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    Structured Review

    Thermo Fisher qrt pcr reaction
    miR-214 attenuated NRK-52E cell apoptosis under hypoxia. a <t>qRT-PCR</t> analysis of miR-214 expression in kidney cortex of I/R-induced rat AKI models after 12, 48 and 72 h of reperfusion. n = 6 rats/group. b qRT-PCR analysis of miR-214 expression at 0, 3 h, 6 h, and 9 h during hypoxic incubation of cultured NRK-52E cells. c qRT-PCR analysis of miR-214 expression in NRK-52E cells after transfection with miR-214, anti-miR-214, or matched controls. d–e Flow cytometry was used to analyze the apoptotic rate of NRK-52E cells after transfection with miR-214, anti-miR-214, or respective controls during hypoxia incubation. f Western blot was performed to detect the protein levels of Bcl-2 and Bax in NRK-52E cells after introduction with miR-214, anti-miR-214, or corresponding controls under hypoxia. Each experiment was independently repeated 3 times. * P
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    1) Product Images from "miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway"

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway

    Journal: Biological Research

    doi: 10.1186/s40659-018-0179-2

    miR-214 attenuated NRK-52E cell apoptosis under hypoxia. a qRT-PCR analysis of miR-214 expression in kidney cortex of I/R-induced rat AKI models after 12, 48 and 72 h of reperfusion. n = 6 rats/group. b qRT-PCR analysis of miR-214 expression at 0, 3 h, 6 h, and 9 h during hypoxic incubation of cultured NRK-52E cells. c qRT-PCR analysis of miR-214 expression in NRK-52E cells after transfection with miR-214, anti-miR-214, or matched controls. d–e Flow cytometry was used to analyze the apoptotic rate of NRK-52E cells after transfection with miR-214, anti-miR-214, or respective controls during hypoxia incubation. f Western blot was performed to detect the protein levels of Bcl-2 and Bax in NRK-52E cells after introduction with miR-214, anti-miR-214, or corresponding controls under hypoxia. Each experiment was independently repeated 3 times. * P
    Figure Legend Snippet: miR-214 attenuated NRK-52E cell apoptosis under hypoxia. a qRT-PCR analysis of miR-214 expression in kidney cortex of I/R-induced rat AKI models after 12, 48 and 72 h of reperfusion. n = 6 rats/group. b qRT-PCR analysis of miR-214 expression at 0, 3 h, 6 h, and 9 h during hypoxic incubation of cultured NRK-52E cells. c qRT-PCR analysis of miR-214 expression in NRK-52E cells after transfection with miR-214, anti-miR-214, or matched controls. d–e Flow cytometry was used to analyze the apoptotic rate of NRK-52E cells after transfection with miR-214, anti-miR-214, or respective controls during hypoxia incubation. f Western blot was performed to detect the protein levels of Bcl-2 and Bax in NRK-52E cells after introduction with miR-214, anti-miR-214, or corresponding controls under hypoxia. Each experiment was independently repeated 3 times. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Incubation, Cell Culture, Transfection, Flow Cytometry, Cytometry, Western Blot

    Dkk3 was a target of miR-214 in NRK-52E cells. a The predictive wild-type miR-214 binding sites in the 3′UTR of Dkk3 and the corresponding mutant binding sites were displayed. b The relative luciferase activity was measured by luciferase reporter assay after NRK-52E cells were cotransfected with Dkk3 -WT or Dkk3 -MUT and miR-214 or miR-con. The mRNA ( c ) and protein ( d , e ) levels of DKK3 were examined by qRT-PCR and western blot in NRK-52E cells introduced with miR-214, anti-miR-214, or matched controls. Each experiment was independently repeated 3 times. * P
    Figure Legend Snippet: Dkk3 was a target of miR-214 in NRK-52E cells. a The predictive wild-type miR-214 binding sites in the 3′UTR of Dkk3 and the corresponding mutant binding sites were displayed. b The relative luciferase activity was measured by luciferase reporter assay after NRK-52E cells were cotransfected with Dkk3 -WT or Dkk3 -MUT and miR-214 or miR-con. The mRNA ( c ) and protein ( d , e ) levels of DKK3 were examined by qRT-PCR and western blot in NRK-52E cells introduced with miR-214, anti-miR-214, or matched controls. Each experiment was independently repeated 3 times. * P

    Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Reporter Assay, Quantitative RT-PCR, Western Blot

    Evaluation of rat AKI model following I/R surgery and NRK-52E cell model following hypoxia treatment. The serum levels of SCr ( a ), BUN ( b ), and urine Kim-1 ( c ) in I/R-induced rat AKI models at 24 h after surgery were measured. d The mRNA expressions of TNF-α, IL-1β, and IL-6 in hypoxia-induced NRK-52E cell I/R model were detected by qRT-PCR. e The protein levels of Bcl-2 and Bax in hypoxia-induced NRK-52E cell I/R models were assessed by western blot. n = 6 rats/group. * P
    Figure Legend Snippet: Evaluation of rat AKI model following I/R surgery and NRK-52E cell model following hypoxia treatment. The serum levels of SCr ( a ), BUN ( b ), and urine Kim-1 ( c ) in I/R-induced rat AKI models at 24 h after surgery were measured. d The mRNA expressions of TNF-α, IL-1β, and IL-6 in hypoxia-induced NRK-52E cell I/R model were detected by qRT-PCR. e The protein levels of Bcl-2 and Bax in hypoxia-induced NRK-52E cell I/R models were assessed by western blot. n = 6 rats/group. * P

    Techniques Used: Quantitative RT-PCR, Western Blot

    miR-214 protected against AKI in vivo through targeting Dkk3 and activating Wnt/β-catenin pathway. miR-214 or miR-con was intraperitoneally injected into mice, followed by ischemic surgery. a qRT-PCR analysis of miR-214 expression in I/R-induced rat AKI model and sham mice. The serum levels of BUN ( b ), SCr ( c ), and Urine Kim-1 ( d ) level in I/R-induced rat AKI model and sham mice. e Western blot analysis of Bcl-2, Bax and fibronectin in I/R-induced rat AKI model and sham group. f The protein levels of DKK3, β-catenin, c-myc, and cyclin D1 in I/R-induced rat AKI model and sham group. Each experiment was independently repeated 3 times. * P
    Figure Legend Snippet: miR-214 protected against AKI in vivo through targeting Dkk3 and activating Wnt/β-catenin pathway. miR-214 or miR-con was intraperitoneally injected into mice, followed by ischemic surgery. a qRT-PCR analysis of miR-214 expression in I/R-induced rat AKI model and sham mice. The serum levels of BUN ( b ), SCr ( c ), and Urine Kim-1 ( d ) level in I/R-induced rat AKI model and sham mice. e Western blot analysis of Bcl-2, Bax and fibronectin in I/R-induced rat AKI model and sham group. f The protein levels of DKK3, β-catenin, c-myc, and cyclin D1 in I/R-induced rat AKI model and sham group. Each experiment was independently repeated 3 times. * P

    Techniques Used: In Vivo, Injection, Mouse Assay, Quantitative RT-PCR, Expressing, Western Blot

    2) Product Images from "Evaluation of glutathione S‐transferase pi 1 expression and gene promoter methylation in Moroccan patients with urothelial bladder cancer, et al. Evaluation of glutathione S‐transferase pi 1 expression and gene promoter methylation in Moroccan patients with urothelial bladder cancer"

    Article Title: Evaluation of glutathione S‐transferase pi 1 expression and gene promoter methylation in Moroccan patients with urothelial bladder cancer, et al. Evaluation of glutathione S‐transferase pi 1 expression and gene promoter methylation in Moroccan patients with urothelial bladder cancer

    Journal: Molecular Genetics & Genomic Medicine

    doi: 10.1002/mgg3.449

    Results of methylation‐specific PCR ( MS ‐ PCR ) for GSTP 1 promoter region. Lanes U and M correspond to amplification with primers recognizing unmethylated (97 bp) and methylated (91 bp) sequences, respectively. DNA from MCF ‐7 cell line was used as positive control for methylation and DNA from BCPAP cell line was used as negative control. Results of normal bladder urotheliums (C1, C2: Control 1, 2) and the 30 BC cases (1–30) are represented. Water was used as negative PCR control (H 2 O). On the right side: the 100 bp DNA ladder. GSTP1 : NM_000852.3 . BC, bladder cancer
    Figure Legend Snippet: Results of methylation‐specific PCR ( MS ‐ PCR ) for GSTP 1 promoter region. Lanes U and M correspond to amplification with primers recognizing unmethylated (97 bp) and methylated (91 bp) sequences, respectively. DNA from MCF ‐7 cell line was used as positive control for methylation and DNA from BCPAP cell line was used as negative control. Results of normal bladder urotheliums (C1, C2: Control 1, 2) and the 30 BC cases (1–30) are represented. Water was used as negative PCR control (H 2 O). On the right side: the 100 bp DNA ladder. GSTP1 : NM_000852.3 . BC, bladder cancer

    Techniques Used: Methylation, Polymerase Chain Reaction, Mass Spectrometry, Amplification, Positive Control, Negative Control

    3) Product Images from "Potent suppression of HIV-1 cell attachment by Kudzu root extract"

    Article Title: Potent suppression of HIV-1 cell attachment by Kudzu root extract

    Journal: Retrovirology

    doi: 10.1186/s12977-018-0446-x

    Kudzu inhibits HIV-1 replication of X4 tropic viruses. a , b Activity of Kudzu in acute infection of HeLa-CD4-LTR-LacZ cells with NL4-3. HeLa-CD4-LTR-LacZ cells were infected with HIV-1 NL4-3 strain in the presence of different dilutions of Kudzu. a β-Gal activity was measured 72 h later. Untreat.: untreated. The mean ± SD of 5 independent experiments is represented. b Viral supernatants recovered 72 h post infection were assayed for their p24 antigen content using a sandwich ELISA kit. Untreat.: untreated. Data is a mean ± SD of 2 independent experiments. c Impact of Kudzu on HIV-1 integration. HeLa-CD4-LTR-LacZ were infected with NL4-3 in presence of compounds for 24 h. Next day, DNA was extracted and provirus integration was quantified by Alu-PCR followed by qPCR. Saquinavir (Saq., a protease inhibitor, 200 nM), Efavirenz (Efav., a reverse transcriptase inhibitor, 200 nM), Raltegravir (Ralt., an integrase inhibitor, 200 nM), and AMD3100 (an entry inhibitor, 10 nM) were used as controls. Kudzu was used at 1:200. The mean ± SD of 5 independent experiments is represented. d Impact of Kudzu on HIV-1 integration in primary CD4 + T cells 24 h post-infection and treatment. A cocktail of antiretrovirals (ARVS: 180 nM AZT, a reverse transcriptase inhibitor, 100 nM Efavirenz and 200 nM Raltegravir) and Enfuvirtide (1 μg/ml) were used as controls. Shown is the mean ± SD of 3 independent experiments. e Activity of Kudzu on reverse-transcription products of HIV-1 10 h post-infection. HeLa-CD4-LTR-LacZ were infected with NL4-3 in presence of compounds for 10 h. Next, DNA was extracted and early and late RT products were measured by qPCR. Kudzu was used at 1:200. Error Bars from qPCR (n = 3) ± SD from 2 independent experiments for early products. The mean ± SD of 4 independent experiments is represented for late products. f Time-of-addition experiment of kudzu in HeLa-CD4-LTR-LacZ infected with NL4-3 strain. Kudzu or control compounds (4 nM AMD3100 and 10 nM Efavirenz) were added at 1, 2, 3, 4, 5 and 6 h postinfection. β-Gal activity was measured 72 h later. Data shown is representative of 2 independent experiments. The two-tailed paired t test was used for statistical comparisons. *: p value
    Figure Legend Snippet: Kudzu inhibits HIV-1 replication of X4 tropic viruses. a , b Activity of Kudzu in acute infection of HeLa-CD4-LTR-LacZ cells with NL4-3. HeLa-CD4-LTR-LacZ cells were infected with HIV-1 NL4-3 strain in the presence of different dilutions of Kudzu. a β-Gal activity was measured 72 h later. Untreat.: untreated. The mean ± SD of 5 independent experiments is represented. b Viral supernatants recovered 72 h post infection were assayed for their p24 antigen content using a sandwich ELISA kit. Untreat.: untreated. Data is a mean ± SD of 2 independent experiments. c Impact of Kudzu on HIV-1 integration. HeLa-CD4-LTR-LacZ were infected with NL4-3 in presence of compounds for 24 h. Next day, DNA was extracted and provirus integration was quantified by Alu-PCR followed by qPCR. Saquinavir (Saq., a protease inhibitor, 200 nM), Efavirenz (Efav., a reverse transcriptase inhibitor, 200 nM), Raltegravir (Ralt., an integrase inhibitor, 200 nM), and AMD3100 (an entry inhibitor, 10 nM) were used as controls. Kudzu was used at 1:200. The mean ± SD of 5 independent experiments is represented. d Impact of Kudzu on HIV-1 integration in primary CD4 + T cells 24 h post-infection and treatment. A cocktail of antiretrovirals (ARVS: 180 nM AZT, a reverse transcriptase inhibitor, 100 nM Efavirenz and 200 nM Raltegravir) and Enfuvirtide (1 μg/ml) were used as controls. Shown is the mean ± SD of 3 independent experiments. e Activity of Kudzu on reverse-transcription products of HIV-1 10 h post-infection. HeLa-CD4-LTR-LacZ were infected with NL4-3 in presence of compounds for 10 h. Next, DNA was extracted and early and late RT products were measured by qPCR. Kudzu was used at 1:200. Error Bars from qPCR (n = 3) ± SD from 2 independent experiments for early products. The mean ± SD of 4 independent experiments is represented for late products. f Time-of-addition experiment of kudzu in HeLa-CD4-LTR-LacZ infected with NL4-3 strain. Kudzu or control compounds (4 nM AMD3100 and 10 nM Efavirenz) were added at 1, 2, 3, 4, 5 and 6 h postinfection. β-Gal activity was measured 72 h later. Data shown is representative of 2 independent experiments. The two-tailed paired t test was used for statistical comparisons. *: p value

    Techniques Used: Activity Assay, Infection, Sandwich ELISA, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Protease Inhibitor, Two Tailed Test

    4) Product Images from "Supplementation with IL-6 and Muscle Cell Culture Conditioned Media Enhances Myogenic Differentiation of Adipose Tissue-Derived Stem Cells through STAT3 Activation"

    Article Title: Supplementation with IL-6 and Muscle Cell Culture Conditioned Media Enhances Myogenic Differentiation of Adipose Tissue-Derived Stem Cells through STAT3 Activation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061557

    Effect of a combination of IL-6 and C2C12 CM on myogenic differentiation. mRNA expression of ( a ) MyoD, ( b ) myogenin (MyoG), and ( c ) myosin heavy chain (MYH) during myogenic differentiation, mRNA expression was analyzed by qRT-PCR. n = 3 independent experiments, each assay was performed in duplicate All values are expressed relative to the gene expression observed at 6 weeks using protocol C. * p
    Figure Legend Snippet: Effect of a combination of IL-6 and C2C12 CM on myogenic differentiation. mRNA expression of ( a ) MyoD, ( b ) myogenin (MyoG), and ( c ) myosin heavy chain (MYH) during myogenic differentiation, mRNA expression was analyzed by qRT-PCR. n = 3 independent experiments, each assay was performed in duplicate All values are expressed relative to the gene expression observed at 6 weeks using protocol C. * p

    Techniques Used: Expressing, Quantitative RT-PCR

    5) Product Images from "In Vitro Anti-Inflammatory and Radical Scavenging Properties of Chinotto (Citrus myrtifolia Raf.) Essential Oils"

    Article Title: In Vitro Anti-Inflammatory and Radical Scavenging Properties of Chinotto (Citrus myrtifolia Raf.) Essential Oils

    Journal: Nutrients

    doi: 10.3390/nu10060783

    Effect of CEO2 on the lipopolysaccharide (LPS)-induced COX2 ( a ), iNOS ( b ), IL-1β ( c ), IL-6 ( d ), MCP-1 ( e ) gene expression in RAW264.7 macrophages. Cells were seeded in 6-well plates at a density of 5 × 10 5 cells mL −1 and, after overnight incubation, were treated for 24 h with LPS (0.5 μg mL −1 ) and with (or without) different concentrations of CEO2. Total RNA was isolated and reverse transcribed, yielding cDNA prior to quantitative real-time PCR. Gene expression fold increase normalized to RPS27A2 is presented as percentages, and LPS stimulation (without CEO2) was fixed at 100%, and represents the mean of three separate experiments (each done in duplicate) ± standard deviation. Mean value was statistically different from control (* p
    Figure Legend Snippet: Effect of CEO2 on the lipopolysaccharide (LPS)-induced COX2 ( a ), iNOS ( b ), IL-1β ( c ), IL-6 ( d ), MCP-1 ( e ) gene expression in RAW264.7 macrophages. Cells were seeded in 6-well plates at a density of 5 × 10 5 cells mL −1 and, after overnight incubation, were treated for 24 h with LPS (0.5 μg mL −1 ) and with (or without) different concentrations of CEO2. Total RNA was isolated and reverse transcribed, yielding cDNA prior to quantitative real-time PCR. Gene expression fold increase normalized to RPS27A2 is presented as percentages, and LPS stimulation (without CEO2) was fixed at 100%, and represents the mean of three separate experiments (each done in duplicate) ± standard deviation. Mean value was statistically different from control (* p

    Techniques Used: Expressing, Incubation, Isolation, Real-time Polymerase Chain Reaction, Standard Deviation

    6) Product Images from "Effect of CD44st and HER2 expression on the postoperative prognosis of breast cancer patients"

    Article Title: Effect of CD44st and HER2 expression on the postoperative prognosis of breast cancer patients

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S180972

    Expression of the CD44 and HER2 proteins in breast cancer tissues detected by using immunohistochemistry method. Notes: ( A ) CD44-negative tissue from breast cancer (blank control ×400); ( B ) HER2-negative tissue from breast cancer (blank control ×400); ( C ) CD44-negative tissue from breast cancer (negative control ×400); ( D ) HER2-negative tissue from breast cancer (negative control ×400); ( E ) CD44-positive tissue from breast cancer; ( F ) HER2-positive tissue from breast cancer.
    Figure Legend Snippet: Expression of the CD44 and HER2 proteins in breast cancer tissues detected by using immunohistochemistry method. Notes: ( A ) CD44-negative tissue from breast cancer (blank control ×400); ( B ) HER2-negative tissue from breast cancer (blank control ×400); ( C ) CD44-negative tissue from breast cancer (negative control ×400); ( D ) HER2-negative tissue from breast cancer (negative control ×400); ( E ) CD44-positive tissue from breast cancer; ( F ) HER2-positive tissue from breast cancer.

