in vitro rna transcription Thermo Fisher Search Results


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  • 99
    Thermo Fisher rna
    miR-21 expression in B6.Sle123 is regulated transcriptionally and correlates with age and disease severity pre-miR-21 and mature miR-21 expression in B6.Sle123 splenic B cells. Twenty micrograms of total splenic B cell <t>RNA</t> from individual age-matched 2, 6 and 12 months old B6.Sle123 and B6 mice were analysed under denaturing conditions, transferred and hybridized with LNA probe complementary to miR-21. For mass-normalization, the same membrane was stripped and re-probed with mouse snoRNA-429 radiolabelled DNA probe. The data were normalized to snoRNA-429 expression and relative fold-expression values calculated by taking the B6.Sle123/B6 ratio. A shorter exposure of the same membrane was used for the detection of the miR-21. B6.Sle123 disease severity correlates with age: BUN of 2, 6 and 12 months old female B6 and B6.Sle123 mice used as a measure of disease severity. Comparison of spleens extracted from 6 months old B6 and B6.Sle123 mice showing correlation of BUN and splenomegaly. <t>qRT-PCR</t> amplification of primary miR-21 (pri-miR-21) using splenic B cell RNA from individual, age-matched 9 months old B6.Sle123 and B6 mice. The average of three individual experiments is shown. Error bars represent SEM.
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    Thermo Fisher capped rna
    miR-21 expression in B6.Sle123 is regulated transcriptionally and correlates with age and disease severity pre-miR-21 and mature miR-21 expression in B6.Sle123 splenic B cells. Twenty micrograms of total splenic B cell <t>RNA</t> from individual age-matched 2, 6 and 12 months old B6.Sle123 and B6 mice were analysed under denaturing conditions, transferred and hybridized with LNA probe complementary to miR-21. For mass-normalization, the same membrane was stripped and re-probed with mouse snoRNA-429 radiolabelled DNA probe. The data were normalized to snoRNA-429 expression and relative fold-expression values calculated by taking the B6.Sle123/B6 ratio. A shorter exposure of the same membrane was used for the detection of the miR-21. B6.Sle123 disease severity correlates with age: BUN of 2, 6 and 12 months old female B6 and B6.Sle123 mice used as a measure of disease severity. Comparison of spleens extracted from 6 months old B6 and B6.Sle123 mice showing correlation of BUN and splenomegaly. <t>qRT-PCR</t> amplification of primary miR-21 (pri-miR-21) using splenic B cell RNA from individual, age-matched 9 months old B6.Sle123 and B6 mice. The average of three individual experiments is shown. Error bars represent SEM.
    Capped Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna to ct kit
    Mosquito cells produce host reverse transcriptase-dependent arbovirus-derived DNA. ( a ) Schematic of CHIKV viral genome. Top arrows indicate the position of the genomic and subgenomic promoters. Bottom arrows indicate the position of the primers used for vDNA detection. ( b ) Kinetics of vDNA synthesis. C6/36, U4.4 and Aag2 cells were infected with CHIKV at a MOI of 0.1 and cells were harvested at the indicated time points. Cells were analysed by <t>PCR</t> (upper panel) for vDNA detection. RT-PCR (lower panel) was used to follow viral infections. Non-infected cells (n.i) were used as a negative control and cellular 18S rRNA was used as a housekeeping gene loading control. ( c ) AZT inhibits vDNA synthesis in vitro . C6/36, U4.4 and Aag2 cells were treated with increasing concentration of AZT for 2 days. At the indicated time point, cells were harvested and vDNA and <t>RNA</t> were assessed as described in b . ( d – e ) Endogenous reverse transcriptase activity in insect cells. ( d ) Dose-dependent AZT inhibition was tested in insect cell extracts and ( e ) quantification of reverse transcriptase inhibition expressed as an activity percentage of non-treated extracts. Heat inactivated samples (heat) were used as negative controls. Drosophila S2 cells were used as a positive control. Each experiment was completed at least 3 times. Error bars correspond to the s.d. t -test with Welch's correction was used to determine statistical significance compared with the untreated control as a reference (** P
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    Thermo Fisher t7 rna
    Mosquito cells produce host reverse transcriptase-dependent arbovirus-derived DNA. ( a ) Schematic of CHIKV viral genome. Top arrows indicate the position of the genomic and subgenomic promoters. Bottom arrows indicate the position of the primers used for vDNA detection. ( b ) Kinetics of vDNA synthesis. C6/36, U4.4 and Aag2 cells were infected with CHIKV at a MOI of 0.1 and cells were harvested at the indicated time points. Cells were analysed by <t>PCR</t> (upper panel) for vDNA detection. RT-PCR (lower panel) was used to follow viral infections. Non-infected cells (n.i) were used as a negative control and cellular 18S rRNA was used as a housekeeping gene loading control. ( c ) AZT inhibits vDNA synthesis in vitro . C6/36, U4.4 and Aag2 cells were treated with increasing concentration of AZT for 2 days. At the indicated time point, cells were harvested and vDNA and <t>RNA</t> were assessed as described in b . ( d – e ) Endogenous reverse transcriptase activity in insect cells. ( d ) Dose-dependent AZT inhibition was tested in insect cell extracts and ( e ) quantification of reverse transcriptase inhibition expressed as an activity percentage of non-treated extracts. Heat inactivated samples (heat) were used as negative controls. Drosophila S2 cells were used as a positive control. Each experiment was completed at least 3 times. Error bars correspond to the s.d. t -test with Welch's correction was used to determine statistical significance compared with the untreated control as a reference (** P
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    Thermo Fisher totalprep rna amplification kit
    Myc regulates the metabolic transcriptome. (A) Unsupervised hierarchical clustering of <t>Illumina</t> bead arrays made from <t>RNA</t> of splenic B cells from three wildtype and four precancerous λ- Myc transgenic mice. See Table S1 for genes used in the clustering. (B) qRT-PCR confirmation of 4 of the 20 most significantly, or most elevated expressed genes, in B220-sorted B cells from λ- Myc transgenic mice. *indicates p
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    Thermo Fisher rna storage solution
    Sequence-specific <t>RNA</t> cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by <t>the</t> arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.
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    Thermo Fisher messageamp rna kit
    Sequence-specific <t>RNA</t> cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by <t>the</t> arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.
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    Thermo Fisher rna precipitation
    MEG3 and <t>PTBP1</t> induce Cyp7a1 and Cyp8b1 expression by downregulating SHP (A–B) qPCR of SHP mRNA in Huh7 cells overexpressed with pcDNA3 (−), MEG3 <t>RNA</t> (A) or PTBP1 (B) for 48 h. (C–D) qPCR of SHP (C), Cyp7a1, Cyp8b1 and FXR mRNA (D) in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA or PTBP1, alone or in combination for 24 h. (E) Western blot of PTBP1 protein in Huh7 cells transfected with control (Con) or siRNA against PTBP1. (F) qPCR of SHP and Cyp8b1 mRNA in Huh7 cells overexpressed with MEG3 in the presence or absence of siPTBP1. Data are shown in mean ± SEM from triplicate assays. * p
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    Thermo Fisher t7 rna synthesis kit
    MEG3 and <t>PTBP1</t> induce Cyp7a1 and Cyp8b1 expression by downregulating SHP (A–B) qPCR of SHP mRNA in Huh7 cells overexpressed with pcDNA3 (−), MEG3 <t>RNA</t> (A) or PTBP1 (B) for 48 h. (C–D) qPCR of SHP (C), Cyp7a1, Cyp8b1 and FXR mRNA (D) in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA or PTBP1, alone or in combination for 24 h. (E) Western blot of PTBP1 protein in Huh7 cells transfected with control (Con) or siRNA against PTBP1. (F) qPCR of SHP and Cyp8b1 mRNA in Huh7 cells overexpressed with MEG3 in the presence or absence of siPTBP1. Data are shown in mean ± SEM from triplicate assays. * p
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    Thermo Fisher in vitro rna transcription
    MEG3 and <t>PTBP1</t> induce Cyp7a1 and Cyp8b1 expression by downregulating SHP (A–B) qPCR of SHP mRNA in Huh7 cells overexpressed with pcDNA3 (−), MEG3 <t>RNA</t> (A) or PTBP1 (B) for 48 h. (C–D) qPCR of SHP (C), Cyp7a1, Cyp8b1 and FXR mRNA (D) in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA or PTBP1, alone or in combination for 24 h. (E) Western blot of PTBP1 protein in Huh7 cells transfected with control (Con) or siRNA against PTBP1. (F) qPCR of SHP and Cyp8b1 mRNA in Huh7 cells overexpressed with MEG3 in the presence or absence of siPTBP1. Data are shown in mean ± SEM from triplicate assays. * p
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    Thermo Fisher t7 rna promoter
    MEG3 and <t>PTBP1</t> induce Cyp7a1 and Cyp8b1 expression by downregulating SHP (A–B) qPCR of SHP mRNA in Huh7 cells overexpressed with pcDNA3 (−), MEG3 <t>RNA</t> (A) or PTBP1 (B) for 48 h. (C–D) qPCR of SHP (C), Cyp7a1, Cyp8b1 and FXR mRNA (D) in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA or PTBP1, alone or in combination for 24 h. (E) Western blot of PTBP1 protein in Huh7 cells transfected with control (Con) or siRNA against PTBP1. (F) qPCR of SHP and Cyp8b1 mRNA in Huh7 cells overexpressed with MEG3 in the presence or absence of siPTBP1. Data are shown in mean ± SEM from triplicate assays. * p
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    Thermo Fisher dephosphorylated rna
    MEG3 and <t>PTBP1</t> induce Cyp7a1 and Cyp8b1 expression by downregulating SHP (A–B) qPCR of SHP mRNA in Huh7 cells overexpressed with pcDNA3 (−), MEG3 <t>RNA</t> (A) or PTBP1 (B) for 48 h. (C–D) qPCR of SHP (C), Cyp7a1, Cyp8b1 and FXR mRNA (D) in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA or PTBP1, alone or in combination for 24 h. (E) Western blot of PTBP1 protein in Huh7 cells transfected with control (Con) or siRNA against PTBP1. (F) qPCR of SHP and Cyp8b1 mRNA in Huh7 cells overexpressed with MEG3 in the presence or absence of siPTBP1. Data are shown in mean ± SEM from triplicate assays. * p
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    Thermo Fisher quant it ribogreen rna assay kit
    MEG3 and <t>PTBP1</t> induce Cyp7a1 and Cyp8b1 expression by downregulating SHP (A–B) qPCR of SHP mRNA in Huh7 cells overexpressed with pcDNA3 (−), MEG3 <t>RNA</t> (A) or PTBP1 (B) for 48 h. (C–D) qPCR of SHP (C), Cyp7a1, Cyp8b1 and FXR mRNA (D) in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA or PTBP1, alone or in combination for 24 h. (E) Western blot of PTBP1 protein in Huh7 cells transfected with control (Con) or siRNA against PTBP1. (F) qPCR of SHP and Cyp8b1 mRNA in Huh7 cells overexpressed with MEG3 in the presence or absence of siPTBP1. Data are shown in mean ± SEM from triplicate assays. * p
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    Thermo Fisher mmachine rna transcription kit
    MEG3 and <t>PTBP1</t> induce Cyp7a1 and Cyp8b1 expression by downregulating SHP (A–B) qPCR of SHP mRNA in Huh7 cells overexpressed with pcDNA3 (−), MEG3 <t>RNA</t> (A) or PTBP1 (B) for 48 h. (C–D) qPCR of SHP (C), Cyp7a1, Cyp8b1 and FXR mRNA (D) in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA or PTBP1, alone or in combination for 24 h. (E) Western blot of PTBP1 protein in Huh7 cells transfected with control (Con) or siRNA against PTBP1. (F) qPCR of SHP and Cyp8b1 mRNA in Huh7 cells overexpressed with MEG3 in the presence or absence of siPTBP1. Data are shown in mean ± SEM from triplicate assays. * p
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    Photomicrographs of brightfield images of sections of pig corpus luteum at day 7 (a and b; scale bars represent 25 μm) and follicle at day 15 (c; scale bar represents 100 μm) of the estrous cycle. An <t>antisense</t> hypoxia inducible factor (HIF)-1α <t>RNA</t> probe was used for hybridization in the ovary. Slides were stained with hematoxylin-eosin (a,b) or periodic acid Schiff stain (c). (a) Hybridization signals, seen as silver grains (black dots), are present in most luteal cells. (b) Hybridization signals were dispersed in the thecal (T) and luteal (L) tissue interface. (c) The intense hybridization signals obscured the granulosa cells (G) and were evident in the thecal layers (T) of a follicle at day 15.
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    Photomicrographs of brightfield images of sections of pig corpus luteum at day 7 (a and b; scale bars represent 25 μm) and follicle at day 15 (c; scale bar represents 100 μm) of the estrous cycle. An <t>antisense</t> hypoxia inducible factor (HIF)-1α <t>RNA</t> probe was used for hybridization in the ovary. Slides were stained with hematoxylin-eosin (a,b) or periodic acid Schiff stain (c). (a) Hybridization signals, seen as silver grains (black dots), are present in most luteal cells. (b) Hybridization signals were dispersed in the thecal (T) and luteal (L) tissue interface. (c) The intense hybridization signals obscured the granulosa cells (G) and were evident in the thecal layers (T) of a follicle at day 15.
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    Photomicrographs of brightfield images of sections of pig corpus luteum at day 7 (a and b; scale bars represent 25 μm) and follicle at day 15 (c; scale bar represents 100 μm) of the estrous cycle. An <t>antisense</t> hypoxia inducible factor (HIF)-1α <t>RNA</t> probe was used for hybridization in the ovary. Slides were stained with hematoxylin-eosin (a,b) or periodic acid Schiff stain (c). (a) Hybridization signals, seen as silver grains (black dots), are present in most luteal cells. (b) Hybridization signals were dispersed in the thecal (T) and luteal (L) tissue interface. (c) The intense hybridization signals obscured the granulosa cells (G) and were evident in the thecal layers (T) of a follicle at day 15.
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    MUC1 is a bona fide target of miR455-3P in BeWo cells. ( a ) Expression of potential miR455 target mRNAs in FSK-treated BeWo cells. Expression of the indicated potential miR455 target mRNAs was analyzed by <t>qRT-PCR.</t> Values were normalized to RPLP0 and displayed relative to DMSO-treated cells. ( b ) MUC1 protein levels are reduced by FSK treatment. MUC1 and TBP protein levels were analyzed by western blotting 48 h after treatment. ( c and d ) MUC1 is repressed by miR455-3P but not miR455-5P. BeWo cells were transfected with synthetic miRNAs at two concentrations (5 and 20 nM). <t>RNA</t> and protein samples were harvested 48 h post-transfection. MUC1 mRNA and protein levels were determined by qRT-PCR ( c ) and western blotting ( d ), respectively. One western blot representative of a transfection with 20 nM synthetic miRNA is presented in d . TBP served as a loading control
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    MUC1 is a bona fide target of miR455-3P in BeWo cells. ( a ) Expression of potential miR455 target mRNAs in FSK-treated BeWo cells. Expression of the indicated potential miR455 target mRNAs was analyzed by <t>qRT-PCR.</t> Values were normalized to RPLP0 and displayed relative to DMSO-treated cells. ( b ) MUC1 protein levels are reduced by FSK treatment. MUC1 and TBP protein levels were analyzed by western blotting 48 h after treatment. ( c and d ) MUC1 is repressed by miR455-3P but not miR455-5P. BeWo cells were transfected with synthetic miRNAs at two concentrations (5 and 20 nM). <t>RNA</t> and protein samples were harvested 48 h post-transfection. MUC1 mRNA and protein levels were determined by qRT-PCR ( c ) and western blotting ( d ), respectively. One western blot representative of a transfection with 20 nM synthetic miRNA is presented in d . TBP served as a loading control
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    Image Search Results


