMLC2, in magenta) in Drosophila larval fat body from h flies expressing either Luciferase or dRhoA RNAi , or dRhoA RNAi and treated with fatostatin (FTS) and i wild-type flies treated with either DMSO or Y-27632. Scale bar, 20 μm. Graphs bars in ( c – f ) represent mean ± s.d. of n = 3 biological replicates. Values in ( d – f ) are expressed as Relative Luminometer Units (RLU). Nuclei in ( h – i ) were stained with HOECHST (in blue). Blots and images are representative of n = 3 biological replicates. P value: * P

    Journal: Nature Communications

    Article Title: Sterol regulatory element binding protein 1 couples mechanical cues and lipid metabolism

    doi: 10.1038/s41467-019-09152-7

    Figure Lengend Snippet: RhoA and acto-myosin regulate the activity of hSREBP1 and dSREBP. a Screening of low density lipoprotein receptor promoter-luciferase activity in MDA-MB-231 cells transfected with constructs expressing firefly (LDLR-Luc) and renilla (Rluc) luciferase and either control siRNA (siCTL) or siRNAs targeting genes encoding geranylgeranyl pyrophosphate transferase I (GGTase1) protein substrates. b Western blot analysis of MDA-MB-231 and Mahlavu cells 48 h after transfection with siCTL or RhoA targeting siRNAs (siR#1 and siR#2). Hsp90 was used as loading control. mSREBP indicates mature protein. c Western blot analyses of immunoprecipitated (IP) RhoA in MCF-10A cells treated DMSO (as control), 1 μM cerivastatin (STAT), 1 μM cerivastatin and 20 μM GGPP (STAT+GGPP), 5 μM GGTI-298 or 5 μM FTI-277. IgGs were used as IP control. d LDLR-Luc assay in MCF-10A cells 12 h after transfection with pcDNA3-GFP control plasmid, pcDNA3-GFP-RhoA G14V construct with a 6 h DMSO treatment, pcDNA3-GFP-RhoA G14V construct with a 6 h Latrunculin A (G14V + Lat.A) treatment, or pcDNA3-GFP-RhoA T19N construct. e LDLR-Luc assay in MCF-10A cells treated with either DMSO as control or C3 for 24 h. f LDLR-Luc assay in MCF-10A cells treated with either DMSO as control, Y-27632, or Blebbistatin (Blebbist.) for 24 h. g BODIPY 493/503 staining of lipid droplets (in red) in Mahlavu cells treated with either DMSO, C3 or Y-27632 for 24 h. Scale bar, 15 μm. h , i BODIPY 493/503 staining of lipid droplets (in red) and immunofluorescence analysis of dSREBP (in green) and phosphorylated myosin light chain (pMLC2, in magenta) in Drosophila larval fat body from h flies expressing either Luciferase or dRhoA RNAi , or dRhoA RNAi and treated with fatostatin (FTS) and i wild-type flies treated with either DMSO or Y-27632. Scale bar, 20 μm. Graphs bars in ( c – f ) represent mean ± s.d. of n = 3 biological replicates. Values in ( d – f ) are expressed as Relative Luminometer Units (RLU). Nuclei in ( h – i ) were stained with HOECHST (in blue). Blots and images are representative of n = 3 biological replicates. P value: * P

    Article Snippet: Transfections siRNA transfections were performed with Lipofectamine RNAi-MAX (Life technologies) in antibiotic-free medium according to manufacturer instructions.

    Techniques: Activity Assay, Luciferase, Multiple Displacement Amplification, Transfection, Construct, Expressing, Western Blot, Immunoprecipitation, Plasmid Preparation, Staining, Immunofluorescence

    AMPK suppresses SREBP1 activation downstream of mechanical inputs. a Western blot analysis of MCF-10A cells 48 h after transfection with either control (siCTL) or RhoA targeting siRNA (siR#1 and siR#2). GAPDH was used as loading control. b Western blot analysis of MCF-10A cells treated with either DMSO, C3 or Y-27632, for 24 h. Hsp90 was used as loading control. c Western blot analysis MCF-10A cells cultured on either stiff (50 kPa elastic modulus) or soft (0.5 kPa elastic modulus) fibronectin-coated hydrogel matrix for 24 h. GAPDH was used as loading control. d Western blot analysis of MCF-10A cells treated with either DMSO, Y-27632 or AICAR, for 24 h. GAPDH was used as loading control. e Western blot analysis of MCF-10A cells cultured on either stiff (50 kPa elastic modulus) or soft (0.5 kPa elastic modulus) fibronectin-coated hydrogel matrix, or soft (0.5 kPa elastic modulus) fibronectin-coated hydrogel matrix with AICAR treatment, for 24 h. Hsp90 was used as loading control. f BODIPY 493/503 staining of lipid droplets (in red) in Mahlavu cells treated with Y-27632 or Y-27632 and AICAR, for 24 h. Nuclei were stained with HOECHST (in blue). Scale bar, 15 μm. For western blots, mSREBP indicates mature protein. Blots and images are representative of n = 3 biological replicates

    Journal: Nature Communications

    Article Title: Sterol regulatory element binding protein 1 couples mechanical cues and lipid metabolism

    doi: 10.1038/s41467-019-09152-7

    Figure Lengend Snippet: AMPK suppresses SREBP1 activation downstream of mechanical inputs. a Western blot analysis of MCF-10A cells 48 h after transfection with either control (siCTL) or RhoA targeting siRNA (siR#1 and siR#2). GAPDH was used as loading control. b Western blot analysis of MCF-10A cells treated with either DMSO, C3 or Y-27632, for 24 h. Hsp90 was used as loading control. c Western blot analysis MCF-10A cells cultured on either stiff (50 kPa elastic modulus) or soft (0.5 kPa elastic modulus) fibronectin-coated hydrogel matrix for 24 h. GAPDH was used as loading control. d Western blot analysis of MCF-10A cells treated with either DMSO, Y-27632 or AICAR, for 24 h. GAPDH was used as loading control. e Western blot analysis of MCF-10A cells cultured on either stiff (50 kPa elastic modulus) or soft (0.5 kPa elastic modulus) fibronectin-coated hydrogel matrix, or soft (0.5 kPa elastic modulus) fibronectin-coated hydrogel matrix with AICAR treatment, for 24 h. Hsp90 was used as loading control. f BODIPY 493/503 staining of lipid droplets (in red) in Mahlavu cells treated with Y-27632 or Y-27632 and AICAR, for 24 h. Nuclei were stained with HOECHST (in blue). Scale bar, 15 μm. For western blots, mSREBP indicates mature protein. Blots and images are representative of n = 3 biological replicates

    Article Snippet: Transfections siRNA transfections were performed with Lipofectamine RNAi-MAX (Life technologies) in antibiotic-free medium according to manufacturer instructions.