    Techniques Used: Expressing, Immunohistochemistry, Negative Control

    7) Product Images from "Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression"

    Article Title: Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02935

    CC10 treatment decreased the expression of TNF-α and IL-1β and inhibited hepatocyte apoptosis in mouse livers. Livers from BALB/cJ mice treated with CC10 protein, and from mice treated with saline were collected 72 h following MHV-3 infection. (A) The levels of TNF- α (a) and IL-1 β (b) in livers were detected using qPCR ( n = 6/group). Values shown are means and standard error. * P
    Figure Legend Snippet: CC10 treatment decreased the expression of TNF-α and IL-1β and inhibited hepatocyte apoptosis in mouse livers. Livers from BALB/cJ mice treated with CC10 protein, and from mice treated with saline were collected 72 h following MHV-3 infection. (A) The levels of TNF- α (a) and IL-1 β (b) in livers were detected using qPCR ( n = 6/group). Values shown are means and standard error. * P

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

    CC10 inhibited Fgl2 expression and decreased fibrin deposition in mice. Livers from BALB/cJ mice treated with CC10 protein, and from mice treated with saline were collected 72 h following MHV-3 infection. (A) Fgl2 expression was measured by qPCR ( n = 6–8/group). Data represent means and standard error of three independent experiments performed in triplicate. * P
    Figure Legend Snippet: CC10 inhibited Fgl2 expression and decreased fibrin deposition in mice. Livers from BALB/cJ mice treated with CC10 protein, and from mice treated with saline were collected 72 h following MHV-3 infection. (A) Fgl2 expression was measured by qPCR ( n = 6–8/group). Data represent means and standard error of three independent experiments performed in triplicate. * P

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

    CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3–infected BALB/cJ mice treated with saline. CC10 protein (2 μg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 μg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days ( n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. *** P
    Figure Legend Snippet: CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3–infected BALB/cJ mice treated with saline. CC10 protein (2 μg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 μg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days ( n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. *** P

    Techniques Used: Mouse Assay, Infection, Injection

    8) Product Images from "SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB"

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

    Journal: Thoracic Cancer

    doi: 10.1111/1759-7714.12895

    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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

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

    9) Product Images from "Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway"

    Article Title: Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1547-8

    DBP induces low sperm motility and reduces sperm flagellum-related genes expression. a – c The number of sperms ( a ) and the percentages of motile sperm ( b ) and progressive sperm ( c ) in offsprings in the administration of DBP treatment, SC79 treatment, or MK-2206 treatment in the embryonic period. d Mean-centered, hierarchical clustering of all genes altered in DBP administration rats. e The classification of enriched pathway in the RNA sequencing results. f Mean-centered, hierarchical clustering of sperm flagellum-related genes in DBP administration rats. g Expression of sperm flagellum-related genes in DBP administration group were detected by qRT-PCR analysis. h Gene GO terms significantly enriched in the DBP stimulation gene subsets. All measurements are shown as the means ± SD from three independent experiments, ** p
    Figure Legend Snippet: DBP induces low sperm motility and reduces sperm flagellum-related genes expression. a – c The number of sperms ( a ) and the percentages of motile sperm ( b ) and progressive sperm ( c ) in offsprings in the administration of DBP treatment, SC79 treatment, or MK-2206 treatment in the embryonic period. d Mean-centered, hierarchical clustering of all genes altered in DBP administration rats. e The classification of enriched pathway in the RNA sequencing results. f Mean-centered, hierarchical clustering of sperm flagellum-related genes in DBP administration rats. g Expression of sperm flagellum-related genes in DBP administration group were detected by qRT-PCR analysis. h Gene GO terms significantly enriched in the DBP stimulation gene subsets. All measurements are shown as the means ± SD from three independent experiments, ** p

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

    miR-29b and DNMT3b are required for DBP-induced PTEN demethylation. a DNMT1, DNMT3a and DNMT3b were detected using qRT-PCR in GC-2 cells after treated with DBP. b Representative WB images showing the expression of DNMT1, DNMT3a, and DNMT3b in GC-2 cells after treated with DBP. c , d qRT-PCR ( c ) and WB ( d ) results showed the expression of DNMT3b and PTEN in the treatment of si-NC or si-DNMT3b in GC-2 cells. e , f qRT-PCR ( e ) and WB ( f ) results showed the expression of DNMT3b and PTEN in GC-2 cells after DNMT3b overexpression. g Percentage of methylation for the CpG island of PTEN in GC-2 cells in the treatment of DMSO, DBP, vector, or DNMT3b overexpression. h miR-29b expression was detected by qRT-PCR in GC-2 cells after treated with DBP. i qRT-PCR showed the expression of miR-29b, DNMT3b, and PTEN in the treatment of miR-NC, miR-29b-mimic, or miR-29b-inhibitor in GC-2 cells. j The position of the binding sites was numbered relative to the first nucleotide of the 3′-UTR. Mutations were introduced into DNMT3b 3′-UTR that matched the seed region of miR-29b as shown in DNMT3b Mu. Luciferase activity was detected using dual-luciferase assay in GC-2 cells co-transfected with luciferase constructs containing the DNMT3b Wt or Mu 3′-UTR and miR-29b mimics or scrambled oligonucleotides as the negative control. k qRT-PCR showed the expression of DNMT3b in the treatment of DMSO, DBP, miR-NC, or miR-29b-inhibitor. All measurements are shown as the means ± SD from three independent experiments, **** p
    Figure Legend Snippet: miR-29b and DNMT3b are required for DBP-induced PTEN demethylation. a DNMT1, DNMT3a and DNMT3b were detected using qRT-PCR in GC-2 cells after treated with DBP. b Representative WB images showing the expression of DNMT1, DNMT3a, and DNMT3b in GC-2 cells after treated with DBP. c , d qRT-PCR ( c ) and WB ( d ) results showed the expression of DNMT3b and PTEN in the treatment of si-NC or si-DNMT3b in GC-2 cells. e , f qRT-PCR ( e ) and WB ( f ) results showed the expression of DNMT3b and PTEN in GC-2 cells after DNMT3b overexpression. g Percentage of methylation for the CpG island of PTEN in GC-2 cells in the treatment of DMSO, DBP, vector, or DNMT3b overexpression. h miR-29b expression was detected by qRT-PCR in GC-2 cells after treated with DBP. i qRT-PCR showed the expression of miR-29b, DNMT3b, and PTEN in the treatment of miR-NC, miR-29b-mimic, or miR-29b-inhibitor in GC-2 cells. j The position of the binding sites was numbered relative to the first nucleotide of the 3′-UTR. Mutations were introduced into DNMT3b 3′-UTR that matched the seed region of miR-29b as shown in DNMT3b Mu. Luciferase activity was detected using dual-luciferase assay in GC-2 cells co-transfected with luciferase constructs containing the DNMT3b Wt or Mu 3′-UTR and miR-29b mimics or scrambled oligonucleotides as the negative control. k qRT-PCR showed the expression of DNMT3b in the treatment of DMSO, DBP, miR-NC, or miR-29b-inhibitor. All measurements are shown as the means ± SD from three independent experiments, **** p

    Techniques Used: Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Methylation, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Transfection, Construct, Negative Control

    DBP induces reproductive toxicity via AKT pathway and PTEN methylation might be involved in it in male mice offsprings. a Bisulfite sequencing PCR results for the CpG island in the PTEN promoter region in DBP administration male mice offsprings. b Quantitative analysis for the results of global methylation in DBP administration group. c qRT-PCR was used to detect miR-29b, DNMT3b, and PTEN levels in DBP administration group. d Representative WB images showing the protein levels of DNMT3b, PTEN and p-AKT in DBP administration group. e – h Pearson correlation test results showing the correlation between PTEN mRNA level and miR-29b level ( e ) and between PTEN mRNA level and DNMT3b mRNA level ( f ) and between DNMT3b mRNA level and miR-29b level ( g ) and between PTEN methylation and p-AKT protein abundances ( h ). i Representative hematoxylin and eosin (h e) staining showed the testicular morphology after DBP treatment, SC79 treatment, or MK-2206 treatment. Representative immunohistochemistry (IHC) images showing BrdU, TUNEL, 8-OHdG, and γ-H2AX positive areas in testicular tissues after DBP treatment, SC79 treatment or MK-2206 treatment. Scale bar, 50 μm. j Representative WB images showing the protein levels of AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR (S2448), Bax, Bcl-2, cleaved caspase-3, γ-H2AX in GC-1 and GC-2 cells after DBP treatment, SC79 treatment, or MK-2206 treatment. Protein loading is indicated by β-actin. All measurements are shown as the means ± SD from three independent experiments, **** p
    Figure Legend Snippet: DBP induces reproductive toxicity via AKT pathway and PTEN methylation might be involved in it in male mice offsprings. a Bisulfite sequencing PCR results for the CpG island in the PTEN promoter region in DBP administration male mice offsprings. b Quantitative analysis for the results of global methylation in DBP administration group. c qRT-PCR was used to detect miR-29b, DNMT3b, and PTEN levels in DBP administration group. d Representative WB images showing the protein levels of DNMT3b, PTEN and p-AKT in DBP administration group. e – h Pearson correlation test results showing the correlation between PTEN mRNA level and miR-29b level ( e ) and between PTEN mRNA level and DNMT3b mRNA level ( f ) and between DNMT3b mRNA level and miR-29b level ( g ) and between PTEN methylation and p-AKT protein abundances ( h ). i Representative hematoxylin and eosin (h e) staining showed the testicular morphology after DBP treatment, SC79 treatment, or MK-2206 treatment. Representative immunohistochemistry (IHC) images showing BrdU, TUNEL, 8-OHdG, and γ-H2AX positive areas in testicular tissues after DBP treatment, SC79 treatment or MK-2206 treatment. Scale bar, 50 μm. j Representative WB images showing the protein levels of AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR (S2448), Bax, Bcl-2, cleaved caspase-3, γ-H2AX in GC-1 and GC-2 cells after DBP treatment, SC79 treatment, or MK-2206 treatment. Protein loading is indicated by β-actin. All measurements are shown as the means ± SD from three independent experiments, **** p

    Techniques Used: Methylation, Mouse Assay, Methylation Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemistry, TUNEL Assay

    DBP-mediated AKT activity by regulating PTEN expression in germ cells. a qRT-PCR was used to detect the efficiency of PTEN knockdown in GC-1 and GC-2 cells. b qRT-PCR was used to detect PTEN expression after treated with DBP in GC-1 and GC-2 cells. c , d GC-1 ( c ) and GC-2 ( d ) cells were transfected with PTEN siRNA or treated with DBP alone, and together, respectively. And then samples were analyzed for PTEN, AKT, p-AKT, PI3K, and p-PI3K expression. All measurements are shown as the means ± SD from three independent experiments, **** p
    Figure Legend Snippet: DBP-mediated AKT activity by regulating PTEN expression in germ cells. a qRT-PCR was used to detect the efficiency of PTEN knockdown in GC-1 and GC-2 cells. b qRT-PCR was used to detect PTEN expression after treated with DBP in GC-1 and GC-2 cells. c , d GC-1 ( c ) and GC-2 ( d ) cells were transfected with PTEN siRNA or treated with DBP alone, and together, respectively. And then samples were analyzed for PTEN, AKT, p-AKT, PI3K, and p-PI3K expression. All measurements are shown as the means ± SD from three independent experiments, **** p

    Techniques Used: Activity Assay, Expressing, Quantitative RT-PCR, Transfection

    10) Product Images from "VirB10 vaccination for protection against Anaplasma phagocytophilum"

    Article Title: VirB10 vaccination for protection against Anaplasma phagocytophilum

    Journal: BMC Microbiology

    doi: 10.1186/s12866-018-1346-x

    Analysis of the recombinant proteins VirB9-1, VirB9-2 and VirB10. SDS-PAGE and Western Blot analysis of recombinant VirB9-1 ( a ), VirB9-2 ( b ), and VirB10 ( c ). Lanes represent the different fractions analyzed during the purification procedure, 1 = Total crude protein, 2 = Filtered supernatant fraction obtained after high-speed centrifugation, 3 = Washed fraction, 4 = Eluted protein. Western Blot analysis was performed using the monoclonal anti-histidine tag antibody reacting with the recombinant protein (red arrows). Predicted molecular weights plus tags for VirB9-1 (33.5KDa), VirB9-2 (31.6KDa) and VirB10 (52KDa)
    Figure Legend Snippet: Analysis of the recombinant proteins VirB9-1, VirB9-2 and VirB10. SDS-PAGE and Western Blot analysis of recombinant VirB9-1 ( a ), VirB9-2 ( b ), and VirB10 ( c ). Lanes represent the different fractions analyzed during the purification procedure, 1 = Total crude protein, 2 = Filtered supernatant fraction obtained after high-speed centrifugation, 3 = Washed fraction, 4 = Eluted protein. Western Blot analysis was performed using the monoclonal anti-histidine tag antibody reacting with the recombinant protein (red arrows). Predicted molecular weights plus tags for VirB9-1 (33.5KDa), VirB9-2 (31.6KDa) and VirB10 (52KDa)

    Techniques Used: Recombinant, SDS Page, Western Blot, Purification, Centrifugation

    Sera from C3H/HeN immunized mice reacted against A. phagocytophilum VirB9-1, VirB9-2, and VirB10 proteins. Proteins from equal amounts (10 8 ) of host cell-free A. phagocytophilum were separated by SDS-PAGE gel electrophoresis. Immunoblots of transferred proteins were reacted with pooled sera from the 3 immunized but not challenged mice (1:1000) and reactions were visualized by chemiluminescence. The sizes of protein standards are indicated on the left in kDa. Predicted molecular weights for A. phagoytophilum VirB9-1 (30.5KDa), VirB9-2 (28.6KDa), and VirB10 (49.2KDa). Bands of similar sizes were not visualized in sera from the pcDNA3.1 /Ovalbumin control group
    Figure Legend Snippet: Sera from C3H/HeN immunized mice reacted against A. phagocytophilum VirB9-1, VirB9-2, and VirB10 proteins. Proteins from equal amounts (10 8 ) of host cell-free A. phagocytophilum were separated by SDS-PAGE gel electrophoresis. Immunoblots of transferred proteins were reacted with pooled sera from the 3 immunized but not challenged mice (1:1000) and reactions were visualized by chemiluminescence. The sizes of protein standards are indicated on the left in kDa. Predicted molecular weights for A. phagoytophilum VirB9-1 (30.5KDa), VirB9-2 (28.6KDa), and VirB10 (49.2KDa). Bands of similar sizes were not visualized in sera from the pcDNA3.1 /Ovalbumin control group

    Techniques Used: Mouse Assay, SDS Page, Nucleic Acid Electrophoresis, Western Blot

    11) Product Images from "Anti-angiogenic effect of Livistona chinensis seed extract in vitro and in vivo"

    Article Title: Anti-angiogenic effect of Livistona chinensis seed extract in vitro and in vivo

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.7075

    Effect of EELC on VEGF-A and VEGFR-2 protein expression. HUVECs were treated with EELC for 24 h. The protein levels of VEGF-A in the supernatant of each group and VEGFR-2 from lysed HUVECs were determined by ELISA. Data are the means ± standard deviation of triplicate experiments. *P
    Figure Legend Snippet: Effect of EELC on VEGF-A and VEGFR-2 protein expression. HUVECs were treated with EELC for 24 h. The protein levels of VEGF-A in the supernatant of each group and VEGFR-2 from lysed HUVECs were determined by ELISA. Data are the means ± standard deviation of triplicate experiments. *P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Effect of EELC on mRNA expression. The mRNA levels of VEGF-A and VEGFR-2 were detected by reverse transcription-semi-quantitative polymerase chain reaction with an internal control of GAPDH. The treated group data were compared with an untreated control. Data are means ± standard deviation of triplicate experiments. *P
    Figure Legend Snippet: Effect of EELC on mRNA expression. The mRNA levels of VEGF-A and VEGFR-2 were detected by reverse transcription-semi-quantitative polymerase chain reaction with an internal control of GAPDH. The treated group data were compared with an untreated control. Data are means ± standard deviation of triplicate experiments. *P

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

    12) Product Images from "Different De Novo Initiation Strategies Are Used by Influenza Virus RNA Polymerase on Its cRNA and Viral RNA Promoters during Viral RNA Replication"

    Article Title: Different De Novo Initiation Strategies Are Used by Influenza Virus RNA Polymerase on Its cRNA and Viral RNA Promoters during Viral RNA Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.80.5.2337-2348.2006

    Internally synthesized pppApG derived from the cRNA promoter can realign to a vRNA template for elongation in vitro. (A) Autoradiograph showing pppApG synthesized internally on the cRNA promoter and realigned onto 3′-terminal residues of the vRNA promoter for extension into a trinucleotide by partially purified influenza A/Turkey/England polymerase. Either 1 mM CTP or 1 mM UTP was added to ATP and radiolabeled GTP as indicated for extension to the third nucleotide on either a vRNA or a cRNA promoter. The synthesized di- or trinucleotides are shown on the left. The solid circle on the right marks an unknown band. (B) Characterization of bands A to H by treatment with T 2 RNase and PEI-cellulose TLC. The positions of pppA*p and pp*pGp are shown. *p, 32 P.
    Figure Legend Snippet: Internally synthesized pppApG derived from the cRNA promoter can realign to a vRNA template for elongation in vitro. (A) Autoradiograph showing pppApG synthesized internally on the cRNA promoter and realigned onto 3′-terminal residues of the vRNA promoter for extension into a trinucleotide by partially purified influenza A/Turkey/England polymerase. Either 1 mM CTP or 1 mM UTP was added to ATP and radiolabeled GTP as indicated for extension to the third nucleotide on either a vRNA or a cRNA promoter. The synthesized di- or trinucleotides are shown on the left. The solid circle on the right marks an unknown band. (B) Characterization of bands A to H by treatment with T 2 RNase and PEI-cellulose TLC. The positions of pppA*p and pp*pGp are shown. *p, 32 P.

    Techniques Used: Synthesized, Derivative Assay, In Vitro, Autoradiography, Purification, Thin Layer Chromatography

    Mutations or deletion of the 3′-terminal residue of the cRNA template are corrected to wild type in an in vivo minireplicon system. (A) Autoradiograph showing primer extension analysis of virus-like CAT RNA transcribed in an in vivo minireplicon system. 293T cells were transfected with four protein expression plasmids encoding the three subunits of the influenza virus RNA polymerase (either wild-type [3P] or inactive mutant [3P-ASM]) and the nucleoprotein (NP), together with a pPOLIcCAT plasmid encoding a CAT reporter gene flanked by either wild-type (WT) or mutant influenza virus cRNA promoter sequences as indicated. RNA was extracted at 41 h posttransfection and analyzed by primer extension with vRNA- or cRNA-specific primers. (B) Sample sequence traces of cDNA amplified by cRNA- or vRNA-specific RT-PCR from virus-like CAT RNA described in A. Extracted RNA was TAP treated and circularized prior to RT-PCR amplification across the junction and cloning of the amplicon. The arrows indicate the mutated 3′-terminal residue (1U→A complement) of the template cRNA (left) and the corrected wild-type 5′-terminal residue (5′A) of the replicated vRNA (right).
    Figure Legend Snippet: Mutations or deletion of the 3′-terminal residue of the cRNA template are corrected to wild type in an in vivo minireplicon system. (A) Autoradiograph showing primer extension analysis of virus-like CAT RNA transcribed in an in vivo minireplicon system. 293T cells were transfected with four protein expression plasmids encoding the three subunits of the influenza virus RNA polymerase (either wild-type [3P] or inactive mutant [3P-ASM]) and the nucleoprotein (NP), together with a pPOLIcCAT plasmid encoding a CAT reporter gene flanked by either wild-type (WT) or mutant influenza virus cRNA promoter sequences as indicated. RNA was extracted at 41 h posttransfection and analyzed by primer extension with vRNA- or cRNA-specific primers. (B) Sample sequence traces of cDNA amplified by cRNA- or vRNA-specific RT-PCR from virus-like CAT RNA described in A. Extracted RNA was TAP treated and circularized prior to RT-PCR amplification across the junction and cloning of the amplicon. The arrows indicate the mutated 3′-terminal residue (1U→A complement) of the template cRNA (left) and the corrected wild-type 5′-terminal residue (5′A) of the replicated vRNA (right).

    Techniques Used: In Vivo, Autoradiography, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction, Clone Assay

    Identification of nucleotide residues required for pppApG synthesis in the 3′ strand of both the cRNA and vRNA promoters. (A) Influenza virus model cRNA promoter in the corkscrew configuration (B) Influenza virus model vRNA promoter in the corkscrew configuration. The prime notation is used to identify nucleotides of the 5′ end of the promoter. (C) Autoradiograph showing pppApG synthesized by 3P with the wild-type (cWT) or mutant 3′ cRNA strand, as indicated, in the presence of the wild-type 5′ cRNA strand. (D) Autoradiograph showing pppApG synthesized by 3P with the wild-type (vWT) or mutant 3′ vRNA strand, as indicated, in the presence of the wild-type 5′ vRNA strand. (E) Autoradiograph showing pppApG synthesized by 3P with the wild-type (vWT) or mutant (single or double) 3′ vRNA strand, as indicated, in the presence of the wild-type 5′ vRNA strand. -ve, 3′ cRNA or vRNA promoter strands alone. *p, 32 P.
    Figure Legend Snippet: Identification of nucleotide residues required for pppApG synthesis in the 3′ strand of both the cRNA and vRNA promoters. (A) Influenza virus model cRNA promoter in the corkscrew configuration (B) Influenza virus model vRNA promoter in the corkscrew configuration. The prime notation is used to identify nucleotides of the 5′ end of the promoter. (C) Autoradiograph showing pppApG synthesized by 3P with the wild-type (cWT) or mutant 3′ cRNA strand, as indicated, in the presence of the wild-type 5′ cRNA strand. (D) Autoradiograph showing pppApG synthesized by 3P with the wild-type (vWT) or mutant 3′ vRNA strand, as indicated, in the presence of the wild-type 5′ vRNA strand. (E) Autoradiograph showing pppApG synthesized by 3P with the wild-type (vWT) or mutant (single or double) 3′ vRNA strand, as indicated, in the presence of the wild-type 5′ vRNA strand. -ve, 3′ cRNA or vRNA promoter strands alone. *p, 32 P.

    Techniques Used: Autoradiography, Synthesized, Mutagenesis

    Identification of sequence requirements in the 5′ and 3′ arms of the cRNA promoter for internal pppApG synthesis by a “transplant” experiment. (A) Model wild-type (WT) or mutant 5′ vRNA strands with the 5′ cRNA-like elements transplanted one by one. The transplanted 5′ cRNA elements are boxed. 10′D, hinge A deleted in the 5′ strand. (B) Autoradiograph showing internal pppApG synthesis activity with the 5′ mutants shown in A in combination with the 3′ cRNA promoter strand containing a mutation at position 1(U→A). (C) Model wild-type (WT) or mutant 3′ vRNA strands with the 3′ cRNA-like elements transplanted one by one. The transplanted 3′ cRNA elements and a mutation at position 1 are boxed. 10I, hinge U inserted in the 3′ strand. (D) Autoradiograph showing internal pppApG synthesis activity with the 5′ wild-type cRNA promoter in combination with the 3′ mutants shown in C. Note that the promoter mutant used in lane 12 differs in sequence only at position 1 from the wild-type cRNA promoter used in lane 2. -ve, 3′ strand of the promoter alone. The position of pppApG is shown. *p, 32 P.
    Figure Legend Snippet: Identification of sequence requirements in the 5′ and 3′ arms of the cRNA promoter for internal pppApG synthesis by a “transplant” experiment. (A) Model wild-type (WT) or mutant 5′ vRNA strands with the 5′ cRNA-like elements transplanted one by one. The transplanted 5′ cRNA elements are boxed. 10′D, hinge A deleted in the 5′ strand. (B) Autoradiograph showing internal pppApG synthesis activity with the 5′ mutants shown in A in combination with the 3′ cRNA promoter strand containing a mutation at position 1(U→A). (C) Model wild-type (WT) or mutant 3′ vRNA strands with the 3′ cRNA-like elements transplanted one by one. The transplanted 3′ cRNA elements and a mutation at position 1 are boxed. 10I, hinge U inserted in the 3′ strand. (D) Autoradiograph showing internal pppApG synthesis activity with the 5′ wild-type cRNA promoter in combination with the 3′ mutants shown in C. Note that the promoter mutant used in lane 12 differs in sequence only at position 1 from the wild-type cRNA promoter used in lane 2. -ve, 3′ strand of the promoter alone. The position of pppApG is shown. *p, 32 P.