    miR-21 expression in B6.Sle123 is regulated transcriptionally and correlates with age and disease severity pre-miR-21 and mature miR-21 expression in B6.Sle123 splenic B cells. Twenty micrograms of total splenic B cell RNA from individual age-matched 2, 6 and 12 months old B6.Sle123 and B6 mice were analysed under denaturing conditions, transferred and hybridized with LNA probe complementary to miR-21. For mass-normalization, the same membrane was stripped and re-probed with mouse snoRNA-429 radiolabelled DNA probe. The data were normalized to snoRNA-429 expression and relative fold-expression values calculated by taking the B6.Sle123/B6 ratio. A shorter exposure of the same membrane was used for the detection of the miR-21. B6.Sle123 disease severity correlates with age: BUN of 2, 6 and 12 months old female B6 and B6.Sle123 mice used as a measure of disease severity. Comparison of spleens extracted from 6 months old B6 and B6.Sle123 mice showing correlation of BUN and splenomegaly. qRT-PCR amplification of primary miR-21 (pri-miR-21) using splenic B cell RNA from individual, age-matched 9 months old B6.Sle123 and B6 mice. The average of three individual experiments is shown. Error bars represent SEM.

    Journal: EMBO Molecular Medicine

    Article Title: Silencing of microRNA-21 in vivo ameliorates autoimmune splenomegaly in lupus mice

    doi: 10.1002/emmm.201100171

    Figure Lengend Snippet: miR-21 expression in B6.Sle123 is regulated transcriptionally and correlates with age and disease severity pre-miR-21 and mature miR-21 expression in B6.Sle123 splenic B cells. Twenty micrograms of total splenic B cell RNA from individual age-matched 2, 6 and 12 months old B6.Sle123 and B6 mice were analysed under denaturing conditions, transferred and hybridized with LNA probe complementary to miR-21. For mass-normalization, the same membrane was stripped and re-probed with mouse snoRNA-429 radiolabelled DNA probe. The data were normalized to snoRNA-429 expression and relative fold-expression values calculated by taking the B6.Sle123/B6 ratio. A shorter exposure of the same membrane was used for the detection of the miR-21. B6.Sle123 disease severity correlates with age: BUN of 2, 6 and 12 months old female B6 and B6.Sle123 mice used as a measure of disease severity. Comparison of spleens extracted from 6 months old B6 and B6.Sle123 mice showing correlation of BUN and splenomegaly. qRT-PCR amplification of primary miR-21 (pri-miR-21) using splenic B cell RNA from individual, age-matched 9 months old B6.Sle123 and B6 mice. The average of three individual experiments is shown. Error bars represent SEM.

    Article Snippet: For miRNA-specific reverse transcription (RT), 20 ng purified total RNA was reverse-transcribed (TaqMan miRNA-specific RT PCR primers, Applied Biosystems). qRT-PCR was performed in triplicates using 1:10 dilution of the RT product (Applied Biosystems 7500 real-time PCR system, according to the manufacturer's instructions). snoRNA-429 expression was used as control.

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, Amplification

    Silencing of miR-21 by tiny LNA-anti-miR in vivo and in vitro derepresses PDCD4 in B6.Sle123 T cells Three groups of two B6.Sle123 mice were treated in vivo with seed-targeting LNA antimiR-21 or LNA scramble control compound daily for 3 days. miR-21 qRT-PCR quantification using total RNA from CD4 + T cells FACS purified from LNA antimiR-21- and LNA scramble compound-treated mice. Mean RQs of three independent experiments. * p = 8.1 × 10 −6 . Western blot analysis of PDCD4 expression in CD4 + T cells derived from in vivo LNA antimiR-21 treated mice and controls. Results are representative of three independent experiments. Unassisted uptake of LNA antimiR-21 by primary B6.Sle123 CD4 + T cells maintained in culture. Cells were purified, cultured in the presence of tiny seed-targeting LNA antimiR-21 or control compound as described in Materials and Methods section. At selected time points, cells were harvested and miR-21 expression was assessed by real-time PCR. PDCD4 expression in CD4 + T cells treated in vitro with seed-targeting LNA antimiR-21 or control compound.

    Journal: EMBO Molecular Medicine

    Article Title: Silencing of microRNA-21 in vivo ameliorates autoimmune splenomegaly in lupus mice

    doi: 10.1002/emmm.201100171

    Figure Lengend Snippet: Silencing of miR-21 by tiny LNA-anti-miR in vivo and in vitro derepresses PDCD4 in B6.Sle123 T cells Three groups of two B6.Sle123 mice were treated in vivo with seed-targeting LNA antimiR-21 or LNA scramble control compound daily for 3 days. miR-21 qRT-PCR quantification using total RNA from CD4 + T cells FACS purified from LNA antimiR-21- and LNA scramble compound-treated mice. Mean RQs of three independent experiments. * p = 8.1 × 10 −6 . Western blot analysis of PDCD4 expression in CD4 + T cells derived from in vivo LNA antimiR-21 treated mice and controls. Results are representative of three independent experiments. Unassisted uptake of LNA antimiR-21 by primary B6.Sle123 CD4 + T cells maintained in culture. Cells were purified, cultured in the presence of tiny seed-targeting LNA antimiR-21 or control compound as described in Materials and Methods section. At selected time points, cells were harvested and miR-21 expression was assessed by real-time PCR. PDCD4 expression in CD4 + T cells treated in vitro with seed-targeting LNA antimiR-21 or control compound.

    Article Snippet: For miRNA-specific reverse transcription (RT), 20 ng purified total RNA was reverse-transcribed (TaqMan miRNA-specific RT PCR primers, Applied Biosystems). qRT-PCR was performed in triplicates using 1:10 dilution of the RT product (Applied Biosystems 7500 real-time PCR system, according to the manufacturer's instructions). snoRNA-429 expression was used as control.

    Techniques: In Vivo, In Vitro, Mouse Assay, Quantitative RT-PCR, FACS, Purification, Western Blot, Expressing, Derivative Assay, Cell Culture, Real-time Polymerase Chain Reaction

    Detection of in vitro-transcribed HDV control RNA by RT-PCR and confirmation that the DNA plasmid template was efficiently removed. (A) Lanes 1 to 10, RT-PCR amplification products from a 10-fold serial dilution of in vitro-transcribed HDV RNA. The dilution series extended from 0.1 μg of RNA per RT-PCR mixture in lane 1 to 0.1 fg in lane 10. The amplification primers were delta-1 and delta-2, which gave a product of 761 bp. Lanes 11 to 20, as for lanes 1 to 10, but without RT of the template RNA to detect any contaminating plasmid template DNA; lane 21, negative control; lane L, 1-kb ladder (Bio-Rad). Sizes (in base pairs) are indicated on the left. A total of 10 μl of each 50-μl PCR mixture was analyzed. (B) As for panel A, but showing the amplification products of a nested PCR performed with the templates described in the legend to panel A and primers delta-3 and delta-4 to obtain a product of 506 bp. The detection limit is shown in lane 8 and is 10 fg of RNA, which corresponds to approximately 10,800 molecules of RNA added to the extract.

