real time pcr total rna  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    Thermo Fisher real time pcr total rna
    Tat-mediated suppression of miR-221/-222 is NF-κB-dependent. Pre-treatment of HUVECs with the IKK2/NF-κB inhibitor SC514 (10 µM) abrogated Tat-mediated down-regulation of miR-221/-222 expression. Total <t>RNA</t> was isolated, and the expression of miR-221/-222 was quantified by real-time <t>RT-PCR.</t> RNU6B (U6) was used as the control. All the data are presented as mean ± SD of three independent experiments. **p
    Real Time Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1670 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time pcr total rna/product/Thermo Fisher
    Average 95 stars, based on 1670 article reviews
    Price from $9.99 to $1999.99
    real time pcr total rna - by Bioz Stars, 2020-08
    95/100 stars

    Images

    1) Product Images from "HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy"

    Article Title: HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060170

    Tat-mediated suppression of miR-221/-222 is NF-κB-dependent. Pre-treatment of HUVECs with the IKK2/NF-κB inhibitor SC514 (10 µM) abrogated Tat-mediated down-regulation of miR-221/-222 expression. Total RNA was isolated, and the expression of miR-221/-222 was quantified by real-time RT-PCR. RNU6B (U6) was used as the control. All the data are presented as mean ± SD of three independent experiments. **p
    Figure Legend Snippet: Tat-mediated suppression of miR-221/-222 is NF-κB-dependent. Pre-treatment of HUVECs with the IKK2/NF-κB inhibitor SC514 (10 µM) abrogated Tat-mediated down-regulation of miR-221/-222 expression. Total RNA was isolated, and the expression of miR-221/-222 was quantified by real-time RT-PCR. RNU6B (U6) was used as the control. All the data are presented as mean ± SD of three independent experiments. **p

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    2) Product Images from "Up-regulation of the Hippo pathway effector TAZ renders lung adenocarcinoma cells harboring EGFR-T790M mutation resistant to gefitinib"

    Article Title: Up-regulation of the Hippo pathway effector TAZ renders lung adenocarcinoma cells harboring EGFR-T790M mutation resistant to gefitinib

    Journal: Cell & Bioscience

    doi: 10.1186/2045-3701-5-7

    Enhanced expression of TAZ in gefitinib-resistant cell. (A) Lysates derived from human bronchial epithelial cell line 16HBE, lung adenocarcinoma cell lines A549, cisplatin-resistant A549/DDP, gefitinib-sensitive PC9 and gefitinib-resistant PC9/GR were analyzed by western blot using anti-TAZ antibodies. The levels of GAPDH were detected as loading controls. (B) Relative mRNAs of TAZ in lung cancer cell lines were examined by real-time PCR. The endogenous b-actin RNA was used as the internal control. (C) Nuclear fractions in PC9 and PC9/GR cell lines were analyzed by western blot. The levels of histone H3 were detected as loading controls. Densitometric evaluation of TAZ:H3 ratios is illustrated in graph beside. Data are shown as means ± SEM. n=3. Statistical analyses were carried out using Student’s t-test. Significance: * P
    Figure Legend Snippet: Enhanced expression of TAZ in gefitinib-resistant cell. (A) Lysates derived from human bronchial epithelial cell line 16HBE, lung adenocarcinoma cell lines A549, cisplatin-resistant A549/DDP, gefitinib-sensitive PC9 and gefitinib-resistant PC9/GR were analyzed by western blot using anti-TAZ antibodies. The levels of GAPDH were detected as loading controls. (B) Relative mRNAs of TAZ in lung cancer cell lines were examined by real-time PCR. The endogenous b-actin RNA was used as the internal control. (C) Nuclear fractions in PC9 and PC9/GR cell lines were analyzed by western blot. The levels of histone H3 were detected as loading controls. Densitometric evaluation of TAZ:H3 ratios is illustrated in graph beside. Data are shown as means ± SEM. n=3. Statistical analyses were carried out using Student’s t-test. Significance: * P

    Techniques Used: Expressing, Derivative Assay, Western Blot, Real-time Polymerase Chain Reaction

    3) Product Images from "Delayed triggering of oestrogen induced apoptosis that contrasts with rapid paclitaxel-induced breast cancer cell death"

    Article Title: Delayed triggering of oestrogen induced apoptosis that contrasts with rapid paclitaxel-induced breast cancer cell death

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2014.50

    Heat map of E 2 -mediated apoptotic genes that are differentially expressed by 36 and 48 h of treatment. Cells were parsed into groups of three replicates per treatment per time point, and then treated with either 0.1% ethanol (control group), 1 n M E 2 (group 1), 1 μ M 4OHT (group 2), in the presence (group 3) or in the absence of E 2 over a period of 48 h. Total RNA was extracted and reverse transcribed as described in Materials and Methods section. Samples were loaded onto customised PCR array plates with primers for indicated apoptotic genes. Gene expression values were obtained and analysed in comparison with the controls at ( A ) 36 h and ( B ) 48 h. The maximum expressed level of any given gene is represented by red colour and minimum levels are represented as green colour.
    Figure Legend Snippet: Heat map of E 2 -mediated apoptotic genes that are differentially expressed by 36 and 48 h of treatment. Cells were parsed into groups of three replicates per treatment per time point, and then treated with either 0.1% ethanol (control group), 1 n M E 2 (group 1), 1 μ M 4OHT (group 2), in the presence (group 3) or in the absence of E 2 over a period of 48 h. Total RNA was extracted and reverse transcribed as described in Materials and Methods section. Samples were loaded onto customised PCR array plates with primers for indicated apoptotic genes. Gene expression values were obtained and analysed in comparison with the controls at ( A ) 36 h and ( B ) 48 h. The maximum expressed level of any given gene is represented by red colour and minimum levels are represented as green colour.

    Techniques Used: Polymerase Chain Reaction, Expressing

    E 2 activates both mitochondrial and extrinsic pathway of apoptosis, whereas paclitaxel activates only the extrinsic pathway. MCF7:5C cells were treated with vehicle (Veh), 1 n M E2, 1 μ M 4OHT or combination treatment of E 2 and 4OHT for 24, 36 and 48 h. Total RNA was reverse transcribed and assessed for ( A ) BIM and ( B ) TNF gene expression. Induction of ( C ) BIM and ( D ) TNF mRNA was determined in MCF7:5C cells treated with either Veh or 1 μ M paclitaxel for 12 and 24 h using RT–PCR. PCR data values are presented as fold difference versus Veh-treated cells±s.e.m. (** P
    Figure Legend Snippet: E 2 activates both mitochondrial and extrinsic pathway of apoptosis, whereas paclitaxel activates only the extrinsic pathway. MCF7:5C cells were treated with vehicle (Veh), 1 n M E2, 1 μ M 4OHT or combination treatment of E 2 and 4OHT for 24, 36 and 48 h. Total RNA was reverse transcribed and assessed for ( A ) BIM and ( B ) TNF gene expression. Induction of ( C ) BIM and ( D ) TNF mRNA was determined in MCF7:5C cells treated with either Veh or 1 μ M paclitaxel for 12 and 24 h using RT–PCR. PCR data values are presented as fold difference versus Veh-treated cells±s.e.m. (** P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    4) Product Images from "Transcriptional Repression by the BRG1-SWI/SNF Complex Affects the Pluripotency of Human Embryonic Stem Cells"

    Article Title: Transcriptional Repression by the BRG1-SWI/SNF Complex Affects the Pluripotency of Human Embryonic Stem Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2014.07.004

    BRG1 Depletion Affects a Broad Range of Biological Processes in hESCs (A) Knockdown of BRG1 in hESCs and mESCs was verified by western blots. (B and C) Quantitative RT-PCR validation for genes differentially expressed in microarray analyses. RNA samples were collected from either mESCs (B) or hESCs (C) transfected with siRNAs against BRG1. (D) Genes affected by BRG1 knockdown in hESCs were analyzed by IPA according to their functions. (E) Venn diagram with the numbers of genes affected by BRG1 knockdown in hESCs and mESCs. The number in the overlapping circles indicates the number of common genes altered in both hESCs and mESCs upon knockdown. (F) Expression levels of the same set of genes upon BRG1 depletion in hESCs, mESCs, or mEpiSCs examined by quantitative RT-PCR (fold changes of expression relative to their own scrambled shRNA controls). All PCR data are presented as the average of six biological replicates ± 1 SEM from three independent experiments; ∗ p
    Figure Legend Snippet: BRG1 Depletion Affects a Broad Range of Biological Processes in hESCs (A) Knockdown of BRG1 in hESCs and mESCs was verified by western blots. (B and C) Quantitative RT-PCR validation for genes differentially expressed in microarray analyses. RNA samples were collected from either mESCs (B) or hESCs (C) transfected with siRNAs against BRG1. (D) Genes affected by BRG1 knockdown in hESCs were analyzed by IPA according to their functions. (E) Venn diagram with the numbers of genes affected by BRG1 knockdown in hESCs and mESCs. The number in the overlapping circles indicates the number of common genes altered in both hESCs and mESCs upon knockdown. (F) Expression levels of the same set of genes upon BRG1 depletion in hESCs, mESCs, or mEpiSCs examined by quantitative RT-PCR (fold changes of expression relative to their own scrambled shRNA controls). All PCR data are presented as the average of six biological replicates ± 1 SEM from three independent experiments; ∗ p

    Techniques Used: Western Blot, Quantitative RT-PCR, Microarray, Transfection, Indirect Immunoperoxidase Assay, Expressing, shRNA, Polymerase Chain Reaction

    5) Product Images from "Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor"

    Article Title: Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1367

    circRNAs are less dynamic and abundant relative to linear isoforms derived from the same host gene. ( A ) A linear diagram exemplifying primer pairs (arrows) for parallel measurements of levels of circular and linear RNA isoforms derived from the same host gene. The ERBB2 (chromosome 17) gene is shown as an example. The respective circRNA (red) spans five internal exons. The divergent primers (red arrows; circular transcript) flank the non-canonical splicing (circularization), whereas the convergent primers (blue arrows) were designed for measuring the corresponding linear isoform. ( B ) PCR and four pairs of primers (designed as in A) were used to follow the response of the indicated linear and circular isoforms of four EGF-regulated genes. Note that mRNAs corresponding to PUS7 and SLC25A32 are induced when MCF10A cells are stimulated with EGF, whereas PCMTD1 and PIK3C2G mRNAs undergo down-regulation under the same conditions. Shown are means ± S.E. of triplicates. P -values were calculated using two-way Anova with time and RNA type as categorical factors. ( C ) A histogram comparing the abundance of circRNAs and the corresponding linear transcripts ( N = 203) of MCF10A mammary cells. Note that circRNAs are, on average, 36 times less abundant than the corresponding linear isoforms ( P = 5.45e-78, t -test, two-tail, paired; A.U., arbitrary units). ( D ) A dot plot presenting the ratio between expression levels of each circRNA of MCF10A cells we characterized and the respective linear transcript, which is derived from the same host gene. The ratios are presented against the abundance of the latter isoform. Note that most circRNAs are expressed at lower levels than the corresponding linear transcripts; only five circRNAs (red dots; identified by names) exceeded the abundance of their respective linear isoforms, but the absolute expression levels of all five circRNAs are relatively low.
    Figure Legend Snippet: circRNAs are less dynamic and abundant relative to linear isoforms derived from the same host gene. ( A ) A linear diagram exemplifying primer pairs (arrows) for parallel measurements of levels of circular and linear RNA isoforms derived from the same host gene. The ERBB2 (chromosome 17) gene is shown as an example. The respective circRNA (red) spans five internal exons. The divergent primers (red arrows; circular transcript) flank the non-canonical splicing (circularization), whereas the convergent primers (blue arrows) were designed for measuring the corresponding linear isoform. ( B ) PCR and four pairs of primers (designed as in A) were used to follow the response of the indicated linear and circular isoforms of four EGF-regulated genes. Note that mRNAs corresponding to PUS7 and SLC25A32 are induced when MCF10A cells are stimulated with EGF, whereas PCMTD1 and PIK3C2G mRNAs undergo down-regulation under the same conditions. Shown are means ± S.E. of triplicates. P -values were calculated using two-way Anova with time and RNA type as categorical factors. ( C ) A histogram comparing the abundance of circRNAs and the corresponding linear transcripts ( N = 203) of MCF10A mammary cells. Note that circRNAs are, on average, 36 times less abundant than the corresponding linear isoforms ( P = 5.45e-78, t -test, two-tail, paired; A.U., arbitrary units). ( D ) A dot plot presenting the ratio between expression levels of each circRNA of MCF10A cells we characterized and the respective linear transcript, which is derived from the same host gene. The ratios are presented against the abundance of the latter isoform. Note that most circRNAs are expressed at lower levels than the corresponding linear transcripts; only five circRNAs (red dots; identified by names) exceeded the abundance of their respective linear isoforms, but the absolute expression levels of all five circRNAs are relatively low.

    Techniques Used: Derivative Assay, Polymerase Chain Reaction, Expressing

    Unlike mRNAs and microRNAs, circular RNAs of mammary cells display minor changes in response to an extracellular cue. ( A ) Venn diagrams presenting genomic origins of circRNAs found in MCF10A mammary cells. The analysis comprises 1498 circRNA molecules we identified in MCF10A cells using RNA-sequencing. Of these, 1451 molecules (97%) overlap known transcripts. The remainder 47 circRNAs are either intergenic (14 transcripts) or antisense to known transcripts ( N = 33). Inner circles represent Quantified fractions, meaning transcripts we followed also by using PCR; For circRNAs derived from known transcripts, the majority ( > 90%) of the Quantified fraction refers to circRNAs, the response of which to EGF was assayed using divergent and convergent sets of primers, while for the remainder of the Quantified fraction, including circRNAs derived from either antisense or intergenic regions, measurements were performed using only divergent sets of primers. ( B ) MCF10A human mammary epithelial cells were starved overnight for serum factors. Thereafter they were treated with EGF (10 ng/ml) for the indicated time intervals. High-throughput PCR and specific primers were applied on isolated RNA samples to amplify 241 of 1498 circRNA species previously identified using RNA sequencing. This group included > 50% of the most abundant candidates, along with dozens of other circRNAs with varying expression levels. The presented heatmap (right panel) depicts time-dependent alterations in expression levels of specific circRNAs. These alterations are compared to mRNA and microRNA alterations we previously observed, using microarrays, while stimulating MCF10A under identical conditions ( 5 , 36 ). Note that all previously analyzed miRNAs are represented, but in order to match the size of the circRNA population, only randomly selected, 16.1% of all MCF10A's mRNA molecules, are depicted in the heatmap. Data were normalized to time zero and ordered according to the time point corresponding to the maximal change. Red squares represent an increase and blue squares represent a decrease, as shown in the scale bar on the right. CircRNA results represent biological duplicates performed in technical triplicates. ( C ) A histogram showing the range of abundance changes of mRNAs, miRNAs and circRNAs ( N = 3608, N = 164 and N = 288, respectively) displayed by EGF-stimulated MCF10A cells. To construct the histogram, the maximal change value (induction or repression) along the stimulation interval (240 min) was found for each RNA molecule. Note that circRNAs exhibit narrower dynamic range (highlighted region) than mRNAs and miRNAs ( P
    Figure Legend Snippet: Unlike mRNAs and microRNAs, circular RNAs of mammary cells display minor changes in response to an extracellular cue. ( A ) Venn diagrams presenting genomic origins of circRNAs found in MCF10A mammary cells. The analysis comprises 1498 circRNA molecules we identified in MCF10A cells using RNA-sequencing. Of these, 1451 molecules (97%) overlap known transcripts. The remainder 47 circRNAs are either intergenic (14 transcripts) or antisense to known transcripts ( N = 33). Inner circles represent Quantified fractions, meaning transcripts we followed also by using PCR; For circRNAs derived from known transcripts, the majority ( > 90%) of the Quantified fraction refers to circRNAs, the response of which to EGF was assayed using divergent and convergent sets of primers, while for the remainder of the Quantified fraction, including circRNAs derived from either antisense or intergenic regions, measurements were performed using only divergent sets of primers. ( B ) MCF10A human mammary epithelial cells were starved overnight for serum factors. Thereafter they were treated with EGF (10 ng/ml) for the indicated time intervals. High-throughput PCR and specific primers were applied on isolated RNA samples to amplify 241 of 1498 circRNA species previously identified using RNA sequencing. This group included > 50% of the most abundant candidates, along with dozens of other circRNAs with varying expression levels. The presented heatmap (right panel) depicts time-dependent alterations in expression levels of specific circRNAs. These alterations are compared to mRNA and microRNA alterations we previously observed, using microarrays, while stimulating MCF10A under identical conditions ( 5 , 36 ). Note that all previously analyzed miRNAs are represented, but in order to match the size of the circRNA population, only randomly selected, 16.1% of all MCF10A's mRNA molecules, are depicted in the heatmap. Data were normalized to time zero and ordered according to the time point corresponding to the maximal change. Red squares represent an increase and blue squares represent a decrease, as shown in the scale bar on the right. CircRNA results represent biological duplicates performed in technical triplicates. ( C ) A histogram showing the range of abundance changes of mRNAs, miRNAs and circRNAs ( N = 3608, N = 164 and N = 288, respectively) displayed by EGF-stimulated MCF10A cells. To construct the histogram, the maximal change value (induction or repression) along the stimulation interval (240 min) was found for each RNA molecule. Note that circRNAs exhibit narrower dynamic range (highlighted region) than mRNAs and miRNAs ( P

    Techniques Used: RNA Sequencing Assay, Polymerase Chain Reaction, Derivative Assay, High Throughput Screening Assay, Isolation, Expressing, Construct

    Analysis of newly transcribed RNAs reveals that circRNAs are more stable and static than the linear isoforms derived from the same host genes. ( A ) MCF10A cells were treated with EGF as in Figure 1B and RNA was simultaneously metabolically labeled using 4-thiouridine (4sU), for the indicated time intervals. RNA was extracted (Total-RNA) with Trizol, biotinylated and purified on streptavidin magnetic beads (denoted 4sU-RNA). Flow-through RNA was also collected (denoted FT-RNA). Thereafter, RNA was reverse transcribed and quantified using high-throughput real time PCR (Fluidigm). The boxplot diagram shows enrichment of newly transcribed RNA (4sU labeled) in linear isoforms (blue dots) relative to the respective circRNA isoforms (red dots; P
    Figure Legend Snippet: Analysis of newly transcribed RNAs reveals that circRNAs are more stable and static than the linear isoforms derived from the same host genes. ( A ) MCF10A cells were treated with EGF as in Figure 1B and RNA was simultaneously metabolically labeled using 4-thiouridine (4sU), for the indicated time intervals. RNA was extracted (Total-RNA) with Trizol, biotinylated and purified on streptavidin magnetic beads (denoted 4sU-RNA). Flow-through RNA was also collected (denoted FT-RNA). Thereafter, RNA was reverse transcribed and quantified using high-throughput real time PCR (Fluidigm). The boxplot diagram shows enrichment of newly transcribed RNA (4sU labeled) in linear isoforms (blue dots) relative to the respective circRNA isoforms (red dots; P

    Techniques Used: Derivative Assay, Metabolic Labelling, Labeling, Purification, Magnetic Beads, Flow Cytometry, High Throughput Screening Assay, Real-time Polymerase Chain Reaction

    6) Product Images from "Activation of Nuclear Factor Kappa B in the Hepatic Stellate Cells of Mice with Schistosomiasis Japonica"

    Article Title: Activation of Nuclear Factor Kappa B in the Hepatic Stellate Cells of Mice with Schistosomiasis Japonica

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104323

    Activation of NF-κB signaling in HSCs in hepatic schistosomiasis. (A) Total protein, cytoplasm protein and nuclear protein were extracted from HSCs and then levels of p65 were detected in each by western bolt; (B) the activity of NF-κB presented by the ratio of nuclear p65 to cytoplasm p65; and (C) the total p65 level. Expression level of (D) Ccl2; (E) Ccl3; (F) Ccl5; (G) Cxcl1; (H) α-SMA; and (I) Colα1 determined from total RNA by real-time PCR. * P
    Figure Legend Snippet: Activation of NF-κB signaling in HSCs in hepatic schistosomiasis. (A) Total protein, cytoplasm protein and nuclear protein were extracted from HSCs and then levels of p65 were detected in each by western bolt; (B) the activity of NF-κB presented by the ratio of nuclear p65 to cytoplasm p65; and (C) the total p65 level. Expression level of (D) Ccl2; (E) Ccl3; (F) Ccl5; (G) Cxcl1; (H) α-SMA; and (I) Colα1 determined from total RNA by real-time PCR. * P

    Techniques Used: Activation Assay, Western Blot, Activity Assay, Expressing, Real-time Polymerase Chain Reaction

    7) Product Images from "Long noncoding RNA lnc-RI is a new regulator of mitosis via targeting miRNA-210-3p to release PLK1 mRNA activity"

    Article Title: Long noncoding RNA lnc-RI is a new regulator of mitosis via targeting miRNA-210-3p to release PLK1 mRNA activity