    Techniques Used: Sequencing, Mutagenesis, Autoradiography, Activity Assay

    Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and vRNA promoters for viral RNA replication. (A) Internal initiation and realignment model for the cRNA promoter. (B) Terminal initiation and elongation model for the vRNA promoter. Base-pairing is shown as dotted lines. The phosphodiester bonds formed within nascent di- or trinucleotides are shown as dashes.
    Figure Legend Snippet: Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and vRNA promoters for viral RNA replication. (A) Internal initiation and realignment model for the cRNA promoter. (B) Terminal initiation and elongation model for the vRNA promoter. Base-pairing is shown as dotted lines. The phosphodiester bonds formed within nascent di- or trinucleotides are shown as dashes.

    Techniques Used:

    13) Product Images from "GSTP1 CpG Island Hypermethylation Is Responsible for the Absence of GSTP1 Expression in Human Prostate Cancer Cells "

    Article Title: GSTP1 CpG Island Hypermethylation Is Responsible for the Absence of GSTP1 Expression in Human Prostate Cancer Cells

    Journal: The American Journal of Pathology

    doi:

    Discrimination of DNA hypermethylation at maternal and paternal GSTP1 alleles using a PCR strategy. DNA from matched normal (normal) and neoplastic (tumor) prostate tissues was left untreated (U; lanes 1 , 4 , 7 , and 10 ), or was treated with Hpa II (H; lanes 2 , 5 , 8 , and 11 ), which cuts CCGG but not C 5-m CGG, or treated with Msp I (M; lanes 3 , 6 , 9 , and 12 ), which cuts CCGG and C 5-m CGG, before being subjected to PCR amplification using oligonucleotide primers targeting a polymorphic [ATAAA] n repeat sequence near the GSTP1 regulatory region. For primer set B, the amplification of polymorphic GSTP1 promoter sequences after Hpa II digestion, but not after Msp I digestion, indicated the presence of CpG dinucleotide methylation at the Hpa II/ Msp I sites in the DNA analyzed.
    Figure Legend Snippet: Discrimination of DNA hypermethylation at maternal and paternal GSTP1 alleles using a PCR strategy. DNA from matched normal (normal) and neoplastic (tumor) prostate tissues was left untreated (U; lanes 1 , 4 , 7 , and 10 ), or was treated with Hpa II (H; lanes 2 , 5 , 8 , and 11 ), which cuts CCGG but not C 5-m CGG, or treated with Msp I (M; lanes 3 , 6 , 9 , and 12 ), which cuts CCGG and C 5-m CGG, before being subjected to PCR amplification using oligonucleotide primers targeting a polymorphic [ATAAA] n repeat sequence near the GSTP1 regulatory region. For primer set B, the amplification of polymorphic GSTP1 promoter sequences after Hpa II digestion, but not after Msp I digestion, indicated the presence of CpG dinucleotide methylation at the Hpa II/ Msp I sites in the DNA analyzed.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Methylation

    14) Product Images from "Role of phospholipase A2 (PLA2) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis"

    Article Title: Role of phospholipase A2 (PLA2) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis

    Journal: Experimental eye research

    doi: 10.1016/j.exer.2013.05.021

    MIP-133 induces cytosolic phospholipase A 2α (cPLA 2α ) up-regulation in HCORN cells. HCORN cells were treated with MIP-133 at doses 7.5, 15, and 50 μg/ml for 6, 12, and 24 hrs following which cells were processed for total RNA isolation and RT-PCR. The amount of mRNA expression was quantified by densitometry of bands in comparison to the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1A ). HCORN cells were homogenized and then cell lysates were used to measure cPLA 2α enzyme analysis (1B) . The data are mean ± SEM of three independent experiments. Asterisk indicates P values
    Figure Legend Snippet: MIP-133 induces cytosolic phospholipase A 2α (cPLA 2α ) up-regulation in HCORN cells. HCORN cells were treated with MIP-133 at doses 7.5, 15, and 50 μg/ml for 6, 12, and 24 hrs following which cells were processed for total RNA isolation and RT-PCR. The amount of mRNA expression was quantified by densitometry of bands in comparison to the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1A ). HCORN cells were homogenized and then cell lysates were used to measure cPLA 2α enzyme analysis (1B) . The data are mean ± SEM of three independent experiments. Asterisk indicates P values

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

    MIP-133 induces up-regulation of CXCL2 in HCORN cells. HCORN cells were exposed to MIP-133 at indicated doses and times, and were then processed for total RNA isolation and RT-PCR analysis. The amount of mRNA expression was quantified by densitometry of bands in comparison to GAPDH (5A) . Supernatants were collected from harvested cells and then processed for protein estimation by ELISA (5B) . The data is mean ± SEM of three independent experiments. Asterisk indicates P values
    Figure Legend Snippet: MIP-133 induces up-regulation of CXCL2 in HCORN cells. HCORN cells were exposed to MIP-133 at indicated doses and times, and were then processed for total RNA isolation and RT-PCR analysis. The amount of mRNA expression was quantified by densitometry of bands in comparison to GAPDH (5A) . Supernatants were collected from harvested cells and then processed for protein estimation by ELISA (5B) . The data is mean ± SEM of three independent experiments. Asterisk indicates P values

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    15) Product Images from "Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle"

    Article Title: Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.6.3495-3504.2003

    Gene expression from in vivo rAAV vector forms. A circle PCR approach was used to clone the predicted in vivo monomeric circular vector form. PCR primers, overlapping the unique Pst I site at their 3′ ends, were used to amplify rAAV vectors from PS-DNase-treated muscle DNA. The resulting PCR products were cloned into a TA-cloning vector for further analysis. To determine whether the cloned vectors were transcriptionally active, they were excised from the TA vector with Pst I, self-ligated, and transfected into HeLa cells. Cell lysates were analyzed by Western blotting with an anti-ova-horseradish peroxidase antibody. Four independently rescued clones (lanes 1 to 4) that express chicken ovalbumin protein are shown. Lane U, untransfected cell lysate; lane C, lysate from cells transfected with a DNA expression plasmid encoding chicken ovalbumin.
    Figure Legend Snippet: Gene expression from in vivo rAAV vector forms. A circle PCR approach was used to clone the predicted in vivo monomeric circular vector form. PCR primers, overlapping the unique Pst I site at their 3′ ends, were used to amplify rAAV vectors from PS-DNase-treated muscle DNA. The resulting PCR products were cloned into a TA-cloning vector for further analysis. To determine whether the cloned vectors were transcriptionally active, they were excised from the TA vector with Pst I, self-ligated, and transfected into HeLa cells. Cell lysates were analyzed by Western blotting with an anti-ova-horseradish peroxidase antibody. Four independently rescued clones (lanes 1 to 4) that express chicken ovalbumin protein are shown. Lane U, untransfected cell lysate; lane C, lysate from cells transfected with a DNA expression plasmid encoding chicken ovalbumin.

    Techniques Used: Expressing, In Vivo, Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, TA Cloning, Transfection, Western Blot

    16) Product Images from "CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies"

    Article Title: CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies

    Journal: Blood

    doi: 10.1182/blood-2017-01-761320

    Loss of CD7 does not alter phenotype or effector function of CAR T cells. (A) T cells were electroporated with Cas9 complexed with CD7-specific or control (CD19 specific) gRNA and transduced with CD19 CAR. Representative dot plots show expression of CD7 and CD19 CAR in T cells electroporated with Cas9+gRNA 7 days posttransduction. Nontreated activated T cells were used as control. Numbers denote frequency of cells in corresponding quadrants. (B) Frequency of naïve-like cells (naïve; CCR7 + CD45RA − ), central memory (CM; CCR7 + CD45RA − ), effector memory (EM; CCR7 − CD45RA − ), and effector memory RA (EMRA; CCR7 − CD45RA + ) in CD19 CAR T cells assessed by flow cytometry on day 7 posttransduction. (C) Frequency of CD4 + and CD8 + CD19 CAR T cells 7 days posttransduction. (D) CD19 CAR T cells were incubated with CD19 + NALM6 cells, and production of TNFα and IFNγ in CD4 + cells was assessed by intracellular cytokine staining. Dot plots represent cytokine production in CD19 CAR T cells in the presence of NALM6 or in media alone. Summarized data from 3 donors are shown on the right. (E) Control or CD7 KO CD19 CAR T cells or control nontransduced T cells were cocultured with GFP + Raji cells at the effector-to-target ratio 1:1 for 72 hours. Dot plots show representative frequency of gated CAR T cells and GFP + tumor cells at the end of coculture. Total numbers of live tumor cells (F) and CD19 CAR T cells (G) were counted by flow cytometry at 72 hours using counting beads. Lines denote individual donors. Data represent 2 independent experiments with n = 3 donors in each. * P
    Figure Legend Snippet: Loss of CD7 does not alter phenotype or effector function of CAR T cells. (A) T cells were electroporated with Cas9 complexed with CD7-specific or control (CD19 specific) gRNA and transduced with CD19 CAR. Representative dot plots show expression of CD7 and CD19 CAR in T cells electroporated with Cas9+gRNA 7 days posttransduction. Nontreated activated T cells were used as control. Numbers denote frequency of cells in corresponding quadrants. (B) Frequency of naïve-like cells (naïve; CCR7 + CD45RA − ), central memory (CM; CCR7 + CD45RA − ), effector memory (EM; CCR7 − CD45RA − ), and effector memory RA (EMRA; CCR7 − CD45RA + ) in CD19 CAR T cells assessed by flow cytometry on day 7 posttransduction. (C) Frequency of CD4 + and CD8 + CD19 CAR T cells 7 days posttransduction. (D) CD19 CAR T cells were incubated with CD19 + NALM6 cells, and production of TNFα and IFNγ in CD4 + cells was assessed by intracellular cytokine staining. Dot plots represent cytokine production in CD19 CAR T cells in the presence of NALM6 or in media alone. Summarized data from 3 donors are shown on the right. (E) Control or CD7 KO CD19 CAR T cells or control nontransduced T cells were cocultured with GFP + Raji cells at the effector-to-target ratio 1:1 for 72 hours. Dot plots show representative frequency of gated CAR T cells and GFP + tumor cells at the end of coculture. Total numbers of live tumor cells (F) and CD19 CAR T cells (G) were counted by flow cytometry at 72 hours using counting beads. Lines denote individual donors. Data represent 2 independent experiments with n = 3 donors in each. * P

    Techniques Used: Transduction, Expressing, Flow Cytometry, Cytometry, Incubation, Staining

    Disruption of CD7 expression with CRISPR/Cas9 restores expansion of CD7 CAR T cells. (A) Representative histogram showing ablation of CD7 expression in T cells after electroporation with CRISPR/cas9 and CD7-specific gRNAs 3 days after electroporation. Numbers denote frequency of CD7-negative cells. T cells electroporated with Cas9 only were used as a negative control. (B) Downregulation of surface CD7 expression in T cells after electroporation with CRISPR/cas9 and gRNA-85. A CD7-negative cell line Raji was used as a negative control. (C) Schematic outline of the optimized protocol for generating CD7-knockout (CD7 KO ) CD7 CAR T cells. (D) Representative dot plots showing expression of CD7 and CD7 CAR in T cells generated with the optimized protocol. Numbers indicate percentage of cells in each quadrant. (E) Total expansion of CD7 CAR T cells with and without CD7 knockout after 14 days of in vitro culture. (F) Viability of CD7 CAR T cells with and without CD7 gene disruption measured at day 6 after transduction by flow cytometry. Lines denote individual donors. Data represent 3 independent experiments with 3 donors in each. h, hour. * P
    Figure Legend Snippet: Disruption of CD7 expression with CRISPR/Cas9 restores expansion of CD7 CAR T cells. (A) Representative histogram showing ablation of CD7 expression in T cells after electroporation with CRISPR/cas9 and CD7-specific gRNAs 3 days after electroporation. Numbers denote frequency of CD7-negative cells. T cells electroporated with Cas9 only were used as a negative control. (B) Downregulation of surface CD7 expression in T cells after electroporation with CRISPR/cas9 and gRNA-85. A CD7-negative cell line Raji was used as a negative control. (C) Schematic outline of the optimized protocol for generating CD7-knockout (CD7 KO ) CD7 CAR T cells. (D) Representative dot plots showing expression of CD7 and CD7 CAR in T cells generated with the optimized protocol. Numbers indicate percentage of cells in each quadrant. (E) Total expansion of CD7 CAR T cells with and without CD7 knockout after 14 days of in vitro culture. (F) Viability of CD7 CAR T cells with and without CD7 gene disruption measured at day 6 after transduction by flow cytometry. Lines denote individual donors. Data represent 3 independent experiments with 3 donors in each. h, hour. * P

    Techniques Used: Expressing, CRISPR, Electroporation, Negative Control, Knock-Out, Generated, In Vitro, Transduction, Flow Cytometry, Cytometry

    17) Product Images from "HER-2 gene amplification in human breast cancer without concurrent HER-2 over-expression"

    Article Title: HER-2 gene amplification in human breast cancer without concurrent HER-2 over-expression

    Journal: SpringerPlus

    doi: 10.1186/2193-1801-2-386

    GRB7 and HER-2 expression in frozen breast tumor tissues. a GRB7 and HER-2 Western Blot Analysis. Densitometric analysis is presented in Additional file 1 : Table S1. b HER-2 IHC testing of HER-2 FISH positive tumors which do not over-express HER-2 protein by Western blot analysis. Sample 012 is positive control with 3+ HER-2 over-expression; sample 002 is negative control with 0 HER-2 expression. Magnification bars represent 50 micron. c GRB7 and HER2 mRNA expression by multiplex RT-PCR. Top panel: GRB7 and β-actin, Bottom panel: HER-2 and β-actin. Amplified: positive control with HER-2 and GRB 7 over-expression. Non-amplified: negative control - without either HER-2 or GRB7 over-expression. H 2 O: negative control without RT product. X: 4 tumors over-expressing GRB7 but not HER-2. O: 1 tumor over-expressing both GRB7 and HER-2.
    Figure Legend Snippet: GRB7 and HER-2 expression in frozen breast tumor tissues. a GRB7 and HER-2 Western Blot Analysis. Densitometric analysis is presented in Additional file 1 : Table S1. b HER-2 IHC testing of HER-2 FISH positive tumors which do not over-express HER-2 protein by Western blot analysis. Sample 012 is positive control with 3+ HER-2 over-expression; sample 002 is negative control with 0 HER-2 expression. Magnification bars represent 50 micron. c GRB7 and HER2 mRNA expression by multiplex RT-PCR. Top panel: GRB7 and β-actin, Bottom panel: HER-2 and β-actin. Amplified: positive control with HER-2 and GRB 7 over-expression. Non-amplified: negative control - without either HER-2 or GRB7 over-expression. H 2 O: negative control without RT product. X: 4 tumors over-expressing GRB7 but not HER-2. O: 1 tumor over-expressing both GRB7 and HER-2.

    Techniques Used: Expressing, Western Blot, Immunohistochemistry, Fluorescence In Situ Hybridization, Positive Control, Over Expression, Negative Control, Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    18) Product Images from "Genetic and epigenetic alterations of steroidogenic factor-1 in ovarian tumors"

    Article Title: Genetic and epigenetic alterations of steroidogenic factor-1 in ovarian tumors

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2012.1758

    Genotyping reveals somatic NR5A1 gene alterations in ovarian tumors. (A) Genotype distribution by sample. Somatic mutations were identified by genotyping matched ovarian tumors and normal tissue from the same individuals by Taqman SNP genotyping asssays for the indicated SNPs. Only tumor genotypes are shown. (B) Genotyping distribution by SNP. Key: LOH, loss of heterozygosity (e.g. AG > AA); gain, gain of heterozygosity (e.g. GG > AG). Unknown, unclear genotype, for either tumor or normal sample.
    Figure Legend Snippet: Genotyping reveals somatic NR5A1 gene alterations in ovarian tumors. (A) Genotype distribution by sample. Somatic mutations were identified by genotyping matched ovarian tumors and normal tissue from the same individuals by Taqman SNP genotyping asssays for the indicated SNPs. Only tumor genotypes are shown. (B) Genotyping distribution by SNP. Key: LOH, loss of heterozygosity (e.g. AG > AA); gain, gain of heterozygosity (e.g. GG > AG). Unknown, unclear genotype, for either tumor or normal sample.

    Techniques Used:

    19) Product Images from "Suggested Role for G4 DNA in Recombinational Switching at the Antigenic Variation Locus of the Lyme Disease Spirochete"

    Article Title: Suggested Role for G4 DNA in Recombinational Switching at the Antigenic Variation Locus of the Lyme Disease Spirochete

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057792

    Precise excision of the vlsE variable region during PCR amplification. A) Schematic showing the variable, constant and 17 bp direct repeats of the vlsE gene. The location of PCR primers used for amplification are shown by arrows below the constant regions. B) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using three different templates and two different primer sets. The templates used were B. burgdorferi B31 5A4 genomic DNA [29] pMBL20, a plasmid carrying the vlsE gene [51] ; the 776 bp or 935 bp PCR products resulting from PCR amplification. The asterisks indicate smaller discrete bands observed in lanes 1–3 and 4–6. M denotes a 100 bp molecular weight ladder marker. PCR samples were run on a 1.2% agarose gel in 1X TAE buffer at 80 V for 1.2 hours. C) Characterization of precise deletions in vlsE . The first entry on the left side of the panel shows the sequence obtained from direct sequencing of the PCR product obtained with either B248 and B249 or B1701 and 1702. The remainder of the lineup shows sequence generated with 10 randomly selected E. coli transformants. The transformants were generated by cloning the 223 bp truncated PCR product generated in vitro with primers B248 and B249. The fragment was gel-excised and cloned into pJET1.2/blunt vector (Fermentas). The alignment shows that all the sequenced vlsE inserts had a precise excision of the 570 bp variable region. D) Schematic showing the precise excision of the vlsE variable region that occurred during PCR amplification.
    Figure Legend Snippet: Precise excision of the vlsE variable region during PCR amplification. A) Schematic showing the variable, constant and 17 bp direct repeats of the vlsE gene. The location of PCR primers used for amplification are shown by arrows below the constant regions. B) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using three different templates and two different primer sets. The templates used were B. burgdorferi B31 5A4 genomic DNA [29] pMBL20, a plasmid carrying the vlsE gene [51] ; the 776 bp or 935 bp PCR products resulting from PCR amplification. The asterisks indicate smaller discrete bands observed in lanes 1–3 and 4–6. M denotes a 100 bp molecular weight ladder marker. PCR samples were run on a 1.2% agarose gel in 1X TAE buffer at 80 V for 1.2 hours. C) Characterization of precise deletions in vlsE . The first entry on the left side of the panel shows the sequence obtained from direct sequencing of the PCR product obtained with either B248 and B249 or B1701 and 1702. The remainder of the lineup shows sequence generated with 10 randomly selected E. coli transformants. The transformants were generated by cloning the 223 bp truncated PCR product generated in vitro with primers B248 and B249. The fragment was gel-excised and cloned into pJET1.2/blunt vector (Fermentas). The alignment shows that all the sequenced vlsE inserts had a precise excision of the 570 bp variable region. D) Schematic showing the precise excision of the vlsE variable region that occurred during PCR amplification.

