lipofectamine rnaimax Thermo Fisher Search Results


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  • 90
    Thermo Fisher transfection reagent
    Knockdown efficiency of c-Jun and CREB mRNA and IL-32 expression induced by IFNγ and H 2 O 2 in HBE cells transfected with c-Jun or CREB siRNAs . c-Jun (A) and CREB (B) mRNA expression levels examined by quantitative real time PCR in HBE cells transfected with indicated siRNAs. Expression levels of c-Jun (A) and CREB (B) by each siRNA <t>transfection</t> were looked by quantitative real time PCR. IL-32 expression was examined by real time PCR in HBE cells transfected with control-siRNA, closed bars, c-Jun-siRNA, open bars, or CREB-siRNA, hatched bars, respectively. Then 48 hours after transfection, cells were stimulated with H 2 O 2 and/or IFNγ, followed by IL-32 quantitative real time PCR of RNA extracted 24 hours after the stimulation (C). The bars represent the means ± SE from 3 different individuals. *p
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    Thermo Fisher lipofectamine rnaimax
    microRNAs 146a, 146b, and 155 are down-regulated in statin-treated Mac. a microRNA array. Total RNA was isolated from macrophages differentiated in the absence or presence of statin (25 cm 2 flasks, 100,000 cells/cm 2 ) with the “RNeasy Plus Mini Kit”. Deep sequencing was performed by the “Core Unit DNA”, Universität Leipzig, using the “TruSeq™Small RNA sample prepkit v2” (illumina, San Diego, USA). The mean of the normalized data of macrophages pretreated without statin and macrophages pretreated with statin was calculated and data of samples with a mean > 100 counts (192 samples; compare Supplementary Table 2 ) were included into the analysis and blotted against each other. The orange line indicates unchanged expression. The red dots mark three selected miRs, which were down-regulated in statin-pretreated macrophages, as compared to Mac prepared in the absence of statin (compare Supplementary Table 2 ). A second array showed a similar result. b Blockade of miR-146a and miR-155 reverses the hypo-responsiveness in macrophages only to some degree. Macrophages were incubated as described in Fig. 1a (6-well plate; 100,000 cells/cm 2 ). The respective “miRCURY LNA™” anti-miR (Exiqon, Qiagen, Vedbaek, Denmark) were prepared in <t>“Lipofectamine</t> <t>RNAiMax”</t> and 250 µl of this solution was added to 2750 µl of culture medium in the absence (−) or presence (+) of statin. After 24 h LPS was added. After further 24 h, the supernatants were harvested and analyzed in ELISA. Four experiments with similar results were performed. Data analysis and color code as in Fig. 1c (“Ctrl” vs. “anti-miR”).
    Lipofectamine Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 38905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine rnaimaxx
    microRNAs 146a, 146b, and 155 are down-regulated in statin-treated Mac. a microRNA array. Total RNA was isolated from macrophages differentiated in the absence or presence of statin (25 cm 2 flasks, 100,000 cells/cm 2 ) with the “RNeasy Plus Mini Kit”. Deep sequencing was performed by the “Core Unit DNA”, Universität Leipzig, using the “TruSeq™Small RNA sample prepkit v2” (illumina, San Diego, USA). The mean of the normalized data of macrophages pretreated without statin and macrophages pretreated with statin was calculated and data of samples with a mean > 100 counts (192 samples; compare Supplementary Table 2 ) were included into the analysis and blotted against each other. The orange line indicates unchanged expression. The red dots mark three selected miRs, which were down-regulated in statin-pretreated macrophages, as compared to Mac prepared in the absence of statin (compare Supplementary Table 2 ). A second array showed a similar result. b Blockade of miR-146a and miR-155 reverses the hypo-responsiveness in macrophages only to some degree. Macrophages were incubated as described in Fig. 1a (6-well plate; 100,000 cells/cm 2 ). The respective “miRCURY LNA™” anti-miR (Exiqon, Qiagen, Vedbaek, Denmark) were prepared in <t>“Lipofectamine</t> <t>RNAiMax”</t> and 250 µl of this solution was added to 2750 µl of culture medium in the absence (−) or presence (+) of statin. After 24 h LPS was added. After further 24 h, the supernatants were harvested and analyzed in ELISA. Four experiments with similar results were performed. Data analysis and color code as in Fig. 1c (“Ctrl” vs. “anti-miR”).
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    Thermo Fisher lipofectamine rnaimix