    Journal: Scientific Reports

    doi: 10.1038/srep25385

    PLK1 is the target of lnc-RI and is involved in mitotic arrest induced by lnc-RI knockdown. ( a ) The functional association of lnc-RI target genes. All 144 candidate target genes were uploaded into STRING 10 ( http://string-db.org/ ), and three networks related to cell cycle, apoptosis, and DNA damage were identified. ( b,c ) Knockdown of lnc-RI reduced PLK1 mRNA and protein expression levels. The HEK293 ( b ) and HeLa cells ( c ) were transfected with lnc-RI RNAi-1, RNAi-2 or Ctrl RNAi. At 48 h post-transfection, total RNA and total protein were extracted. Real-time PCR and western blot were performed to detect the expression of PLK1mRNA (upper) and protein (lower) after lnc-RI knockdown. ( d,e ) PLK1 expression rescued mitotic arrest induced by lnc-RI knockdown. 3 day after infection with LV-shRNA-lnc-RI, HeLa cells were transfected with plasmid expressing PLK1-myc and mitotic index was analyzed 48 h after transfection. *P-value
    Figure Legend Snippet: PLK1 is the target of lnc-RI and is involved in mitotic arrest induced by lnc-RI knockdown. ( a ) The functional association of lnc-RI target genes. All 144 candidate target genes were uploaded into STRING 10 ( http://string-db.org/ ), and three networks related to cell cycle, apoptosis, and DNA damage were identified. ( b,c ) Knockdown of lnc-RI reduced PLK1 mRNA and protein expression levels. The HEK293 ( b ) and HeLa cells ( c ) were transfected with lnc-RI RNAi-1, RNAi-2 or Ctrl RNAi. At 48 h post-transfection, total RNA and total protein were extracted. Real-time PCR and western blot were performed to detect the expression of PLK1mRNA (upper) and protein (lower) after lnc-RI knockdown. ( d,e ) PLK1 expression rescued mitotic arrest induced by lnc-RI knockdown. 3 day after infection with LV-shRNA-lnc-RI, HeLa cells were transfected with plasmid expressing PLK1-myc and mitotic index was analyzed 48 h after transfection. *P-value

    Techniques Used: Functional Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Infection, shRNA, Plasmid Preparation

    8) Product Images from "Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms"

    Article Title: Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw189

    Butyrate transcriptionally upregulates the TLR5 expression in IEC. ( A ) qRT-PCR showing TLR5 mRNA expression in HT29 cells treated with physiological concentration of butyrate (1 to 20 mM) for 24 h (left) or with 4 mM of butyrate for indicated times (right). ( B ) Immunoblot of total lysates from HT29 cells treated with butyrate (4 mM) for indicated times and probed with TLR5 and Tubulin (loading control) antibodies (upper); Histogram showing increment of TLR5 expression in HT29 cells treated with butyrate as above and analyzed by flow cytometry (lower). ( C ) Immunoblot of TLR5 and Tubulin (loading control) in cell lysates of primary colonic epithelial cells isolated from mice fed with PBS (Vehicle) or butyrate (300 µmol/kg body wt.) twice a day at 12 h intervals for 3 days (left); immunohistochemistry showing TLR5 expression in colonic tissues from mice treated as above (right). ( D ) qRT-PCR showing TLR5 mRNA expression in HT29 cells treated with actinomycin D (8 μM) and butyrate, either alone or in combination. Actinomycin D was added 1 h before butyrate. ( E ) ChIP assays showing IgG and RNA polymerase II binding to the TLR5 promoter in HT29 cells. ( F ) qRT-PCR showing TLR5 mRNAs in HT29 cells treated with cycloheximide (CHX) (50 μg/ml) for 1 h followed by butyrate. * P
    Figure Legend Snippet: Butyrate transcriptionally upregulates the TLR5 expression in IEC. ( A ) qRT-PCR showing TLR5 mRNA expression in HT29 cells treated with physiological concentration of butyrate (1 to 20 mM) for 24 h (left) or with 4 mM of butyrate for indicated times (right). ( B ) Immunoblot of total lysates from HT29 cells treated with butyrate (4 mM) for indicated times and probed with TLR5 and Tubulin (loading control) antibodies (upper); Histogram showing increment of TLR5 expression in HT29 cells treated with butyrate as above and analyzed by flow cytometry (lower). ( C ) Immunoblot of TLR5 and Tubulin (loading control) in cell lysates of primary colonic epithelial cells isolated from mice fed with PBS (Vehicle) or butyrate (300 µmol/kg body wt.) twice a day at 12 h intervals for 3 days (left); immunohistochemistry showing TLR5 expression in colonic tissues from mice treated as above (right). ( D ) qRT-PCR showing TLR5 mRNA expression in HT29 cells treated with actinomycin D (8 μM) and butyrate, either alone or in combination. Actinomycin D was added 1 h before butyrate. ( E ) ChIP assays showing IgG and RNA polymerase II binding to the TLR5 promoter in HT29 cells. ( F ) qRT-PCR showing TLR5 mRNAs in HT29 cells treated with cycloheximide (CHX) (50 μg/ml) for 1 h followed by butyrate. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Concentration Assay, Flow Cytometry, Cytometry, Isolation, Mouse Assay, Immunohistochemistry, Chromatin Immunoprecipitation, Binding Assay

    9) Product Images from "Leptin Receptor Metabolism Disorder in Primary Chondrocytes from Adolescent Idiopathic Scoliosis Girls"

    Article Title: Leptin Receptor Metabolism Disorder in Primary Chondrocytes from Adolescent Idiopathic Scoliosis Girls

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17071160

    Effect of the lysosome inhibitor 3MA on Ob-R expression in chondrocytes from the AIS and control groups. RNA and protein samples were extracted from primary chondrocytes from the participants in both the AIS and the control groups. ( A ) RT-PCR was used to detect the Ob-R mRNA expression levels. The Y axis represents the fold change in transcript levels in the AIS and control groups. The AIS group was set to 1.0. The data are displayed as the means ± SDs from 3 experiments; ( B ) Chondrocytes from the AIS and the control groups were incubated with the lysosome inhibitor 3MA (5 mM) or proteasome inhibitor MG132 (2 μM) for 3 h. Total and membrane proteins were extracted. Ob-R expression was analyzed by Western blotting. β-actin was used as an internal reference. Representative Western blots from three independent experiments are shown; ( C ) The quantitation of the total Ob-R/β-actin levels is shown in the graphs, and the mean value for the untreated control groups was set to 1.0. The Y axis represents the fold change between the AIS and control groups. * p
    Figure Legend Snippet: Effect of the lysosome inhibitor 3MA on Ob-R expression in chondrocytes from the AIS and control groups. RNA and protein samples were extracted from primary chondrocytes from the participants in both the AIS and the control groups. ( A ) RT-PCR was used to detect the Ob-R mRNA expression levels. The Y axis represents the fold change in transcript levels in the AIS and control groups. The AIS group was set to 1.0. The data are displayed as the means ± SDs from 3 experiments; ( B ) Chondrocytes from the AIS and the control groups were incubated with the lysosome inhibitor 3MA (5 mM) or proteasome inhibitor MG132 (2 μM) for 3 h. Total and membrane proteins were extracted. Ob-R expression was analyzed by Western blotting. β-actin was used as an internal reference. Representative Western blots from three independent experiments are shown; ( C ) The quantitation of the total Ob-R/β-actin levels is shown in the graphs, and the mean value for the untreated control groups was set to 1.0. The Y axis represents the fold change between the AIS and control groups. * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Western Blot, Quantitation Assay

    10) Product Images from "Targeting PI3Kδ and PI3Kγ signalling disrupts human AML survival and bone marrow stromal cell mediated protection"

    Article Title: Targeting PI3Kδ and PI3Kγ signalling disrupts human AML survival and bone marrow stromal cell mediated protection

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9289

    shRNA knockdown of PI3Kδ and PI3Kγ A. AML cell line (OCI-AML3) was transduced with either PI3Kδ or PI3Kγ-targeted shRNA or a negative-targeted shRNA lentiviral construct for 72 h. RNA was extracted and assessed for PI3Kδ and PI3Kγ mRNA by real-time PCR. mRNA expression was normalized to GAPDH mRNA levels (n=4). B. OCI-AML3 was transduced with either PI3Kδ or PI3Kγ-targeted shRNA lentivirus for up to 7 days. Cell numbers were measured and expressed as a percentage of control transfected cells. C. OCI-AML3 was transduced with either PI3Kδ or PI3Kγ-targeted shRNA lentivirus for 72 h. Calcein-AM was added at 2.5μM for 1 hour, then washed off. The cells were then cultured on top of primary BMSC for 4 hours. Non-adherent cells were removed with gentle washing. Adherent cells were measured using fluorescence plate reader. Cell adhesion was then assessed and expressed as percentage of total cells in the assay. D. OCI-AML3 were transduced with either PI3Kδ or PI3Kγ-targeted shRNA lentivirus for 72 h and then placed in the upper well of a 5.0μM transwell plate. The lower chamber contained 500ul of serum free media supplemented with SDF1 (100 ng/ml) for 3 hours; we then assessed cell numbers using a flow cytometer. Data were normalised to control-KD cells. E. OCI-AML3 were transduced with either PI3Kδ or PI3Kγ-targeted shRNA lentivirus for 72 h and then protein was extracted and analysed for pAKT (s473 and t308) and total AKT using Western blotting. * indicates those results which are statistically significant (P-value
    Figure Legend Snippet: shRNA knockdown of PI3Kδ and PI3Kγ A. AML cell line (OCI-AML3) was transduced with either PI3Kδ or PI3Kγ-targeted shRNA or a negative-targeted shRNA lentiviral construct for 72 h. RNA was extracted and assessed for PI3Kδ and PI3Kγ mRNA by real-time PCR. mRNA expression was normalized to GAPDH mRNA levels (n=4). B. OCI-AML3 was transduced with either PI3Kδ or PI3Kγ-targeted shRNA lentivirus for up to 7 days. Cell numbers were measured and expressed as a percentage of control transfected cells. C. OCI-AML3 was transduced with either PI3Kδ or PI3Kγ-targeted shRNA lentivirus for 72 h. Calcein-AM was added at 2.5μM for 1 hour, then washed off. The cells were then cultured on top of primary BMSC for 4 hours. Non-adherent cells were removed with gentle washing. Adherent cells were measured using fluorescence plate reader. Cell adhesion was then assessed and expressed as percentage of total cells in the assay. D. OCI-AML3 were transduced with either PI3Kδ or PI3Kγ-targeted shRNA lentivirus for 72 h and then placed in the upper well of a 5.0μM transwell plate. The lower chamber contained 500ul of serum free media supplemented with SDF1 (100 ng/ml) for 3 hours; we then assessed cell numbers using a flow cytometer. Data were normalised to control-KD cells. E. OCI-AML3 were transduced with either PI3Kδ or PI3Kγ-targeted shRNA lentivirus for 72 h and then protein was extracted and analysed for pAKT (s473 and t308) and total AKT using Western blotting. * indicates those results which are statistically significant (P-value

    Techniques Used: shRNA, Transduction, Construct, Real-time Polymerase Chain Reaction, Expressing, Transfection, Cell Culture, Fluorescence, Flow Cytometry, Cytometry, Western Blot, Significance Assay

    11) Product Images from "ECAT11/L1td1 Is Enriched in ESCs and Rapidly Activated During iPSCGeneration, but It Is Dispensable for the Maintenance and Induction of Pluripotency"

    Article Title: ECAT11/L1td1 Is Enriched in ESCs and Rapidly Activated During iPSCGeneration, but It Is Dispensable for the Maintenance and Induction of Pluripotency

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020461

    Protein structure andexpression of ECAT11. (A) Amino acid sequences of ECAT11 from various species. The boxes indicate the conserved Transposase_22 motif. Double asterisks indicate equivalent regions of arginin residues responsible for the RNA binding activity of L1ORF1. Black letters on a white background; non-similar residues, blue letters on a cyan background; consensus residues derived from a block of similar residues at a given position, black letters on a green background; consensus residues derived from the occurrence of > 50% of a single residue at a given position, red letters on a yellow background; consensus residues derived from a completely conserved residue at a given position, green letters on a white background; residues weakly similar to the consensus residue at a given position. (B)The expression profiles of mouse (upper)and human (lower) ECAT11. RNA was isolated from ESCs, differentiated ESCs, human ESCs (H9), human dermal fibroblasts (HDF), an embryonic tumor cell line (NCR-G3)and various tissues from adult mice or humans, and were used for an RT-PCR analysis. The differentiation of mouse ESCs was achieved by retinoic acid (RA) treatment (300 nM) for 7 days. The amplification cycles are shown at right. NAT1 was used as a loading control.
    Figure Legend Snippet: Protein structure andexpression of ECAT11. (A) Amino acid sequences of ECAT11 from various species. The boxes indicate the conserved Transposase_22 motif. Double asterisks indicate equivalent regions of arginin residues responsible for the RNA binding activity of L1ORF1. Black letters on a white background; non-similar residues, blue letters on a cyan background; consensus residues derived from a block of similar residues at a given position, black letters on a green background; consensus residues derived from the occurrence of > 50% of a single residue at a given position, red letters on a yellow background; consensus residues derived from a completely conserved residue at a given position, green letters on a white background; residues weakly similar to the consensus residue at a given position. (B)The expression profiles of mouse (upper)and human (lower) ECAT11. RNA was isolated from ESCs, differentiated ESCs, human ESCs (H9), human dermal fibroblasts (HDF), an embryonic tumor cell line (NCR-G3)and various tissues from adult mice or humans, and were used for an RT-PCR analysis. The differentiation of mouse ESCs was achieved by retinoic acid (RA) treatment (300 nM) for 7 days. The amplification cycles are shown at right. NAT1 was used as a loading control.

    Techniques Used: RNA Binding Assay, Activity Assay, Derivative Assay, Blocking Assay, Expressing, Isolation, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    Generation of iPSCs from ECAT11 EGFP/EGFP MEFs. (A) Morphology of an ECAT11 EGFP/EGFP iPSC colony, which was picked on day 23 after induction of four factors (Oct4, Sox2, Klf4 and c-Myc) and cultured on feeder cells for three passages. Scale bars: 100 µm. (B) The expression levels of three pluripotency markers (Nanog, ECAT1 and Zfp42), and four transcription factors (Oct4, Sox2, Klf4 and c-Myc). Total RNA was collected from four clones of ECAT11 EGFP/EGFP iPSCs(686F1, 686I5, 686L5 and 686O2) established using four factors (Oct4, Sox2, Klf4 and c-Myc), and four clones (686E1, 686H7, 686K1 and 686N1)established usingthree factors (Oct4, Sox2 and Klf4). The iPSCs selected with Fbx15 orthe Nanog reporter (20D17), MEFs, and ES cells were also usedas controls. For reprogramming factor detection, RT–PCR analyses were performed with primers that amplified endogenous transcripts only (endo) and transgene transcripts only (tg) to detect the viral vector silencing. (C) Hematoxylin and eosin staining of teratomas generated from ECAT11 EGFP/EGFP iPSCs. Scale bars: 50 µm.
    Figure Legend Snippet: Generation of iPSCs from ECAT11 EGFP/EGFP MEFs. (A) Morphology of an ECAT11 EGFP/EGFP iPSC colony, which was picked on day 23 after induction of four factors (Oct4, Sox2, Klf4 and c-Myc) and cultured on feeder cells for three passages. Scale bars: 100 µm. (B) The expression levels of three pluripotency markers (Nanog, ECAT1 and Zfp42), and four transcription factors (Oct4, Sox2, Klf4 and c-Myc). Total RNA was collected from four clones of ECAT11 EGFP/EGFP iPSCs(686F1, 686I5, 686L5 and 686O2) established using four factors (Oct4, Sox2, Klf4 and c-Myc), and four clones (686E1, 686H7, 686K1 and 686N1)established usingthree factors (Oct4, Sox2 and Klf4). The iPSCs selected with Fbx15 orthe Nanog reporter (20D17), MEFs, and ES cells were also usedas controls. For reprogramming factor detection, RT–PCR analyses were performed with primers that amplified endogenous transcripts only (endo) and transgene transcripts only (tg) to detect the viral vector silencing. (C) Hematoxylin and eosin staining of teratomas generated from ECAT11 EGFP/EGFP iPSCs. Scale bars: 50 µm.

    Techniques Used: Cell Culture, Expressing, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Plasmid Preparation, Staining, Generated

    12) Product Images from "Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD"

    Article Title: Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-017-0341-7

    Differential mRNA expression profiles in PBECs from COPD patients and control subjects. a Schematic representation of the ALI culture model. PBECs from control 7–17 and COPD 6–16 (Table 1 ) were seeded onto transwell inserts and grown to confluence. Thereafter, apical medium was removed to create an ALI. Cells were harvested after 14 days for RNA and DNA analysis. b Expressions of MUC5AC , AGR2 , SPDEF , and FOXA2 were analyzed by real-time quantitative PCR. Significance was tested by the Mann-Whitney U test. Medians are indicated. ns not significant. * p
    Figure Legend Snippet: Differential mRNA expression profiles in PBECs from COPD patients and control subjects. a Schematic representation of the ALI culture model. PBECs from control 7–17 and COPD 6–16 (Table 1 ) were seeded onto transwell inserts and grown to confluence. Thereafter, apical medium was removed to create an ALI. Cells were harvested after 14 days for RNA and DNA analysis. b Expressions of MUC5AC , AGR2 , SPDEF , and FOXA2 were analyzed by real-time quantitative PCR. Significance was tested by the Mann-Whitney U test. Medians are indicated. ns not significant. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    Characterization of the differentiation state of primary bronchial epithelial cells (PBEC) cultured in the air-liquid interface (ALI) model. a Schematic representation of the ALI culture model. PBECs were seeded on to a transwell insert and grown until confluence. Thereafter, apical medium was removed to create an ALI. Cells were harvested after 0, 14, 21, or 28 days for RNA, DNA, and morphology analysis. b – d PBEC from control subjects 1–6 (Table 1 , n = 6) were cultured at ALI with IL-13 stimulation. b Representative images of immunohistochemistry staining on the differentiated ciliated cells and goblet cells at ALI day 21. Ciliated cells were determined by acetylated-α-tubulin antibody staining and specified by arrows in the images; goblet cells were determined by Alcian Blue staining and MUC5AC antibody staining and were specified by arrow heads in the images. mRNA expressions of c MUC5AC , d AGR2 , e SPDEF , and f FOXA2 were analyzed by real-time quantitative PCR at four different time points. Medians are indicated. Significance was tested by the Kruskal-Wallis non-parametric test with Dunn’s posttest data. ns not significant. * p
    Figure Legend Snippet: Characterization of the differentiation state of primary bronchial epithelial cells (PBEC) cultured in the air-liquid interface (ALI) model. a Schematic representation of the ALI culture model. PBECs were seeded on to a transwell insert and grown until confluence. Thereafter, apical medium was removed to create an ALI. Cells were harvested after 0, 14, 21, or 28 days for RNA, DNA, and morphology analysis. b – d PBEC from control subjects 1–6 (Table 1 , n = 6) were cultured at ALI with IL-13 stimulation. b Representative images of immunohistochemistry staining on the differentiated ciliated cells and goblet cells at ALI day 21. Ciliated cells were determined by acetylated-α-tubulin antibody staining and specified by arrows in the images; goblet cells were determined by Alcian Blue staining and MUC5AC antibody staining and were specified by arrow heads in the images. mRNA expressions of c MUC5AC , d AGR2 , e SPDEF , and f FOXA2 were analyzed by real-time quantitative PCR at four different time points. Medians are indicated. Significance was tested by the Kruskal-Wallis non-parametric test with Dunn’s posttest data. ns not significant. * p

    Techniques Used: Cell Culture, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

    13) Product Images from "Smad7 knockdown activates protein kinase RNA-associated eIF2α pathway leading to colon cancer cell death"

    Article Title: Smad7 knockdown activates protein kinase RNA-associated eIF2α pathway leading to colon cancer cell death

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.103

    SMAD7 antisense (AS)-induced PKR phosphorylation in DLD-1 cells does not rely on the modulation of known PKR-activating pathways. ( a–c ) DLD-1 cells were transfected with either Smad7 sense (S) or AS oligonucleotide (both used at 2 μ g/ml). After 24 h, PKR ( a ), caspase-9, procaspase-3 and cleaved caspase-3 ( b ) as well as PACT expression ( c ) was assessed by western blotting. Staurosporine (ST) (1 μ g/ml) was used as a positive control for caspase-3 activation. β -Actin was used as loading control. One of three representative experiments is shown. ( d and e ) DLD-1 cells were transfected with either Smad7 S or AS oligonucleotide (both used at 2 μ g/ml). After 24 h, RNA transcripts for the ER stress-related genes GRP-78 ( d ) and ATF6α ( e ) were determined by quantitative PCR. TM (1 μ g/ml) was used as a positive control. Levels are normalized to β -actin. Values mean±S.E.M. of three independent experiments. ( f ) DLD-1 cells were transfected with either Smad7 S or AS oligonucleotide (both used at 2 μ g/ml). After 24 h, Smad7, p-PKR (Thr-446), GRP-78, ATF6 α and p-IRE1 α expression was evaluated by western blotting. β -Actin was used as a loading control. ( g ) DLD-1 cells were transfected with either CAD-11 AS oligonucleotide (used at 400 nM) or FSTL1 AS oligonucleotide (used at 10 nM) along with the respective negative controls (scrambled). After 24 h, cells were washed with PBS and cultured for further 30 min. CDH-11, FSTL1, p-PKR (Thr-446) and PKR expression was assessed by western blotting. β -Actin was used as a loading control. One of three representative experiments is shown
    Figure Legend Snippet: SMAD7 antisense (AS)-induced PKR phosphorylation in DLD-1 cells does not rely on the modulation of known PKR-activating pathways. ( a–c ) DLD-1 cells were transfected with either Smad7 sense (S) or AS oligonucleotide (both used at 2 μ g/ml). After 24 h, PKR ( a ), caspase-9, procaspase-3 and cleaved caspase-3 ( b ) as well as PACT expression ( c ) was assessed by western blotting. Staurosporine (ST) (1 μ g/ml) was used as a positive control for caspase-3 activation. β -Actin was used as loading control. One of three representative experiments is shown. ( d and e ) DLD-1 cells were transfected with either Smad7 S or AS oligonucleotide (both used at 2 μ g/ml). After 24 h, RNA transcripts for the ER stress-related genes GRP-78 ( d ) and ATF6α ( e ) were determined by quantitative PCR. TM (1 μ g/ml) was used as a positive control. Levels are normalized to β -actin. Values mean±S.E.M. of three independent experiments. ( f ) DLD-1 cells were transfected with either Smad7 S or AS oligonucleotide (both used at 2 μ g/ml). After 24 h, Smad7, p-PKR (Thr-446), GRP-78, ATF6 α and p-IRE1 α expression was evaluated by western blotting. β -Actin was used as a loading control. ( g ) DLD-1 cells were transfected with either CAD-11 AS oligonucleotide (used at 400 nM) or FSTL1 AS oligonucleotide (used at 10 nM) along with the respective negative controls (scrambled). After 24 h, cells were washed with PBS and cultured for further 30 min. CDH-11, FSTL1, p-PKR (Thr-446) and PKR expression was assessed by western blotting. β -Actin was used as a loading control. One of three representative experiments is shown

    Techniques Used: Transfection, Expressing, Western Blot, Positive Control, Activation Assay, Real-time Polymerase Chain Reaction, Cell Culture

    14) Product Images from "Transducin β-like 1 X-linked receptor 1 suppresses cisplatin sensitivity in Nasopharyngeal Carcinoma via activation of NF-κB pathway"

    Article Title: Transducin β-like 1 X-linked receptor 1 suppresses cisplatin sensitivity in Nasopharyngeal Carcinoma via activation of NF-κB pathway

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-195

    TBL1XR1 activates NF-κB signaling pathway. (A) Luciferase-reporter NF-κB activity in indicated cells. (B) Real-time PCR analysis indicating an apparent overlap between NF-κB-dependent gene expression and TBL1XR1-regulated gene expression. The pseudo color represents the intensity scale of TBL1XR1 vs Vector, or TBL1XR1 short hairpin RNA vs RNAi-vector, generated by a log2 transformation. (C) Analysis of expression and correlation of TBL1XR1 with Bcl-2, Bcl-xl, c-FLIP and IκB mRNA expression, as well as NF-κB activity in 10 freshly collected NPC biopsies.
    Figure Legend Snippet: TBL1XR1 activates NF-κB signaling pathway. (A) Luciferase-reporter NF-κB activity in indicated cells. (B) Real-time PCR analysis indicating an apparent overlap between NF-κB-dependent gene expression and TBL1XR1-regulated gene expression. The pseudo color represents the intensity scale of TBL1XR1 vs Vector, or TBL1XR1 short hairpin RNA vs RNAi-vector, generated by a log2 transformation. (C) Analysis of expression and correlation of TBL1XR1 with Bcl-2, Bcl-xl, c-FLIP and IκB mRNA expression, as well as NF-κB activity in 10 freshly collected NPC biopsies.