    Techniques Used: Polymerase Chain Reaction, Amplification, Staining, Agarose Gel Electrophoresis, Plasmid Preparation, Molecular Weight, Marker, Sequencing, Generated, Clone Assay, In Vitro

    20) Product Images from "Role of the DSC1 Channel in Regulating Neuronal Excitability in Drosophila melanogaster: Extending Nervous System Stability under Stress"

    Article Title: Role of the DSC1 Channel in Regulating Neuronal Excitability in Drosophila melanogaster: Extending Nervous System Stability under Stress

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003327

    Method for targeted gene knockout and confirmation by PCR and Southern blot analysis. ( A ) Schematic presentation of the donor construct, homologous recombination and replacement of the endogenous DSC1 sequence with the donor sequences carrying stop codons ( * ). A 6.6-kb DSC1 genomic DNA region was amplified in two 3.3-kb fragments and a stop codon was introduced into the middle of each fragment (i.e., ST1 in the upstream fragment and ST2 in the downstream fragment). The upstream and downstream fragments were then cloned into the pW25 vector (shown at the top in A ). The donor construct was transformed into w 1118 flies to generate donor construct lines. The donor construct lines were crossed with another transgenic line that contains heat-inducible 70FLP recombinase and 70I-Sce I endonuclease genes to induce DSC1 -targeted homologous recombination. ( B ) Confirmation of DSC1 knockout by genomic PCR. Amplification of a 3.6 kb DSC1 genomic fragment using the primer pairs a/c from DSC1 knockout flies, but not from donor flies. ( C ) Southern blot analysis. Genomic DNA from WT (lane 2), a donor construct line (lane 3), and two independent homozygous DSC1 knockout lines (lanes 4 and 5) was digested with the restriction enzyme KpnI and hybridized with two genomic DNA probes made from the two 3.3-kb DSC1 fragments (in A ). As expected, the wild-type 6.9-kb band was converted into two bands of 3.3 kb and 8.1 kb in the DSC1 knockout lines due to homologous recombination.
    Figure Legend Snippet: Method for targeted gene knockout and confirmation by PCR and Southern blot analysis. ( A ) Schematic presentation of the donor construct, homologous recombination and replacement of the endogenous DSC1 sequence with the donor sequences carrying stop codons ( * ). A 6.6-kb DSC1 genomic DNA region was amplified in two 3.3-kb fragments and a stop codon was introduced into the middle of each fragment (i.e., ST1 in the upstream fragment and ST2 in the downstream fragment). The upstream and downstream fragments were then cloned into the pW25 vector (shown at the top in A ). The donor construct was transformed into w 1118 flies to generate donor construct lines. The donor construct lines were crossed with another transgenic line that contains heat-inducible 70FLP recombinase and 70I-Sce I endonuclease genes to induce DSC1 -targeted homologous recombination. ( B ) Confirmation of DSC1 knockout by genomic PCR. Amplification of a 3.6 kb DSC1 genomic fragment using the primer pairs a/c from DSC1 knockout flies, but not from donor flies. ( C ) Southern blot analysis. Genomic DNA from WT (lane 2), a donor construct line (lane 3), and two independent homozygous DSC1 knockout lines (lanes 4 and 5) was digested with the restriction enzyme KpnI and hybridized with two genomic DNA probes made from the two 3.3-kb DSC1 fragments (in A ). As expected, the wild-type 6.9-kb band was converted into two bands of 3.3 kb and 8.1 kb in the DSC1 knockout lines due to homologous recombination.

    Techniques Used: Gene Knockout, Polymerase Chain Reaction, Southern Blot, Construct, Homologous Recombination, Sequencing, Amplification, Clone Assay, Plasmid Preparation, Transformation Assay, Transgenic Assay, Knock-Out

    21) Product Images from "Aberrant Gene Expression and Sexually Incompatible Genomic Imprinting in Oocytes Derived from XY Mouse Embryonic Stem Cells In Vitro"

    Article Title: Aberrant Gene Expression and Sexually Incompatible Genomic Imprinting in Oocytes Derived from XY Mouse Embryonic Stem Cells In Vitro

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058555

    Expression of genes associated with sex determination in ESC-derived FLSs. (A) Semi-quantitative RT-PCR analysis was performed to examine the expression of male GC marker genes ( Nanos2 , Miwi , Dnmt3L ) and sex determination factors of male ( Sry , Sox9 , Dmrt1 ) and female ( Foxl2 ) gonads. Only representative images from three escalated cycles are presented. Total RNA was prepared from RW-4 ESCs, clone-G ESCs, clone-G ESCs-derived FLSs (day 3), adult mouse ovary and testis. β-Actin was analyzed as an internal control and water was used as a negative control. (B) Gene expression in clone-G ESC-derived FLSs (day 3) was compared to those in embryonic (E19.0) and neonatal (P5) ovaries and in neonatal testis by semi-quantitative RT-PCR analysis as shown in (A).
    Figure Legend Snippet: Expression of genes associated with sex determination in ESC-derived FLSs. (A) Semi-quantitative RT-PCR analysis was performed to examine the expression of male GC marker genes ( Nanos2 , Miwi , Dnmt3L ) and sex determination factors of male ( Sry , Sox9 , Dmrt1 ) and female ( Foxl2 ) gonads. Only representative images from three escalated cycles are presented. Total RNA was prepared from RW-4 ESCs, clone-G ESCs, clone-G ESCs-derived FLSs (day 3), adult mouse ovary and testis. β-Actin was analyzed as an internal control and water was used as a negative control. (B) Gene expression in clone-G ESC-derived FLSs (day 3) was compared to those in embryonic (E19.0) and neonatal (P5) ovaries and in neonatal testis by semi-quantitative RT-PCR analysis as shown in (A).

    Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR, Marker, Negative Control

    Gene expression profiles of ESC-derived FLSs. (A) The expression of GCs and folliculogenesis marker genes was examined by semi-quantitative RT-PCR analysis. Only representative images from three escalated cycles are presented. Total RNA was prepared from RW-4 ESCs, clone-G ESCs, clone-G ESC-derived FLSs (day 3), adult mouse ovary and testis. β-Actin was analyzed as an internal control and water was used as a negative control. (B) Gene expression in clone-G ESC-derived FLSs (day 3) was compared to those in embryonic (E19.0) and neonatal (P5) ovaries and in neonatal testis by semi-quantitative RT-PCR analysis as shown in (A).
    Figure Legend Snippet: Gene expression profiles of ESC-derived FLSs. (A) The expression of GCs and folliculogenesis marker genes was examined by semi-quantitative RT-PCR analysis. Only representative images from three escalated cycles are presented. Total RNA was prepared from RW-4 ESCs, clone-G ESCs, clone-G ESC-derived FLSs (day 3), adult mouse ovary and testis. β-Actin was analyzed as an internal control and water was used as a negative control. (B) Gene expression in clone-G ESC-derived FLSs (day 3) was compared to those in embryonic (E19.0) and neonatal (P5) ovaries and in neonatal testis by semi-quantitative RT-PCR analysis as shown in (A).

    Techniques Used: Expressing, Derivative Assay, Marker, Quantitative RT-PCR, Negative Control

    22) Product Images from "Abnormal T regulatory cells (Tregs: FOXP3+, CTLA-4+), myeloid-derived suppressor cells (MDSCs: monocytic, granulocytic) and polarised T helper cell profiles (Th1, Th2, Th17) in women with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC) and surgery: failure of abolition of abnormal treg profile with treatment and correlation of treg levels with pathological response to NAC"

    Article Title: Abnormal T regulatory cells (Tregs: FOXP3+, CTLA-4+), myeloid-derived suppressor cells (MDSCs: monocytic, granulocytic) and polarised T helper cell profiles (Th1, Th2, Th17) in women with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC) and surgery: failure of abolition of abnormal treg profile with treatment and correlation of treg levels with pathological response to NAC

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-11-16

    Gene expression in LLABCs and HFDs , (A) FOXP3, (B) CTLA-4: Mean FOXP3 and CTLA-4 gene expression over β actin in the circulation in women with LLABCs (n = 16); baseline (T1), following eight cycles of NAC (T6) and post-surgery (T7). Patients allocated into groups according to histological responses elicited in the breast cancers after NAC: Group I-cPR; Group II-VGR; Group III-partial response; IV-poor or no response. Statistical analysis: (A) FOXP3 (Group I II): T1 v HFD (p = 0.006); T1 v T6 (NS); T1 v T7 (NS); T6 v HFD (NS); T7 v HFD (p = 0.002). (A) FOXP3 (Group III V): T1 v HFD (p = 0.050); T1 v T6 (NS); T1 v T7 (NS); T6 v HFD (NS); T7 v HFD (p = 0.001). (B) CTLA-4 (Group I II): T1 v HFD (p = 0.011); T1vT6 (NS); T1vT7 (NS); T6 v HFD (NS); T7 v HFD (p = 0.002). (B) CTLA-4 (Group III IV): T1 v HFD (p = 0.038); T1 v T6 (NS); T1 v T7 (NS); T6 v HFD (NS); T7 v HFD (p = 0.041). Group I II (T1) v III IV (T1) (NS). Clear columns are good pathological responders whilst shaded columns are poor pathological responders to NAC.
    Figure Legend Snippet: Gene expression in LLABCs and HFDs , (A) FOXP3, (B) CTLA-4: Mean FOXP3 and CTLA-4 gene expression over β actin in the circulation in women with LLABCs (n = 16); baseline (T1), following eight cycles of NAC (T6) and post-surgery (T7). Patients allocated into groups according to histological responses elicited in the breast cancers after NAC: Group I-cPR; Group II-VGR; Group III-partial response; IV-poor or no response. Statistical analysis: (A) FOXP3 (Group I II): T1 v HFD (p = 0.006); T1 v T6 (NS); T1 v T7 (NS); T6 v HFD (NS); T7 v HFD (p = 0.002). (A) FOXP3 (Group III V): T1 v HFD (p = 0.050); T1 v T6 (NS); T1 v T7 (NS); T6 v HFD (NS); T7 v HFD (p = 0.001). (B) CTLA-4 (Group I II): T1 v HFD (p = 0.011); T1vT6 (NS); T1vT7 (NS); T6 v HFD (NS); T7 v HFD (p = 0.002). (B) CTLA-4 (Group III IV): T1 v HFD (p = 0.038); T1 v T6 (NS); T1 v T7 (NS); T6 v HFD (NS); T7 v HFD (p = 0.041). Group I II (T1) v III IV (T1) (NS). Clear columns are good pathological responders whilst shaded columns are poor pathological responders to NAC.

    Techniques Used: Expressing

    (A) Correlation between % of regulatory FOXP3 + (CD4 + CD25 + CD127 - FOX P3 + ) T cells before and after NAC and subsequent histological response in the breast following NAC (n = 16), graded as complete (cPR, 5), very good (VG, 4), partial (3), poor and no pathological response (2 + 1) in the breast following NAC. Values shown are means ± standard deviations. Statistical analysis: Spearman’s correlation coefficient Rho = −0.620, p = 0.04 (2-tailed) (pre-chemotherapy) and Rho = −0.799, p = 0.05 (2-tailed) (post-chemotherapy) (B) Correlation between % of regulatory CTLA-4 + (CD4 + CD25 + CD152 + ) T cells before and after NAC and subsequent histological response in the breast following NAC (n = 16), graded as complete (cPR, 5), very good (VG, 4), partial (3), poor and no pathological response (2 + 1) in the breast following NAC. Values shown are means ± standard deviations. Statistical analysis: Spearman’s correlation coefficient Rho = 0.260, p = 0.78 (NS) (pre-chemotherapy) and Rho = −0.794, p = 0.01 (2-tailed) (post-chemotherapy).
    Figure Legend Snippet: (A) Correlation between % of regulatory FOXP3 + (CD4 + CD25 + CD127 - FOX P3 + ) T cells before and after NAC and subsequent histological response in the breast following NAC (n = 16), graded as complete (cPR, 5), very good (VG, 4), partial (3), poor and no pathological response (2 + 1) in the breast following NAC. Values shown are means ± standard deviations. Statistical analysis: Spearman’s correlation coefficient Rho = −0.620, p = 0.04 (2-tailed) (pre-chemotherapy) and Rho = −0.799, p = 0.05 (2-tailed) (post-chemotherapy) (B) Correlation between % of regulatory CTLA-4 + (CD4 + CD25 + CD152 + ) T cells before and after NAC and subsequent histological response in the breast following NAC (n = 16), graded as complete (cPR, 5), very good (VG, 4), partial (3), poor and no pathological response (2 + 1) in the breast following NAC. Values shown are means ± standard deviations. Statistical analysis: Spearman’s correlation coefficient Rho = 0.260, p = 0.78 (NS) (pre-chemotherapy) and Rho = −0.794, p = 0.01 (2-tailed) (post-chemotherapy).

    Techniques Used:

    23) Product Images from "Estradiol Represses the GD3 Synthase Gene ST8SIA1 Expression in Human Breast Cancer Cells by Preventing NF?B Binding to ST8SIA1 Promoter"

    Article Title: Estradiol Represses the GD3 Synthase Gene ST8SIA1 Expression in Human Breast Cancer Cells by Preventing NF?B Binding to ST8SIA1 Promoter

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062559

    Identification of human GD3S transcripts in breast cancer tumors and Hs578T cells. (A) Schematic representation of the 5′-RACE strategy. First strand cDNA synthesis was performed with a ST8SIA1 specific primer. cDNA was dC-tailed at 3′end and amplified by PCR using Anchor Primer and GSP1. Nested PCR was performed using AUAP and GSP2 primers. (B) Schematic representation of the main 5′-ends of GD3S transcripts expressed in Hs578T cells and breast cancer tumor samples (#137, #142 and #148). The size of intronic sequences between E2 and the different first exon are shown. Position of PCR primers used for specific amplification of T1 transcript is indicated by black arrows. (C) qPCR analysis of T1 transcript (grey) and total ST8SIA1 (black) expression, related to HPRT , in 20 ER-negative IDC samples and 2 breast cancer cell lines (Hs578T and MCF-7).
    Figure Legend Snippet: Identification of human GD3S transcripts in breast cancer tumors and Hs578T cells. (A) Schematic representation of the 5′-RACE strategy. First strand cDNA synthesis was performed with a ST8SIA1 specific primer. cDNA was dC-tailed at 3′end and amplified by PCR using Anchor Primer and GSP1. Nested PCR was performed using AUAP and GSP2 primers. (B) Schematic representation of the main 5′-ends of GD3S transcripts expressed in Hs578T cells and breast cancer tumor samples (#137, #142 and #148). The size of intronic sequences between E2 and the different first exon are shown. Position of PCR primers used for specific amplification of T1 transcript is indicated by black arrows. (C) qPCR analysis of T1 transcript (grey) and total ST8SIA1 (black) expression, related to HPRT , in 20 ER-negative IDC samples and 2 breast cancer cell lines (Hs578T and MCF-7).

    Techniques Used: Amplification, Polymerase Chain Reaction, Nested PCR, Real-time Polymerase Chain Reaction, Expressing

    24) Product Images from "An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants"

    Article Title: An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-5-30

    Schematic illustration of mutant fragment generation by overlap extension PCR . During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW- attB 1 and GW- attB 2, resulting in a recombination-proficient mutant PCR fragment.
    Figure Legend Snippet: Schematic illustration of mutant fragment generation by overlap extension PCR . During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW- attB 1 and GW- attB 2, resulting in a recombination-proficient mutant PCR fragment.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Purification, In Vitro

    Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome . The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221- Gene ::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW- Gene ::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB -mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.
    Figure Legend Snippet: Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome . The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221- Gene ::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW- Gene ::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB -mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.

    Techniques Used: Clone Assay, Plasmid Preparation, Mutagenesis, Generated, Polymerase Chain Reaction, Homologous Recombination, Marker

    25) Product Images from "Digoxin Downregulates NDRG1 and VEGF through the Inhibition of HIF-1? under Hypoxic Conditions in Human Lung Adenocarcinoma A549 Cells"

    Article Title: Digoxin Downregulates NDRG1 and VEGF through the Inhibition of HIF-1? under Hypoxic Conditions in Human Lung Adenocarcinoma A549 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms14047273

    Digoxin suppresses mRNA and protein expressions of HIF-1α and reduces the HIF-1/DNA complex in A549 cells under hypoxic conditions. ( a ) Under normoxic or hypoxic conditions, mRNA and protein levels of HIF-1α in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were determined. Quantitative data were presented as mean ± SD ( n = 4). * p
    Figure Legend Snippet: Digoxin suppresses mRNA and protein expressions of HIF-1α and reduces the HIF-1/DNA complex in A549 cells under hypoxic conditions. ( a ) Under normoxic or hypoxic conditions, mRNA and protein levels of HIF-1α in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were determined. Quantitative data were presented as mean ± SD ( n = 4). * p

    Techniques Used:

    Digoxin inhibits mRNA and protein expression of NDRG1 in A549 cells under hypoxic conditions. Under normoxic or hypoxic conditions, mRNA and protein levels of NDRG1 in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were measured. Quantitative data were presented as mean ± SD ( n = 4). * p
    Figure Legend Snippet: Digoxin inhibits mRNA and protein expression of NDRG1 in A549 cells under hypoxic conditions. Under normoxic or hypoxic conditions, mRNA and protein levels of NDRG1 in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were measured. Quantitative data were presented as mean ± SD ( n = 4). * p

    Techniques Used: Expressing

    Digoxin inhibits the viability of A549 cells under hypoxic conditions. A549 cell viability was measured by MTT assay and analyzed at different time points (0, 24, 48 and 72 h) with or without various concentrations of digoxin (0.01, 0.1, and 1 μM). Quantitative data are presented as mean ± SD ( n = 4). * p
    Figure Legend Snippet: Digoxin inhibits the viability of A549 cells under hypoxic conditions. A549 cell viability was measured by MTT assay and analyzed at different time points (0, 24, 48 and 72 h) with or without various concentrations of digoxin (0.01, 0.1, and 1 μM). Quantitative data are presented as mean ± SD ( n = 4). * p

    Techniques Used: MTT Assay

    Digoxin attenuates mRNA and protein expression of VEGF in A549 cells under hypoxic conditions. Under normoxic or hypoxic conditions, mRNA and protein levels of VEGF in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were analyzed. Quantitative data were presented as mean ± SD ( n = 4). * p
    Figure Legend Snippet: Digoxin attenuates mRNA and protein expression of VEGF in A549 cells under hypoxic conditions. Under normoxic or hypoxic conditions, mRNA and protein levels of VEGF in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were analyzed. Quantitative data were presented as mean ± SD ( n = 4). * p

    Techniques Used: Expressing

    26) Product Images from "Genomic analysis of heat-shock factor targets in Drosophila"

    Article Title: Genomic analysis of heat-shock factor targets in Drosophila

    Journal: Genome Biology

    doi: 10.1186/gb-2005-6-7-r63

    PCR validation of selected positives from the cDNA arrays. Agarose gels showing the products generated by specific PCRs for each of the indicated genes using preimmune purified (-) or anti-Hsf purified (+) chromatin as an input.
    Figure Legend Snippet: PCR validation of selected positives from the cDNA arrays. Agarose gels showing the products generated by specific PCRs for each of the indicated genes using preimmune purified (-) or anti-Hsf purified (+) chromatin as an input.

    Techniques Used: Polymerase Chain Reaction, Generated, Purification

    Distribution of fragment enrichment with anti-Hsf immunopurified chromatin on the genomic tiling array. The y -axis plots the asinh transformation (approximately equivalent to the log 2 scale) of the ratio of anti-Hsf versus preimmune sera. The x -axis represents each of the 3,444 PCR products, the Adh region, Hsp gene and segmentation gene ( Seg ) sequences are indicated below the x -axis. Strong enrichment of fragments from the Hsp genes is indicated by their high ratio. The signals from l(2)35Bg and PRL-1 in the Adh region are indicated.
    Figure Legend Snippet: Distribution of fragment enrichment with anti-Hsf immunopurified chromatin on the genomic tiling array. The y -axis plots the asinh transformation (approximately equivalent to the log 2 scale) of the ratio of anti-Hsf versus preimmune sera. The x -axis represents each of the 3,444 PCR products, the Adh region, Hsp gene and segmentation gene ( Seg ) sequences are indicated below the x -axis. Strong enrichment of fragments from the Hsp genes is indicated by their high ratio. The signals from l(2)35Bg and PRL-1 in the Adh region are indicated.

    Techniques Used: Transformation Assay, Polymerase Chain Reaction

    27) Product Images from "Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clusters within proteobacteria"

    Article Title: Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clusters within proteobacteria

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-13-79

    Characterization of the czr and ncz promoter regions. ( A ) Beta-galactosidase activity assay of transcription fusions of P czr and P ncz to the lacZ reporter gene. Cells were grown in PYE medium and samples were taken at midlog phase and stationary phase (24 h) for assaying β-galactosidase as described [ 38 ]. The background activity for plasmid alone is around 200 Miller Units. Asterisks indicate results significantly different between the two growth phases within each promoter fusion (p ≤ 0.05). ( B ) Determination of co-transcription of CCNA02805 and CCNA_02806 by amplification with primers RND3 and RND4. Lane 1, PCR amplification using cDNA previously synthesized with Reverse Transcriptase from total RNA from the NA1000 strain; lane 2, PCR amplification from total NA1000 genomic DNA (positive control); lane 3, PCR amplification from total RNA from the NA1000 strain (negative control). The 0.43 kb fragment corresponding to the amplified products is indicated.
    Figure Legend Snippet: Characterization of the czr and ncz promoter regions. ( A ) Beta-galactosidase activity assay of transcription fusions of P czr and P ncz to the lacZ reporter gene. Cells were grown in PYE medium and samples were taken at midlog phase and stationary phase (24 h) for assaying β-galactosidase as described [ 38 ]. The background activity for plasmid alone is around 200 Miller Units. Asterisks indicate results significantly different between the two growth phases within each promoter fusion (p ≤ 0.05). ( B ) Determination of co-transcription of CCNA02805 and CCNA_02806 by amplification with primers RND3 and RND4. Lane 1, PCR amplification using cDNA previously synthesized with Reverse Transcriptase from total RNA from the NA1000 strain; lane 2, PCR amplification from total NA1000 genomic DNA (positive control); lane 3, PCR amplification from total RNA from the NA1000 strain (negative control). The 0.43 kb fragment corresponding to the amplified products is indicated.