    Techniques: Activation Assay, Western Blot, Transfection, Cell Culture, Staining

    Effect of lncRNA H19 expression manipulation on breast cancer cell migration. Notes: ( A and B ) Cell migration after transfection of H19 or H19 siRNA in MCF-7 and MCF-7-R cells (20×, P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA H19 mediated the chemosensitivity of breast cancer cells via Wnt pathway and EMT process

    doi: 10.2147/OTT.S172379

    Figure Lengend Snippet: Effect of lncRNA H19 expression manipulation on breast cancer cell migration. Notes: ( A and B ) Cell migration after transfection of H19 or H19 siRNA in MCF-7 and MCF-7-R cells (20×, P

    Article Snippet: The siRNA transfection reagent (Invitrogen) was used according to the manufacturer’s protocol.

    Techniques: Expressing, Migration, Transfection

    The H19 knockdown deregulated Wnt pathway in tamoxifen-resistant breast cancer cells. Notes: ( A ) H19 siRNA transfection promoted the translocation of β-catenin from nuclear to cytoplasm in tamoxifen-resistant cells. Red staining indicates β-catenin, blue means DAPI, and purple presents the merged color of β-catenin and DAPI. ( B ) Dual-luciferase reporter system and TOP/FOP reporter assays to presents the Wnt pathway transcription activity. ( C ) H19 knockdown decreased β-catenin protein level both in MCF-7-R and SK-BR-3-R cells. ( D ) Quantification of β-catenin protein level after H19 knockdown. ( E – G ) β-catenin protein level response in the presence of H19 knockdown and R-spondin 1. ( H and I ) The response of cell proliferation in the presence of H19 knockdown and R-spondin 1. * P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA H19 mediated the chemosensitivity of breast cancer cells via Wnt pathway and EMT process

    doi: 10.2147/OTT.S172379

    Figure Lengend Snippet: The H19 knockdown deregulated Wnt pathway in tamoxifen-resistant breast cancer cells. Notes: ( A ) H19 siRNA transfection promoted the translocation of β-catenin from nuclear to cytoplasm in tamoxifen-resistant cells. Red staining indicates β-catenin, blue means DAPI, and purple presents the merged color of β-catenin and DAPI. ( B ) Dual-luciferase reporter system and TOP/FOP reporter assays to presents the Wnt pathway transcription activity. ( C ) H19 knockdown decreased β-catenin protein level both in MCF-7-R and SK-BR-3-R cells. ( D ) Quantification of β-catenin protein level after H19 knockdown. ( E – G ) β-catenin protein level response in the presence of H19 knockdown and R-spondin 1. ( H and I ) The response of cell proliferation in the presence of H19 knockdown and R-spondin 1. * P

    Article Snippet: The siRNA transfection reagent (Invitrogen) was used according to the manufacturer’s protocol.

    Techniques: Transfection, Translocation Assay, Staining, Luciferase, Activity Assay

    lncRNA H19 expression manipulates the tamoxifen sensitivity of breast cancer cells. Notes: lncRNA H19 mimic was used to transfect breast cancer cells to increase H19 expression level and H19 siRNA was used to knockdown H19 expression level in breast cancer cell lines. Then, 72 hours after transfection, tamoxifen with dose escalation were added to the media as an anti-tumor agent. After 48 hours co-culture (120 hours post transfection), the proliferation of the cells was determined by CCK8 kit and the apoptotic cells were observed by performing Annexin V and PI double staining. ( A ) Cell survival rate of MCF-7 (* P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA H19 mediated the chemosensitivity of breast cancer cells via Wnt pathway and EMT process

    doi: 10.2147/OTT.S172379

    Figure Lengend Snippet: lncRNA H19 expression manipulates the tamoxifen sensitivity of breast cancer cells. Notes: lncRNA H19 mimic was used to transfect breast cancer cells to increase H19 expression level and H19 siRNA was used to knockdown H19 expression level in breast cancer cell lines. Then, 72 hours after transfection, tamoxifen with dose escalation were added to the media as an anti-tumor agent. After 48 hours co-culture (120 hours post transfection), the proliferation of the cells was determined by CCK8 kit and the apoptotic cells were observed by performing Annexin V and PI double staining. ( A ) Cell survival rate of MCF-7 (* P

    Article Snippet: The siRNA transfection reagent (Invitrogen) was used according to the manufacturer’s protocol.

    Techniques: Expressing, Transfection, Co-Culture Assay, Double Staining

    Knock-down of BC induced G1/S phase arrest and thus inhibited cell proliferation in NIH/3T3 cells. ( A ) Representative fluorescent images of EdU incorporation assay. Blue nuclei represent total cells visualized by UV excitation, while red nuclei represent EdU-positive cells visualized by green light excitation. ( B ) Quantitative analysis of EdU-positive cells (shown in A). A total of > 4000 cells were counted for each group. Data were mean ± s.d. of three independent experiments. ( C ) Flow cytometric analysis of cell cycle in NIH/3T3 cells transfected with nonsense or BC siRNAs at 48 h after siRNA transfection. 2n = cells in G0/G1 phase, and 4n = cells in G2/M phase. ( D ) Quantitative analysis of cell cycle phase distribution (shown in C). Data were mean ± s.d. of at least three independent experiments performed in triplicate. Values shown on top of bars are P values vs nonsense.

    Journal: PLoS ONE

    Article Title: Identification of BC005512 as a DNA Damage Responsive Murine Endogenous Retrovirus of GLN Family Involved in Cell Growth Regulation

    doi: 10.1371/journal.pone.0035010

    Figure Lengend Snippet: Knock-down of BC induced G1/S phase arrest and thus inhibited cell proliferation in NIH/3T3 cells. ( A ) Representative fluorescent images of EdU incorporation assay. Blue nuclei represent total cells visualized by UV excitation, while red nuclei represent EdU-positive cells visualized by green light excitation. ( B ) Quantitative analysis of EdU-positive cells (shown in A). A total of > 4000 cells were counted for each group. Data were mean ± s.d. of three independent experiments. ( C ) Flow cytometric analysis of cell cycle in NIH/3T3 cells transfected with nonsense or BC siRNAs at 48 h after siRNA transfection. 2n = cells in G0/G1 phase, and 4n = cells in G2/M phase. ( D ) Quantitative analysis of cell cycle phase distribution (shown in C). Data were mean ± s.d. of at least three independent experiments performed in triplicate. Values shown on top of bars are P values vs nonsense.

    Article Snippet: Cell cycle analysis At 48 h after siRNA transfection, NIH/3T3 cells were trypsinized, fixed in 70% ethanol, incubated with RNaseA, stained with propidium iodide (Sigma) and analyzed by a FACSCalibur (BD, Franklin Lakes, NJ, USA) instrument.

    Techniques: Flow Cytometry, Transfection

    Down-regulating BC expression suppressed cell growth in several mouse cell lines. ( A, C and E ) Quantitative PCR results showing knock-down efficiency of BC siRNAs in NIH/3T3, Hepa 1–6 or SV40 MES 13 cells at 48 h after siRNA transfection. Data were mean ± s.d. of at least three independent experiments. ( B, D and F ) Cell numbers of NIH/3T3, Hepa 1–6 and SV40 MES 13 cells at indicated times after siRNA transfection. Data were mean ± s.d. of at least three independent experiments performed in triplicate. Values shown on top of bars are P values vs nonsense.