    microRNAs 146a, 146b, and 155 are down-regulated in statin-treated Mac. a microRNA array. Total RNA was isolated from macrophages differentiated in the absence or presence of statin (25 cm 2 flasks, 100,000 cells/cm 2 ) with the “RNeasy Plus Mini Kit”. Deep sequencing was performed by the “Core Unit DNA”, Universität Leipzig, using the “TruSeq™Small RNA sample prepkit v2” (illumina, San Diego, USA). The mean of the normalized data of macrophages pretreated without statin and macrophages pretreated with statin was calculated and data of samples with a mean > 100 counts (192 samples; compare Supplementary Table 2 ) were included into the analysis and blotted against each other. The orange line indicates unchanged expression. The red dots mark three selected miRs, which were down-regulated in statin-pretreated macrophages, as compared to Mac prepared in the absence of statin (compare Supplementary Table 2 ). A second array showed a similar result. b Blockade of miR-146a and miR-155 reverses the hypo-responsiveness in macrophages only to some degree. Macrophages were incubated as described in Fig. 1a (6-well plate; 100,000 cells/cm 2 ). The respective “miRCURY LNA™” anti-miR (Exiqon, Qiagen, Vedbaek, Denmark) were prepared in <t>“Lipofectamine</t> <t>RNAiMax”</t> and 250 µl of this solution was added to 2750 µl of culture medium in the absence (−) or presence (+) of statin. After 24 h LPS was added. After further 24 h, the supernatants were harvested and analyzed in ELISA. Four experiments with similar results were performed. Data analysis and color code as in Fig. 1c (“Ctrl” vs. “anti-miR”).
    Lipofectamine Rnaimix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    microRNAs 146a, 146b, and 155 are down-regulated in statin-treated Mac. a microRNA array. Total RNA was isolated from macrophages differentiated in the absence or presence of statin (25 cm 2 flasks, 100,000 cells/cm 2 ) with the “RNeasy Plus Mini Kit”. Deep sequencing was performed by the “Core Unit DNA”, Universität Leipzig, using the “TruSeq™Small RNA sample prepkit v2” (illumina, San Diego, USA). The mean of the normalized data of macrophages pretreated without statin and macrophages pretreated with statin was calculated and data of samples with a mean > 100 counts (192 samples; compare Supplementary Table 2 ) were included into the analysis and blotted against each other. The orange line indicates unchanged expression. The red dots mark three selected miRs, which were down-regulated in statin-pretreated macrophages, as compared to Mac prepared in the absence of statin (compare Supplementary Table 2 ). A second array showed a similar result. b Blockade of miR-146a and miR-155 reverses the hypo-responsiveness in macrophages only to some degree. Macrophages were incubated as described in Fig. 1a (6-well plate; 100,000 cells/cm 2 ). The respective “miRCURY LNA™” anti-miR (Exiqon, Qiagen, Vedbaek, Denmark) were prepared in <t>“Lipofectamine</t> <t>RNAiMax”</t> and 250 µl of this solution was added to 2750 µl of culture medium in the absence (−) or presence (+) of statin. After 24 h LPS was added. After further 24 h, the supernatants were harvested and analyzed in ELISA. Four experiments with similar results were performed. Data analysis and color code as in Fig. 1c (“Ctrl” vs. “anti-miR”).
    Lipofectamine Rnaimas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine 3000
    microRNAs 146a, 146b, and 155 are down-regulated in statin-treated Mac. a microRNA array. Total RNA was isolated from macrophages differentiated in the absence or presence of statin (25 cm 2 flasks, 100,000 cells/cm 2 ) with the “RNeasy Plus Mini Kit”. Deep sequencing was performed by the “Core Unit DNA”, Universität Leipzig, using the “TruSeq™Small RNA sample prepkit v2” (illumina, San Diego, USA). The mean of the normalized data of macrophages pretreated without statin and macrophages pretreated with statin was calculated and data of samples with a mean > 100 counts (192 samples; compare Supplementary Table 2 ) were included into the analysis and blotted against each other. The orange line indicates unchanged expression. The red dots mark three selected miRs, which were down-regulated in statin-pretreated macrophages, as compared to Mac prepared in the absence of statin (compare Supplementary Table 2 ). A second array showed a similar result. b Blockade of miR-146a and miR-155 reverses the hypo-responsiveness in macrophages only to some degree. Macrophages were incubated as described in Fig. 1a (6-well plate; 100,000 cells/cm 2 ). The respective “miRCURY LNA™” anti-miR (Exiqon, Qiagen, Vedbaek, Denmark) were prepared in <t>“Lipofectamine</t> <t>RNAiMax”</t> and 250 µl of this solution was added to 2750 µl of culture medium in the absence (−) or presence (+) of statin. After 24 h LPS was added. After further 24 h, the supernatants were harvested and analyzed in ELISA. Four experiments with similar results were performed. Data analysis and color code as in Fig. 1c (“Ctrl” vs. “anti-miR”).
    Lipofectamine 3000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 36962 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine ltx