    Techniques Used: Activity Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Synthesized, Positive Control, Negative Control

    28) Product Images from "ARID1A Alterations Are Associated with FGFR3-Wild Type, Poor-Prognosis, Urothelial Bladder Tumors"

    Article Title: ARID1A Alterations Are Associated with FGFR3-Wild Type, Poor-Prognosis, Urothelial Bladder Tumors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062483

    ARID1A mutations and expression in UBC. Panel A . A G > C transversion identified through Solexa resequencing, confirmed by Sanger sequencing of independent PCR products, leading to a predicted Q2210H substitution in VMCUB-3 cells. Panel B . Western blotting analysis in a panel of UBC cell lines identifies a subset with undetectable expression, including VMCUB-3. mRNA expression was analyzed by RT-qPCR; results are shown as values normalized with respect to the housekeeping gene HPRT . Panel C . A C > T mutation in codon 403, leading to a premature stop codon, was identified in a primary T1G3 tumor. The mutation was absent from matched normal leukocyte DNA. Lack of protein expression in the corresponding tumor tissue was confirmed using immunohistochemistry. The red arrowhead points to a tumor cell lacking ARID1A staining, whereas the black arrowhead indicates a positive stromal cell. For comparison, a TaG1 tumor with wild type ARID1A sequence is shown.
    Figure Legend Snippet: ARID1A mutations and expression in UBC. Panel A . A G > C transversion identified through Solexa resequencing, confirmed by Sanger sequencing of independent PCR products, leading to a predicted Q2210H substitution in VMCUB-3 cells. Panel B . Western blotting analysis in a panel of UBC cell lines identifies a subset with undetectable expression, including VMCUB-3. mRNA expression was analyzed by RT-qPCR; results are shown as values normalized with respect to the housekeeping gene HPRT . Panel C . A C > T mutation in codon 403, leading to a premature stop codon, was identified in a primary T1G3 tumor. The mutation was absent from matched normal leukocyte DNA. Lack of protein expression in the corresponding tumor tissue was confirmed using immunohistochemistry. The red arrowhead points to a tumor cell lacking ARID1A staining, whereas the black arrowhead indicates a positive stromal cell. For comparison, a TaG1 tumor with wild type ARID1A sequence is shown.

    Techniques Used: Expressing, Sequencing, Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Mutagenesis, Immunohistochemistry, Staining

    29) Product Images from "Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale"

    Article Title: Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035231

    Discriminating VEGF isoform-specific RT-PCR highlights the importance of primer design in influencing detection of putative VEGFxxxb products. RT-PCR was performed using an exon 3 forward primer together with different VEGFxxxb isoform specific reverse primers designed to detect VEGF188b and/or VEGF164b or VEGF120b. A , RT-PCR using a reverse primer spanning 13 bases across the exon8b/exon7 junction (164b/188b-2) amplified a putative VEGF164b (311 bp) product in wt, VEGF164 VEGF120-expressing tumours and a putative VEGF188b product (383 bp) in VEGF188-expressing tumours. B , More discriminating RT-PCR using a 164b/188b-3 reverse primer spanning only 4 bases across the exon8b/7 splice-junction failed to reveal VEGFxxxb PCR products. The ability to detect VEGFxxx products in reactions using exon8 and 3′UTR-C reverse primers highlights efficient PCR conditions. C , No products corresponding to VEGFxxxb isoforms were generated by RT-PCR using cDNA from mouse fibrosarcoma cells and a reverse primer spanning 5 bases across the exon8b/7 junction (164b/188b-1); VEGF188b (1209 bp) and VEGF164b (1137 bp) products were amplified from pPNT VEGF 164 (V 164 ) pPNT VEGF 188 (V 188 ) ‘knock-in’ plasmid vector cDNA templates [23] . D E , Putative VEGF188b and/or VEGF164b products were detected in RT-PCR reactions using reverse primers spanning 13 bases across the exon8b/7 junction (164b/188b-2 164b/188b-4) in fibrosarcoma cells and pPNT VEGF 164 pPNT VEGF 188 plasmids. F G , Products corresponding to VEGF120b (179 bp) species were readily detected in all fibrosarcoma tumour and cell extracts with a VEGF120-specific reverse primer spanning 16 bases across the exon8b/5 junction (120b-2), whilst no VEGF120b products were generated when the reverse primer spanned only 5 bases across the 8b/5 junction (120b-1).
    Figure Legend Snippet: Discriminating VEGF isoform-specific RT-PCR highlights the importance of primer design in influencing detection of putative VEGFxxxb products. RT-PCR was performed using an exon 3 forward primer together with different VEGFxxxb isoform specific reverse primers designed to detect VEGF188b and/or VEGF164b or VEGF120b. A , RT-PCR using a reverse primer spanning 13 bases across the exon8b/exon7 junction (164b/188b-2) amplified a putative VEGF164b (311 bp) product in wt, VEGF164 VEGF120-expressing tumours and a putative VEGF188b product (383 bp) in VEGF188-expressing tumours. B , More discriminating RT-PCR using a 164b/188b-3 reverse primer spanning only 4 bases across the exon8b/7 splice-junction failed to reveal VEGFxxxb PCR products. The ability to detect VEGFxxx products in reactions using exon8 and 3′UTR-C reverse primers highlights efficient PCR conditions. C , No products corresponding to VEGFxxxb isoforms were generated by RT-PCR using cDNA from mouse fibrosarcoma cells and a reverse primer spanning 5 bases across the exon8b/7 junction (164b/188b-1); VEGF188b (1209 bp) and VEGF164b (1137 bp) products were amplified from pPNT VEGF 164 (V 164 ) pPNT VEGF 188 (V 188 ) ‘knock-in’ plasmid vector cDNA templates [23] . D E , Putative VEGF188b and/or VEGF164b products were detected in RT-PCR reactions using reverse primers spanning 13 bases across the exon8b/7 junction (164b/188b-2 164b/188b-4) in fibrosarcoma cells and pPNT VEGF 164 pPNT VEGF 188 plasmids. F G , Products corresponding to VEGF120b (179 bp) species were readily detected in all fibrosarcoma tumour and cell extracts with a VEGF120-specific reverse primer spanning 16 bases across the exon8b/5 junction (120b-2), whilst no VEGF120b products were generated when the reverse primer spanned only 5 bases across the 8b/5 junction (120b-1).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Polymerase Chain Reaction, Generated, Knock-In, Plasmid Preparation

    A general RT-PCR approach using primers to exon 3 or 4 and 3'UTR failed to detect VEGFxxxb isoforms in mouse and human cDNA extracts respectively. A , PCR products corresponding to VEGF188 (474 bp), VEGF164 (402 bp), VEGF164/120 heteroduplex (arrowed) and VEGF120 (270 bp) are evident in the panel of mouse cDNAs amplified using the 3′UTR reverse primer and a forward primer to exon 3. B C , PCR products corresponding to VEGF189 (371 bp), VEGF165 (299 bp) and VEGF121 (167 bp) are evident in the commercial human tissue cDNAs amplified using the 3′UTR reverse primer (DB 3'UTR) and a forward primer to exon 4 (DB exon4). D , Amplification of the heteroduplex species (arrowed) a lso occurred when VEGF164 and VEGF120 tumour cDNAs were pooled and analysed by RT-PCR using exon3/3'UTR primers (lanes labelled 120+164). E , The same heteroduplex species was generated when cDNAs from VEGF164 and VEGF188 expressing fibrosarcoma cells were pooled and amplified as above (lanes labelled 120+164). M corresponds to a 100 bp ladder, whilst -tem represents a control PCR reaction in which water was used instead of cDNA template. The Figure contains data we published previously [25] .
    Figure Legend Snippet: A general RT-PCR approach using primers to exon 3 or 4 and 3'UTR failed to detect VEGFxxxb isoforms in mouse and human cDNA extracts respectively. A , PCR products corresponding to VEGF188 (474 bp), VEGF164 (402 bp), VEGF164/120 heteroduplex (arrowed) and VEGF120 (270 bp) are evident in the panel of mouse cDNAs amplified using the 3′UTR reverse primer and a forward primer to exon 3. B C , PCR products corresponding to VEGF189 (371 bp), VEGF165 (299 bp) and VEGF121 (167 bp) are evident in the commercial human tissue cDNAs amplified using the 3′UTR reverse primer (DB 3'UTR) and a forward primer to exon 4 (DB exon4). D , Amplification of the heteroduplex species (arrowed) a lso occurred when VEGF164 and VEGF120 tumour cDNAs were pooled and analysed by RT-PCR using exon3/3'UTR primers (lanes labelled 120+164). E , The same heteroduplex species was generated when cDNAs from VEGF164 and VEGF188 expressing fibrosarcoma cells were pooled and amplified as above (lanes labelled 120+164). M corresponds to a 100 bp ladder, whilst -tem represents a control PCR reaction in which water was used instead of cDNA template. The Figure contains data we published previously [25] .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Generated, Expressing, Transmission Electron Microscopy

    C-terminal exon sequences of murine and human VEGF-A genes. Top two panels show the predicted splicing reactions for murine VEGF188b, 164b and 120b (sequence in bold and arrowed), highlighting the exon 7/exon 8b and exon 5/exon 8b splice junction sequences. These splicing reactions would generate putative VEGFxxxb protein isoforms with a PLTGKTD C-terminus. The reverse PCR primers designed for this study are indicated below the exon sequences. The lower panel shows the corresponding sequence of the human VEGF-A gene C-terminus highlighting the exon 7/exon 8b splice junction that generates VEGFxxxb isoforms with a SLTRKD C-terminus. The reverse PCR primers used in the amplification reactions are indicated below the exon sequences and DB 165b/188b-1 and DB 3'UTR are as previously published [26] . The bases highlighted in bold exhibit mismatches from the published human VEGF-A sequence.
    Figure Legend Snippet: C-terminal exon sequences of murine and human VEGF-A genes. Top two panels show the predicted splicing reactions for murine VEGF188b, 164b and 120b (sequence in bold and arrowed), highlighting the exon 7/exon 8b and exon 5/exon 8b splice junction sequences. These splicing reactions would generate putative VEGFxxxb protein isoforms with a PLTGKTD C-terminus. The reverse PCR primers designed for this study are indicated below the exon sequences. The lower panel shows the corresponding sequence of the human VEGF-A gene C-terminus highlighting the exon 7/exon 8b splice junction that generates VEGFxxxb isoforms with a SLTRKD C-terminus. The reverse PCR primers used in the amplification reactions are indicated below the exon sequences and DB 165b/188b-1 and DB 3'UTR are as previously published [26] . The bases highlighted in bold exhibit mismatches from the published human VEGF-A sequence.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

    Effect of IGF-1 and TGFβ-1 on expression of VEGF isoforms in mouse podocytes. Cell lysates were prepared from untreated control (C) podocyte cells or cells treated with either 100 nM IGF-1 (IGF) or 1 nM TGFβ-1 (TGF) for 12 hours in serum free media. Results from three independent experiments are shown. RT-PCR using general primers designed to simultaneously amplify both VEGFxxx and VEGFxxxb isoforms (exon7a/3'UTR) revealed only VEGFxxx (194 bp). Qualitative assessment of the PCR products suggests that treatment with either growth factor increased VEGFxxx expression. The same RT-PCR strategy using three different extracts (1, 2, 3) from HEK 293 cells similarly revealed only VEGFxxx (194 bp). (-tem) corresponds to reactions containing water instead of cDNA. (-RT) corresponds to reactions containing water instead of reverse transcriptase.
    Figure Legend Snippet: Effect of IGF-1 and TGFβ-1 on expression of VEGF isoforms in mouse podocytes. Cell lysates were prepared from untreated control (C) podocyte cells or cells treated with either 100 nM IGF-1 (IGF) or 1 nM TGFβ-1 (TGF) for 12 hours in serum free media. Results from three independent experiments are shown. RT-PCR using general primers designed to simultaneously amplify both VEGFxxx and VEGFxxxb isoforms (exon7a/3'UTR) revealed only VEGFxxx (194 bp). Qualitative assessment of the PCR products suggests that treatment with either growth factor increased VEGFxxx expression. The same RT-PCR strategy using three different extracts (1, 2, 3) from HEK 293 cells similarly revealed only VEGFxxx (194 bp). (-tem) corresponds to reactions containing water instead of cDNA. (-RT) corresponds to reactions containing water instead of reverse transcriptase.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Transmission Electron Microscopy

    A general RT-PCR approach with primers to exon 7a and 3'UTR failed to detect VEGFxxxb isoforms in human and mouse cDNA extracts. RT-PCR reactions were performed using forward/reverse primers designed to amplify both VEGFxxx and VEGFxxxb isoforms from cDNAs extracted from mouse fibrosarcoma cell lines tumours and normal mouse tissues (heart, lung, liver and kidney) as well as commercially sourced human tissue cDNAs. A , A single product corresponding to VEGF164/188 (194 bp) is evident in the panel of mouse cDNAs using forward and reverse primers designed to exon 7 and the 3'UTR (exon7/3′UTR) with no evidence of VEGF164b/188b (128 bp) products. B C , A single product corresponding to VEGF165/189 (194 bp) is evident in human brain, bladder and kidney (AMS Biotechnology Ltd), and prostate and kidney (Primer Design Ltd) using forward and reverse primers designed to exon 7 (DB exon7a) and the 3'UTR (DB 3'UTR) respectively. No products corresponding to VEGF165b/189b (128 bp) are evident in any of these samples.
    Figure Legend Snippet: A general RT-PCR approach with primers to exon 7a and 3'UTR failed to detect VEGFxxxb isoforms in human and mouse cDNA extracts. RT-PCR reactions were performed using forward/reverse primers designed to amplify both VEGFxxx and VEGFxxxb isoforms from cDNAs extracted from mouse fibrosarcoma cell lines tumours and normal mouse tissues (heart, lung, liver and kidney) as well as commercially sourced human tissue cDNAs. A , A single product corresponding to VEGF164/188 (194 bp) is evident in the panel of mouse cDNAs using forward and reverse primers designed to exon 7 and the 3'UTR (exon7/3′UTR) with no evidence of VEGF164b/188b (128 bp) products. B C , A single product corresponding to VEGF165/189 (194 bp) is evident in human brain, bladder and kidney (AMS Biotechnology Ltd), and prostate and kidney (Primer Design Ltd) using forward and reverse primers designed to exon 7 (DB exon7a) and the 3'UTR (DB 3'UTR) respectively. No products corresponding to VEGF165b/189b (128 bp) are evident in any of these samples.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Affinity Magnetic Separation

    30) Product Images from "Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium"

    Article Title: Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002804

    Schematic diagram and reproducibility of M-TraM. (A) Schematic overview of the M-TraM screening. In yellow: inverted terminal repeats (ITRs) of the himar1 transposon with outward-facing T7 promoters; in blue: the gentamicin resistance gene in the transposon. Genomic DNA is isolated from the E. faecium mutant library. DNA is digested with the restriction enzyme Alu I, and the DNA fragments are circularized by self-ligation. The transposon-chromosome junction together with an ITR and a T7 promoter is amplified by PCR with primers (blue arrow) that hybridize to the transposon. To eliminate foreign DNA fragments that ligated into the circularized DNA of transposon-chromosome junctions, the PCR products were re-digested with Alu I. The purified DNA fragments are used as template in the in vitro transcription reaction. The resulting RNA products are reverse transcribed into cDNA. After labelling, the cDNA is used for microarray hybridization. (B) Schematic overview of the screening strategy to identify conditionally essential genes by M-TraM. A chromosomal region encompassing three genes (A, B, and C) from three different mutants (1, 2, and 3) is shown. Each mutant carries a single transposon insertion (blue) that disrupts the function of the gene. Mutant libraries are grown in a control condition ( e.g. , BHI) and a test condition ( e.g. , in the presence of ampicillin). All the three genes are non-essential for growth in the control condition. Gene B is required only for the test condition, so mutant 2 exhibits attenuated growth or poorer survival only in the test condition, and will consequently be reduced or be entirely lost from this library (indicated by light shading). M-TraM samples are generated from the two conditions, labelled with different dyes, and hybridized to a microarray. The DNA probes of gene A and gene C on the microarray will hybridize to the samples generated from both conditions. However, the cDNA sample of gene B will be present at reduced levels only in the test condition. By comparing the signal intensity from the two conditions for each probe, genes involved in growth or survival of the test condition can be identified. (C) Reproducibility of M-TraM. Log-log plot of the microarray signal intensities from two independent experiments of mutant libraries grown under non-selective conditions in BHI broth.
    Figure Legend Snippet: Schematic diagram and reproducibility of M-TraM. (A) Schematic overview of the M-TraM screening. In yellow: inverted terminal repeats (ITRs) of the himar1 transposon with outward-facing T7 promoters; in blue: the gentamicin resistance gene in the transposon. Genomic DNA is isolated from the E. faecium mutant library. DNA is digested with the restriction enzyme Alu I, and the DNA fragments are circularized by self-ligation. The transposon-chromosome junction together with an ITR and a T7 promoter is amplified by PCR with primers (blue arrow) that hybridize to the transposon. To eliminate foreign DNA fragments that ligated into the circularized DNA of transposon-chromosome junctions, the PCR products were re-digested with Alu I. The purified DNA fragments are used as template in the in vitro transcription reaction. The resulting RNA products are reverse transcribed into cDNA. After labelling, the cDNA is used for microarray hybridization. (B) Schematic overview of the screening strategy to identify conditionally essential genes by M-TraM. A chromosomal region encompassing three genes (A, B, and C) from three different mutants (1, 2, and 3) is shown. Each mutant carries a single transposon insertion (blue) that disrupts the function of the gene. Mutant libraries are grown in a control condition ( e.g. , BHI) and a test condition ( e.g. , in the presence of ampicillin). All the three genes are non-essential for growth in the control condition. Gene B is required only for the test condition, so mutant 2 exhibits attenuated growth or poorer survival only in the test condition, and will consequently be reduced or be entirely lost from this library (indicated by light shading). M-TraM samples are generated from the two conditions, labelled with different dyes, and hybridized to a microarray. The DNA probes of gene A and gene C on the microarray will hybridize to the samples generated from both conditions. However, the cDNA sample of gene B will be present at reduced levels only in the test condition. By comparing the signal intensity from the two conditions for each probe, genes involved in growth or survival of the test condition can be identified. (C) Reproducibility of M-TraM. Log-log plot of the microarray signal intensities from two independent experiments of mutant libraries grown under non-selective conditions in BHI broth.

    Techniques Used: Isolation, Mutagenesis, Ligation, Amplification, Polymerase Chain Reaction, Purification, In Vitro, Microarray, Hybridization, Generated

    Footprinting analysis of the transposon mutant library. (A) Schematic overview of the transposon footprinting strategy. PCR is performed using a gene specific primer and a primer corresponding to the transposon sequence. (B) Agarose gel electrophoresis of transposon footprinting on the essential gene ddl (lane 1), and the non-essential genes nox and esp (lane 2 and 3, respectively). Each band represents a PCR product of a different size, corresponding to a transposon insertion in a different position. The red box represents the product size range expected for transposon insertions within the essential ddl gene.
    Figure Legend Snippet: Footprinting analysis of the transposon mutant library. (A) Schematic overview of the transposon footprinting strategy. PCR is performed using a gene specific primer and a primer corresponding to the transposon sequence. (B) Agarose gel electrophoresis of transposon footprinting on the essential gene ddl (lane 1), and the non-essential genes nox and esp (lane 2 and 3, respectively). Each band represents a PCR product of a different size, corresponding to a transposon insertion in a different position. The red box represents the product size range expected for transposon insertions within the essential ddl gene.