    Journal: PLoS ONE

    Article Title: Identification of BC005512 as a DNA Damage Responsive Murine Endogenous Retrovirus of GLN Family Involved in Cell Growth Regulation

    doi: 10.1371/journal.pone.0035010

    Figure Lengend Snippet: Down-regulating BC expression suppressed cell growth in several mouse cell lines. ( A, C and E ) Quantitative PCR results showing knock-down efficiency of BC siRNAs in NIH/3T3, Hepa 1–6 or SV40 MES 13 cells at 48 h after siRNA transfection. Data were mean ± s.d. of at least three independent experiments. ( B, D and F ) Cell numbers of NIH/3T3, Hepa 1–6 and SV40 MES 13 cells at indicated times after siRNA transfection. Data were mean ± s.d. of at least three independent experiments performed in triplicate. Values shown on top of bars are P values vs nonsense.

    Article Snippet: Cell cycle analysis At 48 h after siRNA transfection, NIH/3T3 cells were trypsinized, fixed in 70% ethanol, incubated with RNaseA, stained with propidium iodide (Sigma) and analyzed by a FACSCalibur (BD, Franklin Lakes, NJ, USA) instrument.

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

    a Transfection efficiency was analyzed using real time PCR after treatment of THP differentiated macrophages with mission siRNA (7 pmol) for 48 h. b Immunoblotting image of cyclophilin A protein expression after treatment with mission siRNA to confirm transfection. β-actin was used as endogenous control. Protein densities of immunoreactive bands measured by the Quantity One 1D analysis software program. Data are presented as mean ± SD (n = 3) and asterisk represents p

    Journal: Cardiovascular Diabetology

    Article Title: Cyclophilin A enhances macrophage differentiation and lipid uptake in high glucose conditions: a cellular mechanism for accelerated macro vascular disease in diabetes mellitus

    doi: 10.1186/s12933-016-0467-5

    Figure Lengend Snippet: a Transfection efficiency was analyzed using real time PCR after treatment of THP differentiated macrophages with mission siRNA (7 pmol) for 48 h. b Immunoblotting image of cyclophilin A protein expression after treatment with mission siRNA to confirm transfection. β-actin was used as endogenous control. Protein densities of immunoreactive bands measured by the Quantity One 1D analysis software program. Data are presented as mean ± SD (n = 3) and asterisk represents p

    Article Snippet: Invitro silencing of cyclophilin A gene THP1 cells (2–4 × 105 ) were transfected with mission siRNA (7 pmol) using, mission siRNA Transfection Reagent (Sigma Aldrich) for 48 h and TMN 355 for 6 h at 37 °C.

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Expressing, Software

    PARP3 mRNA expression and protein levels in Saos-2 cells after transfection. (A) Analysis of PARP3 expression levels by qRT-PCR, after shRNA transfection (data are the average of triplicate experiments, media ± standard error). (B) Western-blot assay for testing PARP3 protein levels in Saos-2 cell line (bars are the average of three experiments, media ± standard error).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Poly (ADP-ribose) polymerase 3 (PARP3), a potential repressor of telomerase activity

    doi: 10.1186/1756-9966-33-19

    Figure Lengend Snippet: PARP3 mRNA expression and protein levels in Saos-2 cells after transfection. (A) Analysis of PARP3 expression levels by qRT-PCR, after shRNA transfection (data are the average of triplicate experiments, media ± standard error). (B) Western-blot assay for testing PARP3 protein levels in Saos-2 cell line (bars are the average of three experiments, media ± standard error).

    Article Snippet: shRNA transfection We used the shRNA technology (SureSilencing™ shRNA Plasmids, SABiosciences, Valencia, California) in Saos-2 cells to generate stable transfectants depleted in PARP3.

    Techniques: Expressing, Transfection, Quantitative RT-PCR, shRNA, Western Blot

    Knockdown of Jag1 and Rbpj in cultured granulosa cells results in the retention of proliferative potential. Apoptosis and proliferation assays were done 3 days following siRNA transfection, with a total culture period of 7 days. (A, B) As measured by Annexin V staining and PI exclusion assay, Jag1 or Rbpj knockdown did not result in increased cell death. Instead, Jag1 and Rbpj knockdown led to decreased cellular apoptosis. (C, D) An increased rate of cell proliferation was observed following Jag1 and Rbpj knockdown. n = 3; * P

    Journal: Endocrinology

    Article Title: Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells

    doi: 10.1210/en.2017-00677

    Figure Lengend Snippet: Knockdown of Jag1 and Rbpj in cultured granulosa cells results in the retention of proliferative potential. Apoptosis and proliferation assays were done 3 days following siRNA transfection, with a total culture period of 7 days. (A, B) As measured by Annexin V staining and PI exclusion assay, Jag1 or Rbpj knockdown did not result in increased cell death. Instead, Jag1 and Rbpj knockdown led to decreased cellular apoptosis. (C, D) An increased rate of cell proliferation was observed following Jag1 and Rbpj knockdown. n = 3; * P

    Article Snippet: Cells were allowed to adhere and to reach 90% confluency for 96 hours prior to small interfering RNA (siRNA) transfection. siGENOME SMARTpool (GE Dharmacon, Lafayette, CO) was used at concentrations of 20 nM ( siJag1 , siScramble ) or 40 nM ( siRbpj , siScramble ). siRNA transfection was done using DharmaFect I reagent (GE Dharmacon) in 4F media supplemented with 10% charcoal-stripped fetal bovine serum.

    Techniques: Cell Culture, Transfection, Staining, Exclusion Assay

    Steroid biosynthesis is suppressed following Jag1 and Rbpj knockdown in cultured granulosa cells. (A) The Notch ligand JAG1 and the canonical Notch signaling transcriptional cofactor RBPJ were efficiently and specifically decreased following siRNA transfection. (B) Expression of factors and enzymes involved in the steroid biosynthetic cascade was downregulated following Jag1 and Rbpj knockdown. The dashed line indicates the level of expression in siScramble samples. (C) Jag1 knockdown results in decreased levels of E 2 and P 4 in culture media. (D) Consistent with the less robust knockdown of Rbpj , only E 2 levels were found to be significantly decreased in siRbpj samples. n = 3; * P

    Journal: Endocrinology

    Article Title: Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells

    doi: 10.1210/en.2017-00677

    Figure Lengend Snippet: Steroid biosynthesis is suppressed following Jag1 and Rbpj knockdown in cultured granulosa cells. (A) The Notch ligand JAG1 and the canonical Notch signaling transcriptional cofactor RBPJ were efficiently and specifically decreased following siRNA transfection. (B) Expression of factors and enzymes involved in the steroid biosynthetic cascade was downregulated following Jag1 and Rbpj knockdown. The dashed line indicates the level of expression in siScramble samples. (C) Jag1 knockdown results in decreased levels of E 2 and P 4 in culture media. (D) Consistent with the less robust knockdown of Rbpj , only E 2 levels were found to be significantly decreased in siRbpj samples. n = 3; * P

    Article Snippet: Cells were allowed to adhere and to reach 90% confluency for 96 hours prior to small interfering RNA (siRNA) transfection. siGENOME SMARTpool (GE Dharmacon, Lafayette, CO) was used at concentrations of 20 nM ( siJag1 , siScramble ) or 40 nM ( siRbpj , siScramble ). siRNA transfection was done using DharmaFect I reagent (GE Dharmacon) in 4F media supplemented with 10% charcoal-stripped fetal bovine serum.