    microRNAs 146a, 146b, and 155 are down-regulated in statin-treated Mac. a microRNA array. Total RNA was isolated from macrophages differentiated in the absence or presence of statin (25 cm 2 flasks, 100,000 cells/cm 2 ) with the “RNeasy Plus Mini Kit”. Deep sequencing was performed by the “Core Unit DNA”, Universität Leipzig, using the “TruSeq™Small RNA sample prepkit v2” (illumina, San Diego, USA). The mean of the normalized data of macrophages pretreated without statin and macrophages pretreated with statin was calculated and data of samples with a mean > 100 counts (192 samples; compare Supplementary Table 2 ) were included into the analysis and blotted against each other. The orange line indicates unchanged expression. The red dots mark three selected miRs, which were down-regulated in statin-pretreated macrophages, as compared to Mac prepared in the absence of statin (compare Supplementary Table 2 ). A second array showed a similar result. b Blockade of miR-146a and miR-155 reverses the hypo-responsiveness in macrophages only to some degree. Macrophages were incubated as described in Fig. 1a (6-well plate; 100,000 cells/cm 2 ). The respective “miRCURY LNA™” anti-miR (Exiqon, Qiagen, Vedbaek, Denmark) were prepared in <t>“Lipofectamine</t> <t>RNAiMax”</t> and 250 µl of this solution was added to 2750 µl of culture medium in the absence (−) or presence (+) of statin. After 24 h LPS was added. After further 24 h, the supernatants were harvested and analyzed in ELISA. Four experiments with similar results were performed. Data analysis and color code as in Fig. 1c (“Ctrl” vs. “anti-miR”).
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    Thermo Fisher lipofectamine rnaimax reagent
    Effects of siRNA on FMDV-IRES activity. a siRNA targeting the conserved region of FMDV-IRES was reverse transfected into HEK293 cells (clone B10) using <t>Lipofectamine</t> <t>RNAiMAX</t> and incubated for 48 h. Firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values (* p
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    Thermo Fisher lipofectamine rnaimax kit
    Effects of siRNA on FMDV-IRES activity. a siRNA targeting the conserved region of FMDV-IRES was reverse transfected into HEK293 cells (clone B10) using <t>Lipofectamine</t> <t>RNAiMAX</t> and incubated for 48 h. Firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values (* p
    Lipofectamine Rnaimax Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine rnaimax protocol
    Effects of siRNA on FMDV-IRES activity. a siRNA targeting the conserved region of FMDV-IRES was reverse transfected into HEK293 cells (clone B10) using <t>Lipofectamine</t> <t>RNAiMAX</t> and incubated for 48 h. Firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values (* p
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    Thermo Fisher rnaimax
    Matrigel and spheroid uptake of Opti-MEM-formed siRNA can be improved in the presence of 10% serum. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl Opti-MEM using <t>RNAiMax,</t> but co-incubated in a well containing 1.5 mls DMEM containing 10% serum, at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl DMEM containing 10% serum using RNAiMax, but co-incubated in a well containing 1.5 mls Opti-MEM, at 2, 6 and 24 hours post transfection.
    Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine 2000tm rnaimax
    Matrigel and spheroid uptake of Opti-MEM-formed siRNA can be improved in the presence of 10% serum. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl Opti-MEM using <t>RNAiMax,</t> but co-incubated in a well containing 1.5 mls DMEM containing 10% serum, at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl DMEM containing 10% serum using RNAiMax, but co-incubated in a well containing 1.5 mls Opti-MEM, at 2, 6 and 24 hours post transfection.
    Lipofectamine 2000tm Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine
    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using <t>lipofectamine</t> reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p
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    Thermo Fisher lipofectamine 2000 transfection reagent
    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using <t>lipofectamine</t> reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p
    Lipofectamine 2000 Transfection Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sod2 lipofectamine rnaimax reagent
    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using <t>lipofectamine</t> reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p
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    Thermo Fisher fluorescent oligo lipofectamine rnaimax
    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using <t>lipofectamine</t> reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p
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    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using <t>lipofectamine</t> reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p
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    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using <t>lipofectamine</t> reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p
    Lipofectamine Rnaimax Transfection Regent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using <t>lipofectamine</t> reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p
    Lipofectamine Rnaimax Transfection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using <t>lipofectamine</t> reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p
    Vector Construction Lipofectamine Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using <t>lipofectamine</t> reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p
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    Image Search Results