    Journal: Journal of Clinical Microbiology

    Article Title: Stable and Noncompetitive RNA Internal Control for Routine Clinical Diagnostic Reverse Transcription-PCR

    doi: 10.1128/JCM.42.3.1003-1011.2004

    Figure Lengend Snippet: Detection of in vitro-transcribed HDV control RNA by RT-PCR and confirmation that the DNA plasmid template was efficiently removed. (A) Lanes 1 to 10, RT-PCR amplification products from a 10-fold serial dilution of in vitro-transcribed HDV RNA. The dilution series extended from 0.1 μg of RNA per RT-PCR mixture in lane 1 to 0.1 fg in lane 10. The amplification primers were delta-1 and delta-2, which gave a product of 761 bp. Lanes 11 to 20, as for lanes 1 to 10, but without RT of the template RNA to detect any contaminating plasmid template DNA; lane 21, negative control; lane L, 1-kb ladder (Bio-Rad). Sizes (in base pairs) are indicated on the left. A total of 10 μl of each 50-μl PCR mixture was analyzed. (B) As for panel A, but showing the amplification products of a nested PCR performed with the templates described in the legend to panel A and primers delta-3 and delta-4 to obtain a product of 506 bp. The detection limit is shown in lane 8 and is 10 fg of RNA, which corresponds to approximately 10,800 molecules of RNA added to the extract.

    Article Snippet: A protein coat could be added to modified HDV RNA, possibly by using phage proteins, such as those in Armored RNA (Ambion [Europe Ltd.]) ( ).

    Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Amplification, Serial Dilution, Negative Control, Polymerase Chain Reaction, Nested PCR

    The HDV internal control did not compromise the limit of detection of the assay. Nine plasmids containing each of the first-round PCR amplification products were constructed. A dilution series containing 10 2 to 10 −1 molecules of each plasmid per μl was made, and 1 μl of each dilution was amplified in each nested multiplex PCR. (A) RSV A, RSV B, and HuMV; (B) influenza A and B viruses; (C) PIV-1 to PIV-4. To determine whether the HDV internal control compromised the limit of detection, the PCR mixture also contained the HDV internal control cDNA (reverse-transcribed RNA, not plasmid HDV DNA) at the same concentration used in the diagnostic assay. The virus amplicons are indicated by arrowheads, and the HDV control is indicated by asterisks. As the concentration of the plasmid containing the target virus sequences was increased, the yield of the control amplicon decreased or disappeared completely. For comparison, a PCR mixture containing HDV cDNA alone was performed (lanes δ). neg, negative controls lacking target DNA.

    Journal: Journal of Clinical Microbiology

    Article Title: Stable and Noncompetitive RNA Internal Control for Routine Clinical Diagnostic Reverse Transcription-PCR

    doi: 10.1128/JCM.42.3.1003-1011.2004

    Figure Lengend Snippet: The HDV internal control did not compromise the limit of detection of the assay. Nine plasmids containing each of the first-round PCR amplification products were constructed. A dilution series containing 10 2 to 10 −1 molecules of each plasmid per μl was made, and 1 μl of each dilution was amplified in each nested multiplex PCR. (A) RSV A, RSV B, and HuMV; (B) influenza A and B viruses; (C) PIV-1 to PIV-4. To determine whether the HDV internal control compromised the limit of detection, the PCR mixture also contained the HDV internal control cDNA (reverse-transcribed RNA, not plasmid HDV DNA) at the same concentration used in the diagnostic assay. The virus amplicons are indicated by arrowheads, and the HDV control is indicated by asterisks. As the concentration of the plasmid containing the target virus sequences was increased, the yield of the control amplicon decreased or disappeared completely. For comparison, a PCR mixture containing HDV cDNA alone was performed (lanes δ). neg, negative controls lacking target DNA.

    Article Snippet: A protein coat could be added to modified HDV RNA, possibly by using phage proteins, such as those in Armored RNA (Ambion [Europe Ltd.]) ( ).

    Techniques: Polymerase Chain Reaction, Amplification, Construct, Plasmid Preparation, Multiplex Assay, Concentration Assay, Diagnostic Assay

    Target-driven biogenesis occurs on the rER-attached polysomes. (A) Scheme of digitonin-based membrane isolation from HEK 293 cells. (B) Relative steady-state distribution of miR-122 in total, cytosol, and digitonin-insoluble membranes in HEK 293 cells exogenously expressing the liver miRNA-122 in the presence of its target RL-3xbulge-miR-122 and control RL-con mRNA. Northern blot ( left panel ) where ethidium bromide staining of the gel used for Northern blotting of miRNA and U6 are shown. Quantification of the same ( right panel ) against the U6 RNA has been shown. (C) In vitro RISC cleavage assay with FH-Ago2 IP from cytosolic and membrane fractions. Membrane-associated miRISC isolated from target RNA–expressing cells showed higher specific activity for substrate RNA cleavage. The cleaved RNA products are marked by an arrowhead. (D) Target mRNA–induced de novo miRNPs get enriched on microsomes. Northern blotting was used to detect mature miR-122 and U6. RISC cleavage assay was performed with protein equivalent amounts of FH-Ago2 IP. (E) Target mRNA–driven de novo–formed miRNAs are present in the ER fraction. Relative distribution of newly synthesized miR-122 as well as endogenous let-7a in the presence of miR-122 target mRNA. From the 3–30% OptiPrep gradient, fractions 7, 8, and 9 were pooled to get rER-associated miRNA, whereas fractions 1–3 and fractions 4–6 were enriched, respectively, for endosome/MVBs– and lysosome-associated miRNAs (as described in Fig 1C and D ). Amounts of RNA used for quantification in RT-PCR reaction were identical. (F) Inducible miR-122 associates with microsome in the presence of target mRNA. Real-time quantification of mature miR-122 present in microsomal fraction and in total after 24 h of induction in the presence of RL-3xBulge-miR-122 and control RL-3xBulge-let-7a were measured and plotted. (G, H) Increase in de novo–formed mature miR-122 level with time in the presence of its target mRNA. Levels of miR-122 were quantified and plotted after 0, 12, 16, 20, and 24 h of induction. (G) Values obtained with total cellular RNA were plotted in panel (G). (H) Quantifications of microsome and polysome-associated miR-122 are represented in panel (H). In both cases, the level at 0 h is taken as one unit. (I) De novo–formed miR-122 associates with rER-bound Ago2 in the presence of target mRNA. Tet-On–stable HEK 293 cells were transiently transfected with FH-Ago2, inducible miR-122, and RL-3xbulge-miR-122–expressing plasmids. FH-Ago2 IP from cytosolic and microsome after induction were used for quantification of associated mature miR-122. In all cases, the level at 0 h is taken as one unit. (J) The newly formed miRNPs are functional. Repression kinetics of RL-3xbulge-miR-122 after induction of de novo synthesis of miR-122 was followed and quantified. Tet-On–stable HEK 293 cells were transfected with either RL-con or RL-3xbulge-miR-122 and inducible miR-122 expression plasmids. After 0, 2, 4, 12, and 24 h of induction for miR-122 expression, Renilla Luciferase expression in cells transfected with RL-con or RL-3xbulge-miR-122 was measured in the dual luciferase assay. Firefly luciferase–expressing plasmids were co-transfected in all cases, and relative luciferase activity in 0 h was taken as unit. In all cases, Renilla luciferase (RL) expression were normalized by firefly expression. Paired two-tailed t tests were used for all comparisons. P

    Journal: Life Science Alliance

    Article Title: Retrograde trafficking of Argonaute 2 acts as a rate-limiting step for de novo miRNP formation on endoplasmic reticulum–attached polysomes in mammalian cells

    doi: 10.26508/lsa.201800161

    Figure Lengend Snippet: Target-driven biogenesis occurs on the rER-attached polysomes. (A) Scheme of digitonin-based membrane isolation from HEK 293 cells. (B) Relative steady-state distribution of miR-122 in total, cytosol, and digitonin-insoluble membranes in HEK 293 cells exogenously expressing the liver miRNA-122 in the presence of its target RL-3xbulge-miR-122 and control RL-con mRNA. Northern blot ( left panel ) where ethidium bromide staining of the gel used for Northern blotting of miRNA and U6 are shown. Quantification of the same ( right panel ) against the U6 RNA has been shown. (C) In vitro RISC cleavage assay with FH-Ago2 IP from cytosolic and membrane fractions. Membrane-associated miRISC isolated from target RNA–expressing cells showed higher specific activity for substrate RNA cleavage. The cleaved RNA products are marked by an arrowhead. (D) Target mRNA–induced de novo miRNPs get enriched on microsomes. Northern blotting was used to detect mature miR-122 and U6. RISC cleavage assay was performed with protein equivalent amounts of FH-Ago2 IP. (E) Target mRNA–driven de novo–formed miRNAs are present in the ER fraction. Relative distribution of newly synthesized miR-122 as well as endogenous let-7a in the presence of miR-122 target mRNA. From the 3–30% OptiPrep gradient, fractions 7, 8, and 9 were pooled to get rER-associated miRNA, whereas fractions 1–3 and fractions 4–6 were enriched, respectively, for endosome/MVBs– and lysosome-associated miRNAs (as described in Fig 1C and D ). Amounts of RNA used for quantification in RT-PCR reaction were identical. (F) Inducible miR-122 associates with microsome in the presence of target mRNA. Real-time quantification of mature miR-122 present in microsomal fraction and in total after 24 h of induction in the presence of RL-3xBulge-miR-122 and control RL-3xBulge-let-7a were measured and plotted. (G, H) Increase in de novo–formed mature miR-122 level with time in the presence of its target mRNA. Levels of miR-122 were quantified and plotted after 0, 12, 16, 20, and 24 h of induction. (G) Values obtained with total cellular RNA were plotted in panel (G). (H) Quantifications of microsome and polysome-associated miR-122 are represented in panel (H). In both cases, the level at 0 h is taken as one unit. (I) De novo–formed miR-122 associates with rER-bound Ago2 in the presence of target mRNA. Tet-On–stable HEK 293 cells were transiently transfected with FH-Ago2, inducible miR-122, and RL-3xbulge-miR-122–expressing plasmids. FH-Ago2 IP from cytosolic and microsome after induction were used for quantification of associated mature miR-122. In all cases, the level at 0 h is taken as one unit. (J) The newly formed miRNPs are functional. Repression kinetics of RL-3xbulge-miR-122 after induction of de novo synthesis of miR-122 was followed and quantified. Tet-On–stable HEK 293 cells were transfected with either RL-con or RL-3xbulge-miR-122 and inducible miR-122 expression plasmids. After 0, 2, 4, 12, and 24 h of induction for miR-122 expression, Renilla Luciferase expression in cells transfected with RL-con or RL-3xbulge-miR-122 was measured in the dual luciferase assay. Firefly luciferase–expressing plasmids were co-transfected in all cases, and relative luciferase activity in 0 h was taken as unit. In all cases, Renilla luciferase (RL) expression were normalized by firefly expression. Paired two-tailed t tests were used for all comparisons. P

    Article Snippet: RNA isolation, Northern blotting, and real-time PCR analysis TRIzol reagent (Life Technologies) was used to isolate total RNA.