    Techniques: Cell Culture, Transfection, Expressing

    siRNA-mediated depletion of 4E-T affects mRNA turnover. HeLa cells were transfected with siRNA against 4E-T or a control (Ctrl) siRNA (4AIII inverted). Protein and RNA were harvested for use in Western (C and D) and Northern (A, B, and E) analysis, respectively. (A and B) HeLa cells were transfected with siRNA against 4E-T or with control siRNA and later cotransfected with pTet-β-globin or pTet-β-globin c-fos or GM-CSF ARE and pTet Off. Cells were treated with doxycycline (1 μg/ml) ∼72 h after transfection to block transcription. Total RNA was isolated at 0.5, 2, 3, and 4 h for β-globin/GM-CSF ARE assays (A) and 0.5, 1.5, 3, and 6 h for β-globin/ c-fos ARE assays (B) and analyzed by Northern blot. GAPDH served as a loading control. mRNA half-lives were calculated from Northern blots and normalized against GAPDH levels. (C) Protein extracts were collected at 6 h after treatment with doxycycline (1 μg/ml) from cells that were transfected with the reporter plasmid and pTetOff and resolved by SDS-PAGE. Lanes 1 and 4: glo = β-globin + pTetOff; lanes 2 and 5: fos = β-globin/ c-fos ARE + pTetOff; lanes 3 and 6: GM = β-globin/GMCSF ARE + pTetOff. (D) Protein extracts were collected at the latest time point of actinomycin D treatment and resolved by 10% SDS-PAGE. Western blot analysis was done with anti–4E-T and anti–β-actin antibodies. NT = nontransfected. (E) At 48 h after transfection, the half-lives of p21 mRNA were assessed by using actinomycin D (5 μg/ml) for the indicated amount of time. Total RNA (5 μg) was resolved on 1.3% formaldehyde gel and analyzed by Northern blotting. 28S levels and 18S levels served as a loading marker. mRNA half-lives were calculated from Northern blots and normalized against 32 P-labeled 18S levels and plotted on a graph with the zero time point set at 100.

    Journal: The Journal of Cell Biology

    Article Title: A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay

    doi: 10.1083/jcb.200504039

    Figure Lengend Snippet: siRNA-mediated depletion of 4E-T affects mRNA turnover. HeLa cells were transfected with siRNA against 4E-T or a control (Ctrl) siRNA (4AIII inverted). Protein and RNA were harvested for use in Western (C and D) and Northern (A, B, and E) analysis, respectively. (A and B) HeLa cells were transfected with siRNA against 4E-T or with control siRNA and later cotransfected with pTet-β-globin or pTet-β-globin c-fos or GM-CSF ARE and pTet Off. Cells were treated with doxycycline (1 μg/ml) ∼72 h after transfection to block transcription. Total RNA was isolated at 0.5, 2, 3, and 4 h for β-globin/GM-CSF ARE assays (A) and 0.5, 1.5, 3, and 6 h for β-globin/ c-fos ARE assays (B) and analyzed by Northern blot. GAPDH served as a loading control. mRNA half-lives were calculated from Northern blots and normalized against GAPDH levels. (C) Protein extracts were collected at 6 h after treatment with doxycycline (1 μg/ml) from cells that were transfected with the reporter plasmid and pTetOff and resolved by SDS-PAGE. Lanes 1 and 4: glo = β-globin + pTetOff; lanes 2 and 5: fos = β-globin/ c-fos ARE + pTetOff; lanes 3 and 6: GM = β-globin/GMCSF ARE + pTetOff. (D) Protein extracts were collected at the latest time point of actinomycin D treatment and resolved by 10% SDS-PAGE. Western blot analysis was done with anti–4E-T and anti–β-actin antibodies. NT = nontransfected. (E) At 48 h after transfection, the half-lives of p21 mRNA were assessed by using actinomycin D (5 μg/ml) for the indicated amount of time. Total RNA (5 μg) was resolved on 1.3% formaldehyde gel and analyzed by Northern blotting. 28S levels and 18S levels served as a loading marker. mRNA half-lives were calculated from Northern blots and normalized against 32 P-labeled 18S levels and plotted on a graph with the zero time point set at 100.

    Article Snippet: siRNA transfections siRNA annealed duplexes were purchased from Dharmacon and sequences are listed below.

    Techniques: Transfection, Western Blot, Northern Blot, Blocking Assay, Isolation, Plasmid Preparation, SDS Page, Marker, Labeling

    Depletion of 4E-T from HeLa cells results in disappearance of decapping factors from P-bodies. (A) HeLa cells were transfected with siRNA against 4E-T or control siRNAs (4AIII inverted or 4E-T inverted). 48 h after transfection, cells were split into chamber slides for immunofluorescence analysis [see (C)] and into a 6-cm dish for Western blot analysis. 60 h after transfection, protein cell extracts were prepared and 20 μg of extract was resolved by SDS-10% PAGE. Western blotting was performed with rabbit anti–4E-T and anti-eIF4E antibodies, or mouse anti–β-actin monoclonal antibody (Sigma-Aldrich). 4E-T levels were quantified against β-actin, which served as a loading control, and the level of protein in the negative control (4AIII inverted) transfected cells was set as 100. (B) HeLa extract (20 μg) as in (A) was resolved by 10% SDS-PAGE and Western blotting was performed with rabbit anti-Dcp1a and anti- Xenopus p54 (Xp54) antibodies, or monoclonal mouse anti-eIF4E (SK) and anti–β-actin antibodies. (C) HeLa (CCL2) cells were transfected with control siRNA (4AIII inverted) or 4E-T siRNA and indirect immunofluorescence was performed 60 h after transfection with rabbit anti–4E-T and anti-Dcp1a antibodies, or monoclonal mouse anti-eIF4E antibody. (D) HeLa S3 cells were transfected with 4E-T siRNA or control siRNA (4AIII inverted). 24 h after transfection, cells were transfected with myc-EYFP-Me31B; 36 h later, extracts were collected for SDS-PAGE analysis or fixed for immunofluorescence. Cell extracts were resolved by 10% SDS-PAGE and Western blot analysis with rabbit anti–4E-T, or monoclonal anti-myc (9E10) and anti–β-actin was performed. Indirect immunofluorescence was performed to assess the localization of 4E-T, and the localization of myc-EYFP-Me31B was assessed by direct immunofluorescence. The right most panels show the enlarged bordered image.