    Knockdown efficiency of c-Jun and CREB mRNA and IL-32 expression induced by IFNγ and H 2 O 2 in HBE cells transfected with c-Jun or CREB siRNAs . c-Jun (A) and CREB (B) mRNA expression levels examined by quantitative real time PCR in HBE cells transfected with indicated siRNAs. Expression levels of c-Jun (A) and CREB (B) by each siRNA transfection were looked by quantitative real time PCR. IL-32 expression was examined by real time PCR in HBE cells transfected with control-siRNA, closed bars, c-Jun-siRNA, open bars, or CREB-siRNA, hatched bars, respectively. Then 48 hours after transfection, cells were stimulated with H 2 O 2 and/or IFNγ, followed by IL-32 quantitative real time PCR of RNA extracted 24 hours after the stimulation (C). The bars represent the means ± SE from 3 different individuals. *p

    Journal: Respiratory Research

    Article Title: Oxidative stress induced Interleukin-32 mRNA expression in human bronchial epithelial cells

    doi: 10.1186/1465-9921-13-19

    Figure Lengend Snippet: Knockdown efficiency of c-Jun and CREB mRNA and IL-32 expression induced by IFNγ and H 2 O 2 in HBE cells transfected with c-Jun or CREB siRNAs . c-Jun (A) and CREB (B) mRNA expression levels examined by quantitative real time PCR in HBE cells transfected with indicated siRNAs. Expression levels of c-Jun (A) and CREB (B) by each siRNA transfection were looked by quantitative real time PCR. IL-32 expression was examined by real time PCR in HBE cells transfected with control-siRNA, closed bars, c-Jun-siRNA, open bars, or CREB-siRNA, hatched bars, respectively. Then 48 hours after transfection, cells were stimulated with H 2 O 2 and/or IFNγ, followed by IL-32 quantitative real time PCR of RNA extracted 24 hours after the stimulation (C). The bars represent the means ± SE from 3 different individuals. *p

    Article Snippet: HBE cells were incubated with the indicated siRNA and transfection reagent (Lipofectamine RNAiMAX, Invitrogen) for 24 hours.

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

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/s41419-019-2109-9

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

    Article Snippet: “Lipofectamine® RNAiMAX” (9 µl; ambion™) was added to 150 µl of “OptiMEM®” and 3 µl of the anti-miR (10 µM) was added to another volume of 150 µl “Optimem®”.