    Techniques: Isolation, Expressing, Northern Blot, Staining, In Vitro, Cleavage Assay, Activity Assay, Synthesized, Reverse Transcription Polymerase Chain Reaction, Transfection, Functional Assay, Luciferase, Two Tailed Test

    Defective de novo miRNP formation in mitochondria-depolarized cells. (A) Scheme of the experiment used to determine the de novo rate of Ago2-miR-122 miRNP formation using doxycycline-inducible Tet-On HEK 293 cells. The Tet-ON HEK 293 cells were transfected with doxycycline-inducible pre-miR-122 plasmid construct (ipmiR-122). Doxycycline was added to the cells for 48 h after transfection. The cells were harvested after doxycycline induction of indicated time periods, and immunoprecipitation was performed for endogenous Ago2 protein. miRNA estimation and relative quantification were performed for IPed Ago2-associated miRNA by qRT-PCR. (B, C) Values of Ago2-associated miR-122, mature miR-122, or precursor-miR-122 in Tet-on HEK 293 cells induced with doxycycline for specified time periods. Shown are the mean and SEM values from at least three independent experiments. (B, C) All cells were co-transfected with Renilla-3xbulge-miR-122 reporter, Tet-inducible pre-miR-122 expression plasmid ipmiR-122, and FH-Ucp2 (panel B) or siMfn2 (panel C). IPed Ago2 level, U6 small nuclear RNA level, and 18S ribosomal RNA level were used as internal controls for the estimation of Ago2-associated miR-122, mature miR-122, and precursor-miR-122, respectively. All values have been normalized against values at 0 h. (D) Shown are the mean and SEM from at least four independent experiments with values of Ago2-associated miR-122 in an iodixanol OptiPrepR (3–30%) gradient fractions positive for early endosome (EE) and ER markers. Lysate was obtained from TET-ON HEK 293 cells induced with doxycycline for 24 h. All cells were co-transfected with Renilla-3xbulge-miR-122 reporter, Tet-inducible pre-miR-122 expressing ipmiR-122, and either FH-Ucp2 or siMfn2. miR-122 present in ER fractions from each set were normalized against corresponding EE-associated miR-122 levels. (E, F) Target mRNA–driven miRNA biogenesis is hampered in mitochondria–ER uncoupled condition in Huh7 cells. (E) Scheme of the experiment is shown in panel (E). qRT-PCR data have confirmed no up-regulation of miR-122 biogenesis in the presence of its target under Ucp2 over-expression condition during amino acid re-feeding (F, left panel). CAT-1 mRNA level was also increased after 4 h of amino acid starvation on HA-Ucp2–expressing cells (F, right panel). (G) Western Blot data confirm HA-Ucp2 over-expression on Huh7 cells. (H) Pre-miR-122 expression in starved versus re-fed conditions in HA-Ucp2–expressed cells. Reduced pre-miRNA processing was observed upon re-fed of Ucp2 over-expressed cells compared with control. Paired two-tailed t tests were used for all comparisons. P

    Journal: Life Science Alliance

    Article Title: Retrograde trafficking of Argonaute 2 acts as a rate-limiting step for de novo miRNP formation on endoplasmic reticulum–attached polysomes in mammalian cells

    doi: 10.26508/lsa.201800161

    Figure Lengend Snippet: Defective de novo miRNP formation in mitochondria-depolarized cells. (A) Scheme of the experiment used to determine the de novo rate of Ago2-miR-122 miRNP formation using doxycycline-inducible Tet-On HEK 293 cells. The Tet-ON HEK 293 cells were transfected with doxycycline-inducible pre-miR-122 plasmid construct (ipmiR-122). Doxycycline was added to the cells for 48 h after transfection. The cells were harvested after doxycycline induction of indicated time periods, and immunoprecipitation was performed for endogenous Ago2 protein. miRNA estimation and relative quantification were performed for IPed Ago2-associated miRNA by qRT-PCR. (B, C) Values of Ago2-associated miR-122, mature miR-122, or precursor-miR-122 in Tet-on HEK 293 cells induced with doxycycline for specified time periods. Shown are the mean and SEM values from at least three independent experiments. (B, C) All cells were co-transfected with Renilla-3xbulge-miR-122 reporter, Tet-inducible pre-miR-122 expression plasmid ipmiR-122, and FH-Ucp2 (panel B) or siMfn2 (panel C). IPed Ago2 level, U6 small nuclear RNA level, and 18S ribosomal RNA level were used as internal controls for the estimation of Ago2-associated miR-122, mature miR-122, and precursor-miR-122, respectively. All values have been normalized against values at 0 h. (D) Shown are the mean and SEM from at least four independent experiments with values of Ago2-associated miR-122 in an iodixanol OptiPrepR (3–30%) gradient fractions positive for early endosome (EE) and ER markers. Lysate was obtained from TET-ON HEK 293 cells induced with doxycycline for 24 h. All cells were co-transfected with Renilla-3xbulge-miR-122 reporter, Tet-inducible pre-miR-122 expressing ipmiR-122, and either FH-Ucp2 or siMfn2. miR-122 present in ER fractions from each set were normalized against corresponding EE-associated miR-122 levels. (E, F) Target mRNA–driven miRNA biogenesis is hampered in mitochondria–ER uncoupled condition in Huh7 cells. (E) Scheme of the experiment is shown in panel (E). qRT-PCR data have confirmed no up-regulation of miR-122 biogenesis in the presence of its target under Ucp2 over-expression condition during amino acid re-feeding (F, left panel). CAT-1 mRNA level was also increased after 4 h of amino acid starvation on HA-Ucp2–expressing cells (F, right panel). (G) Western Blot data confirm HA-Ucp2 over-expression on Huh7 cells. (H) Pre-miR-122 expression in starved versus re-fed conditions in HA-Ucp2–expressed cells. Reduced pre-miRNA processing was observed upon re-fed of Ucp2 over-expressed cells compared with control. Paired two-tailed t tests were used for all comparisons. P

    Article Snippet: RNA isolation, Northern blotting, and real-time PCR analysis TRIzol reagent (Life Technologies) was used to isolate total RNA.

    Techniques: Transfection, Plasmid Preparation, Construct, Immunoprecipitation, Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Two Tailed Test

    Attachment of de novo–formed miRNAs with ribosomes attached with the rER membrane. (A, B) Active miRNPs are membrane associated. FH-Ago2 was immunoprecipitated (IP) from total cell lysate as well as digitonin-soluble cytosolic and insoluble membrane fractions after fractionation of cells performed by treating with 50 μg/ml of digitonin. The IPed materials obtained from cell equivalent amount of each fraction were used in the in vitro RISC cleavage assays. Densitometric quantification of the cleaved RNA band was used for calculating specific activity of miR-122 and normalized against the amount of Ago2 IP following procedures described in the Materials and Methods section and plotted. Representative autoradiogram data obtained from one experimental set have been shown in panel (A) along with the Western blot data for IP FH-Ago2. The relative amount of Ago2 present in each reaction is shown in the Western blots showing levels of FH-Ago2 and GAPDH in panel (B). The FH-Ago2 detected with anti-HA antibody. RISC(-) is the reaction done without any addition of RISC. In Total (3×) reaction, threefold excesss of cytosolic fractions was used. (C, D) Subcellular distribution of miRNP components in human cells. FH-Ago2 stable HEK 293 cells transiently expressing miR-122 were lysed under isotonic conditions and subjected to ultracentrifugation on a 3–30% iodixanol gradient for separation of subcellular organelles. Hepatocyte growth actor-regulated tyrosine kinase substrate (HRS), calnexin, LAMP1, COX4, and Rab7 were used as markers of endosomes/multivesicular bodies (MVBs), rER, lysosomes, mitochondria, and late endosomes, respectively. Subcellular distribution of Dicer1 has been observed. FH-Ago2 was IPed from individual fractions using FLAG-specific antibody, and IPed materials were used for in vitro RISC cleavage assay. Specific activity of miR-122 RISC in each fraction has been plotted by normalizing the RISC activity present against the amount of Ago2 precipitated from each fraction. Pre-miR-122 levels in individual fractions have been quantified by qRT-PCR. Values for each fraction have been plotted as percentage of total miR-122 calculated by summing miR-122 present in all the fractions. Positions of ribosomal RNA bands are marked by * in the ethidium bromide–stained gel shown here. (E, F) Mature miRNAs are rER-enriched in HEK 293 cells. Relative enrichment of miR-16, miR-21, and let-7a as determined in isolated microsome by qRT-PCR when compared with equal amount of RNA from total cell lysate. Presence of Ago2 and DICER1 in microsome fractions was determined by Western blot. Absence of GAPDH (cytosolic marker) and β-Actin and presence of calnexin (rER marker) in the microsomal fraction confirmed the purity of isolated microsome as compared with protein equivalent amount of non-microsomal or total cell extract. (G) Schematic presentation of isolation of polysomes using KCl–puromycin based on HEK 293 cells. Microsomes isolated from HEK 293 cells were treated with 500 mM KCl and 1 mM puromycin before ultracentrifugation at 100,000 g for 1 h to separate the ribosomal and non-ribosomal pool. (H) miRNP and target mRNAs are associated with the rER-attached ribosomes. Western blot data show the distribution of Ago2 in the soluble and pellet fractions after treatment and centrifugations. Calnexin was used as the ER marker protein. Mature miR-122 as well as its target mRNA RL-3xbulge-miR-122 distribution in the abovementioned pellet and soluble fractions were determined by qRT-PCR. (I) Exclusiveness of the Ago2 and miRNA extraction with KCl–puromycin. Treated and non-treated extracts were analyzed by Western blots using cell equivalent amounts for each fraction. ER (calnexin), ribosome (S3), and endosomes/MVBs (HRS) marker protein distributions were checked to observe the specificity and purity of the isolation method. Western blot data of HRS protein suggest that minimum contamination of endosomal fractions in the microsome that can account for the observed Ago2 that was found to be extracted with ribosome upon extraction with KCl and puromycin. (J) Relative amount of let-7a miRNA associated with soluble and pellet fractions with or without KCl–puromycin treatment was measured. Paired two-tailed t tests were used for all comparisons. P

    Journal: Life Science Alliance

    Article Title: Retrograde trafficking of Argonaute 2 acts as a rate-limiting step for de novo miRNP formation on endoplasmic reticulum–attached polysomes in mammalian cells

    doi: 10.26508/lsa.201800161

    Figure Lengend Snippet: Attachment of de novo–formed miRNAs with ribosomes attached with the rER membrane. (A, B) Active miRNPs are membrane associated. FH-Ago2 was immunoprecipitated (IP) from total cell lysate as well as digitonin-soluble cytosolic and insoluble membrane fractions after fractionation of cells performed by treating with 50 μg/ml of digitonin. The IPed materials obtained from cell equivalent amount of each fraction were used in the in vitro RISC cleavage assays. Densitometric quantification of the cleaved RNA band was used for calculating specific activity of miR-122 and normalized against the amount of Ago2 IP following procedures described in the Materials and Methods section and plotted. Representative autoradiogram data obtained from one experimental set have been shown in panel (A) along with the Western blot data for IP FH-Ago2. The relative amount of Ago2 present in each reaction is shown in the Western blots showing levels of FH-Ago2 and GAPDH in panel (B). The FH-Ago2 detected with anti-HA antibody. RISC(-) is the reaction done without any addition of RISC. In Total (3×) reaction, threefold excesss of cytosolic fractions was used. (C, D) Subcellular distribution of miRNP components in human cells. FH-Ago2 stable HEK 293 cells transiently expressing miR-122 were lysed under isotonic conditions and subjected to ultracentrifugation on a 3–30% iodixanol gradient for separation of subcellular organelles. Hepatocyte growth actor-regulated tyrosine kinase substrate (HRS), calnexin, LAMP1, COX4, and Rab7 were used as markers of endosomes/multivesicular bodies (MVBs), rER, lysosomes, mitochondria, and late endosomes, respectively. Subcellular distribution of Dicer1 has been observed. FH-Ago2 was IPed from individual fractions using FLAG-specific antibody, and IPed materials were used for in vitro RISC cleavage assay. Specific activity of miR-122 RISC in each fraction has been plotted by normalizing the RISC activity present against the amount of Ago2 precipitated from each fraction. Pre-miR-122 levels in individual fractions have been quantified by qRT-PCR. Values for each fraction have been plotted as percentage of total miR-122 calculated by summing miR-122 present in all the fractions. Positions of ribosomal RNA bands are marked by * in the ethidium bromide–stained gel shown here. (E, F) Mature miRNAs are rER-enriched in HEK 293 cells. Relative enrichment of miR-16, miR-21, and let-7a as determined in isolated microsome by qRT-PCR when compared with equal amount of RNA from total cell lysate. Presence of Ago2 and DICER1 in microsome fractions was determined by Western blot. Absence of GAPDH (cytosolic marker) and β-Actin and presence of calnexin (rER marker) in the microsomal fraction confirmed the purity of isolated microsome as compared with protein equivalent amount of non-microsomal or total cell extract. (G) Schematic presentation of isolation of polysomes using KCl–puromycin based on HEK 293 cells. Microsomes isolated from HEK 293 cells were treated with 500 mM KCl and 1 mM puromycin before ultracentrifugation at 100,000 g for 1 h to separate the ribosomal and non-ribosomal pool. (H) miRNP and target mRNAs are associated with the rER-attached ribosomes. Western blot data show the distribution of Ago2 in the soluble and pellet fractions after treatment and centrifugations. Calnexin was used as the ER marker protein. Mature miR-122 as well as its target mRNA RL-3xbulge-miR-122 distribution in the abovementioned pellet and soluble fractions were determined by qRT-PCR. (I) Exclusiveness of the Ago2 and miRNA extraction with KCl–puromycin. Treated and non-treated extracts were analyzed by Western blots using cell equivalent amounts for each fraction. ER (calnexin), ribosome (S3), and endosomes/MVBs (HRS) marker protein distributions were checked to observe the specificity and purity of the isolation method. Western blot data of HRS protein suggest that minimum contamination of endosomal fractions in the microsome that can account for the observed Ago2 that was found to be extracted with ribosome upon extraction with KCl and puromycin. (J) Relative amount of let-7a miRNA associated with soluble and pellet fractions with or without KCl–puromycin treatment was measured. Paired two-tailed t tests were used for all comparisons. P

    Article Snippet: RNA isolation, Northern blotting, and real-time PCR analysis TRIzol reagent (Life Technologies) was used to isolate total RNA.