    Journal: The Journal of Cell Biology

    Article Title: A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay

    doi: 10.1083/jcb.200504039

    Figure Lengend Snippet: Depletion of 4E-T from HeLa cells results in disappearance of decapping factors from P-bodies. (A) HeLa cells were transfected with siRNA against 4E-T or control siRNAs (4AIII inverted or 4E-T inverted). 48 h after transfection, cells were split into chamber slides for immunofluorescence analysis [see (C)] and into a 6-cm dish for Western blot analysis. 60 h after transfection, protein cell extracts were prepared and 20 μg of extract was resolved by SDS-10% PAGE. Western blotting was performed with rabbit anti–4E-T and anti-eIF4E antibodies, or mouse anti–β-actin monoclonal antibody (Sigma-Aldrich). 4E-T levels were quantified against β-actin, which served as a loading control, and the level of protein in the negative control (4AIII inverted) transfected cells was set as 100. (B) HeLa extract (20 μg) as in (A) was resolved by 10% SDS-PAGE and Western blotting was performed with rabbit anti-Dcp1a and anti- Xenopus p54 (Xp54) antibodies, or monoclonal mouse anti-eIF4E (SK) and anti–β-actin antibodies. (C) HeLa (CCL2) cells were transfected with control siRNA (4AIII inverted) or 4E-T siRNA and indirect immunofluorescence was performed 60 h after transfection with rabbit anti–4E-T and anti-Dcp1a antibodies, or monoclonal mouse anti-eIF4E antibody. (D) HeLa S3 cells were transfected with 4E-T siRNA or control siRNA (4AIII inverted). 24 h after transfection, cells were transfected with myc-EYFP-Me31B; 36 h later, extracts were collected for SDS-PAGE analysis or fixed for immunofluorescence. Cell extracts were resolved by 10% SDS-PAGE and Western blot analysis with rabbit anti–4E-T, or monoclonal anti-myc (9E10) and anti–β-actin was performed. Indirect immunofluorescence was performed to assess the localization of 4E-T, and the localization of myc-EYFP-Me31B was assessed by direct immunofluorescence. The right most panels show the enlarged bordered image.

    Article Snippet: siRNA transfections siRNA annealed duplexes were purchased from Dharmacon and sequences are listed below.

    Techniques: Transfection, Immunofluorescence, Western Blot, Polyacrylamide Gel Electrophoresis, Negative Control, SDS Page

    Calpain-2 is required for vesicular trafficking of internalized CVB. ( A ) Top: Representative images of HBMEC and Caco-2 monolayers transfected with control, calpain-1, or calpain-2 siRNAs and infected with CVB (MOI = 1) for 14 hrs (HBMEC) or 7 hrs (Caco-2). VP1 in green and DAPI-stained nuclei in blue. Bottom: Effect of calpain-1 or calpain-2 siRNA transfection on CVB infection of HBMEC (left) or Caco-2 (right) cells. Shown are the percentage of infected cells (normalized to DAPI-stained nuclei) normalized control siRNA-transfected cells ( B ) HBMEC monolayers were treated with the indicated calpain inhibitors and infected with CVB (MOI = 1) for 14 hrs. Inhibitor was added to cultures 1 hr before infection (pre-treat) or 2 hrs p.i. Dashed line indicates the infection level of control cells. ( C ) Calpain activity was measured in HBMEC infected with CVB (50 PFU/cell) for the indicated times. Dashed line indicates calpain activity in control (no virus) cells. ( D ) Immunofluorescence microscopy in HBMEC (top) and Caco-2 (bottom) exposed to CVB (MOI = 50) for 60 min and treated with DMSO (no inhibitor) or calpain inhibitor III. Green staining represents internalized virus. White arrows denote enlarged virus-contained vesicles in calpain inhibitor III-treated cells. ( E ) Immunofluorescence microscopy in HBMEC exposed to CVB (MOI = 50) for 60 min and treated with either control (No Inh) or with calpain inhibitor III. VP1 (green), calpain-2 (red), and DAPI (blue). White arrows denote enlarged virus-containing vesicles in calpain inhibitor III-treated cells that colocalize with calpain-2. ( F ) Immunofluorescence microscopy in HBMEC exposed to CVB (MOI = 50) and Alexa Fluor-488 conjugated cholera toxin B (CTB) for 60 min and treated with either control (No Inh) or with calpain inhibitor III. CTB (green), VP1 (red), and DAPI (blue). White arrows denote enlarged CTB and virus-containing vesicles in calpain inhibitor III-treated cells that colocalize with calpain-2. ( G ) Immunofluorescence microscopy in HBMEC transfected with control, PLCγ-1, or IP 3 R-3 siRNAs and stained for internalized CVB (MOI = 50, in green) and DAPI (in blue) at 60 minutes p.i. White arrows denote enlarge virus-containing vesicles in PLCγ-1 and IP 3 R-3 siRNA treated cells.

    Journal: PLoS Pathogens

    Article Title: Release of Intracellular Calcium Stores Facilitates Coxsackievirus Entry into Polarized Endothelial Cells

    doi: 10.1371/journal.ppat.1001135

    Figure Lengend Snippet: Calpain-2 is required for vesicular trafficking of internalized CVB. ( A ) Top: Representative images of HBMEC and Caco-2 monolayers transfected with control, calpain-1, or calpain-2 siRNAs and infected with CVB (MOI = 1) for 14 hrs (HBMEC) or 7 hrs (Caco-2). VP1 in green and DAPI-stained nuclei in blue. Bottom: Effect of calpain-1 or calpain-2 siRNA transfection on CVB infection of HBMEC (left) or Caco-2 (right) cells. Shown are the percentage of infected cells (normalized to DAPI-stained nuclei) normalized control siRNA-transfected cells ( B ) HBMEC monolayers were treated with the indicated calpain inhibitors and infected with CVB (MOI = 1) for 14 hrs. Inhibitor was added to cultures 1 hr before infection (pre-treat) or 2 hrs p.i. Dashed line indicates the infection level of control cells. ( C ) Calpain activity was measured in HBMEC infected with CVB (50 PFU/cell) for the indicated times. Dashed line indicates calpain activity in control (no virus) cells. ( D ) Immunofluorescence microscopy in HBMEC (top) and Caco-2 (bottom) exposed to CVB (MOI = 50) for 60 min and treated with DMSO (no inhibitor) or calpain inhibitor III. Green staining represents internalized virus. White arrows denote enlarged virus-contained vesicles in calpain inhibitor III-treated cells. ( E ) Immunofluorescence microscopy in HBMEC exposed to CVB (MOI = 50) for 60 min and treated with either control (No Inh) or with calpain inhibitor III. VP1 (green), calpain-2 (red), and DAPI (blue). White arrows denote enlarged virus-containing vesicles in calpain inhibitor III-treated cells that colocalize with calpain-2. ( F ) Immunofluorescence microscopy in HBMEC exposed to CVB (MOI = 50) and Alexa Fluor-488 conjugated cholera toxin B (CTB) for 60 min and treated with either control (No Inh) or with calpain inhibitor III. CTB (green), VP1 (red), and DAPI (blue). White arrows denote enlarged CTB and virus-containing vesicles in calpain inhibitor III-treated cells that colocalize with calpain-2. ( G ) Immunofluorescence microscopy in HBMEC transfected with control, PLCγ-1, or IP 3 R-3 siRNAs and stained for internalized CVB (MOI = 50, in green) and DAPI (in blue) at 60 minutes p.i. White arrows denote enlarge virus-containing vesicles in PLCγ-1 and IP 3 R-3 siRNA treated cells.