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

    RIG-I and MDA5 agonists stimulate the expression of IFN-β in hNSCs. a , b Human NSCs were transfected with 5′pppRNA or poly(I:C) HMW. Total RNA was collected at 12 and 24 h post-transfection, and RT-qPCR assay was performed to examine the relative amount of IFN-β mRNA ( a ). The expression of phospho-IRF3 and total IRF3 was detected through a western blot assay. β-actin was used as an internal control. ( b ). c Human NSCs were transfected with siRNA specific for RIG-I or MDA5 by Lipofectamine RNAiMAX 2000 and then transfected with 1 μg 5′pppRNA for 24 h. RT-qPCR analysis was performed to detect the expression of IFN-β transcripts while western blot was applied to confirm the knockdown efficiency. d Human NSCs were transfected with siRNA targeting RIG-I or MDA5 and then transfected with 1 μg of poly(I:C) HMW for 24 h. RT-qPCR analysis was performed to detect the expression of IFN-β transcripts while western blot was applied to confirm the knockdown efficiency. The experiments were performed in triplicate, and the error bars represented the SD. Student’s t test was used for statistical analysis. * p

    Journal: Stem Cell Research & Therapy

    Article Title: Activation of type I interferon antiviral response in human neural stem cells

    doi: 10.1186/s13287-019-1521-5

    Figure Lengend Snippet: RIG-I and MDA5 agonists stimulate the expression of IFN-β in hNSCs. a , b Human NSCs were transfected with 5′pppRNA or poly(I:C) HMW. Total RNA was collected at 12 and 24 h post-transfection, and RT-qPCR assay was performed to examine the relative amount of IFN-β mRNA ( a ). The expression of phospho-IRF3 and total IRF3 was detected through a western blot assay. β-actin was used as an internal control. ( b ). c Human NSCs were transfected with siRNA specific for RIG-I or MDA5 by Lipofectamine RNAiMAX 2000 and then transfected with 1 μg 5′pppRNA for 24 h. RT-qPCR analysis was performed to detect the expression of IFN-β transcripts while western blot was applied to confirm the knockdown efficiency. d Human NSCs were transfected with siRNA targeting RIG-I or MDA5 and then transfected with 1 μg of poly(I:C) HMW for 24 h. RT-qPCR analysis was performed to detect the expression of IFN-β transcripts while western blot was applied to confirm the knockdown efficiency. The experiments were performed in triplicate, and the error bars represented the SD. Student’s t test was used for statistical analysis. * p

    Article Snippet: Lipofectamine 2000 RNAiMAX (Thermo-Fisher Scientific, MA, USA) was used for transfecting siRNAs.

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

    Effects of siRNA on FMDV-IRES activity. a siRNA targeting the conserved region of FMDV-IRES was reverse transfected into HEK293 cells (clone B10) using Lipofectamine RNAiMAX and incubated for 48 h. Firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values (* p

    Journal: Virus Genes

    Article Title: Silencing of the foot-and-mouth disease virus internal ribosomal entry site by targeting relatively conserved region among serotypes

    doi: 10.1007/s11262-019-01696-6

    Figure Lengend Snippet: Effects of siRNA on FMDV-IRES activity. a siRNA targeting the conserved region of FMDV-IRES was reverse transfected into HEK293 cells (clone B10) using Lipofectamine RNAiMAX and incubated for 48 h. Firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values (* p

    Article Snippet: After 3–4 weeks, G418-resistant cells were identified as colonies. siRNA (1, 5, or 10 nM) reverse transfection was performed using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s specifications.

    Techniques: Activity Assay, Transfection, Incubation, Luciferase

    Matrigel and spheroid uptake of Opti-MEM-formed siRNA can be improved in the presence of 10% serum. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl Opti-MEM using RNAiMax, but co-incubated in a well containing 1.5 mls DMEM containing 10% serum, at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl DMEM containing 10% serum using RNAiMax, but co-incubated in a well containing 1.5 mls Opti-MEM, at 2, 6 and 24 hours post transfection.

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Matrigel and spheroid uptake of Opti-MEM-formed siRNA can be improved in the presence of 10% serum. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl Opti-MEM using RNAiMax, but co-incubated in a well containing 1.5 mls DMEM containing 10% serum, at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl DMEM containing 10% serum using RNAiMax, but co-incubated in a well containing 1.5 mls Opti-MEM, at 2, 6 and 24 hours post transfection.

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Incubation, Transfection

    Failure of Opti-MEM prepared siRNA to penetrate matrigel or organoids is not cell line or transfection reagent specific. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection. ( C ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using Lipofectamine 2000 at 2, 6 and 24 hours post transfection.