    Techniques Used: Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, shRNA, Generated, Transformation Assay

    15) Product Images from "Interferon lambda inhibits dengue virus replication in epithelial cells"

    Article Title: Interferon lambda inhibits dengue virus replication in epithelial cells

    Journal: Virology Journal

    doi: 10.1186/s12985-015-0383-4

    DENV-2 induces the expression of IFN-III in C33-A cells. C33-A cells were treated with IFN-λ1 (35 ng/ml) infected with DENV-2 (MOI = 0.1) or pre-treated with IFN-λ1 and subsequently infected with DENV-2. Forty-eight h post-infection, total cellular RNA was isolated and used in end-point RT-PCR for qualitative determination of the IFN-λ1, IFN-λ2, IFN-λ3 genes, using HPRT as an endogenous control gene. a Results of RT-PCR amplification of mock-infected and the three experimental conditions are shown. b Extracts of total protein from each condition were used for Western blotting to determine the presence of IFN-λ1 and IFN-λ2 proteins. Actin was the loading control. A semi quantitative analysis of detected bands normalized to actin was performed using ImageJ software ( http://rsb.info.nih.gov/ij/ )
    Figure Legend Snippet: DENV-2 induces the expression of IFN-III in C33-A cells. C33-A cells were treated with IFN-λ1 (35 ng/ml) infected with DENV-2 (MOI = 0.1) or pre-treated with IFN-λ1 and subsequently infected with DENV-2. Forty-eight h post-infection, total cellular RNA was isolated and used in end-point RT-PCR for qualitative determination of the IFN-λ1, IFN-λ2, IFN-λ3 genes, using HPRT as an endogenous control gene. a Results of RT-PCR amplification of mock-infected and the three experimental conditions are shown. b Extracts of total protein from each condition were used for Western blotting to determine the presence of IFN-λ1 and IFN-λ2 proteins. Actin was the loading control. A semi quantitative analysis of detected bands normalized to actin was performed using ImageJ software ( http://rsb.info.nih.gov/ij/ )

    Techniques Used: Expressing, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Software

    16) Product Images from "DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration"

    Article Title: DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration

    Journal: Nature

    doi: 10.1038/nature09830

    Alu RNA accumulation in GA triggered by DICER reduction a, b , dsRNA immunolocalized (blue) in RPE ( a, b ) and sub-RPE deposits (drusen; b ) in human GA. c, d , No staining with isotype antibody in GA RPE ( c ) and with anti-dsRNA antibody in control eye ( d ). Scale bars, ( a–d ), 10 µm. n=10 ( a–d ) e , PCR amplification of immunoprecipitated dsRNA yielded amplicons with homology to Alu in GA RPE but not normal RPE. Water control (−) showed no amplification and recombinant dsRNA (+) showed predicted amplicon. f , Increased Alu RNA in GA RPE compared to control (n=7). P
    Figure Legend Snippet: Alu RNA accumulation in GA triggered by DICER reduction a, b , dsRNA immunolocalized (blue) in RPE ( a, b ) and sub-RPE deposits (drusen; b ) in human GA. c, d , No staining with isotype antibody in GA RPE ( c ) and with anti-dsRNA antibody in control eye ( d ). Scale bars, ( a–d ), 10 µm. n=10 ( a–d ) e , PCR amplification of immunoprecipitated dsRNA yielded amplicons with homology to Alu in GA RPE but not normal RPE. Water control (−) showed no amplification and recombinant dsRNA (+) showed predicted amplicon. f , Increased Alu RNA in GA RPE compared to control (n=7). P

    Techniques Used: Staining, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Recombinant

    17) Product Images from "Requirement of NOX2 and Reactive Oxygen Species for Efficient RIG-I-Mediated Antiviral Response through Regulation of MAVS Expression"

    Article Title: Requirement of NOX2 and Reactive Oxygen Species for Efficient RIG-I-Mediated Antiviral Response through Regulation of MAVS Expression

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000930

    NOX2 downregulation or ROS scavenging diminishes MAVS mRNA expression without affecting its subcellular localization. ( A, B ) WCE derived from A549 ( A ) and NHBE ( B ) cells transfected with control (CTRL) or NOX2 RNAi or treated with vehicle or 3mM Tempol were analyzed by immunoblot using anti-RIG-I, anti-MAVS, anti-TRIM25, anti-TRAF3, anti-TRAF6 and anti-actin antibodies. MAVS was detected as two different splice variants as described in [66] . Representative immunoblots of at least three different experiments are shown. ( C ) Total RNA extracted from A549 transfected with CTRL and NOX2 RNAi were analyzed by real time PCR as described in Figure 1 . MAVS mRNA levels are expressed as absolute values after normalization to S9 mRNA used as a reference gene. (*, p
    Figure Legend Snippet: NOX2 downregulation or ROS scavenging diminishes MAVS mRNA expression without affecting its subcellular localization. ( A, B ) WCE derived from A549 ( A ) and NHBE ( B ) cells transfected with control (CTRL) or NOX2 RNAi or treated with vehicle or 3mM Tempol were analyzed by immunoblot using anti-RIG-I, anti-MAVS, anti-TRIM25, anti-TRAF3, anti-TRAF6 and anti-actin antibodies. MAVS was detected as two different splice variants as described in [66] . Representative immunoblots of at least three different experiments are shown. ( C ) Total RNA extracted from A549 transfected with CTRL and NOX2 RNAi were analyzed by real time PCR as described in Figure 1 . MAVS mRNA levels are expressed as absolute values after normalization to S9 mRNA used as a reference gene. (*, p

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

    Interference with NOX2 expression inhibits SeV-induced IFNβ and IFIT1 genes transactivation. ( A and B ) A549 cells were transfected with control- (CTRL; black bars) or NOX2-specific (white bars) RNAi oligonucleotides. ( A ) Efficiency of NOX2 knock down was monitored by immunoblot (IB) using anti-NOX2 antibodies. Anti-tubulin antibodies were used to control equal loading. ( B ) At 48h post-RNAi transfection, cells were further transfected with the IFNβ-pGL3 or ISG56-pGL3 firefly luciferase and the pRL-null renilla luciferase (internal control) reporter constructs and either left uninfected (NS) or infected with SeV (80 HAU/10 6 cells). Luciferase activities were measured and expressed as described in Figure 1 . ( C ) A549 cells were cotransfected with an empty control plasmid (black bars) or the myc-tagged-NOX2 (white bars) encoding plasmid and the IFNβ-pGL3 firefly luciferase and the pRL-null renilla luciferase (internal control) reporter constructs. At 16h post-transfection, cells were left unstimulated or infected with SeV for 8h and luciferase activities were measured and analyzed as described above. ( D ) Total RNA extracted from CTRL (black bars) and NOX2 RNAi (white bars)-transfected A549 either left uninfected (NS) or infected with SeV (5 HAU/10 6 cells) for 5 hours were analyzed by reverse transcription and real-time PCR using IFNβ-, SeV N, and S9-specific primers. IFNβ mRNA levels are presented as absolute copy numbers normalized versus S9 mRNA used as a reference. SeV N fold expression values were determined using the ΔΔC t method as described in Material and Methods .(*, p
    Figure Legend Snippet: Interference with NOX2 expression inhibits SeV-induced IFNβ and IFIT1 genes transactivation. ( A and B ) A549 cells were transfected with control- (CTRL; black bars) or NOX2-specific (white bars) RNAi oligonucleotides. ( A ) Efficiency of NOX2 knock down was monitored by immunoblot (IB) using anti-NOX2 antibodies. Anti-tubulin antibodies were used to control equal loading. ( B ) At 48h post-RNAi transfection, cells were further transfected with the IFNβ-pGL3 or ISG56-pGL3 firefly luciferase and the pRL-null renilla luciferase (internal control) reporter constructs and either left uninfected (NS) or infected with SeV (80 HAU/10 6 cells). Luciferase activities were measured and expressed as described in Figure 1 . ( C ) A549 cells were cotransfected with an empty control plasmid (black bars) or the myc-tagged-NOX2 (white bars) encoding plasmid and the IFNβ-pGL3 firefly luciferase and the pRL-null renilla luciferase (internal control) reporter constructs. At 16h post-transfection, cells were left unstimulated or infected with SeV for 8h and luciferase activities were measured and analyzed as described above. ( D ) Total RNA extracted from CTRL (black bars) and NOX2 RNAi (white bars)-transfected A549 either left uninfected (NS) or infected with SeV (5 HAU/10 6 cells) for 5 hours were analyzed by reverse transcription and real-time PCR using IFNβ-, SeV N, and S9-specific primers. IFNβ mRNA levels are presented as absolute copy numbers normalized versus S9 mRNA used as a reference. SeV N fold expression values were determined using the ΔΔC t method as described in Material and Methods .(*, p

    Techniques Used: Expressing, Transfection, Luciferase, Construct, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    NADPH oxidase-derived ROS are required for SeV-induced IFNβ and IFIT1 genes regulation. ( A–D ) A549 were transfected with the pRL-null renilla luciferase (internal control) and either the IFNβ-pGL3 or ISG56-pGL3 firefly luciferase reporter constructs. At 16h post-transfection, cells were pretreated with the indicated inhibitors (white bars), 3 mM Tempol, 1–30µM DPI (in B, a 10µM concentration was used) or 0.1–1mM Apo (in B, a 1mM concentration was used) or the corresponding vehicle (black bars), before being left unstimulated (NS) or infected with SeV (80 HAU/10 6 cells) for 8h. Luciferase activities were expressed as fold activation over the corresponding NS condition after normalization with renilla luciferase activities. ( E ) A549 were treated with 30µM DPI (white bars) or the vehicle (black bars) and then left uninfected (NS) or infected with SeV (40 HAU/10 6 cells) for 6h. Total RNA was extracted, subjected to reverse transcription and analyzed by real-time PCR using IFNβ-, ISG56- and actin-specific primers. mRNA levels are presented as absolute copy numbers of the target gene normalized versus actin mRNA used as a reference gene. (*, p
    Figure Legend Snippet: NADPH oxidase-derived ROS are required for SeV-induced IFNβ and IFIT1 genes regulation. ( A–D ) A549 were transfected with the pRL-null renilla luciferase (internal control) and either the IFNβ-pGL3 or ISG56-pGL3 firefly luciferase reporter constructs. At 16h post-transfection, cells were pretreated with the indicated inhibitors (white bars), 3 mM Tempol, 1–30µM DPI (in B, a 10µM concentration was used) or 0.1–1mM Apo (in B, a 1mM concentration was used) or the corresponding vehicle (black bars), before being left unstimulated (NS) or infected with SeV (80 HAU/10 6 cells) for 8h. Luciferase activities were expressed as fold activation over the corresponding NS condition after normalization with renilla luciferase activities. ( E ) A549 were treated with 30µM DPI (white bars) or the vehicle (black bars) and then left uninfected (NS) or infected with SeV (40 HAU/10 6 cells) for 6h. Total RNA was extracted, subjected to reverse transcription and analyzed by real-time PCR using IFNβ-, ISG56- and actin-specific primers. mRNA levels are presented as absolute copy numbers of the target gene normalized versus actin mRNA used as a reference gene. (*, p

    Techniques Used: Derivative Assay, Transfection, Luciferase, Construct, Concentration Assay, Infection, Activation Assay, Real-time Polymerase Chain Reaction

    18) Product Images from "?1 Integrin-Mediated Adhesion Signalling Is Essential for Epidermal Progenitor Cell Expansion"

    Article Title: ?1 Integrin-Mediated Adhesion Signalling Is Essential for Epidermal Progenitor Cell Expansion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005488

    Nonsense-mediated decay (NMD) leads to reduced β1 integrin expression from the hpm KI lox allele. (A) Subconfluent primary keratinocytes isolated from adult hpm KI lox /+ mice were treated with 100 µg/ml emetine to block protein translation and thus the NMD pathway, or (B) treated with 5 µg/ml actinomycin D to block de novo mRNA transcription in the presence or absence of emetine. Cells were lysed at indicated times, RNA isolated and applied as template for quantitative RT-PCR using primers specific for the hpm KI lox cDNA. Quantification of 3 independent experiments is shown. Error bars indicate standard deviations (s.d.). (C) Quantification of RT-PCR analysis using primers specific for total β1 mRNA shows reduced transcript levels in 3-week-old hpm epidermis when normalized to controls. RNA from 5 mice per genotype was used as template for the analysis. Error bars indicate s.d. (D) Representative histogram of flow cytometry analysis reveals that cell surface expression of β1 integrin is reduced on freshly isolated keratinocytes from 3-week-old hpm mice. Blue and black histograms denote control and hpm keratinocytes, respectively, and background fluorescence is shown as dotted line. The flow cytometry experiments were performed with 3 mice of each genotype. (E) hpm mice show progressive hair loss, which is delayed and less severe when compared with K5-β1 mice. The age of the mice is indicated.
    Figure Legend Snippet: Nonsense-mediated decay (NMD) leads to reduced β1 integrin expression from the hpm KI lox allele. (A) Subconfluent primary keratinocytes isolated from adult hpm KI lox /+ mice were treated with 100 µg/ml emetine to block protein translation and thus the NMD pathway, or (B) treated with 5 µg/ml actinomycin D to block de novo mRNA transcription in the presence or absence of emetine. Cells were lysed at indicated times, RNA isolated and applied as template for quantitative RT-PCR using primers specific for the hpm KI lox cDNA. Quantification of 3 independent experiments is shown. Error bars indicate standard deviations (s.d.). (C) Quantification of RT-PCR analysis using primers specific for total β1 mRNA shows reduced transcript levels in 3-week-old hpm epidermis when normalized to controls. RNA from 5 mice per genotype was used as template for the analysis. Error bars indicate s.d. (D) Representative histogram of flow cytometry analysis reveals that cell surface expression of β1 integrin is reduced on freshly isolated keratinocytes from 3-week-old hpm mice. Blue and black histograms denote control and hpm keratinocytes, respectively, and background fluorescence is shown as dotted line. The flow cytometry experiments were performed with 3 mice of each genotype. (E) hpm mice show progressive hair loss, which is delayed and less severe when compared with K5-β1 mice. The age of the mice is indicated.

    Techniques Used: Expressing, Isolation, Mouse Assay, Blocking Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    Keratinocytes escaping NMD increase β1 integrin expression and improve skin defects in old hpm mice. (A) Expression of lacZ and β1 integrin in primary keratinocytes from 3-week-, 2.5-month-, 6.5-month- and 20-month-old control and hpm mice assessed by flow cytometry. Keratinocytes were stained with an anti-β1 integrin antibody and subsequently incubated with a fluorogenic substrate (fluorescein di-β-D-galactopyranoside) to determine β-galactosidase activity. The expression of β1 integrin increases with the age of hpm mice. (B) Quantification of results from flow cytometry from at least 3 mice per genotype and developmental stage. Error bars indicate s.d. (C) Expression of the hpm KI lox transcript determined by quantitative RT-PCR analysis. RNA isolated from epidermal lysates from control and hpm mice at indicated ages was used as template for quantitative RT-PCR analysis with primers specific for the hpm KI lox RNA. Expression of the hpm KI lox RNA in hpm mice is shown relative to the expression levels of the hpm KI lox RNA in control mice. The abundance of the hpm KI lox transcript in hpm mice increases with age relative to control mice. 1 to 4 control and 3 to 5 hpm mice per age were analysed. Error bars indicate s.d.
    Figure Legend Snippet: Keratinocytes escaping NMD increase β1 integrin expression and improve skin defects in old hpm mice. (A) Expression of lacZ and β1 integrin in primary keratinocytes from 3-week-, 2.5-month-, 6.5-month- and 20-month-old control and hpm mice assessed by flow cytometry. Keratinocytes were stained with an anti-β1 integrin antibody and subsequently incubated with a fluorogenic substrate (fluorescein di-β-D-galactopyranoside) to determine β-galactosidase activity. The expression of β1 integrin increases with the age of hpm mice. (B) Quantification of results from flow cytometry from at least 3 mice per genotype and developmental stage. Error bars indicate s.d. (C) Expression of the hpm KI lox transcript determined by quantitative RT-PCR analysis. RNA isolated from epidermal lysates from control and hpm mice at indicated ages was used as template for quantitative RT-PCR analysis with primers specific for the hpm KI lox RNA. Expression of the hpm KI lox RNA in hpm mice is shown relative to the expression levels of the hpm KI lox RNA in control mice. The abundance of the hpm KI lox transcript in hpm mice increases with age relative to control mice. 1 to 4 control and 3 to 5 hpm mice per age were analysed. Error bars indicate s.d.