    Article Snippet: siRNA transfections siRNAs were purchased from Dharmacon.

    Techniques: Transfection, Infection, Staining, Activity Assay, Immunofluorescence, Microscopy, CtB Assay

    PLCγ and IP 3 R-3 are involved in CVB-induced depletion of Ca i 2+ stores. ( A ) HBMEC (left) or Caco-2 (right) cells treated with 2-APB or U73122 1 hour prior to infection (pre-treat) or 2 hours p.i. (post-treat) were infected with CVB (MOI = 1) for 14 hrs (HBMEC) or 7 hrs (Caco-2). Graph represents percentage of total cells expressing VP1 normalized to no-inhibitor controls (dashed line). ( B ) Top: Still images of HBMEC pre-treated with U73122, loaded with Fura-2 AM, and exposed to CVB (MOI = 50, t = 55 seconds). Images were pseudo colored for Ca i 2+ visualization. Bottom: Fluorescence intensity ratio (340/380 nm) of Fura-2AM versus time in HBMEC exposed to CVB in the absence (black) or presence of U73122 (blue). ( C ) Top: Still images captured at the indicated times in HBMEC transfected with control, IP 3 R-3, or PLCγ-1 siRNAs, loaded with Fura-2AM and exposed to CVB (MOI = 50, t = 55 seconds). Bottom: Intensity ratio plot (340/380 nm) of HBMEC loaded with Fura-2AM and transfected with control, IP 3 R-3, or PLCγ -1 siRNA and exposed to CVB (MOI = 50, t = 55 seconds).

    Journal: PLoS Pathogens

    Article Title: Release of Intracellular Calcium Stores Facilitates Coxsackievirus Entry into Polarized Endothelial Cells

    doi: 10.1371/journal.ppat.1001135

    Figure Lengend Snippet: PLCγ and IP 3 R-3 are involved in CVB-induced depletion of Ca i 2+ stores. ( A ) HBMEC (left) or Caco-2 (right) cells treated with 2-APB or U73122 1 hour prior to infection (pre-treat) or 2 hours p.i. (post-treat) were infected with CVB (MOI = 1) for 14 hrs (HBMEC) or 7 hrs (Caco-2). Graph represents percentage of total cells expressing VP1 normalized to no-inhibitor controls (dashed line). ( B ) Top: Still images of HBMEC pre-treated with U73122, loaded with Fura-2 AM, and exposed to CVB (MOI = 50, t = 55 seconds). Images were pseudo colored for Ca i 2+ visualization. Bottom: Fluorescence intensity ratio (340/380 nm) of Fura-2AM versus time in HBMEC exposed to CVB in the absence (black) or presence of U73122 (blue). ( C ) Top: Still images captured at the indicated times in HBMEC transfected with control, IP 3 R-3, or PLCγ-1 siRNAs, loaded with Fura-2AM and exposed to CVB (MOI = 50, t = 55 seconds). Bottom: Intensity ratio plot (340/380 nm) of HBMEC loaded with Fura-2AM and transfected with control, IP 3 R-3, or PLCγ -1 siRNA and exposed to CVB (MOI = 50, t = 55 seconds).

    Article Snippet: siRNA transfections siRNAs were purchased from Dharmacon.

    Techniques: Infection, Expressing, Fluorescence, Transfection

    DLC2 is required for junction maintenance and mitotic spindle stability. ( a ) Control and DLC2-depleted HCE cells stained for the adherens junction proteins E-cadherin (red) and β-catenin (green) and DNA (blue). ( b ) Immunoblot for DLC2 of control and DLC2 siRNA-transfected HCE cells; α-tubulin was used as a loading control. ( c ) Control and DLC2-depleted HCE cells stained for α-tubulin (green), α-catenin (red) and DNA (blue). ( d ) Quantification of cells with bipolar spindles with aligned and misaligned metaphase plates after transfection of control and DLC2 siRNAs (shown are means±1 s.d.; ** P

    Journal: Nature Communications

    Article Title: The tumour suppressor DLC2 ensures mitotic fidelity by coordinating spindle positioning and cell–cell adhesion

    doi: 10.1038/ncomms6826

    Figure Lengend Snippet: DLC2 is required for junction maintenance and mitotic spindle stability. ( a ) Control and DLC2-depleted HCE cells stained for the adherens junction proteins E-cadherin (red) and β-catenin (green) and DNA (blue). ( b ) Immunoblot for DLC2 of control and DLC2 siRNA-transfected HCE cells; α-tubulin was used as a loading control. ( c ) Control and DLC2-depleted HCE cells stained for α-tubulin (green), α-catenin (red) and DNA (blue). ( d ) Quantification of cells with bipolar spindles with aligned and misaligned metaphase plates after transfection of control and DLC2 siRNAs (shown are means±1 s.d.; ** P

    Article Snippet: siRNA transfection For siRNA transfections, INTERFERin (Polyplus transfection) was used as described .

    Techniques: Staining, Transfection

    DLC2 and Kif1B regulate spindle positioning and microtubule growth. ( a ) Hela cells stably expressing mCherry-lifeact and GFP-α-tubulin were transfected with siRNAs and plated onto micropatterned dishes with parallel fibronectin lines. Time-lapse recordings were then made collecting images every minute to follow actin behaviour and spindle orientation (see Supplementary Movies 4–6 and Supplementary Fig. 7 ). The movies were then quantified by calculating the percentages of cells with a specific actin behaviour based on kymographs and spindle orientation (Pol, polarized actin; Circ, circular movement of actin; Abs, absent actin polarization). Blue bars represent cells with mitotic spindles aligned with the fibronectin line; red bars, misaligned spindles. Significance was calculated between corresponding subgroups (shown are means±1 s.d.; n =3 experiments). ( b – e ) HeLa cells stably transfected with GFP-EB3 and transfected with the indicated siRNAs were filmed every 2 s. Microtubule dynamics was then quantified in at least 10 cells, following 10 plus-ends per cell (see Supplementary Movies 7–9 ). The box-and-whisker plots show total distance tracked ( b ) and total time tracked ( c ) ( n =45 microtubule tips). Microtubule tips sliding along the cell cortex were also quantified in 10 cells per type of siRNA transfection ( d ). The still images in panel e , taken every 2 s, show examples of microtubule tips tracked. Scale bars, 1 (upper panel in e ) and 2 μm (lower panels in e ). (* P