    Techniques: Immunoprecipitation, Fractionation, In Vitro, Activity Assay, Western Blot, Expressing, Cleavage Assay, Quantitative RT-PCR, Staining, Isolation, Marker, Two Tailed Test

    Increased target RNA trafficking to rER in Mfn2 negative cells. (A) Decreased association of newely formed miR-122 with Ago2 on mitochondria-defective cells. qRT-PCR data showing Ago2-associated de novo formed miR-122 level decrease over time on Mfn2 −/− cells compared with WT cells was plotted. (B) Decreased de novo–formed target mRNA in Mfn 2−/− MEF cells. qRT-PCR data show reduced target mRNA levels in the presence of its cognate miRNA on Mfn2 −/− cells. (C) The relative amount of the reporter target mRNAs in the absence and presence of miR-122 has been shown in both cell types. All cells were co-transfected with Tet-ON expression plasmid and inducible Renilla-3xbulge-miR-122 reporter plasmid and FF reporter plasmid. In the right panel, relative fold repression of the miR-122 reporter has been studied in the presence and absence of miR-122 expression in wild-type and mitochondria-defective cells. (D, E) Higher microsomal association of target mRNA in Mfn2 −/− MEF cells. Microsomal sequestration of target mRNA in the presence and absence of its cognate miRNA has been plotted (D). The relative level of microsome/rER-associated target mRNA between normal and Mfn2-negative cells expressing pmiR-122 has also been shown (E). Western blot data of ribosomal protein S3 show equal amounts of microsome associated ribosomes were used for estimation of associated mRNAs. (F) Schematic representation of in vitro translation assay showing reisolation of rER used for the assay performed in the presence and absence of wild-type and mutant cell derived mitochondria. Calnexin, Ago2, and COX IV Western blots confirmed equivalent amount of microsome along with mitochondria after reisolation. (G) Relative estimation of RNA from reisolated microsome after incubation in the presence of mitochondria obtained from wild-type and Mfn2 −/− cells. Increased target mRNA sequestration in the presence of cognate miRNA in assays performed with detethered mitochondria was observed. (H) Measurement of miR-122 being formed and became associated with microsome showed reduced miR-122 association for per unit of RL-3xbulge-miR-122 mRNA when rER from wild-type cells were incubated with Mfn2 −/− mitochondria (left panel). Reduced microsomal association of miR-122 has been also observed when normalization was done with endogenous control Ago2 (right panel). In both panles while bars represent WT and black bars denote Mfn2 −/− . Paired two-tailed t tests were used for all comparisons. P

    Journal: Life Science Alliance

    Article Title: Retrograde trafficking of Argonaute 2 acts as a rate-limiting step for de novo miRNP formation on endoplasmic reticulum–attached polysomes in mammalian cells

    doi: 10.26508/lsa.201800161

    Figure Lengend Snippet: Increased target RNA trafficking to rER in Mfn2 negative cells. (A) Decreased association of newely formed miR-122 with Ago2 on mitochondria-defective cells. qRT-PCR data showing Ago2-associated de novo formed miR-122 level decrease over time on Mfn2 −/− cells compared with WT cells was plotted. (B) Decreased de novo–formed target mRNA in Mfn 2−/− MEF cells. qRT-PCR data show reduced target mRNA levels in the presence of its cognate miRNA on Mfn2 −/− cells. (C) The relative amount of the reporter target mRNAs in the absence and presence of miR-122 has been shown in both cell types. All cells were co-transfected with Tet-ON expression plasmid and inducible Renilla-3xbulge-miR-122 reporter plasmid and FF reporter plasmid. In the right panel, relative fold repression of the miR-122 reporter has been studied in the presence and absence of miR-122 expression in wild-type and mitochondria-defective cells. (D, E) Higher microsomal association of target mRNA in Mfn2 −/− MEF cells. Microsomal sequestration of target mRNA in the presence and absence of its cognate miRNA has been plotted (D). The relative level of microsome/rER-associated target mRNA between normal and Mfn2-negative cells expressing pmiR-122 has also been shown (E). Western blot data of ribosomal protein S3 show equal amounts of microsome associated ribosomes were used for estimation of associated mRNAs. (F) Schematic representation of in vitro translation assay showing reisolation of rER used for the assay performed in the presence and absence of wild-type and mutant cell derived mitochondria. Calnexin, Ago2, and COX IV Western blots confirmed equivalent amount of microsome along with mitochondria after reisolation. (G) Relative estimation of RNA from reisolated microsome after incubation in the presence of mitochondria obtained from wild-type and Mfn2 −/− cells. Increased target mRNA sequestration in the presence of cognate miRNA in assays performed with detethered mitochondria was observed. (H) Measurement of miR-122 being formed and became associated with microsome showed reduced miR-122 association for per unit of RL-3xbulge-miR-122 mRNA when rER from wild-type cells were incubated with Mfn2 −/− mitochondria (left panel). Reduced microsomal association of miR-122 has been also observed when normalization was done with endogenous control Ago2 (right panel). In both panles while bars represent WT and black bars denote Mfn2 −/− . Paired two-tailed t tests were used for all comparisons. P

    Article Snippet: RNA isolation, Northern blotting, and real-time PCR analysis TRIzol reagent (Life Technologies) was used to isolate total RNA.

    Techniques: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation, Western Blot, In Vitro, Mutagenesis, Derivative Assay, Incubation, Two Tailed Test

    Plaque morphologies and growth curves of JEV-A90C transcript-derived viruses are similar to those of parental JEV stocks. (A) Immunofluorescence analysis of cells transfected with JEV-A90C RNA transcripts. BHK21 cells were transfected in vitro with transcripts derived from JEV-A90C or mock transfected as a control. Monoclonal antibody against the JEV envelope antigen was used to detect the infected cells by indirect immunofluorescence at 1, 2, 3, and 4 days after transfection with in vitro -derived transcripts. (B) Plaque morphology of virus derived from JEV-A90C transcripts and parental JEV. BHK21 cells were infected with the parental JEV RP9 strain viruses or JEV-A90C transcript-derived viruses, overlaid with methylcellulose, and stained at 3 days postinfection with crystal violet. (C) Growth kinetics of virions derived from JEV-A90C RNA transcripts and parental JEV in BHK21 cells. BHK21 cells were infected with parental JEV stock or with JEV transcript-derived viruses at MOIs of 0.01 and 0.1 PFU/cell. Virus samples from the medium of infected BHK21 cells were harvested daily, and the viral titer of each sample was determined in BHK21 cells. (D) Growth kinetics of virions derived from JEV-A90C RNA transcripts and parental JEV in mosquito cells (C6/36 cells). C6/36 cells were infected with parental JEV virus stock or with JEV-A90C transcript-derived JEV at MOIs of 0.01 and 0.1 PFU/cell. Virus samples from the medium of infected C6/36 cells were harvested daily, and the viral titer of each sample was determined in BHK21 cells. The error bars represent the SEMs from three independent experiments.

    Journal: Journal of Virology

    Article Title: Successful Propagation of Flavivirus Infectious cDNAs by a Novel Method To Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes ▿Successful Propagation of Flavivirus Infectious cDNAs by a Novel Method To Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes ▿ †

    doi: 10.1128/JVI.01986-10

    Figure Lengend Snippet: Plaque morphologies and growth curves of JEV-A90C transcript-derived viruses are similar to those of parental JEV stocks. (A) Immunofluorescence analysis of cells transfected with JEV-A90C RNA transcripts. BHK21 cells were transfected in vitro with transcripts derived from JEV-A90C or mock transfected as a control. Monoclonal antibody against the JEV envelope antigen was used to detect the infected cells by indirect immunofluorescence at 1, 2, 3, and 4 days after transfection with in vitro -derived transcripts. (B) Plaque morphology of virus derived from JEV-A90C transcripts and parental JEV. BHK21 cells were infected with the parental JEV RP9 strain viruses or JEV-A90C transcript-derived viruses, overlaid with methylcellulose, and stained at 3 days postinfection with crystal violet. (C) Growth kinetics of virions derived from JEV-A90C RNA transcripts and parental JEV in BHK21 cells. BHK21 cells were infected with parental JEV stock or with JEV transcript-derived viruses at MOIs of 0.01 and 0.1 PFU/cell. Virus samples from the medium of infected BHK21 cells were harvested daily, and the viral titer of each sample was determined in BHK21 cells. (D) Growth kinetics of virions derived from JEV-A90C RNA transcripts and parental JEV in mosquito cells (C6/36 cells). C6/36 cells were infected with parental JEV virus stock or with JEV-A90C transcript-derived JEV at MOIs of 0.01 and 0.1 PFU/cell. Virus samples from the medium of infected C6/36 cells were harvested daily, and the viral titer of each sample was determined in BHK21 cells. The error bars represent the SEMs from three independent experiments.

    Article Snippet: To transfect the viral RNA into BHK21 cells, approximately 0.5 μg full-length in vitro -transcribed DENV2 or JEV RNA was incubated with 1 μl Lipofectamine 2000 (Invitrogen) in 75 μl Opti-MEM medium.

    Techniques: Derivative Assay, Immunofluorescence, Transfection, In Vitro, Infection, Staining

    Construction of a full-length JEV cDNA clone with the yeast shuttle vector pRS313. (A) The yeast shuttle vector contained a bacterial replication origin (ori) and selection marker (Amp r ) and a yeast replication origin (CEN) and selection marker ( His3 ). (B) JEV cDNA fragment M was amplified from JEV viral RNA by RT-PCR. Fragment M possesses short overlaps with the termini of the linearized pRS313 vector. (C) BamHI-linearized pRS313 was mixed with fragment M and used to transform yeast strain YPH857 to His + . (D) In yeast, recombination occurred between the short homologous regions at the termini of the polylinker of pRS313 with fragment M to generate the pRS/JEVM plasmid. (E, F, and G) ClaI-linearized pRS/JEVM was incubated with the R fragment synthesized by RT-PCR from viral RNA and transformed into yeast cells to generate the pRS/JEVMR plasmid through homologous recombination. (H, I, and J) The L fragment synthesized from viral RNA by RT-PCR was first mutated to create the L* fragment. The L* fragment was incubated with the XhoI-linearized pRS/JEVMR plasmid and transformed into yeast cells to generate the full-length pRS/FLJEV infectious cDNA. Regions of crossing over (X) are indicated.