    Techniques Used: Footprinting, Mutagenesis, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, End-sequence Profiling

    31) Product Images from "M1 Muscarinic Receptor Activation Mediates Cell Death in M1-HEK293 Cells"

    Article Title: M1 Muscarinic Receptor Activation Mediates Cell Death in M1-HEK293 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072011

    M1 mAChR expression and localisation in transfected HEK293 cells. (A) PCR was conducted to assess M1 expression by vector-transfected (HEK293-Vec) and M1 transfected HEK293 cells (HEK293-M1) using primers for M1 mAChR. The integrity of cDNA samples was confirmed using GAPDH. Samples are as follows; HEK293-Vec cDNA in lane 1, HEK293-Vec -RT/RNA in lane 2, HEK293-M1 cDNA in lane 3, and HEK293-M1–RT/RNA in lane 4. Samples are representative of those used in subsequent functional studies. (B) Immunocytochemical localisation of M1 receptors in HEK293-M1 cells using anti-HA.11 antibody (middle panel) and M1 antibody (right-hand panel). Left panel shows no primary control Image. (C) Analysis of M1 receptor cell-surface expression and internalization by carbachol. Cell surface receptors were live-labeled (see methods) using the HA.11antibody prior to stimulation with water-control or carbachol treatment. The M1 mAChRs were typically localised at the plasma membrane after water treatment, but after 1 h carbachol treatment for the M1 mAChRs were internalised, as shown by the increased punctate cytoplasmic staining (arrows) and reduced staining intensity on the cell surfaces. Scale bar: 50 µm. Data are representative of at least three independent experiments. (D) shows the time-course of M1 receptor internalization after carbachol addition using the granularity assay in Metamorph to measure internalized receptors (as intracellular granules). The graph shows that 5–60 minutes after carbachol addition there is internalization of M1 receptors.
    Figure Legend Snippet: M1 mAChR expression and localisation in transfected HEK293 cells. (A) PCR was conducted to assess M1 expression by vector-transfected (HEK293-Vec) and M1 transfected HEK293 cells (HEK293-M1) using primers for M1 mAChR. The integrity of cDNA samples was confirmed using GAPDH. Samples are as follows; HEK293-Vec cDNA in lane 1, HEK293-Vec -RT/RNA in lane 2, HEK293-M1 cDNA in lane 3, and HEK293-M1–RT/RNA in lane 4. Samples are representative of those used in subsequent functional studies. (B) Immunocytochemical localisation of M1 receptors in HEK293-M1 cells using anti-HA.11 antibody (middle panel) and M1 antibody (right-hand panel). Left panel shows no primary control Image. (C) Analysis of M1 receptor cell-surface expression and internalization by carbachol. Cell surface receptors were live-labeled (see methods) using the HA.11antibody prior to stimulation with water-control or carbachol treatment. The M1 mAChRs were typically localised at the plasma membrane after water treatment, but after 1 h carbachol treatment for the M1 mAChRs were internalised, as shown by the increased punctate cytoplasmic staining (arrows) and reduced staining intensity on the cell surfaces. Scale bar: 50 µm. Data are representative of at least three independent experiments. (D) shows the time-course of M1 receptor internalization after carbachol addition using the granularity assay in Metamorph to measure internalized receptors (as intracellular granules). The graph shows that 5–60 minutes after carbachol addition there is internalization of M1 receptors.

    Techniques Used: Expressing, Transfection, Polymerase Chain Reaction, Plasmid Preparation, Functional Assay, Labeling, Staining

    32) Product Images from "Regulation of epithelial differentiation and proliferation by the stroma - a role for the retinoblastoma protein"

    Article Title: Regulation of epithelial differentiation and proliferation by the stroma - a role for the retinoblastoma protein

    Journal: The Journal of investigative dermatology

    doi: 10.1038/jid.2012.201

    Rb-depletion in fibroblasts disrupts differentiation and proliferation of neighbouring epithelium A) Schematic of organotypic cultures comprised of epithelial cells seeded on top of modified fibroblasts. B) Western blots confirming knockdown of Rb and p53 in HFFs compared to scrambled shRNA controls (scram). C) Proliferation in fibroblasts assessed by Brdu incorporation (See materials and methods). D) Immuno-fluorescent detection of differentiation markers and Brdu incorporation in organotypic cultures indicate disruption of differentiation and enhanced proliferation in epithelium cultured with Rb-depleted fibroblasts. Proliferation is quantified in E). Scale bars represent 100 μM. F) KGF, IL1A and IL1B expression in Rb-depleted fibroblasts detected by RT-PCR. G) KGF expression in sub-confluent and mitomycin-C treated fibroblasts. H) ELISA detection of KGF secretion in monolayer co-cultures and I) organotypic cultures.
    Figure Legend Snippet: Rb-depletion in fibroblasts disrupts differentiation and proliferation of neighbouring epithelium A) Schematic of organotypic cultures comprised of epithelial cells seeded on top of modified fibroblasts. B) Western blots confirming knockdown of Rb and p53 in HFFs compared to scrambled shRNA controls (scram). C) Proliferation in fibroblasts assessed by Brdu incorporation (See materials and methods). D) Immuno-fluorescent detection of differentiation markers and Brdu incorporation in organotypic cultures indicate disruption of differentiation and enhanced proliferation in epithelium cultured with Rb-depleted fibroblasts. Proliferation is quantified in E). Scale bars represent 100 μM. F) KGF, IL1A and IL1B expression in Rb-depleted fibroblasts detected by RT-PCR. G) KGF expression in sub-confluent and mitomycin-C treated fibroblasts. H) ELISA detection of KGF secretion in monolayer co-cultures and I) organotypic cultures.

    Techniques Used: Modification, Western Blot, shRNA, BrdU Incorporation Assay, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Rescue of Rb-depletion by adenoviral re-expression restores differentiation and proliferation defects A) Restoration of Rb protein levels by adenoviral overexpression in shRb#2-fibroblasts. Re-expression of Rb also reduced active AKT levels. B) Immuno-fluorescent detection of keratin-1, filaggrin and Brdu identified that re-expression of Rb restored differentiation and reduced proliferation in the epithelium of organotypic cultures. Brdu incorporation is quantified in C). Scale bars represent 100 μM. D) Q-PCR and E) ELISA detection of KGF following re-expression of Rb. * represents p
    Figure Legend Snippet: Rescue of Rb-depletion by adenoviral re-expression restores differentiation and proliferation defects A) Restoration of Rb protein levels by adenoviral overexpression in shRb#2-fibroblasts. Re-expression of Rb also reduced active AKT levels. B) Immuno-fluorescent detection of keratin-1, filaggrin and Brdu identified that re-expression of Rb restored differentiation and reduced proliferation in the epithelium of organotypic cultures. Brdu incorporation is quantified in C). Scale bars represent 100 μM. D) Q-PCR and E) ELISA detection of KGF following re-expression of Rb. * represents p

    Techniques Used: Expressing, Over Expression, BrdU Incorporation Assay, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    33) Product Images from "Species-specific evolution of immune receptor tyrosine based activation motif-containing CEACAM1-related immune receptors in the dog"

    Article Title: Species-specific evolution of immune receptor tyrosine based activation motif-containing CEACAM1-related immune receptors in the dog

    Journal: BMC Evolutionary Biology

    doi: 10.1186/1471-2148-7-196

    Expression pattern of dog CEACAM1-related genes . CEACAM1-related transcripts were identified by RT-PCR using gene-specific primers which are located in the N domain and transmembrane exons. For the detection of CEACAM1 transcripts, primers in the N domain and cytoplasmic domain exon 3 were used. The products were separated by agarose gel electrophoresis in the presence of ethidium bromide and visualized by UV illumination. One-kb and 100-bp DNA fragment ladders were used as markers. The possible domain organization of the proteins encoded by the splice variants (number of Ig domains) is indicated in the right margin. Sequence determination of the CEACAM28 PCR products revealed simultaneous detection of CEACAM28 and CEACAM30 cDNAs (till then unknown). C, CEACAM.
    Figure Legend Snippet: Expression pattern of dog CEACAM1-related genes . CEACAM1-related transcripts were identified by RT-PCR using gene-specific primers which are located in the N domain and transmembrane exons. For the detection of CEACAM1 transcripts, primers in the N domain and cytoplasmic domain exon 3 were used. The products were separated by agarose gel electrophoresis in the presence of ethidium bromide and visualized by UV illumination. One-kb and 100-bp DNA fragment ladders were used as markers. The possible domain organization of the proteins encoded by the splice variants (number of Ig domains) is indicated in the right margin. Sequence determination of the CEACAM28 PCR products revealed simultaneous detection of CEACAM28 and CEACAM30 cDNAs (till then unknown). C, CEACAM.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing, Polymerase Chain Reaction

    34) Product Images from "Evaluation of the OPTC gene in primary open angle glaucoma: functional significance of a silent change"

    Article Title: Evaluation of the OPTC gene in primary open angle glaucoma: functional significance of a silent change

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-21

    Difference in the mRNA secondary structure between c.602C and c.602T variant in OPTC . Both the structures were predicted using the default parameter of RNAdraw (calculation temp. 37°C). Region of the mRNA harboring the variant nucleotide (indicated by arrow) has been enlarged for both the alleles to demonstrate the local structural alteration as predicted by RNAdraw.
    Figure Legend Snippet: Difference in the mRNA secondary structure between c.602C and c.602T variant in OPTC . Both the structures were predicted using the default parameter of RNAdraw (calculation temp. 37°C). Region of the mRNA harboring the variant nucleotide (indicated by arrow) has been enlarged for both the alleles to demonstrate the local structural alteration as predicted by RNAdraw.

    Techniques Used: Variant Assay

    Difference in mRNA and protein expression levels between c.602C and c.602T variants in OPTC . ( A ): Quantiative RT-PCR showing expression of c.602C and c.602T in mRNA level. The difference in mRNA expression is represented by ΔΔC T value. ( B ): Western blot of two variants of Opticin core protein fused with GFP as well as endogenous Opticin in RPE cells. UT represents the untransfected RPE cells. ( C ): Bar diagram showing the mean ± SD of densitometric scanning of western blots done thrice, that indicates much less expression of OPTC-c.602T variant (43%) than the normal one (OPTC-c.602C). Expression of transfected protein (Opticin-GFP) was normalized with endogenous Opticin.
    Figure Legend Snippet: Difference in mRNA and protein expression levels between c.602C and c.602T variants in OPTC . ( A ): Quantiative RT-PCR showing expression of c.602C and c.602T in mRNA level. The difference in mRNA expression is represented by ΔΔC T value. ( B ): Western blot of two variants of Opticin core protein fused with GFP as well as endogenous Opticin in RPE cells. UT represents the untransfected RPE cells. ( C ): Bar diagram showing the mean ± SD of densitometric scanning of western blots done thrice, that indicates much less expression of OPTC-c.602T variant (43%) than the normal one (OPTC-c.602C). Expression of transfected protein (Opticin-GFP) was normalized with endogenous Opticin.

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

    35) Product Images from "Histone Deacetylase HDAC4 Promotes Gastric Cancer SGC-7901 Cells Progression via p21 Repression"

    Article Title: Histone Deacetylase HDAC4 Promotes Gastric Cancer SGC-7901 Cells Progression via p21 Repression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098894

    The expression of HDAC4 in transfected SGC-7901 cells. HDAC4 expression was determined in SGC-7901 cells transfected with empty vector (NC) or HDAC4 ( A ) and ( B ). HDAC4 expression was determined in SGC-7901 cells transfected with siRNA oligos targeting HDAC4 (si-HDAC4) or scrambled siRNA (si-NC) by qRT-PCR and western blot ( C ) and ( D ) (n = 4). Data were expressed as mean ± S.E.M. ***P
    Figure Legend Snippet: The expression of HDAC4 in transfected SGC-7901 cells. HDAC4 expression was determined in SGC-7901 cells transfected with empty vector (NC) or HDAC4 ( A ) and ( B ). HDAC4 expression was determined in SGC-7901 cells transfected with siRNA oligos targeting HDAC4 (si-HDAC4) or scrambled siRNA (si-NC) by qRT-PCR and western blot ( C ) and ( D ) (n = 4). Data were expressed as mean ± S.E.M. ***P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

    p21 knockdown reversed the effect of down-regulated HDAC4 on the inhibition of SGC-7901 cell growth. Expression of p21 was analyzed by western blot in SGC-7901 cells transfected with empty pcDNA3.1(+)-vector (NC) or HDAC4 and scrambled siRNA control (si-NC) or HDAC4 siRNA oligos (si-HDAC4) respectively ( A ). The p21 mRNA level was analyzed by qRT-PCR in SGC-7901 cells transfected with si-NC, si-HDAC4 alone or combination with siRNA p21 (si-p21) ( B ). The cell growth curve was measured by CCK-8 assay ( *P
    Figure Legend Snippet: p21 knockdown reversed the effect of down-regulated HDAC4 on the inhibition of SGC-7901 cell growth. Expression of p21 was analyzed by western blot in SGC-7901 cells transfected with empty pcDNA3.1(+)-vector (NC) or HDAC4 and scrambled siRNA control (si-NC) or HDAC4 siRNA oligos (si-HDAC4) respectively ( A ). The p21 mRNA level was analyzed by qRT-PCR in SGC-7901 cells transfected with si-NC, si-HDAC4 alone or combination with siRNA p21 (si-p21) ( B ). The cell growth curve was measured by CCK-8 assay ( *P

    Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR, CCK-8 Assay

    36) Product Images from "Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach"

    Article Title: Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100611

    Alignment of the mitochondrial cox1 gene and design of primers. Alignment of the mitochondrial cox1 gene derived from T. solium (AB524785), T. saginata (AB645845) and T. asiatica (AB608739). The broken arrows indicate the position of Cest_F (forward primer) and the biotinylated Cest_R (reverse primer) for template amplification. The solid arrow indicates Cest_S (sequencing primer), and the rectangular box shows the position (992–1017) of the target region used for species level identification. The dots indicate identical nucleotides between the sequences.
    Figure Legend Snippet: Alignment of the mitochondrial cox1 gene and design of primers. Alignment of the mitochondrial cox1 gene derived from T. solium (AB524785), T. saginata (AB645845) and T. asiatica (AB608739). The broken arrows indicate the position of Cest_F (forward primer) and the biotinylated Cest_R (reverse primer) for template amplification. The solid arrow indicates Cest_S (sequencing primer), and the rectangular box shows the position (992–1017) of the target region used for species level identification. The dots indicate identical nucleotides between the sequences.

    Techniques Used: Derivative Assay, Amplification, Sequencing

    37) Product Images from "NFAT1 and NFAT3 Cooperate with HDAC4 during Regulation of Alternative Splicing of PMCA Isoforms in PC12 Cells"

    Article Title: NFAT1 and NFAT3 Cooperate with HDAC4 during Regulation of Alternative Splicing of PMCA Isoforms in PC12 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099118

    Alternative splicing of PMCA in PMCA2- or PMCA3-deficient PC12 cells upon NFAT inhibition. Alternative splicing pattern at sites A and C of mRNA transcripts of PMCAs: Atp21b1 (PMCA1) ( A ), Atp21b2 (PMCA2) ( B ), Atp21b3 (PMCA3) ( C ), Atp21b4 (PMCA4) ( D ) was determined by RT-PCR in non-treated and 1 µM 11R-VIVIT-treated PC12 cells. RT-PCR product bands were quantified densitometrically, standardized to Gapdh and normalized to control cells, expressed as y = 1, both for non-treated ( E ) and 11R-VIVIT-treated cells ( F ). Student’s t-test was used for comparison of control cells with PMCA2- or PMCA3-reduced cells (n = 3). Bars represent mean values ± SEM. *P≤0.05. Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3). Black arrows indicate the PCR product bands for PMCA2 site A and PMCA3 site C. White asterisks on the images of gels indicate the PCR product bands generated by alternative splicing that underwent a significant change upon NFAT inhibition with 11R-VIVIT.
    Figure Legend Snippet: Alternative splicing of PMCA in PMCA2- or PMCA3-deficient PC12 cells upon NFAT inhibition. Alternative splicing pattern at sites A and C of mRNA transcripts of PMCAs: Atp21b1 (PMCA1) ( A ), Atp21b2 (PMCA2) ( B ), Atp21b3 (PMCA3) ( C ), Atp21b4 (PMCA4) ( D ) was determined by RT-PCR in non-treated and 1 µM 11R-VIVIT-treated PC12 cells. RT-PCR product bands were quantified densitometrically, standardized to Gapdh and normalized to control cells, expressed as y = 1, both for non-treated ( E ) and 11R-VIVIT-treated cells ( F ). Student’s t-test was used for comparison of control cells with PMCA2- or PMCA3-reduced cells (n = 3). Bars represent mean values ± SEM. *P≤0.05. Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3). Black arrows indicate the PCR product bands for PMCA2 site A and PMCA3 site C. White asterisks on the images of gels indicate the PCR product bands generated by alternative splicing that underwent a significant change upon NFAT inhibition with 11R-VIVIT.

    Techniques Used: Inhibition, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Generated

    38) Product Images from "Limbic Epileptogenesis in a Mouse Model of Fragile X Syndrome"

    Article Title: Limbic Epileptogenesis in a Mouse Model of Fragile X Syndrome

    Journal: Cerebral Cortex (New York, NY)

    doi: 10.1093/cercor/bhn163

    Fluoro-Jade B and Nissl staining reveal no obvious neuronal cell loss in Fmr1 KO mice after kindling. ( A ) Fluoro-Jade B staining. A WT hippocampal section 24 h after pilocarpine-induced seizure (340 mg/kg, intraperitoneally) with significant cell death in the hilus of the DG is shown as a positive control ( n = 3). No cell death is detected in sections from sham WT ( n = 3), kindled WT ( n = 6), or kindled Fmr1 KO ( n = 6) mice 24 h after the third consecutive class 5 seizure. Scale bar: 400 μm. ( B ) Nissl staining. Nissl-stained brain sections from sham WT mice, kindled WT mice, sham Fmr1 KO mice, and kindled Fmr1 KO mice reveal similar cell densities in the CA1, CA3, cortex, and hilus of the DG. Scale bars: top, 500 μm for upper one row; bottom, 250 μm for other rows. ( C ) Statistical analysis of cell number in the hilus. No significant difference was found among the 4 groups. Bars represent mean ± standard error of the mean; 1-way ANOVA, post hoc Dunnett's test.
    Figure Legend Snippet: Fluoro-Jade B and Nissl staining reveal no obvious neuronal cell loss in Fmr1 KO mice after kindling. ( A ) Fluoro-Jade B staining. A WT hippocampal section 24 h after pilocarpine-induced seizure (340 mg/kg, intraperitoneally) with significant cell death in the hilus of the DG is shown as a positive control ( n = 3). No cell death is detected in sections from sham WT ( n = 3), kindled WT ( n = 6), or kindled Fmr1 KO ( n = 6) mice 24 h after the third consecutive class 5 seizure. Scale bar: 400 μm. ( B ) Nissl staining. Nissl-stained brain sections from sham WT mice, kindled WT mice, sham Fmr1 KO mice, and kindled Fmr1 KO mice reveal similar cell densities in the CA1, CA3, cortex, and hilus of the DG. Scale bars: top, 500 μm for upper one row; bottom, 250 μm for other rows. ( C ) Statistical analysis of cell number in the hilus. No significant difference was found among the 4 groups. Bars represent mean ± standard error of the mean; 1-way ANOVA, post hoc Dunnett's test.

    Techniques Used: Staining, Mouse Assay, Positive Control

    FMRP expression is upregulated after seizure activity. ( A ) Representative western blot showing a transient increase of FMRP in the forebrain of kindled mice after an evoked seizure. Two weeks after fully kindling, a single class 5 seizure was induced in WT mice and forebrains were isolated 3 h or 12 h later. Each lane was loaded with an equal amount of protein extract from a single forebrain sample (mouse #1–6). Lane 1 and 2, unstimulated fully kindled WT mice; lane 3 and 4, fully kindled mice 3 h after a class 5 seizure; lane 5 and 6, fully kindled mice 12 h after a class 5 seizure. After a class 5 seizure, FMRP expression increases before returning to baseline levels by 12 h. ( B ) Quantitative analysis of western blot band intensities. FMRP immunoreactivity was normalized to Gapdh immunoreactivity and mean ± standard error of the mean (SEM) values are presented as a percentage of the mean level in unstimulated fully kindled mice; * P
    Figure Legend Snippet: FMRP expression is upregulated after seizure activity. ( A ) Representative western blot showing a transient increase of FMRP in the forebrain of kindled mice after an evoked seizure. Two weeks after fully kindling, a single class 5 seizure was induced in WT mice and forebrains were isolated 3 h or 12 h later. Each lane was loaded with an equal amount of protein extract from a single forebrain sample (mouse #1–6). Lane 1 and 2, unstimulated fully kindled WT mice; lane 3 and 4, fully kindled mice 3 h after a class 5 seizure; lane 5 and 6, fully kindled mice 12 h after a class 5 seizure. After a class 5 seizure, FMRP expression increases before returning to baseline levels by 12 h. ( B ) Quantitative analysis of western blot band intensities. FMRP immunoreactivity was normalized to Gapdh immunoreactivity and mean ± standard error of the mean (SEM) values are presented as a percentage of the mean level in unstimulated fully kindled mice; * P

    Techniques Used: Expressing, Activity Assay, Western Blot, Mouse Assay, Isolation

    Axon projections of granule cells in the hippocampus of sham-stimulated and fully kindled WT and Fmr1 KO mice. ( A ) Representative Timm staining of horizontal brain sections of sham-stimulated (sham) WT mice (a, g); sham Fmr1 KO mice (d, j); fully kindled (kindled) WT mice 5 weeks (b, h) or 28 weeks (c, i) after 22 daily stimulation; kindled Fmr1 KO mice 5 weeks (e, k) or 28 weeks (f, l) after 22 daily stimulations. Under control conditions (sham), Fmr1 KO mice show slightly more Timm-stained granules in the granule cell body layer compared with WT mice. Five weeks after 22 daily stimulations, MFS is obvious in the IML of the DG in Fmr1 KO mice. Twenty-eight weeks after 22 daily stimulations, MFS is dramatically more severe in Fmr1 KO mice. In contrast, WT mice show little MFS 28 weeks after 22 daily stimulations. Scale bars: upper 2 panels, 250 μm; lower 2 panels, 50 μm. ( B ) Statistical analysis of Timm index in the IML of the DG. Bars represent mean ± standard error of the mean. *** P
    Figure Legend Snippet: Axon projections of granule cells in the hippocampus of sham-stimulated and fully kindled WT and Fmr1 KO mice. ( A ) Representative Timm staining of horizontal brain sections of sham-stimulated (sham) WT mice (a, g); sham Fmr1 KO mice (d, j); fully kindled (kindled) WT mice 5 weeks (b, h) or 28 weeks (c, i) after 22 daily stimulation; kindled Fmr1 KO mice 5 weeks (e, k) or 28 weeks (f, l) after 22 daily stimulations. Under control conditions (sham), Fmr1 KO mice show slightly more Timm-stained granules in the granule cell body layer compared with WT mice. Five weeks after 22 daily stimulations, MFS is obvious in the IML of the DG in Fmr1 KO mice. Twenty-eight weeks after 22 daily stimulations, MFS is dramatically more severe in Fmr1 KO mice. In contrast, WT mice show little MFS 28 weeks after 22 daily stimulations. Scale bars: upper 2 panels, 250 μm; lower 2 panels, 50 μm. ( B ) Statistical analysis of Timm index in the IML of the DG. Bars represent mean ± standard error of the mean. *** P