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Staining, Incubation, Activity Assay, Quantitative RT-PCR, Isolation, RNA Expression

    19) Product Images from "p38? Mitogen-Activated Protein Kinase Is a Key Regulator in Skeletal Muscle Metabolic Adaptation in Mice"

    Article Title: p38? Mitogen-Activated Protein Kinase Is a Key Regulator in Skeletal Muscle Metabolic Adaptation in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007934

    Overexpression of dominant negative p38γ, but not that of p38α and p38β, blocks contractility-induced Pgc-1α mRNA expression. Plasmid DNA (100 µg) containing an empty vector (pCI-neo) or FLAG-tagged dominant negative forms of p38α (p38α (AF)), p38β (p38β(AF)), or p38γ (p38γ(AF)) were injected into right and left tibialis anterior muscles followed by electric pulse-mediated gene transfer (described in Materials and Methods ). After 10 days of recovery, motor nerve stimulation (10 Hz, 2 hours) was performed for the left tibialis anterior muscle, and both the stimulated and the contralateral control tibialis anterior muscles were harvested for analyses for transgene expression, signaling molecule activation and Pgc-1α mRNA expression. A) Fluorescence image showing the efficacy (∼60%) of gene transfer in tibialis anterior muscle (outline in red). The scale bar equals 200 µ; B) Immunoblot analysis for the tibialis anterior muscles 10 days after gene transfer showing expression of FLAG-tagged p38α (AF), p38β(AF), and p38γ(AF) compared with the tibialis anterior muscle transfected with pCI-neo control plasmid. Lines divide images from different gels. Non-specific bands (NB) were labeled; and C) Real-time PCR analysis showing that motor nerve stimulation results in significant induction of Pgc-1α mRNA in tibialis anterior muscles transfected with pCI-neo, p38α (AF) or p38β(AF), which is blocked by overexpression of p38γ(AF). The data was normalized by 18S ribosomal RNA (n = 6–12). *, ** and *** denote p
    Figure Legend Snippet: Overexpression of dominant negative p38γ, but not that of p38α and p38β, blocks contractility-induced Pgc-1α mRNA expression. Plasmid DNA (100 µg) containing an empty vector (pCI-neo) or FLAG-tagged dominant negative forms of p38α (p38α (AF)), p38β (p38β(AF)), or p38γ (p38γ(AF)) were injected into right and left tibialis anterior muscles followed by electric pulse-mediated gene transfer (described in Materials and Methods ). After 10 days of recovery, motor nerve stimulation (10 Hz, 2 hours) was performed for the left tibialis anterior muscle, and both the stimulated and the contralateral control tibialis anterior muscles were harvested for analyses for transgene expression, signaling molecule activation and Pgc-1α mRNA expression. A) Fluorescence image showing the efficacy (∼60%) of gene transfer in tibialis anterior muscle (outline in red). The scale bar equals 200 µ; B) Immunoblot analysis for the tibialis anterior muscles 10 days after gene transfer showing expression of FLAG-tagged p38α (AF), p38β(AF), and p38γ(AF) compared with the tibialis anterior muscle transfected with pCI-neo control plasmid. Lines divide images from different gels. Non-specific bands (NB) were labeled; and C) Real-time PCR analysis showing that motor nerve stimulation results in significant induction of Pgc-1α mRNA in tibialis anterior muscles transfected with pCI-neo, p38α (AF) or p38β(AF), which is blocked by overexpression of p38γ(AF). The data was normalized by 18S ribosomal RNA (n = 6–12). *, ** and *** denote p

    Techniques Used: Over Expression, Dominant Negative Mutation, Pyrolysis Gas Chromatography, Expressing, Plasmid Preparation, Injection, Activation Assay, Fluorescence, Transfection, Labeling, Real-time Polymerase Chain Reaction

    Muscle-specific deletion of the p38γ gene results in alterations in endurance exercise-induced gene expression. Wild type and p38γ KO mice were subjected to motor nerve stimulation (10 Hz, 2 hours) via the deep peroneal nerve, which innervates the tibialis anterior muscle. One hour following motor nerve stimulation, both the stimulated tibialis anterior and the contralateral control tibialis anterior muscles were harvested for total RNA isolation and analyzed by real-time PCR and Affymetrix microarray analyses. A) Real-time PCR analysis for Pgc-1α, Pgc-1β, Vegf, Nrf-1 , Nrf-2 and Dscr1 mRNA (n = 5–6). *, ** and *** denote p
    Figure Legend Snippet: Muscle-specific deletion of the p38γ gene results in alterations in endurance exercise-induced gene expression. Wild type and p38γ KO mice were subjected to motor nerve stimulation (10 Hz, 2 hours) via the deep peroneal nerve, which innervates the tibialis anterior muscle. One hour following motor nerve stimulation, both the stimulated tibialis anterior and the contralateral control tibialis anterior muscles were harvested for total RNA isolation and analyzed by real-time PCR and Affymetrix microarray analyses. A) Real-time PCR analysis for Pgc-1α, Pgc-1β, Vegf, Nrf-1 , Nrf-2 and Dscr1 mRNA (n = 5–6). *, ** and *** denote p

    Techniques Used: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Microarray, Pyrolysis Gas Chromatography

    20) Product Images from "Glucose-Regulated Protein 78 (Grp78) Confers Chemoresistance to Tumor Endothelial Cells under Acidic Stress"

    Article Title: Glucose-Regulated Protein 78 (Grp78) Confers Chemoresistance to Tumor Endothelial Cells under Acidic Stress

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0101053

    In A , LCM retrieval of endothelial cells from paraffin embedded tissue sections (a - before dissection, b - removal of blood cells, c- capture of endothelial cells). B , RNA purity control, using VEGFR2, a marker for endothelial cells, and E-cadherin, a marker for cells of ectodermal origin. C, D, E , real-time PCR to quantify Grp78, ATF4 and CHOP expression in TEC compared to NEC and primary endothelial cells. Specimens were obtained from human oral squamous cell carcinoma biopsies. Data presented from real-time PCR experiments reflect the expression levels of Grp78, ATF4 and CHOP normalized to β-tubulin. Values are means ± SD. Columns, means of individual experiments; Bars, SD. ***p
    Figure Legend Snippet: In A , LCM retrieval of endothelial cells from paraffin embedded tissue sections (a - before dissection, b - removal of blood cells, c- capture of endothelial cells). B , RNA purity control, using VEGFR2, a marker for endothelial cells, and E-cadherin, a marker for cells of ectodermal origin. C, D, E , real-time PCR to quantify Grp78, ATF4 and CHOP expression in TEC compared to NEC and primary endothelial cells. Specimens were obtained from human oral squamous cell carcinoma biopsies. Data presented from real-time PCR experiments reflect the expression levels of Grp78, ATF4 and CHOP normalized to β-tubulin. Values are means ± SD. Columns, means of individual experiments; Bars, SD. ***p

    Techniques Used: Laser Capture Microdissection, Dissection, Marker, Real-time Polymerase Chain Reaction, Expressing

    Tumor conditioned medium induces UPR in endothelial cells. Primary human endothelial cells, HDMECs, are maintained in conditioned medium obtained from oral squamous cell carcinoma UM-SCC-81B cell line ( A, B, C ) or U-87 glioblastoma cell line ( D, E, F ), HDMECs kept at regular medium served as control. Cell lysates were collected for western blot analysis, β-actin expression was used as a loading control. A, D , Treatment with CM increased protein expression of UPR markers: Grp78, ATF4, p -elf2α at 48 hours (western blot analysis). B, E , Exposure to CM increased XBP1 mRNA splicing levels at 48 hours. C, F , CM treatment upregulated CHOP mRNA levels. RNA was collected using Qiagen RNAeasy kit, reverse transcribed and analyzed using RT-PCR. Values are means ± SEM. Columns, means of individual experiments; Bars, SEM.**p
    Figure Legend Snippet: Tumor conditioned medium induces UPR in endothelial cells. Primary human endothelial cells, HDMECs, are maintained in conditioned medium obtained from oral squamous cell carcinoma UM-SCC-81B cell line ( A, B, C ) or U-87 glioblastoma cell line ( D, E, F ), HDMECs kept at regular medium served as control. Cell lysates were collected for western blot analysis, β-actin expression was used as a loading control. A, D , Treatment with CM increased protein expression of UPR markers: Grp78, ATF4, p -elf2α at 48 hours (western blot analysis). B, E , Exposure to CM increased XBP1 mRNA splicing levels at 48 hours. C, F , CM treatment upregulated CHOP mRNA levels. RNA was collected using Qiagen RNAeasy kit, reverse transcribed and analyzed using RT-PCR. Values are means ± SEM. Columns, means of individual experiments; Bars, SEM.**p

    Techniques Used: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

    Acidic stress induces UPR in endothelial cells. Primary human endothelial cells, HDMECs, were maintained in acidic cultures for 48 hours; pH was adjusted by lactic acid ( A, B, C ) or HCl ( D, E, F ), HDMECs kept at regular pH 7.5 medium served as control. Cell lysates were collected for western blot analysis, β-actin expression was used as a loading control. A, D , Treatment with low pH increased protein expression of UPR markers: Grp78, ATF4, and p -elf2α at 48 hours (western blot analysis). B, E , Exposure to low pH increased XBP1 mRNA splicing levels at 48 hours. C, F . Acidic stress upregulated CHOP mRNA levels. RNA was collected using Qiagen RNAeasy kit, reverse transcribed and analyzed using RT-PCR. Values are means ± SEM. Columns, means of individual experiments; Bars, SEM.***p
    Figure Legend Snippet: Acidic stress induces UPR in endothelial cells. Primary human endothelial cells, HDMECs, were maintained in acidic cultures for 48 hours; pH was adjusted by lactic acid ( A, B, C ) or HCl ( D, E, F ), HDMECs kept at regular pH 7.5 medium served as control. Cell lysates were collected for western blot analysis, β-actin expression was used as a loading control. A, D , Treatment with low pH increased protein expression of UPR markers: Grp78, ATF4, and p -elf2α at 48 hours (western blot analysis). B, E , Exposure to low pH increased XBP1 mRNA splicing levels at 48 hours. C, F . Acidic stress upregulated CHOP mRNA levels. RNA was collected using Qiagen RNAeasy kit, reverse transcribed and analyzed using RT-PCR. Values are means ± SEM. Columns, means of individual experiments; Bars, SEM.***p

    Techniques Used: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

    21) Product Images from "STAT3 activity is necessary and sufficient for the development of immune-mediated myocarditis in mice and promotes progression to dilated cardiomyopathy"

    Article Title: STAT3 activity is necessary and sufficient for the development of immune-mediated myocarditis in mice and promotes progression to dilated cardiomyopathy

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201201876

    Stat3 C/C mice are more prone to Th17 differentiation. A. Cytofluorimetric analysis of IL-17 producing CD4 + T cells in the spleen of Stat3 WT/WT (white bars, n = 6) and Stat3 C/C (black bars, n = 6) mice. Data are shown as mean ± SEM. * p = 0.015. B. In vitro differentiation of Th17 cells. Naïve CD4 + cells were purified from spleens of Stat3 WT/WT (white bars, n = 6) and Stat3 C/C (black bars, n = 10) mice and treated or not with TGF-β, IL-6 and anti-IFN-γ, followed by cytofluorimetric analysis for CD4 and IL-17. Data are shown as mean ± SEM. ** p = 0.001. C. In vitro differentiation of Th17 cells without IL-6. Naïve CD4 + cells were purified from spleens of Stat3 WT/WT (white bars, n = 6) and Stat3 C/C (black bars, n = 6) mice, treated or not with TGF-β and anti-IFN-γ and analysed as above. Note the different scales between (B) and (C). Data are shown as mean ± SEM. ** p = 0.006 (Stat3 WT/WT versus Stat3 C/C cells); * p = 0.025 (untreated versus treated Stat3 C/C cells). D. Taqman RT-PCR analysis on total RNA from the spleen of Stat3 WT/WT (white bars, n = 7) and Stat3 C/C (black bars, n = 7) mice. Data are shown as mean ± SEM. * p = 0.018 (RORγt); ** p = 0.0057 (IL-23). E. IL-17 (red) and CD4 (green) IF staining of hearts from Stat3 WT/WT (WT) and Stat3 C/C (C) mice at the indicated weeks of age. Representative samples of three mice per group are shown. Scale bar: 50 µm. F. Kaplan-Meier survival curves of Stat3 C/C mice treated with anti-IL-17 neutralizing mAbs (red line, n = 7) or with total IgG as controls (black line, n = 4). G. IL-17 (red) and CD4 (green) IF staining of hearts from anti IL-17-treated and control mice as above. Representative samples from survived ( n = 2) or diseased ( n = 5) mice are shown. Scale bar: 50 µm. H. Kaplan-Meier survival curves of Stat3 C/C mice treated with anti-CD4 neutralizing mAbs (red line, n = 13) or with total IgGs as controls (black line, n = 10). * p = 0.026. I. The hearts of mice from (H) were analysed as indicated. IgG, control mice; for the aCD4-treated mice, samples from survived mice are shown on the left, and those from dead mice on the right. Scale bar: 50 µm.
    Figure Legend Snippet: Stat3 C/C mice are more prone to Th17 differentiation. A. Cytofluorimetric analysis of IL-17 producing CD4 + T cells in the spleen of Stat3 WT/WT (white bars, n = 6) and Stat3 C/C (black bars, n = 6) mice. Data are shown as mean ± SEM. * p = 0.015. B. In vitro differentiation of Th17 cells. Naïve CD4 + cells were purified from spleens of Stat3 WT/WT (white bars, n = 6) and Stat3 C/C (black bars, n = 10) mice and treated or not with TGF-β, IL-6 and anti-IFN-γ, followed by cytofluorimetric analysis for CD4 and IL-17. Data are shown as mean ± SEM. ** p = 0.001. C. In vitro differentiation of Th17 cells without IL-6. Naïve CD4 + cells were purified from spleens of Stat3 WT/WT (white bars, n = 6) and Stat3 C/C (black bars, n = 6) mice, treated or not with TGF-β and anti-IFN-γ and analysed as above. Note the different scales between (B) and (C). Data are shown as mean ± SEM. ** p = 0.006 (Stat3 WT/WT versus Stat3 C/C cells); * p = 0.025 (untreated versus treated Stat3 C/C cells). D. Taqman RT-PCR analysis on total RNA from the spleen of Stat3 WT/WT (white bars, n = 7) and Stat3 C/C (black bars, n = 7) mice. Data are shown as mean ± SEM. * p = 0.018 (RORγt); ** p = 0.0057 (IL-23). E. IL-17 (red) and CD4 (green) IF staining of hearts from Stat3 WT/WT (WT) and Stat3 C/C (C) mice at the indicated weeks of age. Representative samples of three mice per group are shown. Scale bar: 50 µm. F. Kaplan-Meier survival curves of Stat3 C/C mice treated with anti-IL-17 neutralizing mAbs (red line, n = 7) or with total IgG as controls (black line, n = 4). G. IL-17 (red) and CD4 (green) IF staining of hearts from anti IL-17-treated and control mice as above. Representative samples from survived ( n = 2) or diseased ( n = 5) mice are shown. Scale bar: 50 µm. H. Kaplan-Meier survival curves of Stat3 C/C mice treated with anti-CD4 neutralizing mAbs (red line, n = 13) or with total IgGs as controls (black line, n = 10). * p = 0.026. I. The hearts of mice from (H) were analysed as indicated. IgG, control mice; for the aCD4-treated mice, samples from survived mice are shown on the left, and those from dead mice on the right. Scale bar: 50 µm.

    Techniques Used: Mouse Assay, In Vitro, Purification, Reverse Transcription Polymerase Chain Reaction, Staining

    22) Product Images from "Metabolic reprogramming during neuronal differentiation"

    Article Title: Metabolic reprogramming during neuronal differentiation

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2016.36

    In vitro terminal differentiation of cortical neurons is associated with mitochondrial biogenesis. ( a ) Relative quantification of mtDNA copy number during differentiation of cortical neurons. Real-time PCR was performed with primers against a single-copy nuclear gene succinate dehydrogenase complex, subunit A and the mitochondrially encoded NADH dehydrogenase 5 gene. ( b ) Expression of the different complexes of ETC increases along the differentiation. Western blotting was performed with MitoProfile Total OXPHOS. ( c ) Transmission electron microscopy analysis of DIV1 and DIV7 neurons. The mitochondria at DIV1 are small and rounded, with a dense matrix. At DIV7, the mitochondria have a much less dense matrix and the percentage of mitochondria with elongated shape increase, as shown in the graph (right panel). For comparison, we restricted imaging to the perinuclear/Golgi region. ( d ) Western blotting analysis of the transcription factors TFAM, PGC-1 α , MEF-2 and NRF-1 during in vitro terminal differentiation of cortical neurons. Numbers indicate densitometric analysis of a representative experiment. ( e and f ) RNA levels of the indicated PGC-1 α and NRF-1 target genes during in vitro terminal differentiation of cortical neurons, respectively. RNA levels were assessed by real-time PCR
    Figure Legend Snippet: In vitro terminal differentiation of cortical neurons is associated with mitochondrial biogenesis. ( a ) Relative quantification of mtDNA copy number during differentiation of cortical neurons. Real-time PCR was performed with primers against a single-copy nuclear gene succinate dehydrogenase complex, subunit A and the mitochondrially encoded NADH dehydrogenase 5 gene. ( b ) Expression of the different complexes of ETC increases along the differentiation. Western blotting was performed with MitoProfile Total OXPHOS. ( c ) Transmission electron microscopy analysis of DIV1 and DIV7 neurons. The mitochondria at DIV1 are small and rounded, with a dense matrix. At DIV7, the mitochondria have a much less dense matrix and the percentage of mitochondria with elongated shape increase, as shown in the graph (right panel). For comparison, we restricted imaging to the perinuclear/Golgi region. ( d ) Western blotting analysis of the transcription factors TFAM, PGC-1 α , MEF-2 and NRF-1 during in vitro terminal differentiation of cortical neurons. Numbers indicate densitometric analysis of a representative experiment. ( e and f ) RNA levels of the indicated PGC-1 α and NRF-1 target genes during in vitro terminal differentiation of cortical neurons, respectively. RNA levels were assessed by real-time PCR

    Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Imaging, Pyrolysis Gas Chromatography

    23) Product Images from "Effect of Porphyromonas gingivalis infection on post-transcriptional regulation of the low-density lipoprotein receptor in mice"

    Article Title: Effect of Porphyromonas gingivalis infection on post-transcriptional regulation of the low-density lipoprotein receptor in mice

    Journal: Lipids in Health and Disease

    doi: 10.1186/1476-511X-11-121

    Effect of P. gingivalis infection on PCSK9 and LDLR gene and protein expression in the liver. Total RNA and protein were extracted from the P. gingivalis -infected and sham-infected mice, and the samples were analyzed by quantitative RT-PCR or western blotting. Pcsk9 and Ldlr gene expression was significantly higher in the P. gingivalis -infected mice compared with the sham-infected mice (** P
    Figure Legend Snippet: Effect of P. gingivalis infection on PCSK9 and LDLR gene and protein expression in the liver. Total RNA and protein were extracted from the P. gingivalis -infected and sham-infected mice, and the samples were analyzed by quantitative RT-PCR or western blotting. Pcsk9 and Ldlr gene expression was significantly higher in the P. gingivalis -infected mice compared with the sham-infected mice (** P

    Techniques Used: Infection, Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot

    24) Product Images from "Vascular Endothelium Derived Endothelin-1 Is Required for Normal Heart Function after Chronic Pressure Overload in Mice"

    Article Title: Vascular Endothelium Derived Endothelin-1 Is Required for Normal Heart Function after Chronic Pressure Overload in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088730

    Gene expression level of (A) ET-1, (B) TNF-α, (C) ANP, (D) BNP, (E) bcl2, (F) bax,(H) caspase 3 and (I) caspase 8. Messenger RNA expression levels were determined by real time-PCR analysis and normalized to actin expression using the ΔΔC T method. (G) The expression ratio bax/bcl2 was also calculated. Values are mean±sem, n = 6–9, Student's T-test: * p
    Figure Legend Snippet: Gene expression level of (A) ET-1, (B) TNF-α, (C) ANP, (D) BNP, (E) bcl2, (F) bax,(H) caspase 3 and (I) caspase 8. Messenger RNA expression levels were determined by real time-PCR analysis and normalized to actin expression using the ΔΔC T method. (G) The expression ratio bax/bcl2 was also calculated. Values are mean±sem, n = 6–9, Student's T-test: * p

    Techniques Used: Expressing, Aqueous Normal-phase Chromatography, RNA Expression, Real-time Polymerase Chain Reaction

    25) Product Images from "ALS-Linked P56S-VAPB Mutation Impairs the Formation of Multinuclear Myotube in C2C12 Cells"

    Article Title: ALS-Linked P56S-VAPB Mutation Impairs the Formation of Multinuclear Myotube in C2C12 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms160818628

    P56S mutation leads to aberrant aggregation of VAPB in C2C12 cells. ( A ) VAPB mRNA expression was analyzed by RT-PCR. Total RNA was collected from the indicated tissues of mice. N.C. indicates negative control (sample in which no reverse transcriptase was added); ( B ) C2C12 cells were transfected with the indicated plasmids and fixed either before inducing differentiation ( upper : Myoblast) or five days after differentiation ( lower : Myotube). The distribution of the VAPB protein is indicated by GFP expression; ( C ) C2C12 cells were co-transfected with vectors encoding C-terminally GFP-fused wt-VAPB and Ds-Red-fused P56S-VAPB, followed by fixation 24 h after transfection. Scale bar = 20 μm. The merged image is shown on the bottom. Examples of co-localization are indicated with arrowheads. These images are representative of three similar experiments.
    Figure Legend Snippet: P56S mutation leads to aberrant aggregation of VAPB in C2C12 cells. ( A ) VAPB mRNA expression was analyzed by RT-PCR. Total RNA was collected from the indicated tissues of mice. N.C. indicates negative control (sample in which no reverse transcriptase was added); ( B ) C2C12 cells were transfected with the indicated plasmids and fixed either before inducing differentiation ( upper : Myoblast) or five days after differentiation ( lower : Myotube). The distribution of the VAPB protein is indicated by GFP expression; ( C ) C2C12 cells were co-transfected with vectors encoding C-terminally GFP-fused wt-VAPB and Ds-Red-fused P56S-VAPB, followed by fixation 24 h after transfection. Scale bar = 20 μm. The merged image is shown on the bottom. Examples of co-localization are indicated with arrowheads. These images are representative of three similar experiments.

    Techniques Used: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Negative Control, Transfection

    26) Product Images from "The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC"

    Article Title: The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2014.560

    BMP activation influences Wnt target gene expression to a greater extent in SMAD4-deficient cells. HCT116 and HCT116 SMAD4−/− cells were transfected with either BMPR2 (to activate BMP signalling) or the empty control vector pcDNA4/TO. After 24 h, RNA was isolated and a Human WNT Signalling Targets RT 2 Profiler PCR Array from SABIOsciences was used. The graphs show the expression patterns of the control samples (x axis) versus the BMPR2 transfected samples (y axis). The lines indicate a 2-fold change compared to controls.
    Figure Legend Snippet: BMP activation influences Wnt target gene expression to a greater extent in SMAD4-deficient cells. HCT116 and HCT116 SMAD4−/− cells were transfected with either BMPR2 (to activate BMP signalling) or the empty control vector pcDNA4/TO. After 24 h, RNA was isolated and a Human WNT Signalling Targets RT 2 Profiler PCR Array from SABIOsciences was used. The graphs show the expression patterns of the control samples (x axis) versus the BMPR2 transfected samples (y axis). The lines indicate a 2-fold change compared to controls.