    Journal: Journal of Virology

    Article Title: Successful Propagation of Flavivirus Infectious cDNAs by a Novel Method To Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes ▿Successful Propagation of Flavivirus Infectious cDNAs by a Novel Method To Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes ▿ †

    doi: 10.1128/JVI.01986-10

    Figure Lengend Snippet: Construction of a full-length JEV cDNA clone with the yeast shuttle vector pRS313. (A) The yeast shuttle vector contained a bacterial replication origin (ori) and selection marker (Amp r ) and a yeast replication origin (CEN) and selection marker ( His3 ). (B) JEV cDNA fragment M was amplified from JEV viral RNA by RT-PCR. Fragment M possesses short overlaps with the termini of the linearized pRS313 vector. (C) BamHI-linearized pRS313 was mixed with fragment M and used to transform yeast strain YPH857 to His + . (D) In yeast, recombination occurred between the short homologous regions at the termini of the polylinker of pRS313 with fragment M to generate the pRS/JEVM plasmid. (E, F, and G) ClaI-linearized pRS/JEVM was incubated with the R fragment synthesized by RT-PCR from viral RNA and transformed into yeast cells to generate the pRS/JEVMR plasmid through homologous recombination. (H, I, and J) The L fragment synthesized from viral RNA by RT-PCR was first mutated to create the L* fragment. The L* fragment was incubated with the XhoI-linearized pRS/JEVMR plasmid and transformed into yeast cells to generate the full-length pRS/FLJEV infectious cDNA. Regions of crossing over (X) are indicated.

    Article Snippet: To transfect the viral RNA into BHK21 cells, approximately 0.5 μg full-length in vitro -transcribed DENV2 or JEV RNA was incubated with 1 μl Lipofectamine 2000 (Invitrogen) in 75 μl Opti-MEM medium.

    Techniques: Plasmid Preparation, Selection, Marker, Amplification, Reverse Transcription Polymerase Chain Reaction, Incubation, Synthesized, Transformation Assay, Homologous Recombination

    Mosquito cells produce host reverse transcriptase-dependent arbovirus-derived DNA. ( a ) Schematic of CHIKV viral genome. Top arrows indicate the position of the genomic and subgenomic promoters. Bottom arrows indicate the position of the primers used for vDNA detection. ( b ) Kinetics of vDNA synthesis. C6/36, U4.4 and Aag2 cells were infected with CHIKV at a MOI of 0.1 and cells were harvested at the indicated time points. Cells were analysed by PCR (upper panel) for vDNA detection. RT-PCR (lower panel) was used to follow viral infections. Non-infected cells (n.i) were used as a negative control and cellular 18S rRNA was used as a housekeeping gene loading control. ( c ) AZT inhibits vDNA synthesis in vitro . C6/36, U4.4 and Aag2 cells were treated with increasing concentration of AZT for 2 days. At the indicated time point, cells were harvested and vDNA and RNA were assessed as described in b . ( d – e ) Endogenous reverse transcriptase activity in insect cells. ( d ) Dose-dependent AZT inhibition was tested in insect cell extracts and ( e ) quantification of reverse transcriptase inhibition expressed as an activity percentage of non-treated extracts. Heat inactivated samples (heat) were used as negative controls. Drosophila S2 cells were used as a positive control. Each experiment was completed at least 3 times. Error bars correspond to the s.d. t -test with Welch's correction was used to determine statistical significance compared with the untreated control as a reference (** P

    Journal: Nature Communications

    Article Title: Virus-derived DNA drives mosquito vector tolerance to arboviral infection

    doi: 10.1038/ncomms12410

    Figure Lengend Snippet: Mosquito cells produce host reverse transcriptase-dependent arbovirus-derived DNA. ( a ) Schematic of CHIKV viral genome. Top arrows indicate the position of the genomic and subgenomic promoters. Bottom arrows indicate the position of the primers used for vDNA detection. ( b ) Kinetics of vDNA synthesis. C6/36, U4.4 and Aag2 cells were infected with CHIKV at a MOI of 0.1 and cells were harvested at the indicated time points. Cells were analysed by PCR (upper panel) for vDNA detection. RT-PCR (lower panel) was used to follow viral infections. Non-infected cells (n.i) were used as a negative control and cellular 18S rRNA was used as a housekeeping gene loading control. ( c ) AZT inhibits vDNA synthesis in vitro . C6/36, U4.4 and Aag2 cells were treated with increasing concentration of AZT for 2 days. At the indicated time point, cells were harvested and vDNA and RNA were assessed as described in b . ( d – e ) Endogenous reverse transcriptase activity in insect cells. ( d ) Dose-dependent AZT inhibition was tested in insect cell extracts and ( e ) quantification of reverse transcriptase inhibition expressed as an activity percentage of non-treated extracts. Heat inactivated samples (heat) were used as negative controls. Drosophila S2 cells were used as a positive control. Each experiment was completed at least 3 times. Error bars correspond to the s.d. t -test with Welch's correction was used to determine statistical significance compared with the untreated control as a reference (** P

    Article Snippet: Before qRT-PCR analysis, all RNA samples were treated for 30 min with DNase I (Roche) and the DNase was inactivated by incubation at 75 °C for 30 min. qRT-PCR analysis was performed using the RNA-to-Ct kit (Applied Biosystems, Foster, CA, USA), the forward primer 5′-TCACTCCCTGCTGGACTTGATAGA-3′ and reverse primer 5′-TTGACGAACAGAGTTAGGAACATACC-3′, and FAM-labeled probe 5′-AGGTACGCGCTTCAAGTTCGGCG-3′ as previously described .

    Techniques: Derivative Assay, Infection, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, In Vitro, Concentration Assay, Activity Assay, Inhibition, Positive Control

    Myc regulates the metabolic transcriptome. (A) Unsupervised hierarchical clustering of Illumina bead arrays made from RNA of splenic B cells from three wildtype and four precancerous λ- Myc transgenic mice. See Table S1 for genes used in the clustering. (B) qRT-PCR confirmation of 4 of the 20 most significantly, or most elevated expressed genes, in B220-sorted B cells from λ- Myc transgenic mice. *indicates p

    Journal: PLoS Genetics

    Article Title: Mouse Genetics Suggests Cell-Context Dependency for Myc-Regulated Metabolic Enzymes during Tumorigenesis

    doi: 10.1371/journal.pgen.1002573

    Figure Lengend Snippet: Myc regulates the metabolic transcriptome. (A) Unsupervised hierarchical clustering of Illumina bead arrays made from RNA of splenic B cells from three wildtype and four precancerous λ- Myc transgenic mice. See Table S1 for genes used in the clustering. (B) qRT-PCR confirmation of 4 of the 20 most significantly, or most elevated expressed genes, in B220-sorted B cells from λ- Myc transgenic mice. *indicates p

    Article Snippet: For in vitro transcription amplification, 200 ng of RNA was used with the Illumina RNA Amplification Kit (Ambion).

    Techniques: Transgenic Assay, Mouse Assay, Quantitative RT-PCR

    Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.

    Journal: Journal of Bacteriology

    Article Title: Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum

    doi: 10.1128/JB.00400-12

    Figure Lengend Snippet: Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.

    Article Snippet: For 5′ RACE (rapid amplification of cDNA ends) analysis, Ambion SUPERase-In, Ambion diethyl pyrocarbonate (DEPC)-treated double-distilled water (ddH2 O), Ambion THE RNA storage solution, and the Topo TA cloning kit for sequencing with One Shot chemically competent T10 E. coli cells were purchased from Life Technologies (Darmstadt, Germany); Roti-Aqua-P/C/I (phenol-chloroform-isoamyl alcohol, 25:24:1, pH 4.5 to 5) was purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany); T4 RNA ligase was purchased from NEB (Ipswich, MA); tobacco acid phosphatase (TAP) was purchased from Epicentre (Madison, WI); and the first-strand cDNA synthesis kit for reverse transcription-PCR (RT-PCR) was purchased from Roche (Mannheim, Germany).

    Techniques: Sequencing, Purification, SDS Page, Affinity Chromatography, In Vitro, Labeling, Incubation

    MEG3 and PTBP1 induce Cyp7a1 and Cyp8b1 expression by downregulating SHP (A–B) qPCR of SHP mRNA in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA (A) or PTBP1 (B) for 48 h. (C–D) qPCR of SHP (C), Cyp7a1, Cyp8b1 and FXR mRNA (D) in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA or PTBP1, alone or in combination for 24 h. (E) Western blot of PTBP1 protein in Huh7 cells transfected with control (Con) or siRNA against PTBP1. (F) qPCR of SHP and Cyp8b1 mRNA in Huh7 cells overexpressed with MEG3 in the presence or absence of siPTBP1. Data are shown in mean ± SEM from triplicate assays. * p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: LncRNA MEG3 induces cholestatic liver injury by interaction with PTBP1 to facilitate Shp mRNA decay

    doi: 10.1002/hep.28882

    Figure Lengend Snippet: MEG3 and PTBP1 induce Cyp7a1 and Cyp8b1 expression by downregulating SHP (A–B) qPCR of SHP mRNA in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA (A) or PTBP1 (B) for 48 h. (C–D) qPCR of SHP (C), Cyp7a1, Cyp8b1 and FXR mRNA (D) in Huh7 cells overexpressed with pcDNA3 (−), MEG3 RNA or PTBP1, alone or in combination for 24 h. (E) Western blot of PTBP1 protein in Huh7 cells transfected with control (Con) or siRNA against PTBP1. (F) qPCR of SHP and Cyp8b1 mRNA in Huh7 cells overexpressed with MEG3 in the presence or absence of siPTBP1. Data are shown in mean ± SEM from triplicate assays. * p

    Article Snippet: The following antibodies were used for RNA precipitation and Western blot (WB): antibodies against PTBP1 (ThermoFisher, 32-4800), β-actin (Sigma, A-1978), and CREB (Santa cruz, sc-69374).

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

    MEG3 decreases SHP mRNA stability by enhancing PTBP1 binding (A) Schematic of predicted PTBP1 binding sites ( UCUU ) within the CDS and 3′UTR of SHP mRNA. (B) RNA IP to determine the interaction between SHP mRNA (full length) and PTBP1 protein using anti-PTBP1 or IgG (negative control) in HEK293T (left), Hepa-1 (middle), and Huh7 (right) cells in the presence (+) or absence (−) of MEG3 RNA overexpression. (C) RNA pull-down followed by Western blot. Cell lysates of HEK293T, Hepa-1 and Huh7 were incubated with biotin-labeled CDS, 3′UTR of SHP mRNA, or a negative control transcript. After pull-down, the recruitment of PTBP1 to SHP CDS and 3′UTR was examined by Western blot with anti-PTBP1 antibody. (D) Hepa-1 (left) and Huh7 (right) cells transfected with pcDNA3 or MEG3 RNA with or without PTBP1 were treated with actinomycin D (5 μg/ml). RNA was extracted at different time points (0, 30, 60 mins) and SHP mRNA was analyzed by qPCR and normalized to β-actin. Data are shown in mean ± SEM from triplicate assays. * p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: LncRNA MEG3 induces cholestatic liver injury by interaction with PTBP1 to facilitate Shp mRNA decay

    doi: 10.1002/hep.28882

    Figure Lengend Snippet: MEG3 decreases SHP mRNA stability by enhancing PTBP1 binding (A) Schematic of predicted PTBP1 binding sites ( UCUU ) within the CDS and 3′UTR of SHP mRNA. (B) RNA IP to determine the interaction between SHP mRNA (full length) and PTBP1 protein using anti-PTBP1 or IgG (negative control) in HEK293T (left), Hepa-1 (middle), and Huh7 (right) cells in the presence (+) or absence (−) of MEG3 RNA overexpression. (C) RNA pull-down followed by Western blot. Cell lysates of HEK293T, Hepa-1 and Huh7 were incubated with biotin-labeled CDS, 3′UTR of SHP mRNA, or a negative control transcript. After pull-down, the recruitment of PTBP1 to SHP CDS and 3′UTR was examined by Western blot with anti-PTBP1 antibody. (D) Hepa-1 (left) and Huh7 (right) cells transfected with pcDNA3 or MEG3 RNA with or without PTBP1 were treated with actinomycin D (5 μg/ml). RNA was extracted at different time points (0, 30, 60 mins) and SHP mRNA was analyzed by qPCR and normalized to β-actin. Data are shown in mean ± SEM from triplicate assays. * p

    Article Snippet: The following antibodies were used for RNA precipitation and Western blot (WB): antibodies against PTBP1 (ThermoFisher, 32-4800), β-actin (Sigma, A-1978), and CREB (Santa cruz, sc-69374).