    Techniques Used: Mouse Assay, Staining

    Effects of the NMDA antagonist MK801 on kindling development in WT and Fmr1 KO mice. ( A ) Behavioral seizure intensities evoked by amygdala stimulation at the predetermined EST, data presented as mean ± standard error of the mean (SEM) of saline-treated WT ( n = 25) and MK801-treated WT mice ( n = 13). ( B ) Behavioral seizure intensities evoked by amygdala stimulation at the predetermined EST, data presented as mean ± SEM of saline-treated ( n = 31) and MK801-treated Fmr1 KO mice ( n = 13). MK801 significantly slowed kindling development in both WT mice and Fmr1 KO mice. Saline or MK801 was administered 30 min before each stimulation. Asterisks indicate statistically significant differences in the average behavior seizure class at the indicated time point between the 2 presented groups. * P
    Figure Legend Snippet: Effects of the NMDA antagonist MK801 on kindling development in WT and Fmr1 KO mice. ( A ) Behavioral seizure intensities evoked by amygdala stimulation at the predetermined EST, data presented as mean ± standard error of the mean (SEM) of saline-treated WT ( n = 25) and MK801-treated WT mice ( n = 13). ( B ) Behavioral seizure intensities evoked by amygdala stimulation at the predetermined EST, data presented as mean ± SEM of saline-treated ( n = 31) and MK801-treated Fmr1 KO mice ( n = 13). MK801 significantly slowed kindling development in both WT mice and Fmr1 KO mice. Saline or MK801 was administered 30 min before each stimulation. Asterisks indicate statistically significant differences in the average behavior seizure class at the indicated time point between the 2 presented groups. * P

    Techniques Used: Mouse Assay

    Striking acceleration of kindling development in Fmr1 KO mice. ( A ) No significant difference between WT and Fmr1 KO in mean EST is observed; P = 0.77, unpaired t -test. ( B ) Behavioral seizure intensities (class 1–5) evoked by amygdala stimulation at the predetermined EST, presented as mean ± SEM of WT mice ( n = 25) and Fmr1 KO mice ( n = 31). ( C ) Number of stimulations required to provoke the first episode of class 4/5 seizure and third consecutive episode of class 4/5 seizure in WT mice ( n = 25) and Fmr1 KO mice ( n = 31) mice; *** P
    Figure Legend Snippet: Striking acceleration of kindling development in Fmr1 KO mice. ( A ) No significant difference between WT and Fmr1 KO in mean EST is observed; P = 0.77, unpaired t -test. ( B ) Behavioral seizure intensities (class 1–5) evoked by amygdala stimulation at the predetermined EST, presented as mean ± SEM of WT mice ( n = 25) and Fmr1 KO mice ( n = 31). ( C ) Number of stimulations required to provoke the first episode of class 4/5 seizure and third consecutive episode of class 4/5 seizure in WT mice ( n = 25) and Fmr1 KO mice ( n = 31) mice; *** P

    Techniques Used: Mouse Assay

    Fmr1 KO mice exhibit prolonged electrographic seizures. ( A ) AD durations evoked by amygdala stimulation at the predetermined EST. The progressive prolongation of AD durations was significantly accelerated in Fmr1 KO mice compared with WT mice. Data presented as mean ± standard error of the mean of WT mice ( n = 25) and Fmr1 KO mice ( n = 31). ( B ) Representative EEG recordings of WT and Fmr1 KO mice at the third stimulation, first class 5 seizure-inducing stimulation and third consecutive class 5 seizure-inducing stimulation. Arrows indicate the application of stimulation, and the arrowheads indicate the termination point of the electrographic seizure.
    Figure Legend Snippet: Fmr1 KO mice exhibit prolonged electrographic seizures. ( A ) AD durations evoked by amygdala stimulation at the predetermined EST. The progressive prolongation of AD durations was significantly accelerated in Fmr1 KO mice compared with WT mice. Data presented as mean ± standard error of the mean of WT mice ( n = 25) and Fmr1 KO mice ( n = 31). ( B ) Representative EEG recordings of WT and Fmr1 KO mice at the third stimulation, first class 5 seizure-inducing stimulation and third consecutive class 5 seizure-inducing stimulation. Arrows indicate the application of stimulation, and the arrowheads indicate the termination point of the electrographic seizure.

    Techniques Used: Mouse Assay

    Kindling upregulates Fmr1 mRNA in the forebrain. ( A ) Quantitative real-time PCR (lower) and RT-PCR (upper) analysis of Fmr1 mRNA expression. Total RNA was isolated from forebrains of fully kindled WT mice 3 h after an evoked class 5 seizure (Stim.) or without any further stimulation (Unstim.). Fmr1 mRNA was normalized to G apdh mRNA levels and mean ± standard error of the mean values are presented as a percentage of unstimulated controls; * P
    Figure Legend Snippet: Kindling upregulates Fmr1 mRNA in the forebrain. ( A ) Quantitative real-time PCR (lower) and RT-PCR (upper) analysis of Fmr1 mRNA expression. Total RNA was isolated from forebrains of fully kindled WT mice 3 h after an evoked class 5 seizure (Stim.) or without any further stimulation (Unstim.). Fmr1 mRNA was normalized to G apdh mRNA levels and mean ± standard error of the mean values are presented as a percentage of unstimulated controls; * P

    Techniques Used: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Mouse Assay

    Effects of the mGluR5 antagonist MPEP on kindling development in WT and Fmr1 KO mice. ( A ) Behavioral seizure class evoked by amygdala stimulation at the predetermined EST, data presented as mean ± standard error of the mean (SEM) of saline-treated ( n = 25) and MPEP-treated WT mice ( n = 15). There is no significant difference between kindling development in WT mice treated with MPEP compared with controls. ( B ) Behavioral seizure class evoked by amygdala stimulation at the predetermined EST, data presented as mean ± SEM of saline-treated ( n = 31) and MPEP-treated Fmr1 KO mice treated ( n = 13). Administering MPEP to Fmr1 KO mice significantly represses seizure intensities at the fourth through eighth stimulation point. Saline or MK801 was administered 30 min before each stimulation. Asterisks indicate statistically significant differences in the average behavior seizure class at the indicated time point between the 2 presented groups. * P
    Figure Legend Snippet: Effects of the mGluR5 antagonist MPEP on kindling development in WT and Fmr1 KO mice. ( A ) Behavioral seizure class evoked by amygdala stimulation at the predetermined EST, data presented as mean ± standard error of the mean (SEM) of saline-treated ( n = 25) and MPEP-treated WT mice ( n = 15). There is no significant difference between kindling development in WT mice treated with MPEP compared with controls. ( B ) Behavioral seizure class evoked by amygdala stimulation at the predetermined EST, data presented as mean ± SEM of saline-treated ( n = 31) and MPEP-treated Fmr1 KO mice treated ( n = 13). Administering MPEP to Fmr1 KO mice significantly represses seizure intensities at the fourth through eighth stimulation point. Saline or MK801 was administered 30 min before each stimulation. Asterisks indicate statistically significant differences in the average behavior seizure class at the indicated time point between the 2 presented groups. * P

    Techniques Used: Mouse Assay

    39) Product Images from "GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway"

    Article Title: GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-11-78

    Expression of GDNF and its receptor subunits in cultured ISCs and TM4 cells . (A) RT-PCR results exhibited the expression of GDNF, GFRα1 and NCAM but not RET mRNAs in cultured ISCs and TM4 cells. G3PDH and the Sertoli cell-specific gene CLU were used as positive controls. (B) RET expression in SSCs but not cultured ISCs or TM4 cells was detected by RT-PCR. (C-E) Western blotting demonstrated the expression of NCAM (C) and GFRα1 (D) but not RET (E) proteins in cultured ISCs and TM4 cells. The SY5Y cell line was used as the positive control for the RET protein. The β-actin protein was used as a positive control for each sample.
    Figure Legend Snippet: Expression of GDNF and its receptor subunits in cultured ISCs and TM4 cells . (A) RT-PCR results exhibited the expression of GDNF, GFRα1 and NCAM but not RET mRNAs in cultured ISCs and TM4 cells. G3PDH and the Sertoli cell-specific gene CLU were used as positive controls. (B) RET expression in SSCs but not cultured ISCs or TM4 cells was detected by RT-PCR. (C-E) Western blotting demonstrated the expression of NCAM (C) and GFRα1 (D) but not RET (E) proteins in cultured ISCs and TM4 cells. The SY5Y cell line was used as the positive control for the RET protein. The β-actin protein was used as a positive control for each sample.

    Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control

    GDNF enhances the proliferation of cultured ISCs . (A) The identity and purity of cultured ISCs was confirmed by immunostaining with an antibody against Sertoli cell-specific vimentin protein. (B-C) BrdU-positive ISCs in control (B) and GDNF treated (C) groups. (D) Quantitative analysis of ISC proliferation as indicated by the percentage of BrdU-positive cells in control and GDNF treated groups. (E) Quantitative analysis of TM4 cell proliferation as indicated by the percentages of BrdU-positive cells in GDNF treated and control groups. Statistically significant differences ( p
    Figure Legend Snippet: GDNF enhances the proliferation of cultured ISCs . (A) The identity and purity of cultured ISCs was confirmed by immunostaining with an antibody against Sertoli cell-specific vimentin protein. (B-C) BrdU-positive ISCs in control (B) and GDNF treated (C) groups. (D) Quantitative analysis of ISC proliferation as indicated by the percentage of BrdU-positive cells in control and GDNF treated groups. (E) Quantitative analysis of TM4 cell proliferation as indicated by the percentages of BrdU-positive cells in GDNF treated and control groups. Statistically significant differences ( p

    Techniques Used: Cell Culture, Immunostaining

    40) Product Images from "TXNL6 Is a Novel Oxidative Stress-Induced Reducing System for Methionine Sulfoxide Reductase A Repair of ?-Crystallin and Cytochrome C in the Eye Lens"

    Article Title: TXNL6 Is a Novel Oxidative Stress-Induced Reducing System for Methionine Sulfoxide Reductase A Repair of ?-Crystallin and Cytochrome C in the Eye Lens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015421

    TXNL6 is present in protein extracts prepared from the cytoplasm and mitochondria of human lens epithelial cells. SDS-PAGE and immunoblotting of Panel A - TXNL6, Panel B - MsrA, Panel C - Trx 1, and Panel D -Trx 2 in 5 µg of protein extracts from the cytoplasm and mitochondria of HLE-B3 lens epithelial cells using specific antibodies.
    Figure Legend Snippet: TXNL6 is present in protein extracts prepared from the cytoplasm and mitochondria of human lens epithelial cells. SDS-PAGE and immunoblotting of Panel A - TXNL6, Panel B - MsrA, Panel C - Trx 1, and Panel D -Trx 2 in 5 µg of protein extracts from the cytoplasm and mitochondria of HLE-B3 lens epithelial cells using specific antibodies.

    Techniques Used: SDS Page

    Mitochondrial and cytosolic localization of TXNL6 in human lens epithelial cells. Co-localization of Panel A - TXNL6 (green), Panel B - Trx 1 (green) and Panel C - Trx 2 (green), mitotracker red – a specific mitochondrial marker and merging of the two images (orange/yellow) in HLE-B3 lens epithelial cells by immunofluoresence microscopy.
    Figure Legend Snippet: Mitochondrial and cytosolic localization of TXNL6 in human lens epithelial cells. Co-localization of Panel A - TXNL6 (green), Panel B - Trx 1 (green) and Panel C - Trx 2 (green), mitotracker red – a specific mitochondrial marker and merging of the two images (orange/yellow) in HLE-B3 lens epithelial cells by immunofluoresence microscopy.

    Techniques Used: Marker, Microscopy

    TXNL6 transcript is detected in the human lens epithelium and lens fiber cells and in other human tissues. Ethidium bromide stained agarose gels show Panel A - TXNL6, Panel B - Trx 1, Panel C - Trx 2 and Panel D GAPDH (control) transcript from 200 ng of RNA obtained from human tissues - 1. Lens epithelium, 2. Lens Fiber, 3. Retina, 4. Stomach, 5. Kidney, 6.Heart, 7. Colon, and 8. Spleen.
    Figure Legend Snippet: TXNL6 transcript is detected in the human lens epithelium and lens fiber cells and in other human tissues. Ethidium bromide stained agarose gels show Panel A - TXNL6, Panel B - Trx 1, Panel C - Trx 2 and Panel D GAPDH (control) transcript from 200 ng of RNA obtained from human tissues - 1. Lens epithelium, 2. Lens Fiber, 3. Retina, 4. Stomach, 5. Kidney, 6.Heart, 7. Colon, and 8. Spleen.

    Techniques Used: Staining

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    Synthesized:

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway
    Article Snippet: .. For the measurement of Dkk3 mRNA expression, the first-strand cDNA was synthesized from 1 μg of total RNA by a High-Capacity cDNA Archive Kit (Applied Biosystems) and RT-PCR was conducted using a SYBR Premix Ex Taq II Kit (Applied Biosystems). qRT-PCR reaction was carried out using a 7500 Fast Real-Time Sequence detection system (Applied Biosystems). .. The relative gene expression of miR-214 and Dkk3 was calculated using the 2−ΔΔCt method.

    Mutagenesis:

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Isolation:

    Article Title: KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport
    Article Snippet: .. Isolated RNA was reverse transcribed in cDNA using the RevertAid first strand cDNA synthesis kit (Fermentas). ..

    Article Title: Complement Receptor C5aR1 Inhibition Reduces Pyroptosis in hDPP4-Transgenic Mice Infected with MERS-CoV
    Article Snippet: .. Isolation of RNA and Proteins THP-1 differentiated macrophages were lysed in TRIzol™ Reagent (Life Technologies, Carlsbad, CA, USA) at 24 h post-infection with MERS-CoV. .. Total RNA and proteins were isolated according to the reagent user guide.

    Multiplex Assay:

    Article Title: Decidual Interleukin-22-Producing CD4+ T Cells (Th17/Th0/IL-22+ and Th17/Th2/IL-22+, Th2/IL-22+, Th0/IL-22+), Which Also Produce IL-4, Are Involved in the Success of Pregnancy
    Article Snippet: .. Briefly, the mRNA expression of IL-4, IL-17A, IL-17F, IL-23R, IFN-γ, RORC, GATA3, AHR, IL-22, and Actb (high expression housekeeping gene) was measured using the QuantiGene multiplex assay (Thermo Fisher, Waltham, MA, USA). .. Samples (biopsies of decidua from those experiencing successful pregnancy and tubal biopsies of those experiencing ectopic pregnancies at the implantation site and away from the implantation site) were lysed after treatment in a lysis mixture; mRNA expression in lysates was detected and measured according to the manufacturer’s instructions.

    Construct:

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer
    Article Snippet: .. To generate A549 cells stably overexpressing circPTK2, we cotransfected the above-mentioned construct or empty vector with packaging plasmids psPAX2 and pMD2.G (Geneseed Biotech) into HEK 293 T cells using Lipofectamine 2000 (Invitrogen). .. After HEK 293 T cells were cultured for 48 h, the packaged lentiviruses were harvested.

    Article Title: SMA1, a homolog of the splicing factor Prp28, has a multifaceted role in miRNA biogenesis in Arabidopsis
    Article Snippet: .. Plasmid construction The protein encoding region of SMA1 was PCR-amplified with primers SMA1CDS-F and SMA1CDS-R, cloned into PCR8/GW/TOPO cloning vector (Invitrogen) and subsequently subcloned into pMDC43 binary vector to generate 35S::GFP-SMA1 construct. .. To generate the bimolecular fluorescence complementation (BiFC) construct for SMA1, SMA1 cDNA was inserted into the multiple clone sites of pSAT4-cCFP-C vector.

    Sequencing:

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway
    Article Snippet: .. For the measurement of Dkk3 mRNA expression, the first-strand cDNA was synthesized from 1 μg of total RNA by a High-Capacity cDNA Archive Kit (Applied Biosystems) and RT-PCR was conducted using a SYBR Premix Ex Taq II Kit (Applied Biosystems). qRT-PCR reaction was carried out using a 7500 Fast Real-Time Sequence detection system (Applied Biosystems). .. The relative gene expression of miR-214 and Dkk3 was calculated using the 2−ΔΔCt method.

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Polymerase Chain Reaction:

    Article Title: SMA1, a homolog of the splicing factor Prp28, has a multifaceted role in miRNA biogenesis in Arabidopsis
    Article Snippet: .. Plasmid construction The protein encoding region of SMA1 was PCR-amplified with primers SMA1CDS-F and SMA1CDS-R, cloned into PCR8/GW/TOPO cloning vector (Invitrogen) and subsequently subcloned into pMDC43 binary vector to generate 35S::GFP-SMA1 construct. .. To generate the bimolecular fluorescence complementation (BiFC) construct for SMA1, SMA1 cDNA was inserted into the multiple clone sites of pSAT4-cCFP-C vector.

    Quantitative RT-PCR:

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway
    Article Snippet: .. For the measurement of Dkk3 mRNA expression, the first-strand cDNA was synthesized from 1 μg of total RNA by a High-Capacity cDNA Archive Kit (Applied Biosystems) and RT-PCR was conducted using a SYBR Premix Ex Taq II Kit (Applied Biosystems). qRT-PCR reaction was carried out using a 7500 Fast Real-Time Sequence detection system (Applied Biosystems). .. The relative gene expression of miR-214 and Dkk3 was calculated using the 2−ΔΔCt method.

    Expressing:

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway
    Article Snippet: .. For the measurement of Dkk3 mRNA expression, the first-strand cDNA was synthesized from 1 μg of total RNA by a High-Capacity cDNA Archive Kit (Applied Biosystems) and RT-PCR was conducted using a SYBR Premix Ex Taq II Kit (Applied Biosystems). qRT-PCR reaction was carried out using a 7500 Fast Real-Time Sequence detection system (Applied Biosystems). .. The relative gene expression of miR-214 and Dkk3 was calculated using the 2−ΔΔCt method.

    Article Title: Decidual Interleukin-22-Producing CD4+ T Cells (Th17/Th0/IL-22+ and Th17/Th2/IL-22+, Th2/IL-22+, Th0/IL-22+), Which Also Produce IL-4, Are Involved in the Success of Pregnancy
    Article Snippet: .. Briefly, the mRNA expression of IL-4, IL-17A, IL-17F, IL-23R, IFN-γ, RORC, GATA3, AHR, IL-22, and Actb (high expression housekeeping gene) was measured using the QuantiGene multiplex assay (Thermo Fisher, Waltham, MA, USA). .. Samples (biopsies of decidua from those experiencing successful pregnancy and tubal biopsies of those experiencing ectopic pregnancies at the implantation site and away from the implantation site) were lysed after treatment in a lysis mixture; mRNA expression in lysates was detected and measured according to the manufacturer’s instructions.

    High Throughput Screening Assay:

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Plasmid Preparation:

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer
    Article Snippet: .. To generate A549 cells stably overexpressing circPTK2, we cotransfected the above-mentioned construct or empty vector with packaging plasmids psPAX2 and pMD2.G (Geneseed Biotech) into HEK 293 T cells using Lipofectamine 2000 (Invitrogen). .. After HEK 293 T cells were cultured for 48 h, the packaged lentiviruses were harvested.

    Article Title: SMA1, a homolog of the splicing factor Prp28, has a multifaceted role in miRNA biogenesis in Arabidopsis
    Article Snippet: .. Plasmid construction The protein encoding region of SMA1 was PCR-amplified with primers SMA1CDS-F and SMA1CDS-R, cloned into PCR8/GW/TOPO cloning vector (Invitrogen) and subsequently subcloned into pMDC43 binary vector to generate 35S::GFP-SMA1 construct. .. To generate the bimolecular fluorescence complementation (BiFC) construct for SMA1, SMA1 cDNA was inserted into the multiple clone sites of pSAT4-cCFP-C vector.