    Journal: Nature Communications

    Article Title: The tumour suppressor DLC2 ensures mitotic fidelity by coordinating spindle positioning and cell–cell adhesion

    doi: 10.1038/ncomms6826

    Figure Lengend Snippet: DLC2 and Kif1B regulate spindle positioning and microtubule growth. ( a ) Hela cells stably expressing mCherry-lifeact and GFP-α-tubulin were transfected with siRNAs and plated onto micropatterned dishes with parallel fibronectin lines. Time-lapse recordings were then made collecting images every minute to follow actin behaviour and spindle orientation (see Supplementary Movies 4–6 and Supplementary Fig. 7 ). The movies were then quantified by calculating the percentages of cells with a specific actin behaviour based on kymographs and spindle orientation (Pol, polarized actin; Circ, circular movement of actin; Abs, absent actin polarization). Blue bars represent cells with mitotic spindles aligned with the fibronectin line; red bars, misaligned spindles. Significance was calculated between corresponding subgroups (shown are means±1 s.d.; n =3 experiments). ( b – e ) HeLa cells stably transfected with GFP-EB3 and transfected with the indicated siRNAs were filmed every 2 s. Microtubule dynamics was then quantified in at least 10 cells, following 10 plus-ends per cell (see Supplementary Movies 7–9 ). The box-and-whisker plots show total distance tracked ( b ) and total time tracked ( c ) ( n =45 microtubule tips). Microtubule tips sliding along the cell cortex were also quantified in 10 cells per type of siRNA transfection ( d ). The still images in panel e , taken every 2 s, show examples of microtubule tips tracked. Scale bars, 1 (upper panel in e ) and 2 μm (lower panels in e ). (* P

    Article Snippet: siRNA transfection For siRNA transfections, INTERFERin (Polyplus transfection) was used as described .

    Techniques: Stable Transfection, Expressing, Transfection, Whisker Assay

    Depletion of V-ATPase subunit D1 inhibits HPV16 infectivity. (a) HeLa cells were transfected for 48 h with different siRNAs targeting the V-ATPase subunit D1 or a control siRNA and subsequently infected for 24 h with HPV16 pseudovirions. Depicted are the means ± SD relative to the control siRNA ( n = three independent experiments). (b) At 48 h after siRNA transfection, HeLa cells were analyzed by Western blotting for the levels of V-ATPase D1 and, as a loading control, tubulin.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibition by Cellular Vacuolar ATPase Impairs Human Papillomavirus Uncoating and Infection

    doi: 10.1128/AAC.02284-13

    Figure Lengend Snippet: Depletion of V-ATPase subunit D1 inhibits HPV16 infectivity. (a) HeLa cells were transfected for 48 h with different siRNAs targeting the V-ATPase subunit D1 or a control siRNA and subsequently infected for 24 h with HPV16 pseudovirions. Depicted are the means ± SD relative to the control siRNA ( n = three independent experiments). (b) At 48 h after siRNA transfection, HeLa cells were analyzed by Western blotting for the levels of V-ATPase D1 and, as a loading control, tubulin.

    Article Snippet: Knockdown of ATP6V0D1 was confirmed 48 h after siRNA transfection by Western blotting using a specific monoclonal antibody (sc-81887; Santa Cruz).

    Techniques: Infection, Transfection, Western Blot

    Down-regulation of CRLR expression in HO8910 cells inhibited influence of exogenous AM on cell migration . Reduced CRLR mRNA expression (A) and protein expression (B) were determined by real-time PCR analysis or western blot in CRLR siRNA transfected cells, compared with scrambled siRNA transfected cells. After cells were transfected by CRLR siRNA, the effect of AM on cells migration was decreased consequently (C).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Adrenomedullin expression in epithelial ovarian cancers and promotes HO8910 cell migration associated with upregulating integrin ?5?1 and phosphorylating FAK and paxillin

    doi: 10.1186/1756-9966-31-19

    Figure Lengend Snippet: Down-regulation of CRLR expression in HO8910 cells inhibited influence of exogenous AM on cell migration . Reduced CRLR mRNA expression (A) and protein expression (B) were determined by real-time PCR analysis or western blot in CRLR siRNA transfected cells, compared with scrambled siRNA transfected cells. After cells were transfected by CRLR siRNA, the effect of AM on cells migration was decreased consequently (C).

    Article Snippet: CRLR knockdown with siRNA The CRLR-specific small interfering RNA (siRNA) (#42272) and scrambled siRNA (#4611) were designed and synthesized by Ambion (USA).

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

    Effect of HMGI-C siRNA on the MDA-MB-468 cells. 48 h after transfection with HMGI-C siRNA (40, 60, 80 pmol), cytotoxicity of treatments was determined by MTT assay. The results were expressed as mean ± SD (n = 3); **P

    Journal: Cell Cycle

    Article Title: HMGI-C suppressing induces P53/caspase9 axis to regulate apoptosis in breast adenocarcinoma cells

    doi: 10.1080/15384101.2016.1190892

    Figure Lengend Snippet: Effect of HMGI-C siRNA on the MDA-MB-468 cells. 48 h after transfection with HMGI-C siRNA (40, 60, 80 pmol), cytotoxicity of treatments was determined by MTT assay. The results were expressed as mean ± SD (n = 3); **P

    Article Snippet: Negative control siRNA (NC siRNA) and pooled human HMGI-C siRNA (a pool of 3 different siRNA duplexes sequences including siRNA duplex A (sc-37994A) Sense: GCACUUUCAAUCUCAAUCUtt and Antisense: AGAUUGAGAUUGAAAGUGCtt, siRNA duplex B( sc-37994B) Sense: GUGACCACUUAUUCUGUAUtt and Antisense: AUACAGAAUAAGUGGUCACtt, siRNA duplex C (sc-37994C) Sense: GAGACGAAAUGCUGAUGUAtt and Antisense: UACAUCAGCAUUUCGUCUCtt), goat polyclonal anti HMGI-C anti-body, monoclonal β-actin antibody, siRNA transfection reagent and siRNA transfection medium were purchased from Santa Cruz Biotechnology (California, USA).

    Techniques: Multiple Displacement Amplification, Transfection, MTT Assay

    Low-dose pemetrexed plus cisplatin synergistically up-regulates NOXA expression and down-regulates Mcl-1 expression in choroidal melanoma cells. (A) To analyze the synergistic effect of the co-treatment, the indicated cells were treated with the indicated concentrations of the chemotherapeutic drugs for 24 hours and then harvested for western blotting. NOXA and Mcl-1 expression was quantified using Image J software and analyzed with GraphPad Prism 5.0 software. OCM1 and M619 cells were seeded in 6-well plates and transfected with control or NOXA siRNA on the second day. (B, C, D)At 48 hours after transfection, the cells were treated with 1.25 μmol/L pemetrexed combined with 5 μmol/L cisplatin for another 24 hours and then harvested for western blotting and apoptosis analysis. CF: cleaved form. All data are presented as the mean ± S.D.