    Techniques: Binding Assay, Negative Control, Over Expression, Western Blot, Incubation, Labeling, Transfection, Real-time Polymerase Chain Reaction

    PTBP1 serves as a MEG3 binding protein (A) Biotin-labeled sense and antisense (negative control) MEG3 RNAs were synthesized by in vitro transcription. (B) In vitro RNA pull-down assay. HEK293T cell lysates were incubated with biotin-labeled oligos. After pull-down, proteins were subjected to SDS-PAGE and stained by coomassie brilliant blue. The band indicated by arrow was subjected to mass spectrometry. (C) Western blot to determine the specific interaction of sense MEG3 RNA with PTBP1 protein, but not with PCNA protein (negative control). (D) RNA immunoprecipitation (RIP) of PTBP1 interaction with MEG3 RNA in vivo in HEK293T cells. Protein-RNA complexes immunoprecipited by anti-PTBP1 or IgG were determined by RT-PCR using specific primers for MEG3 or Hprt1 (negative control).

    Journal: Hepatology (Baltimore, Md.)

    Article Title: LncRNA MEG3 induces cholestatic liver injury by interaction with PTBP1 to facilitate Shp mRNA decay

    doi: 10.1002/hep.28882

    Figure Lengend Snippet: PTBP1 serves as a MEG3 binding protein (A) Biotin-labeled sense and antisense (negative control) MEG3 RNAs were synthesized by in vitro transcription. (B) In vitro RNA pull-down assay. HEK293T cell lysates were incubated with biotin-labeled oligos. After pull-down, proteins were subjected to SDS-PAGE and stained by coomassie brilliant blue. The band indicated by arrow was subjected to mass spectrometry. (C) Western blot to determine the specific interaction of sense MEG3 RNA with PTBP1 protein, but not with PCNA protein (negative control). (D) RNA immunoprecipitation (RIP) of PTBP1 interaction with MEG3 RNA in vivo in HEK293T cells. Protein-RNA complexes immunoprecipited by anti-PTBP1 or IgG were determined by RT-PCR using specific primers for MEG3 or Hprt1 (negative control).

    Article Snippet: The following antibodies were used for RNA precipitation and Western blot (WB): antibodies against PTBP1 (ThermoFisher, 32-4800), β-actin (Sigma, A-1978), and CREB (Santa cruz, sc-69374).

    Techniques: Binding Assay, Labeling, Negative Control, Synthesized, In Vitro, Pull Down Assay, Incubation, SDS Page, Staining, Mass Spectrometry, Western Blot, Immunoprecipitation, In Vivo, Reverse Transcription Polymerase Chain Reaction

    MEG3 expression is transcriptionally inhibited by SHP (A) Integrated genome browser visualization of RNA-Seq read coverage for liver MEG3 in WT mice at E14, E18 and P60. (B) Left: Genome browser view of RNA-seq reads in MEG3 loci in Shp −/− and WT liver. Right: qPCR of hepatic MEG3 RNA in Shp −/− (red) and WT (blue) liver. Samples were collected over a 24 hr light/dark cycle. (C) Left: qPCR of MEG3 RNA in Huh7 cells transfected with transcription factors CREB, SREBP1c or pcDNA3, respectively. Middle: Luciferase reporter assay in HEK293T cells transfected with human MEG3 promoter reporter and expression plasmids for SHP and CREB. Top right: Western blot of liver CREB protein in Shp −/− or WT liver. (D) qPCR of MEG3 RNA and PTBP1 mRNA in human liver specimens (normal n=6; steatosis n=8; fibrosis n=6; NASH cirrhosis n=8). Data are shown in mean ± SEM from triplicate assays. * p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: LncRNA MEG3 induces cholestatic liver injury by interaction with PTBP1 to facilitate Shp mRNA decay

    doi: 10.1002/hep.28882

    Figure Lengend Snippet: MEG3 expression is transcriptionally inhibited by SHP (A) Integrated genome browser visualization of RNA-Seq read coverage for liver MEG3 in WT mice at E14, E18 and P60. (B) Left: Genome browser view of RNA-seq reads in MEG3 loci in Shp −/− and WT liver. Right: qPCR of hepatic MEG3 RNA in Shp −/− (red) and WT (blue) liver. Samples were collected over a 24 hr light/dark cycle. (C) Left: qPCR of MEG3 RNA in Huh7 cells transfected with transcription factors CREB, SREBP1c or pcDNA3, respectively. Middle: Luciferase reporter assay in HEK293T cells transfected with human MEG3 promoter reporter and expression plasmids for SHP and CREB. Top right: Western blot of liver CREB protein in Shp −/− or WT liver. (D) qPCR of MEG3 RNA and PTBP1 mRNA in human liver specimens (normal n=6; steatosis n=8; fibrosis n=6; NASH cirrhosis n=8). Data are shown in mean ± SEM from triplicate assays. * p

    Article Snippet: The following antibodies were used for RNA precipitation and Western blot (WB): antibodies against PTBP1 (ThermoFisher, 32-4800), β-actin (Sigma, A-1978), and CREB (Santa cruz, sc-69374).

    Techniques: Expressing, RNA Sequencing Assay, Mouse Assay, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Reporter Assay, Western Blot

    Photomicrographs of brightfield images of sections of pig corpus luteum at day 7 (a and b; scale bars represent 25 μm) and follicle at day 15 (c; scale bar represents 100 μm) of the estrous cycle. An antisense hypoxia inducible factor (HIF)-1α RNA probe was used for hybridization in the ovary. Slides were stained with hematoxylin-eosin (a,b) or periodic acid Schiff stain (c). (a) Hybridization signals, seen as silver grains (black dots), are present in most luteal cells. (b) Hybridization signals were dispersed in the thecal (T) and luteal (L) tissue interface. (c) The intense hybridization signals obscured the granulosa cells (G) and were evident in the thecal layers (T) of a follicle at day 15.

    Journal: Canadian Journal of Veterinary Research

    Article Title: Expression and localization of hypoxia inducible factor-1? mRNA in the porcine ovary

    doi:

    Figure Lengend Snippet: Photomicrographs of brightfield images of sections of pig corpus luteum at day 7 (a and b; scale bars represent 25 μm) and follicle at day 15 (c; scale bar represents 100 μm) of the estrous cycle. An antisense hypoxia inducible factor (HIF)-1α RNA probe was used for hybridization in the ovary. Slides were stained with hematoxylin-eosin (a,b) or periodic acid Schiff stain (c). (a) Hybridization signals, seen as silver grains (black dots), are present in most luteal cells. (b) Hybridization signals were dispersed in the thecal (T) and luteal (L) tissue interface. (c) The intense hybridization signals obscured the granulosa cells (G) and were evident in the thecal layers (T) of a follicle at day 15.

    Article Snippet: For in situ hybridization, 35 S-labeled cRNA antisense and sense probes were prepared using an RNA transcription kit (MAXIscript SP6/T7 Kit; Ambion, Austin, Texas, USA), as previously described ( ).

    Techniques: Hybridization, Staining

    Sections of pig ovary at days 7 (a to d) and day 15 (e and f) of the estrous cycle. Left and right panels are brightfield and darkfield images, respectively, of hybridization in the ovary using an antisense hypoxia inducible factor (HIF)-1α probe (a,b and e,f) or a vascular endothelial growth factor (VEGF) RNA probe (c,d). Hybridization signals for HIF-1α mRNA are seen predominantly in the corpora lutea (CL) and granulosa compartment, and to a lesser extent in the theca compartment of morphologically non-atretic follicles (a,b and e,f). Hybridization signals for VEGF mRNA (c,d) are seen in similar areas as the HIF-1α signals. F — follicle; F(a) — atretic follicle; G — granulosa compartment; S — stroma; T — theca compartment. Scale bars represent 100 μm.

    Journal: Canadian Journal of Veterinary Research

    Article Title: Expression and localization of hypoxia inducible factor-1? mRNA in the porcine ovary

    doi:

    Figure Lengend Snippet: Sections of pig ovary at days 7 (a to d) and day 15 (e and f) of the estrous cycle. Left and right panels are brightfield and darkfield images, respectively, of hybridization in the ovary using an antisense hypoxia inducible factor (HIF)-1α probe (a,b and e,f) or a vascular endothelial growth factor (VEGF) RNA probe (c,d). Hybridization signals for HIF-1α mRNA are seen predominantly in the corpora lutea (CL) and granulosa compartment, and to a lesser extent in the theca compartment of morphologically non-atretic follicles (a,b and e,f). Hybridization signals for VEGF mRNA (c,d) are seen in similar areas as the HIF-1α signals. F — follicle; F(a) — atretic follicle; G — granulosa compartment; S — stroma; T — theca compartment. Scale bars represent 100 μm.

    Article Snippet: For in situ hybridization, 35 S-labeled cRNA antisense and sense probes were prepared using an RNA transcription kit (MAXIscript SP6/T7 Kit; Ambion, Austin, Texas, USA), as previously described ( ).

    Techniques: Hybridization

    Sections of pig ovary at day 4 of the estrous cycle. (a) and (b) are brightfield and darkfield images, respectively, of hybridization in the ovary using a sense hypoxia inducible factor (HIF)-1α RNA probe. (c) and (d) are brightfield and darkfield views, respectively, of adjacent sections hybridized with an antisense HIF-1α RNA probe. High amounts of hybridization signals for HIF-1α mRNA predominantly were observed in granulosa-derived luteal tissues. CL — corpus luteum; S — luteal stroma; A — antrum. Scale bars represent 100 μm.

    Journal: Canadian Journal of Veterinary Research

    Article Title: Expression and localization of hypoxia inducible factor-1? mRNA in the porcine ovary

    doi:

    Figure Lengend Snippet: Sections of pig ovary at day 4 of the estrous cycle. (a) and (b) are brightfield and darkfield images, respectively, of hybridization in the ovary using a sense hypoxia inducible factor (HIF)-1α RNA probe. (c) and (d) are brightfield and darkfield views, respectively, of adjacent sections hybridized with an antisense HIF-1α RNA probe. High amounts of hybridization signals for HIF-1α mRNA predominantly were observed in granulosa-derived luteal tissues. CL — corpus luteum; S — luteal stroma; A — antrum. Scale bars represent 100 μm.

    Article Snippet: For in situ hybridization, 35 S-labeled cRNA antisense and sense probes were prepared using an RNA transcription kit (MAXIscript SP6/T7 Kit; Ambion, Austin, Texas, USA), as previously described ( ).