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    Thermo Fisher taq dna polymerase
    Conditional mutagenesis pipeline. Upon deciding which exon to flox, we recommend sequencing the target sites to identify polymorphisms compared to reference genome. Next, sgRNAs should be designed and tested by either direct sequencing of PCR fragments, T7 endonuclease assay or loss of a restriction enzyme site on bulk <t>DNA</t> from pooled embryos. Once active sgRNAs have been identified, experiments integrating the first loxP site should be performed. In the absence of conclusive data that certain HDR template performs significantly better than others (such experiments are not practical at the only level that matters–germline transmission), we recommend using the design we successfully used to integrate loxP into fleer , aldh1a2 and tcf21 : antisense to PAM, with 49-base 5’ homology arm and 21-base 3’ homology arm, with 3-nucleotide spacers flanking loxP site. As injected embryos are being raised, we then recommend to optimize nested PCR screening conditions DNA from pools of injected embryos. We found “plain” <t>Taq</t> polymerases (NEB #M0270, Thermofisher Scientific 2x PCR Master Mix Cat# AB-0575/DC and #EP0402, or similar) to be most suitable for nested PCR. In contrast, high-performance mixes such as Platinum Taq (Thermofisher #10966026) or Kapa 2G Fast ReadyMix + dye (Kapa Biosystems- KM5101) yield very high background and may only be used for the second (nested) reaction. It is also very helpful if primers for one end of the nested PCR are anchored within an exon. We recommend generating a deletion allele in parallel with integration of the first loxP site. Once highly active sgRNAs are identified, we recommend injecting a pair of sgRNAs flanking the exon to be floxed in order to confirm that removal of selected exon will yield an overt phenotype. We have been able to very efficiently delete exon 8 of aldh1a2 using sgRNAs spaced just over 450 base pairs, but larger deletions are certainly feasible too (1, 2). An additional benefit of a deletion allele is that it can be crossed to Cre drivers of interest, eliminating the need to back-cross floxed allele to obtain homozygotes. Screening for germline transmission should be performed by nested PCR on pools of embryos obtained from incross. Positive crosses should be analyzed by performing short flanking PCR (ideally under 400 base pairs) on DNA from individual embryos. Bands corresponding to loxP-containing allele should be extracted from gel and sequenced to ensure presence of intact loxP site. Siblings of screened embryos should be raised to adulthood and loxP-positive F1s should be identified by flanking PCR as well. Two strategies can be used for integration of the second loxP site. If speed is the main priority, loxP-positive F1s can be in-crossed and second sgRNA/HDR oligonucleotide can be injected. The main drawback of this strategy that there is only 50% likelihood that the second loxP site will integrate into a chromosome already containing the first loxP. It is therefore necessary to genotype adults for presence of the first loxP site before out-crossing. Even though we successfully used this strategy to engineer a floxed allele of tbx20 , we consider it impractical and would generally recommend to first generate adults homozygous for the first loxP site, incross them and then inject the second sgRNA/HDR oligonucleotide.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr reagents
    Example gel electrophoresis image of a successful 16S rRNA gene <t>PCR</t> amplification from fall 2015. Lanes labeled according to contents: “Sample A11-22” is the amplicon from isolate <t>DNA</t> (expected size 1466 bp); “Ladder” is Lambda HindIII digest ladder (NEB N3012S), with the lowest visible band at 2027 bp; “Control” is the negative control (water).
    Pcr Reagents, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr reagents/product/Thermo Fisher
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    Thermo Fisher gene exp plin2 hs00605340 m1
    Localization of ATGL, <t>PLIN2</t> and PLIN3 in term placenta tissue. ( a ) ATGL was localized exclusively in the syncytiotrophoblast layer. ( b ) PLIN2 was detectable on the syncytiotrophoblast by a clear punctate staining. ( c ) PLIN3 was mainly localized in the syncytiotrophoblast. ( d ) Cytokeratin 7 was used as positive control for the syncytiotrophoblast layer. Negative control staining for rabbit IgG ( e ) and mouse IgG ( f ) was performed with equal IgG concentrations. Scale bar, 50 μm.
    Gene Exp Plin2 Hs00605340 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Conditional mutagenesis pipeline. Upon deciding which exon to flox, we recommend sequencing the target sites to identify polymorphisms compared to reference genome. Next, sgRNAs should be designed and tested by either direct sequencing of PCR fragments, T7 endonuclease assay or loss of a restriction enzyme site on bulk DNA from pooled embryos. Once active sgRNAs have been identified, experiments integrating the first loxP site should be performed. In the absence of conclusive data that certain HDR template performs significantly better than others (such experiments are not practical at the only level that matters–germline transmission), we recommend using the design we successfully used to integrate loxP into fleer , aldh1a2 and tcf21 : antisense to PAM, with 49-base 5’ homology arm and 21-base 3’ homology arm, with 3-nucleotide spacers flanking loxP site. As injected embryos are being raised, we then recommend to optimize nested PCR screening conditions DNA from pools of injected embryos. We found “plain” Taq polymerases (NEB #M0270, Thermofisher Scientific 2x PCR Master Mix Cat# AB-0575/DC and #EP0402, or similar) to be most suitable for nested PCR. In contrast, high-performance mixes such as Platinum Taq (Thermofisher #10966026) or Kapa 2G Fast ReadyMix + dye (Kapa Biosystems- KM5101) yield very high background and may only be used for the second (nested) reaction. It is also very helpful if primers for one end of the nested PCR are anchored within an exon. We recommend generating a deletion allele in parallel with integration of the first loxP site. Once highly active sgRNAs are identified, we recommend injecting a pair of sgRNAs flanking the exon to be floxed in order to confirm that removal of selected exon will yield an overt phenotype. We have been able to very efficiently delete exon 8 of aldh1a2 using sgRNAs spaced just over 450 base pairs, but larger deletions are certainly feasible too (1, 2). An additional benefit of a deletion allele is that it can be crossed to Cre drivers of interest, eliminating the need to back-cross floxed allele to obtain homozygotes. Screening for germline transmission should be performed by nested PCR on pools of embryos obtained from incross. Positive crosses should be analyzed by performing short flanking PCR (ideally under 400 base pairs) on DNA from individual embryos. Bands corresponding to loxP-containing allele should be extracted from gel and sequenced to ensure presence of intact loxP site. Siblings of screened embryos should be raised to adulthood and loxP-positive F1s should be identified by flanking PCR as well. Two strategies can be used for integration of the second loxP site. If speed is the main priority, loxP-positive F1s can be in-crossed and second sgRNA/HDR oligonucleotide can be injected. The main drawback of this strategy that there is only 50% likelihood that the second loxP site will integrate into a chromosome already containing the first loxP. It is therefore necessary to genotype adults for presence of the first loxP site before out-crossing. Even though we successfully used this strategy to engineer a floxed allele of tbx20 , we consider it impractical and would generally recommend to first generate adults homozygous for the first loxP site, incross them and then inject the second sgRNA/HDR oligonucleotide.

    Journal: PLoS Genetics

    Article Title: Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish

    doi: 10.1371/journal.pgen.1007754

    Figure Lengend Snippet: Conditional mutagenesis pipeline. Upon deciding which exon to flox, we recommend sequencing the target sites to identify polymorphisms compared to reference genome. Next, sgRNAs should be designed and tested by either direct sequencing of PCR fragments, T7 endonuclease assay or loss of a restriction enzyme site on bulk DNA from pooled embryos. Once active sgRNAs have been identified, experiments integrating the first loxP site should be performed. In the absence of conclusive data that certain HDR template performs significantly better than others (such experiments are not practical at the only level that matters–germline transmission), we recommend using the design we successfully used to integrate loxP into fleer , aldh1a2 and tcf21 : antisense to PAM, with 49-base 5’ homology arm and 21-base 3’ homology arm, with 3-nucleotide spacers flanking loxP site. As injected embryos are being raised, we then recommend to optimize nested PCR screening conditions DNA from pools of injected embryos. We found “plain” Taq polymerases (NEB #M0270, Thermofisher Scientific 2x PCR Master Mix Cat# AB-0575/DC and #EP0402, or similar) to be most suitable for nested PCR. In contrast, high-performance mixes such as Platinum Taq (Thermofisher #10966026) or Kapa 2G Fast ReadyMix + dye (Kapa Biosystems- KM5101) yield very high background and may only be used for the second (nested) reaction. It is also very helpful if primers for one end of the nested PCR are anchored within an exon. We recommend generating a deletion allele in parallel with integration of the first loxP site. Once highly active sgRNAs are identified, we recommend injecting a pair of sgRNAs flanking the exon to be floxed in order to confirm that removal of selected exon will yield an overt phenotype. We have been able to very efficiently delete exon 8 of aldh1a2 using sgRNAs spaced just over 450 base pairs, but larger deletions are certainly feasible too (1, 2). An additional benefit of a deletion allele is that it can be crossed to Cre drivers of interest, eliminating the need to back-cross floxed allele to obtain homozygotes. Screening for germline transmission should be performed by nested PCR on pools of embryos obtained from incross. Positive crosses should be analyzed by performing short flanking PCR (ideally under 400 base pairs) on DNA from individual embryos. Bands corresponding to loxP-containing allele should be extracted from gel and sequenced to ensure presence of intact loxP site. Siblings of screened embryos should be raised to adulthood and loxP-positive F1s should be identified by flanking PCR as well. Two strategies can be used for integration of the second loxP site. If speed is the main priority, loxP-positive F1s can be in-crossed and second sgRNA/HDR oligonucleotide can be injected. The main drawback of this strategy that there is only 50% likelihood that the second loxP site will integrate into a chromosome already containing the first loxP. It is therefore necessary to genotype adults for presence of the first loxP site before out-crossing. Even though we successfully used this strategy to engineer a floxed allele of tbx20 , we consider it impractical and would generally recommend to first generate adults homozygous for the first loxP site, incross them and then inject the second sgRNA/HDR oligonucleotide.

    Article Snippet: All PCR reactions were performed using either NEB 2X Taq Master Mix (M0270L), Thermo Scientific Taq (EP0402 and 2x PCR Master Mix Cat# AB-0575/DC), or Kapa 2G Fast ReadyMix +dye (Kapa Biosystems-KM5101).

    Techniques: Mutagenesis, Sequencing, Polymerase Chain Reaction, Transmission Assay, Injection, Nested PCR

    A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Journal: PLoS ONE

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    doi: 10.1371/journal.pone.0184162

    Figure Lengend Snippet: A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Techniques: Mobility Shift, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Journal: PLoS ONE

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    doi: 10.1371/journal.pone.0184162

    Figure Lengend Snippet: Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Techniques: Polymerase Chain Reaction, Amplification, Marker

    Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Journal: PLoS ONE

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    doi: 10.1371/journal.pone.0184162

    Figure Lengend Snippet: Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Techniques: Activity Assay

    Example gel electrophoresis image of a successful 16S rRNA gene PCR amplification from fall 2015. Lanes labeled according to contents: “Sample A11-22” is the amplicon from isolate DNA (expected size 1466 bp); “Ladder” is Lambda HindIII digest ladder (NEB N3012S), with the lowest visible band at 2027 bp; “Control” is the negative control (water).

    Journal: bioRxiv

    Article Title: The CURE for Cultivating Fastidious Microbes

    doi: 10.1101/167130

    Figure Lengend Snippet: Example gel electrophoresis image of a successful 16S rRNA gene PCR amplification from fall 2015. Lanes labeled according to contents: “Sample A11-22” is the amplicon from isolate DNA (expected size 1466 bp); “Ladder” is Lambda HindIII digest ladder (NEB N3012S), with the lowest visible band at 2027 bp; “Control” is the negative control (water).

    Article Snippet: Segment 3 (blue ) We recommend that instructors aliquot the required amount of DNA extraction reagents ( Appendix 5 -Power Water DNA Isolation Kit; Mo Bio Laboratories) and PCR reagents ( Appendix 6-Taq, MgCl2, and buffer-ThermoFisher; 10mM AMRESCO dNTPs-VWR Life Sciences; 27F/1492R primers) for each group to prevent cross-contamination.

    Techniques: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Labeling, Negative Control

    Grade distributions for two sections of mCURE students during each of two semesters in the 2015-2016 school year. Fall 2015 consisted of ~50 Honors College students majoring in biology. The topics for the five quizzes (Q1-Q5) were as follows: Q1 = Safety, Controls; Q2 = Experimental design, Scientific writing; Q3 = DNA extraction; Q4 = PCR; Q5 = Gel electrophoresis, Purpose of sequencing, Primer design. Spring 2016 consisted of ~60 mostly non-biology major students. The topics for the five quizzes (Q1-Q5) were as follows: Q1 = Dilutions, Pipetting, Safety, Controls, Scientific writing; Q2 = Experimental design, Dilution, Pipetting, Controls; Q3 = DNA extraction; Q4 = PCR, Primer selection/design, Gel electrophoresis; Q5 = Purpose of sequencing, Sequence analysis. The grades for both semesters were assigned based on the following score criteria: A = 90-100%; B = 80-90%; C = 70-80%; D = 60-70%; F =

    Journal: bioRxiv

    Article Title: The CURE for Cultivating Fastidious Microbes

    doi: 10.1101/167130

    Figure Lengend Snippet: Grade distributions for two sections of mCURE students during each of two semesters in the 2015-2016 school year. Fall 2015 consisted of ~50 Honors College students majoring in biology. The topics for the five quizzes (Q1-Q5) were as follows: Q1 = Safety, Controls; Q2 = Experimental design, Scientific writing; Q3 = DNA extraction; Q4 = PCR; Q5 = Gel electrophoresis, Purpose of sequencing, Primer design. Spring 2016 consisted of ~60 mostly non-biology major students. The topics for the five quizzes (Q1-Q5) were as follows: Q1 = Dilutions, Pipetting, Safety, Controls, Scientific writing; Q2 = Experimental design, Dilution, Pipetting, Controls; Q3 = DNA extraction; Q4 = PCR, Primer selection/design, Gel electrophoresis; Q5 = Purpose of sequencing, Sequence analysis. The grades for both semesters were assigned based on the following score criteria: A = 90-100%; B = 80-90%; C = 70-80%; D = 60-70%; F =

    Article Snippet: Segment 3 (blue ) We recommend that instructors aliquot the required amount of DNA extraction reagents ( Appendix 5 -Power Water DNA Isolation Kit; Mo Bio Laboratories) and PCR reagents ( Appendix 6-Taq, MgCl2, and buffer-ThermoFisher; 10mM AMRESCO dNTPs-VWR Life Sciences; 27F/1492R primers) for each group to prevent cross-contamination.

    Techniques: DNA Extraction, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Sequencing, Selection

    Localization of ATGL, PLIN2 and PLIN3 in term placenta tissue. ( a ) ATGL was localized exclusively in the syncytiotrophoblast layer. ( b ) PLIN2 was detectable on the syncytiotrophoblast by a clear punctate staining. ( c ) PLIN3 was mainly localized in the syncytiotrophoblast. ( d ) Cytokeratin 7 was used as positive control for the syncytiotrophoblast layer. Negative control staining for rabbit IgG ( e ) and mouse IgG ( f ) was performed with equal IgG concentrations. Scale bar, 50 μm.

    Journal: International Journal of Obesity (2005)

    Article Title: Maternal obesity modulates intracellular lipid turnover in the human term placenta

    doi: 10.1038/ijo.2016.188

    Figure Lengend Snippet: Localization of ATGL, PLIN2 and PLIN3 in term placenta tissue. ( a ) ATGL was localized exclusively in the syncytiotrophoblast layer. ( b ) PLIN2 was detectable on the syncytiotrophoblast by a clear punctate staining. ( c ) PLIN3 was mainly localized in the syncytiotrophoblast. ( d ) Cytokeratin 7 was used as positive control for the syncytiotrophoblast layer. Negative control staining for rabbit IgG ( e ) and mouse IgG ( f ) was performed with equal IgG concentrations. Scale bar, 50 μm.

    Article Snippet: Quantitative real-time PCR (qRT-PCR) gene expression assays and antibodies for protein analysis Gene expression assays were purchased from Applied Biosystems (Darmstadt, Germany) and included the following genes: adipose triglyceride lipase ATGL (PNPLA2, gene ID 57104, Taq man assay no. Hs00386101_m1), hormone sensitive lipase, HSL (LIPE, gene ID 3991, Taq man assay no. Hs00193510_m1), α/β hydrolase domain containing 5, CGI-58, (ABHD5, gene ID 51099, Taq man assay no. HS01104373_m1), perilipin 1 (PLIN1, gene ID 5346, Taq man assay no. Hs00160173_m1), perilipin 2, ADRP (PLIN2, gene ID 123, Taq man assay no. HS00605340_m1), perilipin 3, TIP47 (PLIN3, gene ID 10226, Taq man assay no. HS00998416_m1), perilipin 4 (PLIN4, gene ID 729359, Taq man assay no. Hs00287411_m1), perilipin 5, OXPAT (PLIN5, gene ID 440503, Taq man assay no. Hs00965990_m1), TATA box-binding protein (TBP, gene ID 6908, Taq man assay no. Hs00427620_m1).

    Techniques: Staining, Positive Control, Negative Control

    Placental CGI-58 is regulated by maternal pre-pregnancy obesity. Lower panel: representative western blots ( n =4 per group) of total placental tissue. ( a ) ATGL protein expression. ( b ) PLIN2 protein expression. ( c ) CGI-58 protein expression. All protein signals were quantitated by densitometry, normalized to β-actin as loading control and to one protein sample which was used on each blot to correct for inter blot variations. Sum rank test was performed, differences between the lean (BMI

    Journal: International Journal of Obesity (2005)

    Article Title: Maternal obesity modulates intracellular lipid turnover in the human term placenta

    doi: 10.1038/ijo.2016.188

    Figure Lengend Snippet: Placental CGI-58 is regulated by maternal pre-pregnancy obesity. Lower panel: representative western blots ( n =4 per group) of total placental tissue. ( a ) ATGL protein expression. ( b ) PLIN2 protein expression. ( c ) CGI-58 protein expression. All protein signals were quantitated by densitometry, normalized to β-actin as loading control and to one protein sample which was used on each blot to correct for inter blot variations. Sum rank test was performed, differences between the lean (BMI

    Article Snippet: Quantitative real-time PCR (qRT-PCR) gene expression assays and antibodies for protein analysis Gene expression assays were purchased from Applied Biosystems (Darmstadt, Germany) and included the following genes: adipose triglyceride lipase ATGL (PNPLA2, gene ID 57104, Taq man assay no. Hs00386101_m1), hormone sensitive lipase, HSL (LIPE, gene ID 3991, Taq man assay no. Hs00193510_m1), α/β hydrolase domain containing 5, CGI-58, (ABHD5, gene ID 51099, Taq man assay no. HS01104373_m1), perilipin 1 (PLIN1, gene ID 5346, Taq man assay no. Hs00160173_m1), perilipin 2, ADRP (PLIN2, gene ID 123, Taq man assay no. HS00605340_m1), perilipin 3, TIP47 (PLIN3, gene ID 10226, Taq man assay no. HS00998416_m1), perilipin 4 (PLIN4, gene ID 729359, Taq man assay no. Hs00287411_m1), perilipin 5, OXPAT (PLIN5, gene ID 440503, Taq man assay no. Hs00965990_m1), TATA box-binding protein (TBP, gene ID 6908, Taq man assay no. Hs00427620_m1).

    Techniques: Western Blot, Expressing

    Association of placental CGI-58 with maternal metabolic parameters. Correlation analysis was performed between CGI-58 mRNA or protein expression in placenta tissue and maternal pre-pregnancy BMI ( a ) or maternal plasma insulin ( b ). PLIN2 mRNA and protein levels were correlated with ( c ) maternal pre-pregnancy BMI and maternal plasma insulin levels ( d ). Black circles (•) protein expression on thr open circles (○) mRNA expression. Spearman correlation was defined as significant if P -values were

    Journal: International Journal of Obesity (2005)

    Article Title: Maternal obesity modulates intracellular lipid turnover in the human term placenta

    doi: 10.1038/ijo.2016.188

    Figure Lengend Snippet: Association of placental CGI-58 with maternal metabolic parameters. Correlation analysis was performed between CGI-58 mRNA or protein expression in placenta tissue and maternal pre-pregnancy BMI ( a ) or maternal plasma insulin ( b ). PLIN2 mRNA and protein levels were correlated with ( c ) maternal pre-pregnancy BMI and maternal plasma insulin levels ( d ). Black circles (•) protein expression on thr open circles (○) mRNA expression. Spearman correlation was defined as significant if P -values were

    Article Snippet: Quantitative real-time PCR (qRT-PCR) gene expression assays and antibodies for protein analysis Gene expression assays were purchased from Applied Biosystems (Darmstadt, Germany) and included the following genes: adipose triglyceride lipase ATGL (PNPLA2, gene ID 57104, Taq man assay no. Hs00386101_m1), hormone sensitive lipase, HSL (LIPE, gene ID 3991, Taq man assay no. Hs00193510_m1), α/β hydrolase domain containing 5, CGI-58, (ABHD5, gene ID 51099, Taq man assay no. HS01104373_m1), perilipin 1 (PLIN1, gene ID 5346, Taq man assay no. Hs00160173_m1), perilipin 2, ADRP (PLIN2, gene ID 123, Taq man assay no. HS00605340_m1), perilipin 3, TIP47 (PLIN3, gene ID 10226, Taq man assay no. HS00998416_m1), perilipin 4 (PLIN4, gene ID 729359, Taq man assay no. Hs00287411_m1), perilipin 5, OXPAT (PLIN5, gene ID 440503, Taq man assay no. Hs00965990_m1), TATA box-binding protein (TBP, gene ID 6908, Taq man assay no. Hs00427620_m1).

    Techniques: Expressing, IF-P