    Journal: PLoS ONE

    Article Title: c-FLIP and the NOXA/Mcl-1 axis participate in the synergistic effect of pemetrexed plus cisplatin in human choroidal melanoma cells

    doi: 10.1371/journal.pone.0184135

    Figure Lengend Snippet: Low-dose pemetrexed plus cisplatin synergistically up-regulates NOXA expression and down-regulates Mcl-1 expression in choroidal melanoma cells. (A) To analyze the synergistic effect of the co-treatment, the indicated cells were treated with the indicated concentrations of the chemotherapeutic drugs for 24 hours and then harvested for western blotting. NOXA and Mcl-1 expression was quantified using Image J software and analyzed with GraphPad Prism 5.0 software. OCM1 and M619 cells were seeded in 6-well plates and transfected with control or NOXA siRNA on the second day. (B, C, D)At 48 hours after transfection, the cells were treated with 1.25 μmol/L pemetrexed combined with 5 μmol/L cisplatin for another 24 hours and then harvested for western blotting and apoptosis analysis. CF: cleaved form. All data are presented as the mean ± S.D.

    Article Snippet: SiRNA transfection was conducted using jetPRIME Transfection Reagent (Polyplus Transfection SA, Illkirch, France) following the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Software, Transfection

    Pemetrexed plus cisplatin synergistically induces CHOP expression. (A) To analyze the synergistic effects of the co-treatment, the indicated cells were treated with the indicated concentrations of chemotherapeutic drugs for 24 hours and then harvested for western blotting analysis. CHOP expression was quantified using Image J software and analyzed with GraphPad Prism 5.0 software. OCM1 and M619 cells were seeded in 6-well plates and transfected with control or CHOP siRNA on the second day. (B, C) At 48 hours after transfection, the cells were treated with 1.25 μmol/L pemetrexed combined with 5 μmol/L cisplatin for another 24 hours and then harvested for western blotting and apoptosis analysis. CF: cleaved form. All data are presented as the mean ± S.D.

    Journal: PLoS ONE

    Article Title: c-FLIP and the NOXA/Mcl-1 axis participate in the synergistic effect of pemetrexed plus cisplatin in human choroidal melanoma cells

    doi: 10.1371/journal.pone.0184135

    Figure Lengend Snippet: Pemetrexed plus cisplatin synergistically induces CHOP expression. (A) To analyze the synergistic effects of the co-treatment, the indicated cells were treated with the indicated concentrations of chemotherapeutic drugs for 24 hours and then harvested for western blotting analysis. CHOP expression was quantified using Image J software and analyzed with GraphPad Prism 5.0 software. OCM1 and M619 cells were seeded in 6-well plates and transfected with control or CHOP siRNA on the second day. (B, C) At 48 hours after transfection, the cells were treated with 1.25 μmol/L pemetrexed combined with 5 μmol/L cisplatin for another 24 hours and then harvested for western blotting and apoptosis analysis. CF: cleaved form. All data are presented as the mean ± S.D.

    Article Snippet: SiRNA transfection was conducted using jetPRIME Transfection Reagent (Polyplus Transfection SA, Illkirch, France) following the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Software, Transfection

    Embryo like cells decreased after CCAAT/enhancer binding protein-α (C/EBPα) was downregulation. (A) The C/EBPα downregulation in endometrial polyp stem cells after siRNA transfection (p

    Journal: International Journal of Biological Sciences

    Article Title: Dedifferentiation of human uterine polyp stem cells into embryo-like cells during inducing pluripotency

    doi: 10.7150/ijbs.23401

    Figure Lengend Snippet: Embryo like cells decreased after CCAAT/enhancer binding protein-α (C/EBPα) was downregulation. (A) The C/EBPα downregulation in endometrial polyp stem cells after siRNA transfection (p

    Article Snippet: SiRNA transfection All siRNA transfections were performed using Dharmafect transfection reagent (GE Healthcare, Buckinghamshire, UK) following the manufacturer's protocol.

    Techniques: Binding Assay, Transfection

    PDZ-RhoGEF/LARG is associated with the increase of ABCA1 protein levels. A , knockdown of RhoGEF expression by siRNA suppressed the apoA-I-mediated ABCA1 stabilization in primary human fibroblast. The cells were transfected with either a nontargeting pool of siRNA duplexes (negative control), siRNA duplex targeting PDZ-RhoGEF, or LARG, or a mixture of siRNA duplexes targeting both PDZ-RhoGEF and LARG. After 48 h transfection, the cells were incubated with apoA-I for 1 h. The level of ABCA1 protein was quantified using a LAS-3000 imager. B , PDZ-RhoGEF expression increased ABCA1 protein level in cells expressing wild-type ( WT ) ABCA1 but in cells expressing the ABCA1-Δ4 mutant, which disrupts the ABCA1 PDZ-binding motif. 293 cells were transfected with wild-type or Δ4 mutant of ABCA1 cDNA either alone or together with PDZ-RhoGEF cDNA. The results are representatives of two or more experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Binding of PDZ-RhoGEF to ATP-binding Cassette Transporter A1 (ABCA1) Induces Cholesterol Efflux through RhoA Activation and Prevention of Transporter Degradation *

    doi: 10.1074/jbc.M109.061424

    Figure Lengend Snippet: PDZ-RhoGEF/LARG is associated with the increase of ABCA1 protein levels. A , knockdown of RhoGEF expression by siRNA suppressed the apoA-I-mediated ABCA1 stabilization in primary human fibroblast. The cells were transfected with either a nontargeting pool of siRNA duplexes (negative control), siRNA duplex targeting PDZ-RhoGEF, or LARG, or a mixture of siRNA duplexes targeting both PDZ-RhoGEF and LARG. After 48 h transfection, the cells were incubated with apoA-I for 1 h. The level of ABCA1 protein was quantified using a LAS-3000 imager. B , PDZ-RhoGEF expression increased ABCA1 protein level in cells expressing wild-type ( WT ) ABCA1 but in cells expressing the ABCA1-Δ4 mutant, which disrupts the ABCA1 PDZ-binding motif. 293 cells were transfected with wild-type or Δ4 mutant of ABCA1 cDNA either alone or together with PDZ-RhoGEF cDNA. The results are representatives of two or more experiments.

    Article Snippet: The siRNA transfection was performed by nucleofection using a human dermal fibroblast Nucleofector kit (Amaxa Biosystems, Cologne, Germany) according to the manufacturer's protocols.

    Techniques: Expressing, Transfection, Negative Control, Incubation, Mutagenesis, Binding Assay