    Techniques: Hybridization, Derivative Assay

    MUC1 is a bona fide target of miR455-3P in BeWo cells. ( a ) Expression of potential miR455 target mRNAs in FSK-treated BeWo cells. Expression of the indicated potential miR455 target mRNAs was analyzed by qRT-PCR. Values were normalized to RPLP0 and displayed relative to DMSO-treated cells. ( b ) MUC1 protein levels are reduced by FSK treatment. MUC1 and TBP protein levels were analyzed by western blotting 48 h after treatment. ( c and d ) MUC1 is repressed by miR455-3P but not miR455-5P. BeWo cells were transfected with synthetic miRNAs at two concentrations (5 and 20 nM). RNA and protein samples were harvested 48 h post-transfection. MUC1 mRNA and protein levels were determined by qRT-PCR ( c ) and western blotting ( d ), respectively. One western blot representative of a transfection with 20 nM synthetic miRNA is presented in d . TBP served as a loading control

    Journal: Cell Death & Disease

    Article Title: miR455 is linked to hypoxia signaling and is deregulated in preeclampsia

    doi: 10.1038/cddis.2014.368

    Figure Lengend Snippet: MUC1 is a bona fide target of miR455-3P in BeWo cells. ( a ) Expression of potential miR455 target mRNAs in FSK-treated BeWo cells. Expression of the indicated potential miR455 target mRNAs was analyzed by qRT-PCR. Values were normalized to RPLP0 and displayed relative to DMSO-treated cells. ( b ) MUC1 protein levels are reduced by FSK treatment. MUC1 and TBP protein levels were analyzed by western blotting 48 h after treatment. ( c and d ) MUC1 is repressed by miR455-3P but not miR455-5P. BeWo cells were transfected with synthetic miRNAs at two concentrations (5 and 20 nM). RNA and protein samples were harvested 48 h post-transfection. MUC1 mRNA and protein levels were determined by qRT-PCR ( c ) and western blotting ( d ), respectively. One western blot representative of a transfection with 20 nM synthetic miRNA is presented in d . TBP served as a loading control

    Article Snippet: To evaluate miRNA and pri-miRNA expression, RT- PCR was performed using a TaqMan MicroRNA reverse transcription kit and a High Capacity RNA to cDNA kit, respectively, followed by a TaqMan Universal Master Mix, no UNG (Life Technologies).

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

    miR455 is induced upon in vitro syncytialization. ( a ) Schematic diagram of cyto- (CT) to syncytiotrophoblast (SCT) differentiation. ( b ) Fusion of BeWo cells following FSK treatment. Cells were fixed and immunostained using anti-E-cadherin antibody (green). Nuclei were counterstained with DAPI (blue). Left panel: BeWo cells were treated with vehicle (DMSO) for 48 h and remained as mononucleated (CT) cells. Right panel: cells started to fuse after 48 h of FSK treatment, producing multinucleated (SCT) cells (white arrow). Scale bar, 100 μ m ( c ) Expression of SCT markers. RNA was extracted at different time points after FSK treatment. mRNA levels of three SCT-specific genes were measured by qRT-PCR. Data were normalized to RPLP0 mRNA and compared to control treatments (median value ± S.E.M. of three independent experiments). ( d ) miR455 induction in SCT versus CT. After 48 h of control or FSK treatment, total RNA was extracted and small RNAs subjected to high-throughput sequencing. Normalized miRNA levels of control- and FSK-treated cells are plotted as a log 2 scale on the x and y axes, respectively. Each miRNA is represented by a cross. Deregulated miR455 miRNAs are indicated by red crosses. The result of one representative biological replicate is shown. ( e and f ) Fold induction of miR455-3P (E) and -5P (F) after 48 h of FSK treatment was determined by small RNA sequencing or qRT-PCR (median value ± S.E.M. of four and three independent experiments, respectively). ( g ) Hairpin structure of the pre-miR455 precursor transcript. ( h ) Expression analysis of pri-miR455, miR455, and of the host gene COL27A1 . RNA was extracted at the indicated time points after FSK treatment and analyzed by qRT-PCR using TaqMan assays. Data were normalized to U6 snRNA or RPLP0 mRNA and compared to control treatments (median value ± S.E.M. of three independent experiments)

    Journal: Cell Death & Disease

    Article Title: miR455 is linked to hypoxia signaling and is deregulated in preeclampsia

    doi: 10.1038/cddis.2014.368

    Figure Lengend Snippet: miR455 is induced upon in vitro syncytialization. ( a ) Schematic diagram of cyto- (CT) to syncytiotrophoblast (SCT) differentiation. ( b ) Fusion of BeWo cells following FSK treatment. Cells were fixed and immunostained using anti-E-cadherin antibody (green). Nuclei were counterstained with DAPI (blue). Left panel: BeWo cells were treated with vehicle (DMSO) for 48 h and remained as mononucleated (CT) cells. Right panel: cells started to fuse after 48 h of FSK treatment, producing multinucleated (SCT) cells (white arrow). Scale bar, 100 μ m ( c ) Expression of SCT markers. RNA was extracted at different time points after FSK treatment. mRNA levels of three SCT-specific genes were measured by qRT-PCR. Data were normalized to RPLP0 mRNA and compared to control treatments (median value ± S.E.M. of three independent experiments). ( d ) miR455 induction in SCT versus CT. After 48 h of control or FSK treatment, total RNA was extracted and small RNAs subjected to high-throughput sequencing. Normalized miRNA levels of control- and FSK-treated cells are plotted as a log 2 scale on the x and y axes, respectively. Each miRNA is represented by a cross. Deregulated miR455 miRNAs are indicated by red crosses. The result of one representative biological replicate is shown. ( e and f ) Fold induction of miR455-3P (E) and -5P (F) after 48 h of FSK treatment was determined by small RNA sequencing or qRT-PCR (median value ± S.E.M. of four and three independent experiments, respectively). ( g ) Hairpin structure of the pre-miR455 precursor transcript. ( h ) Expression analysis of pri-miR455, miR455, and of the host gene COL27A1 . RNA was extracted at the indicated time points after FSK treatment and analyzed by qRT-PCR using TaqMan assays. Data were normalized to U6 snRNA or RPLP0 mRNA and compared to control treatments (median value ± S.E.M. of three independent experiments)

    Article Snippet: To evaluate miRNA and pri-miRNA expression, RT- PCR was performed using a TaqMan MicroRNA reverse transcription kit and a High Capacity RNA to cDNA kit, respectively, followed by a TaqMan Universal Master Mix, no UNG (Life Technologies).

    Techniques: In Vitro, Expressing, Quantitative RT-PCR, Next-Generation Sequencing, RNA Sequencing Assay

    miR455 and miR210 are deregulated in preeclampsia (PE) placentas. ( a ) Schematic diagram of placenta processing. Circa 3–4 pieces (red box) were dissected from the inner part of each placenta (X) to limit maternal contamination. Total RNA was extracted from each placenta piece (X.1 to X.n) and miRNA expression levels measured in three technical replicates by qRT-PCR using TaqMan assays. ( b ) U6 snRNA expression in PE and control (Ctrl) placentas. U6 snRNA levels were measured by TaqMan qRT-PCR. The cycle threshold (Ct) value obtained for U6 snRNA is plotted as a whiskers box plot 5th–95th percentile representation. The P -value was calculated by Mann–Whitney test. ( c ) miRNAs expressed in placentas. The expression of six selected miRNAs was determined by TaqMan qRT-PCR in control placentas. For each miRNA, expression was normalized to U6 snRNA and plotted on a log2 scale using a whiskers box plot 5th–95th percentile representation. ( d ) MicroRNAs that are not expressed differentially in control versus PE placentas. ( e ) miR-210 levels are higher in PE than control placentas. ( f ) miR-455 levels are lower in PE than control placentas. ( d–f ): P -values were calculated by Mann–Whitney test

    Journal: Cell Death & Disease

    Article Title: miR455 is linked to hypoxia signaling and is deregulated in preeclampsia

    doi: 10.1038/cddis.2014.368

    Figure Lengend Snippet: miR455 and miR210 are deregulated in preeclampsia (PE) placentas. ( a ) Schematic diagram of placenta processing. Circa 3–4 pieces (red box) were dissected from the inner part of each placenta (X) to limit maternal contamination. Total RNA was extracted from each placenta piece (X.1 to X.n) and miRNA expression levels measured in three technical replicates by qRT-PCR using TaqMan assays. ( b ) U6 snRNA expression in PE and control (Ctrl) placentas. U6 snRNA levels were measured by TaqMan qRT-PCR. The cycle threshold (Ct) value obtained for U6 snRNA is plotted as a whiskers box plot 5th–95th percentile representation. The P -value was calculated by Mann–Whitney test. ( c ) miRNAs expressed in placentas. The expression of six selected miRNAs was determined by TaqMan qRT-PCR in control placentas. For each miRNA, expression was normalized to U6 snRNA and plotted on a log2 scale using a whiskers box plot 5th–95th percentile representation. ( d ) MicroRNAs that are not expressed differentially in control versus PE placentas. ( e ) miR-210 levels are higher in PE than control placentas. ( f ) miR-455 levels are lower in PE than control placentas. ( d–f ): P -values were calculated by Mann–Whitney test

    Article Snippet: To evaluate miRNA and pri-miRNA expression, RT- PCR was performed using a TaqMan MicroRNA reverse transcription kit and a High Capacity RNA to cDNA kit, respectively, followed by a TaqMan Universal Master Mix, no UNG (Life Technologies).

    Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY

    Gemcitabine induces IL-8 expression in pancreatic cancer cells A. Colo-357 cells were treated with gemcitabine (10 μM) for 8 h. Subsequently, RNA was isolated, cDNA was prepared and cytokines/growth factors profiling was performed using qRT-PCR. B. Colo-357 and MiaPaCa cells were treated with gemcitabine (10 μM) for 8 h. Post treatment, media was replaced with fresh culture medium and incubated for next 24 h. Thereafter, total protein was isolated and subjected to immunoblot analysis to examine IL-8 expression using specific antibody. β-actin was used as a loading control. C. Level of IL-8 in conditioned media of vehicle or gemcitabine treated PC cells was measured using ELISA as described in materials and methods. Data is presented as mean ± SD; n = 3 .* p

    Journal: Oncotarget

    Article Title: Gemcitabine triggers angiogenesis-promoting molecular signals in pancreatic cancer cells: Therapeutic implications

    doi:

    Figure Lengend Snippet: Gemcitabine induces IL-8 expression in pancreatic cancer cells A. Colo-357 cells were treated with gemcitabine (10 μM) for 8 h. Subsequently, RNA was isolated, cDNA was prepared and cytokines/growth factors profiling was performed using qRT-PCR. B. Colo-357 and MiaPaCa cells were treated with gemcitabine (10 μM) for 8 h. Post treatment, media was replaced with fresh culture medium and incubated for next 24 h. Thereafter, total protein was isolated and subjected to immunoblot analysis to examine IL-8 expression using specific antibody. β-actin was used as a loading control. C. Level of IL-8 in conditioned media of vehicle or gemcitabine treated PC cells was measured using ELISA as described in materials and methods. Data is presented as mean ± SD; n = 3 .* p

    Article Snippet: Reagents and antibodies Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI-1640), Kaighn's Modification of Ham's F-12 Medium (F12K), penicillin-streptomycin (Invitrogen, Carlsbad, CA); Fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA); WST-1 proliferation assay kit (Roche, Indianapolis, IN); High-Capacity RNA-to-cDNA™ Kit and SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA); Diff-Quick cell staining kit (Dade Behring, Inc., Newark, DE); In vitro Angiogenesis kit (EMD Millipore, Temecula, CA); anti-human IL-8 ELISA Kit (R & D Systems Inc., Minneapolis, MN); Gemcitabine (Sigma-Aldrich, St. Louis MO); Western blotting SuperSignal West Femto Maximum sensitivity substrate kit (Thermo Scientific, Logan, UT); goat anti-IL-8 antibody (Abcam, Cambridge, MA); biotinylated anti-β-actin (Sigma-Aldrich) and horseradish peroxidase (HRP) labelled secondary antibodies (1:2000; Santa Cruz Biotechnology).

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Incubation, Enzyme-linked Immunosorbent Assay