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Failure of Opti-MEM prepared siRNA to penetrate matrigel or organoids is not cell line or transfection reagent specific. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection. ( C ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using Lipofectamine 2000 at 2, 6 and 24 hours post transfection.

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection

    10 kDa dialyzed serum is also efficient for matrigel and spheroid uptake of siRNA. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed with ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% dialyzed serum using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of dialyzed serum. Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by *** P

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: 10 kDa dialyzed serum is also efficient for matrigel and spheroid uptake of siRNA. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed with ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% dialyzed serum using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of dialyzed serum. Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by *** P

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection, Fluorescence

    Primary organoid uptake of siRNA is more efficient when complexes are formed with serum. Representative confocal images of VilCre ER Apc fl/fl Kras G12D/+ primary mouse organoids showing localization of control-Cy3 siRNA 2, 6 and 24 hours post transfection with siRNA formed in ( A ) Opti-MEM or ( B ) 10% serum using RNAiMax. White scale bars indicate 150 μM. Representative confocal images of primary wild type mouse organoids showing localization of control-Cy3 siRNA 2, 6 and 24 hours post transfection with siRNA formed in ( C ) Opti-MEM or ( D ) 10% serum using RNAiMax. White scale bar indicates 80 μM except for 24 hours where it indicates 130 μM. White/black arrows indicate the location of the matrigel boundary.

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Primary organoid uptake of siRNA is more efficient when complexes are formed with serum. Representative confocal images of VilCre ER Apc fl/fl Kras G12D/+ primary mouse organoids showing localization of control-Cy3 siRNA 2, 6 and 24 hours post transfection with siRNA formed in ( A ) Opti-MEM or ( B ) 10% serum using RNAiMax. White scale bars indicate 150 μM. Representative confocal images of primary wild type mouse organoids showing localization of control-Cy3 siRNA 2, 6 and 24 hours post transfection with siRNA formed in ( C ) Opti-MEM or ( D ) 10% serum using RNAiMax. White scale bar indicates 80 μM except for 24 hours where it indicates 130 μM. White/black arrows indicate the location of the matrigel boundary.

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection

    Optimal siRNA uptake by matrigel and spheroids occurs with a 1–10% serum concentration. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed with ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% serum using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of serum. Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by * P

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Optimal siRNA uptake by matrigel and spheroids occurs with a 1–10% serum concentration. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed with ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% serum using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of serum. Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by * P

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Concentration Assay, Transfection, Fluorescence

    Spheroid uptake of siRNA is more efficient when complexes are formed with serum. ( A ) Workflow of experimental timeframe for spheroid transfection and analysis. ( B ) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. ( C ) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection. White scale bar indicates 250 μM and white/black arrows indicate the location of the matrigel boundary. Summary graphs indicating the level of CTNNB1 mRNA in ( D ) 3D, and ( E ) 2D SW1463 cell cultures, at 48 hours post transfection with control or CTNNB1 siRNA complexes formed in Opti-MEM or 10% serum using RNAiMax. Levels are normalized to the housekeeping gene TATA-binding protein ( TBP ). Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by ** P

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Spheroid uptake of siRNA is more efficient when complexes are formed with serum. ( A ) Workflow of experimental timeframe for spheroid transfection and analysis. ( B ) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. ( C ) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection. White scale bar indicates 250 μM and white/black arrows indicate the location of the matrigel boundary. Summary graphs indicating the level of CTNNB1 mRNA in ( D ) 3D, and ( E ) 2D SW1463 cell cultures, at 48 hours post transfection with control or CTNNB1 siRNA complexes formed in Opti-MEM or 10% serum using RNAiMax. Levels are normalized to the housekeeping gene TATA-binding protein ( TBP ). Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by ** P

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection, Binding Assay

    BSA can facilitate matrigel entry of siRNA, but not spheroid uptake. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed in ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% BSA (no serum present) using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of BSA. Data represents mean ± 1 s.d ( n = 3).

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: BSA can facilitate matrigel entry of siRNA, but not spheroid uptake. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed in ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% BSA (no serum present) using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of BSA. Data represents mean ± 1 s.d ( n = 3).