    Techniques Used: Activation Assay, Expressing, Transfection, Plasmid Preparation, Isolation, Polymerase Chain Reaction

    27) Product Images from "A LUHMES 3D dopaminergic neuronal model for neurotoxicity testing allowing long-term exposure and cellular resilience analysis"

    Article Title: A LUHMES 3D dopaminergic neuronal model for neurotoxicity testing allowing long-term exposure and cellular resilience analysis

    Journal: Archives of Toxicology

    doi: 10.1007/s00204-015-1637-z

    Enhanced neuronal maturation in 3D + T10 cultures. a MAP2 staining of representative aggregates differentiated for 12 days following either 3D +T10 ( first panel ) or 3D diff. ( second panel ) protocols. The nuclei were visualized with Hoechst 33342 staining. b Higher magnification (63×) of representative aggregates differentiated for 12 days under 3D + T10 conditions and stained with synaptophysin ( green ), NF200 ( red ) in the first slide and MAP2 ( green ), NF200 ( red ) in the second slide. The nuclei were visualized with Hoechst 33342 staining. Scale bars are 50 μm. Real-Time RT-PCR of genes involved in LUHMES neuronal differentiation and maturation. LUHMES were differentiated in 3D + T10 ( c ) and 2D monolayer cultures ( d ). RNA samples were collected on days 3, 6, 9, 12, 15, and 21 of differentiation and prior induction of differentiation (day 0) as a control. Data represent mean of log 2 (fold change) ± SEM normalized to d0 from at least four independent experiments. Statistical significance was calculated using one-way ANOVA test followed by Dunnett’s post hoc test. Expression of all genes was significantly ( p
    Figure Legend Snippet: Enhanced neuronal maturation in 3D + T10 cultures. a MAP2 staining of representative aggregates differentiated for 12 days following either 3D +T10 ( first panel ) or 3D diff. ( second panel ) protocols. The nuclei were visualized with Hoechst 33342 staining. b Higher magnification (63×) of representative aggregates differentiated for 12 days under 3D + T10 conditions and stained with synaptophysin ( green ), NF200 ( red ) in the first slide and MAP2 ( green ), NF200 ( red ) in the second slide. The nuclei were visualized with Hoechst 33342 staining. Scale bars are 50 μm. Real-Time RT-PCR of genes involved in LUHMES neuronal differentiation and maturation. LUHMES were differentiated in 3D + T10 ( c ) and 2D monolayer cultures ( d ). RNA samples were collected on days 3, 6, 9, 12, 15, and 21 of differentiation and prior induction of differentiation (day 0) as a control. Data represent mean of log 2 (fold change) ± SEM normalized to d0 from at least four independent experiments. Statistical significance was calculated using one-way ANOVA test followed by Dunnett’s post hoc test. Expression of all genes was significantly ( p

    Techniques Used: Staining, Quantitative RT-PCR, Expressing

    28) Product Images from "Isobavachalcone sensitizes cells to E2‐induced paclitaxel resistance by down‐regulating CD44 expression in ER+ breast cancer cells, et al. Isobavachalcone sensitizes cells to E2‐induced paclitaxel resistance by down‐regulating CD44 expression in ER+ breast cancer cells"

    Article Title: Isobavachalcone sensitizes cells to E2‐induced paclitaxel resistance by down‐regulating CD44 expression in ER+ breast cancer cells, et al. Isobavachalcone sensitizes cells to E2‐induced paclitaxel resistance by down‐regulating CD44 expression in ER+ breast cancer cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13719

    E 2 could stimulate CD 44 expression in ER + breast cancer cells. A, CD 44 transcript level was detected by real‐time PCR in ER + ZR ‐75‐1 cells treated with the indicated concentrations of E 2 for 72 h. B, CD 44 transcript level was detected by real‐time PCR in ER + MCF ‐7 cells treated with the indicated concentrations of E 2 for 72 h. C, CD 44 transcript level was detected by real‐time PCR in ER ‐ MDA ‐ MB ‐231 cells treated with the indicated concentrations of E 2 for 72 h. D, Real‐time PCR analysis of CD 24 expression in ER + ZR ‐75‐1 cells treated with the indicated concentrations of E 2 for 72 h. E, Real‐time PCR analysis of CD 24 expression in ER + MCF ‐7 cells treated with the indicated concentrations of E 2 for 72 h. F, Real‐time PCR analysis of CD 24 expression in ER + MCF ‐7 cells treated with the indicated concentrations of E 2 for 72 h. G, Western blot analysis of CD 44 protein expression in breast cancer cells treated with E 2 (1 μmol/L) for 72 h. H, Western blot analysis of CD 44 protein expression in ER + MCF ‐7 cells transfected with CD 44‐targeting si RNA . H, Western blot analysis of CD 44 protein expression in ER + MCF ‐7 cells transfected with CD 44‐targeting si RNA . I, MCF ‐7 cells described in Figure 1 were treated with paclitaxel (5 nmol/L) for 14 d before being subjected to colony formation assay. * P
    Figure Legend Snippet: E 2 could stimulate CD 44 expression in ER + breast cancer cells. A, CD 44 transcript level was detected by real‐time PCR in ER + ZR ‐75‐1 cells treated with the indicated concentrations of E 2 for 72 h. B, CD 44 transcript level was detected by real‐time PCR in ER + MCF ‐7 cells treated with the indicated concentrations of E 2 for 72 h. C, CD 44 transcript level was detected by real‐time PCR in ER ‐ MDA ‐ MB ‐231 cells treated with the indicated concentrations of E 2 for 72 h. D, Real‐time PCR analysis of CD 24 expression in ER + ZR ‐75‐1 cells treated with the indicated concentrations of E 2 for 72 h. E, Real‐time PCR analysis of CD 24 expression in ER + MCF ‐7 cells treated with the indicated concentrations of E 2 for 72 h. F, Real‐time PCR analysis of CD 24 expression in ER + MCF ‐7 cells treated with the indicated concentrations of E 2 for 72 h. G, Western blot analysis of CD 44 protein expression in breast cancer cells treated with E 2 (1 μmol/L) for 72 h. H, Western blot analysis of CD 44 protein expression in ER + MCF ‐7 cells transfected with CD 44‐targeting si RNA . H, Western blot analysis of CD 44 protein expression in ER + MCF ‐7 cells transfected with CD 44‐targeting si RNA . I, MCF ‐7 cells described in Figure 1 were treated with paclitaxel (5 nmol/L) for 14 d before being subjected to colony formation assay. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Western Blot, Transfection, Colony Assay

    29) Product Images from "The Food Additive Maltodextrin Promotes Endoplasmic Reticulum Stress–Driven Mucus Depletion and Exacerbates Intestinal Inflammation"

    Article Title: The Food Additive Maltodextrin Promotes Endoplasmic Reticulum Stress–Driven Mucus Depletion and Exacerbates Intestinal Inflammation

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2018.09.002

    MDX induces IRE1β expression via P38 MAPK. ( A ) MDX induces Ern-2 expression in the HT29-MTX cell line. Cells were either left untreated (UT) or cultured with increasing concentrations of MDX for 1 hour. Ern-2 RNA transcripts were assessed by real-time PCR. Data are means ± SD of 10 samples per group derived from 4 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (** P ≤ .01, * P ≤ .05). ( B ) HT29-MTX cells were either left UT or stimulated with 5% MDX for the indicated time points. p-p38, p38, phosphorylated extracellular signal–regulated kinase 1/2 (p-ERK1/2), phosphorylated c-Jun N-terminal kinase (p-JNK), and β-actin (β-act) expression were analyzed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with densitometry analysis ( lower panel ). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05, ** P ≤ .01, *** P ≤ .001). ( C ) Effect of the p38 inhibitor (p38i) on MDX-mediated p38 activation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or dimethyl sulfoxide (DMSO) (vehicle). p-p38 and p38 expression was assessed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with the densitometry analysis ( lower panel ). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (*** P ≤ .001). Right : Effect of p38i on MDX-mediated Ern-2 up-regulation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or DMSO (vehicle) and Ern-2 RNA transcripts evaluated by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). ( D ) Effect of p38 knock-down on MDX-mediated Ern-2 up-regulation. HT29-MTX cells were transfected with either control or p38 siRNA (CTR or p38 siRNA, respectively) for 18 hours and then stimulated or not with 5% MDX for 1 hour. Representative Western blot for p-p38, p38, and β-actin expression are shown in the left panels together with the densitometry analysis. Data are means ± SD of 3 independent experiments. Differences between groups were compared using the 2-tailed Student t test (** P ≤ .001). Right : Expression of Ern-2 RNA transcripts assessed by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). ( E ) Immunofluorescence analysis of p-p38 (green) in colon samples isolated from MDX-fed mice and controls killed on day 45. The figure is representative of 3 separate experiments in which similar results were obtained. Right panels show the number and intensity of p-p38–expressing cells per field of colon section. Data are expressed as means ± SD and were generated using 3–5 mice per group from 3 independent experiments. Differences between groups were compared using the 2-tailed Student t test (** P ≤ .01). mRNA, messenger RNA; p38 tot, total p38.
    Figure Legend Snippet: MDX induces IRE1β expression via P38 MAPK. ( A ) MDX induces Ern-2 expression in the HT29-MTX cell line. Cells were either left untreated (UT) or cultured with increasing concentrations of MDX for 1 hour. Ern-2 RNA transcripts were assessed by real-time PCR. Data are means ± SD of 10 samples per group derived from 4 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (** P ≤ .01, * P ≤ .05). ( B ) HT29-MTX cells were either left UT or stimulated with 5% MDX for the indicated time points. p-p38, p38, phosphorylated extracellular signal–regulated kinase 1/2 (p-ERK1/2), phosphorylated c-Jun N-terminal kinase (p-JNK), and β-actin (β-act) expression were analyzed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with densitometry analysis ( lower panel ). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05, ** P ≤ .01, *** P ≤ .001). ( C ) Effect of the p38 inhibitor (p38i) on MDX-mediated p38 activation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or dimethyl sulfoxide (DMSO) (vehicle). p-p38 and p38 expression was assessed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with the densitometry analysis ( lower panel ). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (*** P ≤ .001). Right : Effect of p38i on MDX-mediated Ern-2 up-regulation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or DMSO (vehicle) and Ern-2 RNA transcripts evaluated by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). ( D ) Effect of p38 knock-down on MDX-mediated Ern-2 up-regulation. HT29-MTX cells were transfected with either control or p38 siRNA (CTR or p38 siRNA, respectively) for 18 hours and then stimulated or not with 5% MDX for 1 hour. Representative Western blot for p-p38, p38, and β-actin expression are shown in the left panels together with the densitometry analysis. Data are means ± SD of 3 independent experiments. Differences between groups were compared using the 2-tailed Student t test (** P ≤ .001). Right : Expression of Ern-2 RNA transcripts assessed by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). ( E ) Immunofluorescence analysis of p-p38 (green) in colon samples isolated from MDX-fed mice and controls killed on day 45. The figure is representative of 3 separate experiments in which similar results were obtained. Right panels show the number and intensity of p-p38–expressing cells per field of colon section. Data are expressed as means ± SD and were generated using 3–5 mice per group from 3 independent experiments. Differences between groups were compared using the 2-tailed Student t test (** P ≤ .01). mRNA, messenger RNA; p38 tot, total p38.

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Derivative Assay, Activated Clotting Time Assay, Western Blot, Activation Assay, Transfection, Immunofluorescence, Isolation, Mouse Assay, Generated

    MDX-enriched diet exacerbates intestinal inflammation. ( A ) Wild-type mice were exposed to 5% MDX, 0.5% PG, or 5 g/L GEL, all diluted in drinking water, over a period of 45 days and challenged with DSS (1.75% in drinking water) starting from day 35 of diet. Body weight was recorded daily from day 35 until death (day 45). Data were generated using 10–12 mice per group from 3 independent experiments and expressed as means ± SEM. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test. DSS vs DSS + MDX, ** P ≤ .01. ( B and C ) Representative H E staining and histologic score of colon sections taken from mice treated with DSS either alone or in combination with MDX, PG, or GEL, as indicated in panel A , and killed on day 45. Data were generated using 10–12 mice per group from 3 independent experiments and expressed as means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test. DSS vs DSS + MDX, ** P ≤ .01. ( B ) Scale bars : 100 μm. ( D and E ) IL1β and Lcn-2 RNA expression was assessed by real-time PCR in colonic tissues taken from mice treated as indicated in panel A and killed on day 45. Data were generated using 10–12 mice per group from 3 independent experiments. Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value. Differences among groups were compared using the Kruskal–Wallis test. DSS + MDX vs DSS, ** P ≤ .01. ( F and G ) Representative H E staining and histologic score of colon sections taken from mice treated with DSS either alone or in combination with increasing concentrations of MDX, as indicated in panel A , and killed on day 45. Data were generated using 5 mice per group from 2 independent experiments and expressed as means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test. 5% DSS + MDX vs DSS, ** P ≤ .01. ( H and I ) Representative H E staining and histologic score of ileum sections taken from wild-type mice exposed to 5% MDX diluted in drinking water for 35 days, and then treated with a single subcutaneous injection of indomethacin (5 mg/kg). Mice were killed 24 hours after indomethacin injection and tissues were collected for histopathologic analysis. Data were generated using 10–12 mice per group of 3 independent experiments and expressed as means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). Scale bars : 100 μm. mRNA, messenger RNA.
    Figure Legend Snippet: MDX-enriched diet exacerbates intestinal inflammation. ( A ) Wild-type mice were exposed to 5% MDX, 0.5% PG, or 5 g/L GEL, all diluted in drinking water, over a period of 45 days and challenged with DSS (1.75% in drinking water) starting from day 35 of diet. Body weight was recorded daily from day 35 until death (day 45). Data were generated using 10–12 mice per group from 3 independent experiments and expressed as means ± SEM. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test. DSS vs DSS + MDX, ** P ≤ .01. ( B and C ) Representative H E staining and histologic score of colon sections taken from mice treated with DSS either alone or in combination with MDX, PG, or GEL, as indicated in panel A , and killed on day 45. Data were generated using 10–12 mice per group from 3 independent experiments and expressed as means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test. DSS vs DSS + MDX, ** P ≤ .01. ( B ) Scale bars : 100 μm. ( D and E ) IL1β and Lcn-2 RNA expression was assessed by real-time PCR in colonic tissues taken from mice treated as indicated in panel A and killed on day 45. Data were generated using 10–12 mice per group from 3 independent experiments. Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value. Differences among groups were compared using the Kruskal–Wallis test. DSS + MDX vs DSS, ** P ≤ .01. ( F and G ) Representative H E staining and histologic score of colon sections taken from mice treated with DSS either alone or in combination with increasing concentrations of MDX, as indicated in panel A , and killed on day 45. Data were generated using 5 mice per group from 2 independent experiments and expressed as means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test. 5% DSS + MDX vs DSS, ** P ≤ .01. ( H and I ) Representative H E staining and histologic score of ileum sections taken from wild-type mice exposed to 5% MDX diluted in drinking water for 35 days, and then treated with a single subcutaneous injection of indomethacin (5 mg/kg). Mice were killed 24 hours after indomethacin injection and tissues were collected for histopathologic analysis. Data were generated using 10–12 mice per group of 3 independent experiments and expressed as means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). Scale bars : 100 μm. mRNA, messenger RNA.

    Techniques Used: Mouse Assay, Generated, Staining, RNA Expression, Real-time Polymerase Chain Reaction, Injection

    MDX up-regulates the IRE1-dependent UPR pathway in intestinal epithelial cells. ( A ) Volcano plot and heat map showing microarray-based differential expression, log 2 (fold change) of genes related to lipid and carbohydrate metabolism, protein glycosylation, and UPR pathway of colon samples isolated from mice exposed to drinking water in the presence or absence of 5% MDX for 45 days without DSS. ( B ) Ern-2 , Ern-1 , and Xbp1s RNA expression was assessed by real-time PCR in total colonic samples. Data were generated using 11–12 mice per group from 3 independent experiments (* P ≤ .05, ** P ≤ .01). Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value. Differences among groups were compared using the Mann–Whitney U test. ( C ) Scatter plots showing Grp78 , Atf6 , Atf4 , and Chop RNA transcripts in colonic samples isolated from mice treated as indicated in panel A . Data were generated using 7–8 mice per group of 3 independent experiments. Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value or means ± SD. Differences among groups were compared using the Mann–Whitney U test or the 2-tailed Student t test. ( D ) Representative histograms showing Ern-2 and Ern-1 RNA expression in IECs and LPMCs isolated from a pool of 3–4 mice treated as indicated in panel A . The example is representative of 2 independent experiments in which similar results were obtained. ( E ) Scatter plots showing Ern-2 , Ern-1 , and Xbp1s RNA expression in intestinal crypts isolated from the colons of untreated mice and cultured in the presence or absence of MDX for 30 minutes. Data were generated using crypts isolated from 6–7 mice from 3 independent experiments (* P ≤ .05, ** P ≤ .01). Left : Horizontal bars indicate median value and differences between groups were compared using 2-tailed Mann–Whitney U test. Middle and right : Horizontal bars indicate means ± SD and differences between groups were compared using the Student t test. mRNA, messenger RNA; UT, untreated.
    Figure Legend Snippet: MDX up-regulates the IRE1-dependent UPR pathway in intestinal epithelial cells. ( A ) Volcano plot and heat map showing microarray-based differential expression, log 2 (fold change) of genes related to lipid and carbohydrate metabolism, protein glycosylation, and UPR pathway of colon samples isolated from mice exposed to drinking water in the presence or absence of 5% MDX for 45 days without DSS. ( B ) Ern-2 , Ern-1 , and Xbp1s RNA expression was assessed by real-time PCR in total colonic samples. Data were generated using 11–12 mice per group from 3 independent experiments (* P ≤ .05, ** P ≤ .01). Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value. Differences among groups were compared using the Mann–Whitney U test. ( C ) Scatter plots showing Grp78 , Atf6 , Atf4 , and Chop RNA transcripts in colonic samples isolated from mice treated as indicated in panel A . Data were generated using 7–8 mice per group of 3 independent experiments. Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value or means ± SD. Differences among groups were compared using the Mann–Whitney U test or the 2-tailed Student t test. ( D ) Representative histograms showing Ern-2 and Ern-1 RNA expression in IECs and LPMCs isolated from a pool of 3–4 mice treated as indicated in panel A . The example is representative of 2 independent experiments in which similar results were obtained. ( E ) Scatter plots showing Ern-2 , Ern-1 , and Xbp1s RNA expression in intestinal crypts isolated from the colons of untreated mice and cultured in the presence or absence of MDX for 30 minutes. Data were generated using crypts isolated from 6–7 mice from 3 independent experiments (* P ≤ .05, ** P ≤ .01). Left : Horizontal bars indicate median value and differences between groups were compared using 2-tailed Mann–Whitney U test. Middle and right : Horizontal bars indicate means ± SD and differences between groups were compared using the Student t test. mRNA, messenger RNA; UT, untreated.

    Techniques Used: Microarray, Expressing, Isolation, Mouse Assay, RNA Expression, Real-time Polymerase Chain Reaction, Generated, MANN-WHITNEY, Cell Culture

    ER stress inhibition reduces MDX-mediated Ern-2 up-regulation and improves colitis in MDX-fed mice. ( A ) HT29-MTX cells were pretreated with TUDCA (10 μmol/L) or dimethyl sulfoxide (vehicle) and then stimulated with MDX for 1 hour. Ern-2 RNA transcripts were analyzed by real-time PCR. Data are means ± SD of 4 independent experiments. Differences between groups were compared using the 2-tailed Student t test (* P ≤ .05). ( B and C ) Wild-type mice were exposed to drinking water supplemented with 5% MDX for 45 days and injected or not with TUDCA (250 mg/kg intraperitoneally) every other day starting from day 21. Mice were killed on day 45, colonic tissues were isolated, and ( B ) Ern-2 , Ern-1 , and Xbp1s RNA transcripts and ( C ) Muc-2 protein expression were evaluated by real-time PCR and immunofluorescence, respectively. ( B ) Data were generated using 7–10 mice per group from 3 independent experiments. Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value. Differences between groups were compared using the Mann–Whitney U test (* P ≤ .05). ( C ) Pictures are representative of 4 separate experiments in which similar results were obtained. Scale bars : 25 μm. ( D ) Wild-type mice were exposed to drinking water supplemented with 5% MDX for 45 days and injected or not with TUDCA (250 mg/kg intraperitoneally) every other day starting from day 21. Mice were exposed to 1.75% DSS to induce colitis starting from day 35 until death (day 45), and body weight was recorded every other day. Data were generated using 8–9 mice per group from 3 independent experiments and expressed as means ± SEM. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (** P ≤ .01). ( E and F ) Representative H E staining of colon sections of mice treated as indicated in panel D and killed on day 45. ( F ) Scatter plot shows the histologic score. Data were generated using 8–9 mice per group from 3 independent experiments and expressed as means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05; ** P ≤ .01). ( G and H ) Representative scatter plots showing ( G ) IL1β and ( H ) Lcn-2 RNA expression in colon tissues taken from mice treated as indicated in panel D and killed on day 45. Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value or means ± SD. Data were generated using 8–9 mice per group from 3 independent experiments. Differences among groups were compared using the Kruskal–Wallis test or 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). DAPI, 4′,6-diamidino-2-phenylindole; mRNA, messenger RNA.
    Figure Legend Snippet: ER stress inhibition reduces MDX-mediated Ern-2 up-regulation and improves colitis in MDX-fed mice. ( A ) HT29-MTX cells were pretreated with TUDCA (10 μmol/L) or dimethyl sulfoxide (vehicle) and then stimulated with MDX for 1 hour. Ern-2 RNA transcripts were analyzed by real-time PCR. Data are means ± SD of 4 independent experiments. Differences between groups were compared using the 2-tailed Student t test (* P ≤ .05). ( B and C ) Wild-type mice were exposed to drinking water supplemented with 5% MDX for 45 days and injected or not with TUDCA (250 mg/kg intraperitoneally) every other day starting from day 21. Mice were killed on day 45, colonic tissues were isolated, and ( B ) Ern-2 , Ern-1 , and Xbp1s RNA transcripts and ( C ) Muc-2 protein expression were evaluated by real-time PCR and immunofluorescence, respectively. ( B ) Data were generated using 7–10 mice per group from 3 independent experiments. Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value. Differences between groups were compared using the Mann–Whitney U test (* P ≤ .05). ( C ) Pictures are representative of 4 separate experiments in which similar results were obtained. Scale bars : 25 μm. ( D ) Wild-type mice were exposed to drinking water supplemented with 5% MDX for 45 days and injected or not with TUDCA (250 mg/kg intraperitoneally) every other day starting from day 21. Mice were exposed to 1.75% DSS to induce colitis starting from day 35 until death (day 45), and body weight was recorded every other day. Data were generated using 8–9 mice per group from 3 independent experiments and expressed as means ± SEM. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (** P ≤ .01). ( E and F ) Representative H E staining of colon sections of mice treated as indicated in panel D and killed on day 45. ( F ) Scatter plot shows the histologic score. Data were generated using 8–9 mice per group from 3 independent experiments and expressed as means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05; ** P ≤ .01). ( G and H ) Representative scatter plots showing ( G ) IL1β and ( H ) Lcn-2 RNA expression in colon tissues taken from mice treated as indicated in panel D and killed on day 45. Each point in the graph indicates the RNA expression of the specific transcript in the colon of a single mouse; horizontal bars indicate median value or means ± SD. Data were generated using 8–9 mice per group from 3 independent experiments. Differences among groups were compared using the Kruskal–Wallis test or 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). DAPI, 4′,6-diamidino-2-phenylindole; mRNA, messenger RNA.

    Techniques Used: Inhibition, Mouse Assay, Real-time Polymerase Chain Reaction, Injection, Isolation, Expressing, Immunofluorescence, Generated, RNA Expression, MANN-WHITNEY, Staining

    30) Product Images from "In vivo regulation of the heme oxygenase-1 gene in humanized transgenic mice"

    Article Title: In vivo regulation of the heme oxygenase-1 gene in humanized transgenic mice

    Journal: Kidney International

    doi: 10.1038/ki.2012.102

    HO-1 protein and mRNA are overexpressed in organs of HO-1 BAC transgenic mice (A) Average body weights of HO-1 BAC mice (n=5, solid square) and HO-1 +/+ mice (n=4, open diamond) that were monitored for 19 months. (B) Total RNA was isolated from indicated organs and HO-1 mRNA expression level was quantified using real-time PCR. GAPDH was used to normalize HO-1 mRNA levels. Results are shown as fold-increase versus HO-1 +/+ from 3 independent experiments performed in triplicate each time (Mean ± SEM). * p
    Figure Legend Snippet: HO-1 protein and mRNA are overexpressed in organs of HO-1 BAC transgenic mice (A) Average body weights of HO-1 BAC mice (n=5, solid square) and HO-1 +/+ mice (n=4, open diamond) that were monitored for 19 months. (B) Total RNA was isolated from indicated organs and HO-1 mRNA expression level was quantified using real-time PCR. GAPDH was used to normalize HO-1 mRNA levels. Results are shown as fold-increase versus HO-1 +/+ from 3 independent experiments performed in triplicate each time (Mean ± SEM). * p

    Techniques Used: BAC Assay, Transgenic Assay, Mouse Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction

    31) Product Images from "ABCG1 rs57137919G > A Polymorphism Is Functionally Associated with Varying Gene Expression and Apoptosis of Macrophages"

    Article Title: ABCG1 rs57137919G > A Polymorphism Is Functionally Associated with Varying Gene Expression and Apoptosis of Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097044

    Apoptosis-associated genes Bok and Bid mRNA expression in monocyte-derived macrophages. PBMCs-derived macrophages from three genotypes were exposed to 50 µg/mL ox-LDL for 24 hours and RNA was extracted using TRIzol regent. Bid and Bok mRNA levels were detected by quantitative real-time PCR. Data are expressed as mean ± SD from nine donor samples in triplicate. * P
    Figure Legend Snippet: Apoptosis-associated genes Bok and Bid mRNA expression in monocyte-derived macrophages. PBMCs-derived macrophages from three genotypes were exposed to 50 µg/mL ox-LDL for 24 hours and RNA was extracted using TRIzol regent. Bid and Bok mRNA levels were detected by quantitative real-time PCR. Data are expressed as mean ± SD from nine donor samples in triplicate. * P

    Techniques Used: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction

    32) Product Images from "Mycobacterium tuberculosis evades macrophage defenses by inhibiting plasma membrane repair"

    Article Title: Mycobacterium tuberculosis evades macrophage defenses by inhibiting plasma membrane repair

    Journal: Nature immunology

    doi: 10.1038/ni.1758

    PGE 2 regulates Syt-7 expression in murine Mφ Dose and time response of ( a ) Syt-7 and ( b ) LAMP1 mRNA expression in naïve wild-type Mφ treated with PGE 2 . ( c ) Syt-7 and LAMP1 expression in wild-type Mφ infected or not with H37Rv after addition of PGE 2 (1 μM). ( d ) Syt-7 and LAMP1 mRNA expression in Alox5 −/− , Ptges −/− and wild-type (WT) Mφ at indicated times after infection with H37Rv. ( e ) Wild-type mice were infected or not by the aerosol route with a low dose (~ 100 CFU) of H37Rv or H37Ra. After 7 days of infection, RNA was extracted from the whole lung and the expression of Syt-7 and LAMP1 mRNA was measured by real-time PCR. The expression of Syt-7 or LAMP1 was normalized to β-Actin. Results are from one representative of 2 ( a, b, c , and f ) and 3 ( d, e ) independent experiments. n=3 mice per group; (error bars, s.e.m.) *, p
    Figure Legend Snippet: PGE 2 regulates Syt-7 expression in murine Mφ Dose and time response of ( a ) Syt-7 and ( b ) LAMP1 mRNA expression in naïve wild-type Mφ treated with PGE 2 . ( c ) Syt-7 and LAMP1 expression in wild-type Mφ infected or not with H37Rv after addition of PGE 2 (1 μM). ( d ) Syt-7 and LAMP1 mRNA expression in Alox5 −/− , Ptges −/− and wild-type (WT) Mφ at indicated times after infection with H37Rv. ( e ) Wild-type mice were infected or not by the aerosol route with a low dose (~ 100 CFU) of H37Rv or H37Ra. After 7 days of infection, RNA was extracted from the whole lung and the expression of Syt-7 and LAMP1 mRNA was measured by real-time PCR. The expression of Syt-7 or LAMP1 was normalized to β-Actin. Results are from one representative of 2 ( a, b, c , and f ) and 3 ( d, e ) independent experiments. n=3 mice per group; (error bars, s.e.m.) *, p

    Techniques Used: Expressing, Infection, Mouse Assay, Real-time Polymerase Chain Reaction

    33) Product Images from "Increased Iron Stores Correlate with Worse Disease Outcomes in a Mouse Model of Schistosomiasis Infection"

    Article Title: Increased Iron Stores Correlate with Worse Disease Outcomes in a Mouse Model of Schistosomiasis Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009594

    Interaction of iron status, inflammation, and iron regulatory genes. The response of inflammatory markers and iron regulatory genes was assessed in schistosomiasis infected and control wild-type and Tfr2 −/− mice 6 weeks post infection date. Granuloma size was estimated by point-counting stereology on liver sections, and gene expression was assessed by quantitative real-time PCR from liver RNA. A. Comparison of the average granuloma area within wild-type and Tfr2 −/− mice shows no difference in response in infected animals. B and C. Hepatic inflammatory markers Serum Amyloid-A and Orosomucoid show a significant response to infection, but no difference in the levels of response between wild-type and Tfr2 −/− animals reflecting granuloma size. D and E. Hamp 1 shows a trend and Hamp 2 shows significant down-regulation in response infection. This pattern of regulation is consistent with the identified iron status of the animals and appears unaffected by the inflammatory response identified (*P
    Figure Legend Snippet: Interaction of iron status, inflammation, and iron regulatory genes. The response of inflammatory markers and iron regulatory genes was assessed in schistosomiasis infected and control wild-type and Tfr2 −/− mice 6 weeks post infection date. Granuloma size was estimated by point-counting stereology on liver sections, and gene expression was assessed by quantitative real-time PCR from liver RNA. A. Comparison of the average granuloma area within wild-type and Tfr2 −/− mice shows no difference in response in infected animals. B and C. Hepatic inflammatory markers Serum Amyloid-A and Orosomucoid show a significant response to infection, but no difference in the levels of response between wild-type and Tfr2 −/− animals reflecting granuloma size. D and E. Hamp 1 shows a trend and Hamp 2 shows significant down-regulation in response infection. This pattern of regulation is consistent with the identified iron status of the animals and appears unaffected by the inflammatory response identified (*P

    Techniques Used: Infection, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    34) Product Images from "microRNA-214-mediated UBC9 expression in glioma"

    Article Title: microRNA-214-mediated UBC9 expression in glioma

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2012.45.11.097

    miR-214 down-regulates UBC9 by targeting the 3'UTR of UBC9 . (A) Schematic representation of the miR-214 binding site in the 3’UTR of UBC9 . The mutant sequence of the miR-214 binding site is identical to the wild-type sequence marked by asterisks. (B) A real-time PCR analysis of the miR-214 level in the same two normal brain tissues (N1, N2) and nine glioma tissues (T1-T3 grade II, T4-T6 grade III and T7-T9 grade IV). The RNA input was normalized to U6 . (C) A real-time PCR analysis of miR-214 level in HEB and T98G cells. RNA input was normalized to U6 . (D) Overexpression of miR-214 in 293ET cells was measured by real-time PCR 48 h after transfection with the miR-214 expression plasmid. RNA input was normalized to U6 . (E) Luciferase reporter assay. 293ET cells were transfected with the luciferase reporter constructs containing a wild-type or mutant sequence in the miR-214 binding site and the miR-214 expression plasmid. The relative luciferase activities were normalized with the Renilla luciferase activities. The values are expressed as percentages of relative luciferase activity of the pcDNA3.1-luc plasmid. (F) A representative western blot of endogenous UBC9 protein in T98G cells transfected with miR-214 mimics. β-actin was used as a loading control. (G) A real-time PCR analysis of the effect of miR-214 on UBC9 mRNA. RNA input was normalized to GAPDH . The data are presented as the mean ± standard deviation (SD) of triplicate wells. The asterisks indicate *P
    Figure Legend Snippet: miR-214 down-regulates UBC9 by targeting the 3'UTR of UBC9 . (A) Schematic representation of the miR-214 binding site in the 3’UTR of UBC9 . The mutant sequence of the miR-214 binding site is identical to the wild-type sequence marked by asterisks. (B) A real-time PCR analysis of the miR-214 level in the same two normal brain tissues (N1, N2) and nine glioma tissues (T1-T3 grade II, T4-T6 grade III and T7-T9 grade IV). The RNA input was normalized to U6 . (C) A real-time PCR analysis of miR-214 level in HEB and T98G cells. RNA input was normalized to U6 . (D) Overexpression of miR-214 in 293ET cells was measured by real-time PCR 48 h after transfection with the miR-214 expression plasmid. RNA input was normalized to U6 . (E) Luciferase reporter assay. 293ET cells were transfected with the luciferase reporter constructs containing a wild-type or mutant sequence in the miR-214 binding site and the miR-214 expression plasmid. The relative luciferase activities were normalized with the Renilla luciferase activities. The values are expressed as percentages of relative luciferase activity of the pcDNA3.1-luc plasmid. (F) A representative western blot of endogenous UBC9 protein in T98G cells transfected with miR-214 mimics. β-actin was used as a loading control. (G) A real-time PCR analysis of the effect of miR-214 on UBC9 mRNA. RNA input was normalized to GAPDH . The data are presented as the mean ± standard deviation (SD) of triplicate wells. The asterisks indicate *P

    Techniques Used: Binding Assay, Mutagenesis, Sequencing, Real-time Polymerase Chain Reaction, Over Expression, Transfection, Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Construct, Activity Assay, Western Blot, Standard Deviation

    UBC9 is up-regulated in glioma cells. (A) A representative western blot showing UBC9 protein levels in two normal brain tissues (N1, N2) and nine glioma tissues (T1-T3 grade II, T4-T6 grade III and T7-T9 grade IV). β-actin was used as a loading control. (B) A representative western blot showing the UBC9 protein level in normal glial HEB cells and glioma T98G cells. β-actin was used as a loading control. (C) Real-time PCR analysis of the UBC9 mRNA level in HEB and T98G cells. RNA input was normalized to human GAPDH . The values represented the mean ± standard deviation (SD) of triplicate wells.
    Figure Legend Snippet: UBC9 is up-regulated in glioma cells. (A) A representative western blot showing UBC9 protein levels in two normal brain tissues (N1, N2) and nine glioma tissues (T1-T3 grade II, T4-T6 grade III and T7-T9 grade IV). β-actin was used as a loading control. (B) A representative western blot showing the UBC9 protein level in normal glial HEB cells and glioma T98G cells. β-actin was used as a loading control. (C) Real-time PCR analysis of the UBC9 mRNA level in HEB and T98G cells. RNA input was normalized to human GAPDH . The values represented the mean ± standard deviation (SD) of triplicate wells.

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    miR-214 affects cell proliferation and apoptosis. (A) A real-time PCR analysis of miR-214 overexpression in T98G cells 48 h post-transfection. RNA input was normalized to U6 . (B) An MTT assay of T98G cells after treatment with control or miR-214 mimics as above. (C) TUNEL assays were performed 48 h post-transfection with control or miR-214 as above. (D) Colony-forming assay. T98G cells were transfected with control or miR-214 mimics as above. The histogram shows the relative colony number compared to the control cells. The values represented the mean ± standard deviation (SD) of triplicate wells. The asterisks indicate *P < 0.05 compared to control.
    Figure Legend Snippet: miR-214 affects cell proliferation and apoptosis. (A) A real-time PCR analysis of miR-214 overexpression in T98G cells 48 h post-transfection. RNA input was normalized to U6 . (B) An MTT assay of T98G cells after treatment with control or miR-214 mimics as above. (C) TUNEL assays were performed 48 h post-transfection with control or miR-214 as above. (D) Colony-forming assay. T98G cells were transfected with control or miR-214 mimics as above. The histogram shows the relative colony number compared to the control cells. The values represented the mean ± standard deviation (SD) of triplicate wells. The asterisks indicate *P < 0.05 compared to control.

    Techniques Used: Real-time Polymerase Chain Reaction, Over Expression, Transfection, MTT Assay, TUNEL Assay, Standard Deviation

    35) Product Images from "HIV Vpr protein upregulates microRNA-122 expression and stimulates hepatitis C virus replication"

    Article Title: HIV Vpr protein upregulates microRNA-122 expression and stimulates hepatitis C virus replication

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.000169

    MiR-122 is essential for the effect of Vpr on HCV 5′ UTR activity, HCV RNA replication and HCV protein expression in the JFH1 infection model and OR6 cell lines. (a) qRT-PCR assay of the expression levels of miR-122 in miR-122-silence Huh7.5.1 cell lines and controls (miR-con). Results were normalized with respect to U6 mRNA. (b) Dual luciferase assay of p5′ UTR-luc in miR-122-silence Huh7.5.1 cell lines transfected with Vpr and controls. (c) Quantitative HCV RNA assay in miR-122-silence Huh7.5.1 cell lines transfected with Vpr and controls. (d) Western blot analysis in miR-122-silence Huh7.5.1 cell lines confirmed the expression of β-actin, NS5A and Vpr. (e) Renilla luciferase assay in miR-122-silence OR6 cell lines transfected with Vpr and controls. (f) Western blot analysis in miR-122-silence OR6 cell lines confirmed the expression of β-actin, NS5A and Vpr. The data are presented as mean ± sd. ** P
    Figure Legend Snippet: MiR-122 is essential for the effect of Vpr on HCV 5′ UTR activity, HCV RNA replication and HCV protein expression in the JFH1 infection model and OR6 cell lines. (a) qRT-PCR assay of the expression levels of miR-122 in miR-122-silence Huh7.5.1 cell lines and controls (miR-con). Results were normalized with respect to U6 mRNA. (b) Dual luciferase assay of p5′ UTR-luc in miR-122-silence Huh7.5.1 cell lines transfected with Vpr and controls. (c) Quantitative HCV RNA assay in miR-122-silence Huh7.5.1 cell lines transfected with Vpr and controls. (d) Western blot analysis in miR-122-silence Huh7.5.1 cell lines confirmed the expression of β-actin, NS5A and Vpr. (e) Renilla luciferase assay in miR-122-silence OR6 cell lines transfected with Vpr and controls. (f) Western blot analysis in miR-122-silence OR6 cell lines confirmed the expression of β-actin, NS5A and Vpr. The data are presented as mean ± sd. ** P

    Techniques Used: Activity Assay, Expressing, Infection, Quantitative RT-PCR, Luciferase, Transfection, Western Blot

    Vpr activates HCV replication in stable Vpr-expressing cells. (a) Quantitative HCV RNA assay in stable vector-Huh7.5.1, Vpr-Huh7.5.1 and Huh7.5.1 cells at 48 h, 72 h and 30 days post-JFH1 infection. (b) Relative fold change of HCV RNA levels. (c) Quantitative PCR for intracellular HCV RNA levels in the stable vector-Huh7.5.1, Vpr-Huh7.5.1 and Huh7.5.1 cells at 30 days post-JFH1 infection. (d, e) Western blotting confirmed the expression of β-actin, NS5A and Vpr in stable Huh7.5.1 cell lines at 72 h (d) and 30 days post-JFH1 infection (e). The data are presented as mean ± sd. ** P
    Figure Legend Snippet: Vpr activates HCV replication in stable Vpr-expressing cells. (a) Quantitative HCV RNA assay in stable vector-Huh7.5.1, Vpr-Huh7.5.1 and Huh7.5.1 cells at 48 h, 72 h and 30 days post-JFH1 infection. (b) Relative fold change of HCV RNA levels. (c) Quantitative PCR for intracellular HCV RNA levels in the stable vector-Huh7.5.1, Vpr-Huh7.5.1 and Huh7.5.1 cells at 30 days post-JFH1 infection. (d, e) Western blotting confirmed the expression of β-actin, NS5A and Vpr in stable Huh7.5.1 cell lines at 72 h (d) and 30 days post-JFH1 infection (e). The data are presented as mean ± sd. ** P

    Techniques Used: Expressing, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Western Blot

    Vpr stimulates HCV replication in JFH1 infection model and OR6 cell lines. (a) Quantitative HCV RNA assay in cell culture supernatants from Huh7.5.1 cells transfected with Vpr and then infected with JFH1 at different times. (b) The relative fold change of HCV RNA levels in cell culture supernatants from Huh7.5.1 cells was measured, with the control non-transfected JFH1-infected Huh7.5.1 cells at 24 h being assigned a value of 1. (c) Quantitative PCR for intracellular HCV RNA levels in the transient Vpr or vector-transfected Huh7.5.1 cells at 72 h post-JFH1 infection. (d) Western blotting confirmed the expression of β-actin, NS5A and Vpr in Huh7.5.1 cell lines infected with JFH1. (e) Renilla luciferase assay in OR6 cell lines transfected with Vpr and controls. (f) Western blot analysis in OR6 cell lines confirmed the expression of β-actin, NS5A and Vpr. The data are presented as mean ± sd. ** P
    Figure Legend Snippet: Vpr stimulates HCV replication in JFH1 infection model and OR6 cell lines. (a) Quantitative HCV RNA assay in cell culture supernatants from Huh7.5.1 cells transfected with Vpr and then infected with JFH1 at different times. (b) The relative fold change of HCV RNA levels in cell culture supernatants from Huh7.5.1 cells was measured, with the control non-transfected JFH1-infected Huh7.5.1 cells at 24 h being assigned a value of 1. (c) Quantitative PCR for intracellular HCV RNA levels in the transient Vpr or vector-transfected Huh7.5.1 cells at 72 h post-JFH1 infection. (d) Western blotting confirmed the expression of β-actin, NS5A and Vpr in Huh7.5.1 cell lines infected with JFH1. (e) Renilla luciferase assay in OR6 cell lines transfected with Vpr and controls. (f) Western blot analysis in OR6 cell lines confirmed the expression of β-actin, NS5A and Vpr. The data are presented as mean ± sd. ** P

    Techniques Used: Infection, Cell Culture, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Western Blot, Expressing, Luciferase

    36) Product Images from "Modulation of Bcl-x Alternative Splicing Induces Apoptosis of Human Hepatic Stellate Cells"

    Article Title: Modulation of Bcl-x Alternative Splicing Induces Apoptosis of Human Hepatic Stellate Cells