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection, Fluorescence

    (A) Uninfected macrophages were treated with vehicle, Tat, gp120, or RNA40 for 24 h. (Left) Representative Western blots of TREM1 with antibodies against TREM1 and ACTB. (Right) Densitometric analysis of blots, n = 4. (B) Uninfected macrophages were transfected with si RELA or siNS for 48 h. (Left) Representative Western blots of RELA with antibodies against RELA and ACTB. (Right) Densitometric analysis of blots, n = 4. (C) The transfection efficiency of Lipofectamine RNAiMAX in macrophages was assessed using BLOCK-iT Alexa Fluor red fluorescent control. Cells were harvested 24 h posttransfection and analyzed by flow cytometry. (Left) A representative histogram is shown. (Right) Percentages of cells positive for BLOCK-iT Alexa Fluor red, n = 8. (D) Uninfected macrophages were transfected with si RELA or siNS for 48 h and then treated with vehicle, Tat, gp120, RNA40, RNA41 (R41), or staurosporine (STS) for 24 h. Apoptotic ssDNA damage was quantified by ELISA, n = 4. (E) Uninfected macrophages were transfected with si RELA or siNS for 48 h and then treated with vehicle, Tat, gp120, RNA40, or RNA41 (R41) for 24 h. (Left) Representative Western blots of TREM1 with antibodies against TREM1 and ACTB. (Right) Densitometric analysis of blots, n = 4.

    Journal: mBio

    Article Title: TREM-1 Protects HIV-1-Infected Macrophages from Apoptosis through Maintenance of Mitochondrial Function

    doi: 10.1128/mBio.02638-19

    Figure Lengend Snippet: (A) Uninfected macrophages were treated with vehicle, Tat, gp120, or RNA40 for 24 h. (Left) Representative Western blots of TREM1 with antibodies against TREM1 and ACTB. (Right) Densitometric analysis of blots, n = 4. (B) Uninfected macrophages were transfected with si RELA or siNS for 48 h. (Left) Representative Western blots of RELA with antibodies against RELA and ACTB. (Right) Densitometric analysis of blots, n = 4. (C) The transfection efficiency of Lipofectamine RNAiMAX in macrophages was assessed using BLOCK-iT Alexa Fluor red fluorescent control. Cells were harvested 24 h posttransfection and analyzed by flow cytometry. (Left) A representative histogram is shown. (Right) Percentages of cells positive for BLOCK-iT Alexa Fluor red, n = 8. (D) Uninfected macrophages were transfected with si RELA or siNS for 48 h and then treated with vehicle, Tat, gp120, RNA40, RNA41 (R41), or staurosporine (STS) for 24 h. Apoptotic ssDNA damage was quantified by ELISA, n = 4. (E) Uninfected macrophages were transfected with si RELA or siNS for 48 h and then treated with vehicle, Tat, gp120, RNA40, or RNA41 (R41) for 24 h. (Left) Representative Western blots of TREM1 with antibodies against TREM1 and ACTB. (Right) Densitometric analysis of blots, n = 4.

    Article Snippet: Macrophages were transfected with Thermo Fisher Silencer Select BCL2 (identifier [ID] s1916), RELA (ID s11915), TREM1 (ID s28910), or control (catalog no. 4390846) siRNA (siNS) using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher) in Opti-MEM (Gibco) according to the manufacturer’s instructions.

    Techniques: Western Blot, Transfection, Blocking Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using lipofectamine reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p

    Journal: Nutrition & Metabolism

    Article Title: Mice subjected to aP2-Cre mediated ablation of microsomal triglyceride transfer protein are resistant to high fat diet induced obesity

    doi: 10.1186/s12986-016-0061-6

    Figure Lengend Snippet: RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using lipofectamine reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p

    Article Snippet: Cell pellets were resuspended in 200 μl of OptiMEM (Gibco, Life Technologies) and incubated at 37 °C with equal volume of lipofectamine complexes (RNAiMax; Life Technologies) containing siRNA (100 nM) or plasmid DNA (1 μg/ml).

    Techniques: Over Expression, Transfection, Expressing, Activity Assay, Plasmid Preparation, Homogenization