    Journal: BioMed Research International

    doi: 10.1155/2016/7478650

    Gene expression of Bcl-x S and Bcl-x L in human HSCs. Real-time PCR analysis of RNA extracted from human HSCs of different passages. (a) The gene expression of Bcl-2, Bcl-x L , and Bcl-x S was compared in activated human HSCs (passage 7). (b) The relative expression ratio of Bcl-x L and Bcl-x S was compared in human hepatocytes and human HSCs at different passages.
    Figure Legend Snippet: Gene expression of Bcl-x S and Bcl-x L in human HSCs. Real-time PCR analysis of RNA extracted from human HSCs of different passages. (a) The gene expression of Bcl-2, Bcl-x L , and Bcl-x S was compared in activated human HSCs (passage 7). (b) The relative expression ratio of Bcl-x L and Bcl-x S was compared in human hepatocytes and human HSCs at different passages.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    37) Product Images from "Advanced Glycation End-Products Induce Apoptosis of Vascular Smooth Muscle Cells: A Mechanism for Vascular Calcification"

    Article Title: Advanced Glycation End-Products Induce Apoptosis of Vascular Smooth Muscle Cells: A Mechanism for Vascular Calcification

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17091567

    AGE3-BSA-induced apoptosis is mediated through Nox4 or p22 phox . ( a ) Nox4 or p22 phox siRNA was transfected to A7r5 cells, total RNA was isolated three days after transfection, and Nox4 and p22 phox mRNA levels were measured by real-time PCR, as described in the Method section. GAPDH mRNA was used as loading control. A scramble siRNA was used as a negative control (NC); ( b ) AGE3-BSA-induced apoptosis was inhibited by silencing of Nox4 and p22 phox mRNA. Apoptosis was evaluated by cell death detection ELISA kit. The absorbance in cells treated with AGE3-BSA was compared with that in cells treated with cBSA. The ratio was demonstrated after correction with each cBSA. Results were analyzed by one-way ANOVA and LDS post-hoc test. ** p
    Figure Legend Snippet: AGE3-BSA-induced apoptosis is mediated through Nox4 or p22 phox . ( a ) Nox4 or p22 phox siRNA was transfected to A7r5 cells, total RNA was isolated three days after transfection, and Nox4 and p22 phox mRNA levels were measured by real-time PCR, as described in the Method section. GAPDH mRNA was used as loading control. A scramble siRNA was used as a negative control (NC); ( b ) AGE3-BSA-induced apoptosis was inhibited by silencing of Nox4 and p22 phox mRNA. Apoptosis was evaluated by cell death detection ELISA kit. The absorbance in cells treated with AGE3-BSA was compared with that in cells treated with cBSA. The ratio was demonstrated after correction with each cBSA. Results were analyzed by one-way ANOVA and LDS post-hoc test. ** p

    Techniques Used: Transfection, Isolation, Real-time Polymerase Chain Reaction, Negative Control, Enzyme-linked Immunosorbent Assay

    38) Product Images from "Functional Roles of Pattern Recognition Receptors That Recognize Virus Nucleic Acids in Human Adipose-Derived Mesenchymal Stem Cells"

    Article Title: Functional Roles of Pattern Recognition Receptors That Recognize Virus Nucleic Acids in Human Adipose-Derived Mesenchymal Stem Cells

    Journal: BioMed Research International

    doi: 10.1155/2016/9872138

    HSV60-induced immune responses. (a) Upregulation of IFI16. hAD-MSCs were stimulated with 5 μ g/mL HSV60 at indicated time points. Relative mRNA levels of IFI16 were determined by real-time PCR. (b) The protein levels of IFI16 were determined by Western blot. hAD-MSCs were lysed 24 h after HSV60 stimulation. β -Actin was used as loading control. (c) Time-dependent IFN- α and IFN- β expression. Total mRNA was extracted from hAD-MSCs at the indicated time points post HSV60 stimulation. Relative mRNA levels of IFN- α and IFN- β were determined using real-time PCR by normalizing to β -actin. (d) Dose-dependent IFN- α and IFN- β expression. hAD-MSCs were stimulated with indicated dose of HSV60 for 6 h. Relative mRNA levels of IFN- α and IFN- β were determined by real-time PCR. (e) IFN- α and IFN- β secretion. hAD-MSCs were stimulated with HSV60. After 24 h, IFN- α and IFN- β levels in culture medium were measured using ELISA. (f) Expression of antiviral proteins in mRNA levels after HSV60 stimulation. Total RNA was extracted from hAD-MSCs at the different times points after stimulation with HSV60. Relative mRNA levels of ISG15, OAS1, and Mx1 were determined using real-time PCR. (g) Expression of antiviral proteins in protein levels. hAD-MSCs were stimulated with HSV60. After 24 h, the cell lysates were extracted to Western blot to probe antiviral proteins. The cells treated with LyoVec alone served as Ctrl. Western blot images represent at least three experiments. Data are presented as the mean ± SEM of three experiments. ∗ P
    Figure Legend Snippet: HSV60-induced immune responses. (a) Upregulation of IFI16. hAD-MSCs were stimulated with 5 μ g/mL HSV60 at indicated time points. Relative mRNA levels of IFI16 were determined by real-time PCR. (b) The protein levels of IFI16 were determined by Western blot. hAD-MSCs were lysed 24 h after HSV60 stimulation. β -Actin was used as loading control. (c) Time-dependent IFN- α and IFN- β expression. Total mRNA was extracted from hAD-MSCs at the indicated time points post HSV60 stimulation. Relative mRNA levels of IFN- α and IFN- β were determined using real-time PCR by normalizing to β -actin. (d) Dose-dependent IFN- α and IFN- β expression. hAD-MSCs were stimulated with indicated dose of HSV60 for 6 h. Relative mRNA levels of IFN- α and IFN- β were determined by real-time PCR. (e) IFN- α and IFN- β secretion. hAD-MSCs were stimulated with HSV60. After 24 h, IFN- α and IFN- β levels in culture medium were measured using ELISA. (f) Expression of antiviral proteins in mRNA levels after HSV60 stimulation. Total RNA was extracted from hAD-MSCs at the different times points after stimulation with HSV60. Relative mRNA levels of ISG15, OAS1, and Mx1 were determined using real-time PCR. (g) Expression of antiviral proteins in protein levels. hAD-MSCs were stimulated with HSV60. After 24 h, the cell lysates were extracted to Western blot to probe antiviral proteins. The cells treated with LyoVec alone served as Ctrl. Western blot images represent at least three experiments. Data are presented as the mean ± SEM of three experiments. ∗ P

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

    Poly(I:C)-induced immune responses. (a) Upregulation of TLR3 and RIG-I. hAD-MSCs were stimulated with 5 μ g/mL poly(I:C) at indicated time. Relative mRNA levels of TLR3 and RIG-I were determined by real-time RCR at different time points. (b) The protein levels of TLR3 and RIG-I were determined by Western blot. hAD-MSCs were lysed 24 h after poly(I:C) stimulation. β -Actin was used as loading controls. (c) Poly(I:C) induced the expression of IFN- α and IFN- β in a time-dependent manner. Total RNA was extracted from hAD-MSCs, which were stimulated 5 μ g/mL poly(I:C) in different time. Relative mRNA levels of IFN- α and IFN- β were determined using real-time PCR by normalizing to β -actin. (d) Poly(I:C) induced the expression of IFN- α and IFN- β in a dose-dependent manner. Total RNA were stimulated with the indicated dose of poly(I:C) for 6 h. Relative mRNA levels of IFN- α and IFN- β were determined by real-time PCR. (e) The secretion of IFN- α and IFN- β . hAD-MSCs were stimulated with poly(I:C). The expression levels of IFN- α and IFN- β in culture medium were measured by ELISA. (f) Expression of antiviral proteins in mRNA level. Total RNA was extracted from hAD-MSCs at the different time points after poly(I:C) stimulation. Relative mRNA levels of Mx1, OAS1, and ISG15 were determined using real-time PCR. (g) Expression of antiviral proteins in protein levels. The cell lysates of hAD-MSCs were to probe antiviral proteins by Western blot using specific antibodies after stimulation with poly(I:C) 24 h. The cells treated with LyoVec along served as control (Ctrl). Western blot images are representatives of at least three experiments. Data are presented as the mean ± SEM of three experiments. ∗ P
    Figure Legend Snippet: Poly(I:C)-induced immune responses. (a) Upregulation of TLR3 and RIG-I. hAD-MSCs were stimulated with 5 μ g/mL poly(I:C) at indicated time. Relative mRNA levels of TLR3 and RIG-I were determined by real-time RCR at different time points. (b) The protein levels of TLR3 and RIG-I were determined by Western blot. hAD-MSCs were lysed 24 h after poly(I:C) stimulation. β -Actin was used as loading controls. (c) Poly(I:C) induced the expression of IFN- α and IFN- β in a time-dependent manner. Total RNA was extracted from hAD-MSCs, which were stimulated 5 μ g/mL poly(I:C) in different time. Relative mRNA levels of IFN- α and IFN- β were determined using real-time PCR by normalizing to β -actin. (d) Poly(I:C) induced the expression of IFN- α and IFN- β in a dose-dependent manner. Total RNA were stimulated with the indicated dose of poly(I:C) for 6 h. Relative mRNA levels of IFN- α and IFN- β were determined by real-time PCR. (e) The secretion of IFN- α and IFN- β . hAD-MSCs were stimulated with poly(I:C). The expression levels of IFN- α and IFN- β in culture medium were measured by ELISA. (f) Expression of antiviral proteins in mRNA level. Total RNA was extracted from hAD-MSCs at the different time points after poly(I:C) stimulation. Relative mRNA levels of Mx1, OAS1, and ISG15 were determined using real-time PCR. (g) Expression of antiviral proteins in protein levels. The cell lysates of hAD-MSCs were to probe antiviral proteins by Western blot using specific antibodies after stimulation with poly(I:C) 24 h. The cells treated with LyoVec along served as control (Ctrl). Western blot images are representatives of at least three experiments. Data are presented as the mean ± SEM of three experiments. ∗ P

    Techniques Used: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Expression of PRRs recognized viral RNA and DNA. (a) Total RNA was extracted from human adipose-derived mesenchymal stem cells (hAD-MSCs) and THP1 cell lines (THP1). Relative mRNA levels of PRRs recognized viral RNA nucleic acids, including TLR3, TLR7, TLR8, MDA5, and RIG-I, and RPRs recognized viral DNA nucleic acids, including TLR9, IFI16, DAI, cGAS, and polIII, were examined using real-time PCR by normalizing to GAPDH in the upper panel. The ΔCt values of genes expression in hAD-MSCs were shown in the lower panel. (b) The protein level of PRRs recognized viral nucleic acids in hAD-MSCs and THP1 were determined by Western blot using specific antibodies. β -Actin was used as loading control. (c) Distribution of TLR3, RIG-I, and IFI16. Indirect immunofluorescence staining using specific antibody, respectively, against TLR3, RIG-I, and IFI16 was performed in hAD-MSCs (left panels). The cells were staining with DAPI (middle panels). Images of TLR3, RIG-I, and IFI16 and DAPI staining were merged, respectively (right panels). Data are present as the mean ± SEM of three experiments. Images represent at least three experiments. Scale bar = 20 μ m.
    Figure Legend Snippet: Expression of PRRs recognized viral RNA and DNA. (a) Total RNA was extracted from human adipose-derived mesenchymal stem cells (hAD-MSCs) and THP1 cell lines (THP1). Relative mRNA levels of PRRs recognized viral RNA nucleic acids, including TLR3, TLR7, TLR8, MDA5, and RIG-I, and RPRs recognized viral DNA nucleic acids, including TLR9, IFI16, DAI, cGAS, and polIII, were examined using real-time PCR by normalizing to GAPDH in the upper panel. The ΔCt values of genes expression in hAD-MSCs were shown in the lower panel. (b) The protein level of PRRs recognized viral nucleic acids in hAD-MSCs and THP1 were determined by Western blot using specific antibodies. β -Actin was used as loading control. (c) Distribution of TLR3, RIG-I, and IFI16. Indirect immunofluorescence staining using specific antibody, respectively, against TLR3, RIG-I, and IFI16 was performed in hAD-MSCs (left panels). The cells were staining with DAPI (middle panels). Images of TLR3, RIG-I, and IFI16 and DAPI staining were merged, respectively (right panels). Data are present as the mean ± SEM of three experiments. Images represent at least three experiments. Scale bar = 20 μ m.

    Techniques Used: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining

    39) Product Images from "TAGAP instructs Th17 differentiation by bridging Dectin activation to EPHB2 signaling in innate antifungal response"

    Article Title: TAGAP instructs Th17 differentiation by bridging Dectin activation to EPHB2 signaling in innate antifungal response

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15564-7

    TAGAP is required for Dectin-1 ligand-induced signaling activation. a Real-time PCR was done from different organs (upper panel) and cell types (lower panel) from three mice, and the result was shown. b , c BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with Curdlan (100 μg/ml) for the indicated times, followed by western blot or real-time PCR analysis of indicated proteins and gene expression. d BMDMs from heterozygous control or TAGAP-deficient mice were stimulated with heat-killed C. albicans sc-5314 (upper panel, MOI = 2) or d -zymosan (lower panel, 100 μg/ml) for the indicated times, followed by western blot analysis of indicated proteins. e BMDMs from heterozygous control or TAGAP-deficient mice were stimulated with heat-killed C. albicans sc-5314 for 3 and 6 h, followed by real-time PCR analysis of indicated gene expression. f Control- gRNA or TAGAP-gRNA knocked down THP-1 cells were stimulated with heat-killed C. albicans sc-5314 (MOI = 2) for indicated times, followed by western blot analysis of indicated proteins. g BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with heat-killed C. albicans sc-5314 for indicated times, followed by western blot analysis of indicated proteins. h BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with Curdlan (100 μg/ml), followed by RNA-Seq analysis of gene expression. Heat-map of RNA-Seq data was shown. Arrow indicated top-changed gene expression in cytokine and chemokine groups. The scale bar representing fold induction was shown. * P
    Figure Legend Snippet: TAGAP is required for Dectin-1 ligand-induced signaling activation. a Real-time PCR was done from different organs (upper panel) and cell types (lower panel) from three mice, and the result was shown. b , c BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with Curdlan (100 μg/ml) for the indicated times, followed by western blot or real-time PCR analysis of indicated proteins and gene expression. d BMDMs from heterozygous control or TAGAP-deficient mice were stimulated with heat-killed C. albicans sc-5314 (upper panel, MOI = 2) or d -zymosan (lower panel, 100 μg/ml) for the indicated times, followed by western blot analysis of indicated proteins. e BMDMs from heterozygous control or TAGAP-deficient mice were stimulated with heat-killed C. albicans sc-5314 for 3 and 6 h, followed by real-time PCR analysis of indicated gene expression. f Control- gRNA or TAGAP-gRNA knocked down THP-1 cells were stimulated with heat-killed C. albicans sc-5314 (MOI = 2) for indicated times, followed by western blot analysis of indicated proteins. g BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with heat-killed C. albicans sc-5314 for indicated times, followed by western blot analysis of indicated proteins. h BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with Curdlan (100 μg/ml), followed by RNA-Seq analysis of gene expression. Heat-map of RNA-Seq data was shown. Arrow indicated top-changed gene expression in cytokine and chemokine groups. The scale bar representing fold induction was shown. * P

    Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Mouse Assay, Western Blot, Expressing, RNA Sequencing Assay

    40) Product Images from "INPP4B: the New Kid on the PI3K Block"

    Article Title: INPP4B: the New Kid on the PI3K Block

    Journal: Oncotarget

    doi:

    Regulation of the INPP4B expression A. Interrogation of the AR and ER recruitment sites in the vicinity of the INPP4B locus. Note AR recruitment in LNCaP cells upstream of the INPP4B promoter and in intron 2. Recruitment site for ERα in MCF7 cells is over 200 kb upstream and is immediately upstream of USP36 promoter as marked by the promoter methylation signature (H3K4Me). B. INPP4B expression is not hormonally induced in MCF-7 cells. MCF-7 cells grown in 10% charcoal stripped serum (CSS) for 48 hours were treated with ethanol (control), 10 nM estradiol (E2), 10 nM R5020, or 10 nM R1881. Cells were harvested 24 hours post-treatment, RNA was extracted, and INPP4B and 18S expression was analyzed by quantitative RT-PCR. C. To control for estradiol gene regulation, GREB1 expression was analyzed by quantitative RT-PCR (error bars denote ± S. E.) and normalized by 18S expression.
    Figure Legend Snippet: Regulation of the INPP4B expression A. Interrogation of the AR and ER recruitment sites in the vicinity of the INPP4B locus. Note AR recruitment in LNCaP cells upstream of the INPP4B promoter and in intron 2. Recruitment site for ERα in MCF7 cells is over 200 kb upstream and is immediately upstream of USP36 promoter as marked by the promoter methylation signature (H3K4Me). B. INPP4B expression is not hormonally induced in MCF-7 cells. MCF-7 cells grown in 10% charcoal stripped serum (CSS) for 48 hours were treated with ethanol (control), 10 nM estradiol (E2), 10 nM R5020, or 10 nM R1881. Cells were harvested 24 hours post-treatment, RNA was extracted, and INPP4B and 18S expression was analyzed by quantitative RT-PCR. C. To control for estradiol gene regulation, GREB1 expression was analyzed by quantitative RT-PCR (error bars denote ± S. E.) and normalized by 18S expression.

    Techniques Used: Expressing, Methylation, Quantitative RT-PCR

    INPP4B expression is not induced by testosterone in mice A. Four month old male castrated mice were treated with vehicle (V) (n=5) or 1 μg testosterone (T) (n=9). Prostates were isolated 24 hours following treatment, RNA extracted and Inpp4b and Krt18 expression was analyzed by quantitative RT-PCR. Inpp4b expression was correlated to Krt18 , an epithelial specific marker and expression normalized to the castrated group. B. Brain tissue from the same mice were collected in parallel and analyzed for Inpp4b and 18S expression. C. To control for testosterone gene regulation, Msmb expression was analyzed by quantitative RT-PCR (error bars denote ± S. E.).
    Figure Legend Snippet: INPP4B expression is not induced by testosterone in mice A. Four month old male castrated mice were treated with vehicle (V) (n=5) or 1 μg testosterone (T) (n=9). Prostates were isolated 24 hours following treatment, RNA extracted and Inpp4b and Krt18 expression was analyzed by quantitative RT-PCR. Inpp4b expression was correlated to Krt18 , an epithelial specific marker and expression normalized to the castrated group. B. Brain tissue from the same mice were collected in parallel and analyzed for Inpp4b and 18S expression. C. To control for testosterone gene regulation, Msmb expression was analyzed by quantitative RT-PCR (error bars denote ± S. E.).

    Techniques Used: Expressing, Mouse Assay, Isolation, Quantitative RT-PCR, Marker

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Sphingomyelin synthase 2 promotes an aggressive breast cancer phenotype by disrupting the homoeostasis of ceramide and sphingomyelin
    Article Snippet: .. RNA isolation, reverse transcription and quantitative real-time PCR Total RNA was extracted using Trizol (Invitrogen; Carlsbad, CA). .. To quantify the expression of SGMS2, the total RNA was subjected to polyadenylation and reverse transcription (RT) using a ThermoScriptTM RT-PCR System (Invitrogen).

    Article Title: Kaposi's Sarcoma Herpesvirus Upregulates Aurora A Expression to Promote p53 Phosphorylation and Ubiquitylation
    Article Snippet: .. Quantitative real-time PCR Total RNA from cells was extracted using Trizol reagent and cDNA was made with a Superscript II reverse transcription kit (Invitrogen, Inc., Carlsbad, CA). .. The primers for real-time PCR were as followings: for Aurora A: 5′-GGAGAGCTTAAAATTGCAGATTTTG-3′ and 5′-GGCAAACACATACCAAGAGA CCT-3′ ; for LANA: 5′-CATACGAACTCCAGGTCTGTG-3′ and 5′-GGTGGAAGAGCC CATAATCT-3′ ; for vCyclinD: 5′-TAATAGAGGCGGGCAATGAG-3′ and 5′-ACTCCTTT TCCCGCCAAGAAC-3′ ; for vFLIP: 5′-GTGTAAGAATGTCTGTGGTGTGC-3′ and 5′- GCGGAATGTCTGTTTCGTGC-3′ ; and for GAPDH: 5′-CTCCTCTGACTTCAACAGC G-3′ and 5′-GC CAAATTCGTTGTCATACCAG-3′ .

    Article Title: Orphan nuclear receptor HNF4G promotes bladder cancer growth and invasion through the regulation of the hyaluronan synthase 2 gene
    Article Snippet: .. Quantitative real-time PCR Total RNA was isolated from bladder tissues and cells using the TRIzol reagent (Invitrogen ) according to the manufacturer's instructions. .. Single-stranded cDNAs were synthesized using Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.

    Article Title: Delayed triggering of oestrogen induced apoptosis that contrasts with rapid paclitaxel-induced breast cancer cell death
    Article Snippet: .. RNA isolation and real-time PCR Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNAeasy kit according to the manufacturer's instructions. ..

    Article Title: Up-regulation of the Hippo pathway effector TAZ renders lung adenocarcinoma cells harboring EGFR-T790M mutation resistant to gefitinib
    Article Snippet: .. Real-time PCR Total RNA was extracted from cells with TRIzol reagent (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. cDNA was synthesized with the PrimeScript RT reagent kit (TaKaRa, Dalian, China). .. Quantitative RT-PCR was carried out with a SYBR Premix Ex Taq kit (TaKaRa) on a 7500 real time PCR system (ABI) as follows: 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 30 s. The primers used were as follows: TAZ forward 5’-AGTACCCTGAGCCAGCAGAA-3’, reverse 5’-GATTCTCTGAAGCCGCAGTT-3’; CTGF forward 5’- CCCTCGCGGCTTACCGACTGG-3’, reverse 5’-CACAGGTCTTGGAACAGGCGC-3’; AXL forward 5’-TTTCCTGAGTGAAGCGGTCT-3’, reverse 5’-CATCTGAGTGGGCAGGTACA-3’; b-actin forward 5’-TGACGTGGACATCCGCAAAG-3’, reverse 5’-CTGGAAGGTGGACAGCGAGG-3’.

    Article Title: Different Patterns of Expression and of IL-10 Modulation of Inflammatory Mediators from Macrophages of Lyme Disease-Resistant and -Susceptible Mice
    Article Snippet: .. Quantitative real-time PCR Total RNA was isolated from BMDM treated with L-OspA or with live Bb using Trizol reagent (Invitrogen). .. Complementary DNA (cDNA) was produced from quality RNA (125 ng) samples using an RT2 first strand kit as per the manufacturer's instructions (SABiosciences, Frederick, MD).

    Article Title: Interleukin-4 and melatonin ameliorate high glucose and interleukin-1? stimulated inflammatory reaction in human retinal endothelial cells and retinal pigment epithelial cells
    Article Snippet: .. Evaluation of gene expression using quantitative real-time PCR Total RNA was extracted from cultured RECs and RPE cells using TRIzol (Invitrogen Life Technologies, Grand Island, NY) and was reverse transcribed with the TaKaRa First Strand Synthesis kit (TaKaRa, Dalian, China). .. Quantitative real-time PCR (qPCR) was conducted on an ABI Prism 7000 system with a SYBR Green PCR kit (TaKaRa) by denaturing at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C, annealing at 60 °C, and extension at 72 °C for 10 s, respectively.

    Article Title: HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy
    Article Snippet: .. Reverse Transcription and Real-time PCR Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, as previously reported . .. For in vivo RNA isolation endpoints, total RNA was isolated from the right superior lobe using an AllPrep DNA/RNA/protein kit (Qiagen, Valencia, CA, USA). cDNA was synthesized from total RNA in a 20 µl final reaction volume using a Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Isolation:

    Article Title: Sphingomyelin synthase 2 promotes an aggressive breast cancer phenotype by disrupting the homoeostasis of ceramide and sphingomyelin
    Article Snippet: .. RNA isolation, reverse transcription and quantitative real-time PCR Total RNA was extracted using Trizol (Invitrogen; Carlsbad, CA). .. To quantify the expression of SGMS2, the total RNA was subjected to polyadenylation and reverse transcription (RT) using a ThermoScriptTM RT-PCR System (Invitrogen).

    Article Title: Orphan nuclear receptor HNF4G promotes bladder cancer growth and invasion through the regulation of the hyaluronan synthase 2 gene
    Article Snippet: .. Quantitative real-time PCR Total RNA was isolated from bladder tissues and cells using the TRIzol reagent (Invitrogen ) according to the manufacturer's instructions. .. Single-stranded cDNAs were synthesized using Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.

    Article Title: Delayed triggering of oestrogen induced apoptosis that contrasts with rapid paclitaxel-induced breast cancer cell death
    Article Snippet: .. RNA isolation and real-time PCR Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNAeasy kit according to the manufacturer's instructions. ..

    Article Title: Different Patterns of Expression and of IL-10 Modulation of Inflammatory Mediators from Macrophages of Lyme Disease-Resistant and -Susceptible Mice
    Article Snippet: .. Quantitative real-time PCR Total RNA was isolated from BMDM treated with L-OspA or with live Bb using Trizol reagent (Invitrogen). .. Complementary DNA (cDNA) was produced from quality RNA (125 ng) samples using an RT2 first strand kit as per the manufacturer's instructions (SABiosciences, Frederick, MD).

    Expressing:

    Article Title: Interleukin-4 and melatonin ameliorate high glucose and interleukin-1? stimulated inflammatory reaction in human retinal endothelial cells and retinal pigment epithelial cells
    Article Snippet: .. Evaluation of gene expression using quantitative real-time PCR Total RNA was extracted from cultured RECs and RPE cells using TRIzol (Invitrogen Life Technologies, Grand Island, NY) and was reverse transcribed with the TaKaRa First Strand Synthesis kit (TaKaRa, Dalian, China). .. Quantitative real-time PCR (qPCR) was conducted on an ABI Prism 7000 system with a SYBR Green PCR kit (TaKaRa) by denaturing at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C, annealing at 60 °C, and extension at 72 °C for 10 s, respectively.

    Cell Culture:

    Article Title: Interleukin-4 and melatonin ameliorate high glucose and interleukin-1? stimulated inflammatory reaction in human retinal endothelial cells and retinal pigment epithelial cells
    Article Snippet: .. Evaluation of gene expression using quantitative real-time PCR Total RNA was extracted from cultured RECs and RPE cells using TRIzol (Invitrogen Life Technologies, Grand Island, NY) and was reverse transcribed with the TaKaRa First Strand Synthesis kit (TaKaRa, Dalian, China). .. Quantitative real-time PCR (qPCR) was conducted on an ABI Prism 7000 system with a SYBR Green PCR kit (TaKaRa) by denaturing at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C, annealing at 60 °C, and extension at 72 °C for 10 s, respectively.

    Synthesized:

    Article Title: Up-regulation of the Hippo pathway effector TAZ renders lung adenocarcinoma cells harboring EGFR-T790M mutation resistant to gefitinib
    Article Snippet: .. Real-time PCR Total RNA was extracted from cells with TRIzol reagent (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. cDNA was synthesized with the PrimeScript RT reagent kit (TaKaRa, Dalian, China). .. Quantitative RT-PCR was carried out with a SYBR Premix Ex Taq kit (TaKaRa) on a 7500 real time PCR system (ABI) as follows: 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 30 s. The primers used were as follows: TAZ forward 5’-AGTACCCTGAGCCAGCAGAA-3’, reverse 5’-GATTCTCTGAAGCCGCAGTT-3’; CTGF forward 5’- CCCTCGCGGCTTACCGACTGG-3’, reverse 5’-CACAGGTCTTGGAACAGGCGC-3’; AXL forward 5’-TTTCCTGAGTGAAGCGGTCT-3’, reverse 5’-CATCTGAGTGGGCAGGTACA-3’; b-actin forward 5’-TGACGTGGACATCCGCAAAG-3’, reverse 5’-CTGGAAGGTGGACAGCGAGG-3’.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Thermo Fisher quantitative rt pcr analysis total rna
    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger <t>RNA</t> (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time <t>(qRT)‐PCR.</t> ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P
    Quantitative Rt Pcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative rt pcr analysis total rna/product/Thermo Fisher
    Average 94 stars, based on 169 article reviews
    Price from $9.99 to $1999.99
    quantitative rt pcr analysis total rna - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    94
    Thermo Fisher quantitative real time polymerase chain reaction qrt pcr total rna
    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by <t>qRT-PCR;</t> b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative real time polymerase chain reaction qrt pcr total rna/product/Thermo Fisher
    Average 94 stars, based on 424 article reviews
    Price from $9.99 to $1999.99
    quantitative real time polymerase chain reaction qrt pcr total rna - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    95
    Thermo Fisher rna
    MD001 stimulates fatty acid oxidation in vitro . HepG2 ( A ), differentiated 3T3-L1 adipocyte ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle or MD001 (10 μM) for 24 h and total <t>RNA</t> was isolated for <t>cDNA</t> synthesis. Relative gene expressions were analysed by qRT-PCR. HepG2 ( D ), differentiated 3T3-L1 adipocytes ( E ), and differentiated C2C12 myotubes ( F ) were treated with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h and the fatty acid oxidation rate was analysed as described in the Methods section. To confirm whether the fatty acid oxidation rate enhanced by MD001 is mediated by PPARα, HepG2 ( G ), differentiated 3T3-L1 ( H ), and differentiated C2C12 myotubes ( I ) were transfected with control or PPARα siRNA (20 nM) for 48 h, followed by treatment with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h. WY14643 was treated as positive control. *, **, vs. vehicle; † and ‡ , vs. vehicle/control siRNA; § , vs. WY/control siRNA; ¶ , vs. MD001/control siRNA. Data represent the mean ± SD of three independent experiments. * P
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 3653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna/product/Thermo Fisher
    Average 95 stars, based on 3653 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-08
    95/100 stars
      Buy from Supplier

    92
    Thermo Fisher real time quantitative pcr qpcr total rna
    Pcf11 Expression Is Regulated by IPA (A) Schematic of mouse Pcf11 gene. Multiple PASs are indicated. EPA, internal exonic polyadenylation; IPA, intronic polyadenylation; TPA, 3′ terminal exon polyadenylation. 3′READS data of brain, heart, and testis are shown. (B) Features of intron 1 compared with other introns in the mouse genome. Each feature has ten bins, and the bin containing intron 1 of Pcf11 is highlighted in red. Distance to TSS is based on all IPA sites in PolyA_DB. (C) <t>RT-qPCR</t> analysis of IPA and TPA isoform expression in siPcf11 versus siCtrl cells. Error bars are SD on the basis of three replicates. (D) Top: schematic showing two sgRNAs used to remove a region flanking the IPA site. Bottom: validation of IPA site knockout (IPA Pcf11 -KO) by genomic DNA <t>PCR</t> using primers flanking the KO region. WT, wild-type 4T1 cells. (E) RT-qPCR analysis of Pcf11 isoform expression in WT and IPA Pcf11 -KO cells. Error bars are SD on the basis of three replicates. (F) Western blot analysis of PCF11 in WT and IPA Pcf11 -KO cells. (G) 3′UTR APA changes in IPA Pcf11 -KO cells. (H) IPA changes in IPA Pcf11 -KO cells. IPA isoforms (all combined) were compared with TPA isoforms (all combined) in analysis. (I) IPA distribution maps of activated IPA events in IPA Pcf11 -KO cells. (J) Left: schematic of PASs around the transcription start site (TSS); right: metagene analysis of PAS usage around the TSS in WT and IPA Pcf1 1 -KO cells. (K) Gene expression changes in IPA Pcf11 -KO cells for different groups of genes on the basis of IPA site frequency in PolyA_DB. ***p
    Real Time Quantitative Pcr Qpcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time quantitative pcr qpcr total rna/product/Thermo Fisher
    Average 92 stars, based on 101 article reviews
    Price from $9.99 to $1999.99
    real time quantitative pcr qpcr total rna - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    Image Search Results


    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Migration, In Vitro, Quantitative RT-PCR, Western Blot

    Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Migration, In Vitro, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot

    MD001 stimulates fatty acid oxidation in vitro . HepG2 ( A ), differentiated 3T3-L1 adipocyte ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle or MD001 (10 μM) for 24 h and total RNA was isolated for cDNA synthesis. Relative gene expressions were analysed by qRT-PCR. HepG2 ( D ), differentiated 3T3-L1 adipocytes ( E ), and differentiated C2C12 myotubes ( F ) were treated with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h and the fatty acid oxidation rate was analysed as described in the Methods section. To confirm whether the fatty acid oxidation rate enhanced by MD001 is mediated by PPARα, HepG2 ( G ), differentiated 3T3-L1 ( H ), and differentiated C2C12 myotubes ( I ) were transfected with control or PPARα siRNA (20 nM) for 48 h, followed by treatment with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h. WY14643 was treated as positive control. *, **, vs. vehicle; † and ‡ , vs. vehicle/control siRNA; § , vs. WY/control siRNA; ¶ , vs. MD001/control siRNA. Data represent the mean ± SD of three independent experiments. * P

    Journal: Scientific Reports

    Article Title: MD001, a Novel Peroxisome Proliferator-activated Receptor α/γ Agonist, Improves Glucose and Lipid Metabolism

    doi: 10.1038/s41598-018-38281-0

    Figure Lengend Snippet: MD001 stimulates fatty acid oxidation in vitro . HepG2 ( A ), differentiated 3T3-L1 adipocyte ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle or MD001 (10 μM) for 24 h and total RNA was isolated for cDNA synthesis. Relative gene expressions were analysed by qRT-PCR. HepG2 ( D ), differentiated 3T3-L1 adipocytes ( E ), and differentiated C2C12 myotubes ( F ) were treated with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h and the fatty acid oxidation rate was analysed as described in the Methods section. To confirm whether the fatty acid oxidation rate enhanced by MD001 is mediated by PPARα, HepG2 ( G ), differentiated 3T3-L1 ( H ), and differentiated C2C12 myotubes ( I ) were transfected with control or PPARα siRNA (20 nM) for 48 h, followed by treatment with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h. WY14643 was treated as positive control. *, **, vs. vehicle; † and ‡ , vs. vehicle/control siRNA; § , vs. WY/control siRNA; ¶ , vs. MD001/control siRNA. Data represent the mean ± SD of three independent experiments. * P

    Article Snippet: Complementary DNA (cDNA) was synthesised by reverse transcription using 0.5 μg of total RNA, and quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (ABI Thermo Fisher Scientific, Foster City, CA, USA) on a StepOne 48-well real-time PCR system (ABI/Thermo Fisher Scientific).

    Techniques: In Vitro, Isolation, Quantitative RT-PCR, Transfection, Positive Control

    MD001 induces the expression of target genes through PPARα and PPARγ activation. HEK293 cells were transiently co-transfected with human HA-PPARα ( A ) and HA-PPARγ ( B ) expression vectors along with the reporter plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) with Renilla vector for 24 h. Cells were treated with MD001 (10 μM), rosiglitazone (rosi) (10 μM) or WY14643 (WY) (10 μM) for 24 h. Luciferase activity was normalised to Renilla luciferase activity as described in the Methods section. **, vs. vehicle. ( C ) HepG2 cells were treated with MD001 (10 μM) for 24 h, and total RNA was isolated and synthesised into cDNA. Relative expression was quantitated using qRT-PCR. **, vs. vehicle. ( D , E ) HepG2 cells were transfected with control siRNA, PPARα siRNA ( D ), or PPARγ siRNA ( E ) for 48 h and treated with vehicle, WY14643 (10 μM), rosiglitazone (10 μM) or MD001 (10 μM) for 24 h. Subsequently, cells were harvested for total RNA isolation. Relative expression of target genes was quantitated using qRT-PCR. # , vs. control siRNA; ‡ , vs. control siRNA/vehicle; § , vs. control siRNA/WY; * and **, vs. control siRNA/MD001. Data represent the mean ± SD of three independent experiments. * P

    Journal: Scientific Reports

    Article Title: MD001, a Novel Peroxisome Proliferator-activated Receptor α/γ Agonist, Improves Glucose and Lipid Metabolism

    doi: 10.1038/s41598-018-38281-0

    Figure Lengend Snippet: MD001 induces the expression of target genes through PPARα and PPARγ activation. HEK293 cells were transiently co-transfected with human HA-PPARα ( A ) and HA-PPARγ ( B ) expression vectors along with the reporter plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) with Renilla vector for 24 h. Cells were treated with MD001 (10 μM), rosiglitazone (rosi) (10 μM) or WY14643 (WY) (10 μM) for 24 h. Luciferase activity was normalised to Renilla luciferase activity as described in the Methods section. **, vs. vehicle. ( C ) HepG2 cells were treated with MD001 (10 μM) for 24 h, and total RNA was isolated and synthesised into cDNA. Relative expression was quantitated using qRT-PCR. **, vs. vehicle. ( D , E ) HepG2 cells were transfected with control siRNA, PPARα siRNA ( D ), or PPARγ siRNA ( E ) for 48 h and treated with vehicle, WY14643 (10 μM), rosiglitazone (10 μM) or MD001 (10 μM) for 24 h. Subsequently, cells were harvested for total RNA isolation. Relative expression of target genes was quantitated using qRT-PCR. # , vs. control siRNA; ‡ , vs. control siRNA/vehicle; § , vs. control siRNA/WY; * and **, vs. control siRNA/MD001. Data represent the mean ± SD of three independent experiments. * P

    Article Snippet: Complementary DNA (cDNA) was synthesised by reverse transcription using 0.5 μg of total RNA, and quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (ABI Thermo Fisher Scientific, Foster City, CA, USA) on a StepOne 48-well real-time PCR system (ABI/Thermo Fisher Scientific).

    Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Isolation, Quantitative RT-PCR

    MD001 promotes glucose consumption by induction of GLUT expression. HepG2 ( A ), differentiated 3T3-L1 adipocytes ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle, MD001 (10, 50 μM), or rosiglitazone (rosi, 10 μM) for 24 h; glucose consumption was examined as described in the Methods section. Cells were harvested for total RNA isolation. Relative expression levels of GLUT2 ( D ) and GLUT4 ( E , F ) were quantitated using qRT-PCR. *, **, and † vs. vehicle. Data represent the mean ± SD of three independent experiments. * P

    Journal: Scientific Reports

    Article Title: MD001, a Novel Peroxisome Proliferator-activated Receptor α/γ Agonist, Improves Glucose and Lipid Metabolism

    doi: 10.1038/s41598-018-38281-0

    Figure Lengend Snippet: MD001 promotes glucose consumption by induction of GLUT expression. HepG2 ( A ), differentiated 3T3-L1 adipocytes ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle, MD001 (10, 50 μM), or rosiglitazone (rosi, 10 μM) for 24 h; glucose consumption was examined as described in the Methods section. Cells were harvested for total RNA isolation. Relative expression levels of GLUT2 ( D ) and GLUT4 ( E , F ) were quantitated using qRT-PCR. *, **, and † vs. vehicle. Data represent the mean ± SD of three independent experiments. * P

    Article Snippet: Complementary DNA (cDNA) was synthesised by reverse transcription using 0.5 μg of total RNA, and quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (ABI Thermo Fisher Scientific, Foster City, CA, USA) on a StepOne 48-well real-time PCR system (ABI/Thermo Fisher Scientific).

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    Pcf11 Expression Is Regulated by IPA (A) Schematic of mouse Pcf11 gene. Multiple PASs are indicated. EPA, internal exonic polyadenylation; IPA, intronic polyadenylation; TPA, 3′ terminal exon polyadenylation. 3′READS data of brain, heart, and testis are shown. (B) Features of intron 1 compared with other introns in the mouse genome. Each feature has ten bins, and the bin containing intron 1 of Pcf11 is highlighted in red. Distance to TSS is based on all IPA sites in PolyA_DB. (C) RT-qPCR analysis of IPA and TPA isoform expression in siPcf11 versus siCtrl cells. Error bars are SD on the basis of three replicates. (D) Top: schematic showing two sgRNAs used to remove a region flanking the IPA site. Bottom: validation of IPA site knockout (IPA Pcf11 -KO) by genomic DNA PCR using primers flanking the KO region. WT, wild-type 4T1 cells. (E) RT-qPCR analysis of Pcf11 isoform expression in WT and IPA Pcf11 -KO cells. Error bars are SD on the basis of three replicates. (F) Western blot analysis of PCF11 in WT and IPA Pcf11 -KO cells. (G) 3′UTR APA changes in IPA Pcf11 -KO cells. (H) IPA changes in IPA Pcf11 -KO cells. IPA isoforms (all combined) were compared with TPA isoforms (all combined) in analysis. (I) IPA distribution maps of activated IPA events in IPA Pcf11 -KO cells. (J) Left: schematic of PASs around the transcription start site (TSS); right: metagene analysis of PAS usage around the TSS in WT and IPA Pcf1 1 -KO cells. (K) Gene expression changes in IPA Pcf11 -KO cells for different groups of genes on the basis of IPA site frequency in PolyA_DB. ***p

    Journal: Cell reports

    Article Title: Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes

    doi: 10.1016/j.celrep.2019.02.049

    Figure Lengend Snippet: Pcf11 Expression Is Regulated by IPA (A) Schematic of mouse Pcf11 gene. Multiple PASs are indicated. EPA, internal exonic polyadenylation; IPA, intronic polyadenylation; TPA, 3′ terminal exon polyadenylation. 3′READS data of brain, heart, and testis are shown. (B) Features of intron 1 compared with other introns in the mouse genome. Each feature has ten bins, and the bin containing intron 1 of Pcf11 is highlighted in red. Distance to TSS is based on all IPA sites in PolyA_DB. (C) RT-qPCR analysis of IPA and TPA isoform expression in siPcf11 versus siCtrl cells. Error bars are SD on the basis of three replicates. (D) Top: schematic showing two sgRNAs used to remove a region flanking the IPA site. Bottom: validation of IPA site knockout (IPA Pcf11 -KO) by genomic DNA PCR using primers flanking the KO region. WT, wild-type 4T1 cells. (E) RT-qPCR analysis of Pcf11 isoform expression in WT and IPA Pcf11 -KO cells. Error bars are SD on the basis of three replicates. (F) Western blot analysis of PCF11 in WT and IPA Pcf11 -KO cells. (G) 3′UTR APA changes in IPA Pcf11 -KO cells. (H) IPA changes in IPA Pcf11 -KO cells. IPA isoforms (all combined) were compared with TPA isoforms (all combined) in analysis. (I) IPA distribution maps of activated IPA events in IPA Pcf11 -KO cells. (J) Left: schematic of PASs around the transcription start site (TSS); right: metagene analysis of PAS usage around the TSS in WT and IPA Pcf1 1 -KO cells. (K) Gene expression changes in IPA Pcf11 -KO cells for different groups of genes on the basis of IPA site frequency in PolyA_DB. ***p

    Article Snippet: Real-time quantitative PCR (qPCR) Total RNA was extracted using TRIzol (Thermo Fisher) and treated with Turbo DNase (Thermo Fisher). cDNA was synthesized using oligo(dT) and M-MLV reverse transcriptase with 1.5–2 μg of RNA. cDNA was then mixed with real-time PCR primers and Luna qPCR master mix (NEB).

    Techniques: Expressing, Indirect Immunoperoxidase Assay, Quantitative RT-PCR, Knock-Out, Polymerase Chain Reaction, Western Blot