real time quantitative reverse transcription polymerase chain reaction qrt pcr  (Thermo Fisher)


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    Structured Review

    Thermo Fisher real time quantitative reverse transcription polymerase chain reaction qrt pcr
    Confirmation of gapmer activity on intended targets. Knockdown of ( A ) Pcsk9 mRNA and ( B ) Apob mRNA in mouse liver as measured by <t>qRT-PCR</t> following three 10 mg/kg doses on consecutive days of gapmers P1 and P2 (targeted to Pcsk9 ), or 10 and 5 mg/kg, respectively, of A1 and A2 (targeted to Apob ). Data represent the average ± one standard deviation relative to saline-treated controls (Ctrl) with n = 5 per treatment group. Significance of differences in average transcript levels between groups evaluated by Student's t -test. * P
    Real Time Quantitative Reverse Transcription Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 11824 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice"

    Article Title: Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky397

    Confirmation of gapmer activity on intended targets. Knockdown of ( A ) Pcsk9 mRNA and ( B ) Apob mRNA in mouse liver as measured by qRT-PCR following three 10 mg/kg doses on consecutive days of gapmers P1 and P2 (targeted to Pcsk9 ), or 10 and 5 mg/kg, respectively, of A1 and A2 (targeted to Apob ). Data represent the average ± one standard deviation relative to saline-treated controls (Ctrl) with n = 5 per treatment group. Significance of differences in average transcript levels between groups evaluated by Student's t -test. * P
    Figure Legend Snippet: Confirmation of gapmer activity on intended targets. Knockdown of ( A ) Pcsk9 mRNA and ( B ) Apob mRNA in mouse liver as measured by qRT-PCR following three 10 mg/kg doses on consecutive days of gapmers P1 and P2 (targeted to Pcsk9 ), or 10 and 5 mg/kg, respectively, of A1 and A2 (targeted to Apob ). Data represent the average ± one standard deviation relative to saline-treated controls (Ctrl) with n = 5 per treatment group. Significance of differences in average transcript levels between groups evaluated by Student's t -test. * P

    Techniques Used: Activity Assay, Quantitative RT-PCR, Standard Deviation

    Evaluation of gapmer activity on intended and unintended targets for five gapmers with the same target regions but different binding affinities. ( A ) Expected binding regions in Tradd (intended target) and two unintended targets Ptprd and Adipor1 . Mismatched bases in the five gapmers T1–T5 are indicated in red. Gray lines indicate gapmer binding region. For gapmer sequences, uppercase bold indicates LNA and lowercase indicates DNA. ( B ) Knockdown of the intended target, Tradd mRNA and two unintended targets, Ptprd and Adipor1 mRNA, in mouse liver following five 15 mg/kg doses of gapmers T1–T5 over 2 weeks as measured by qRT-PCR. ( C ) Levels of ALT in mouse serum following five 15 mg/kg doses of gapmers T1–T5 over 2 weeks. Data represent the average ± one standard deviation relative to saline-treated controls (Ctrl) with n = 5 per treatment group. Significance of differences in average transcript levels between groups were evaluated by Student’s t -test. * P
    Figure Legend Snippet: Evaluation of gapmer activity on intended and unintended targets for five gapmers with the same target regions but different binding affinities. ( A ) Expected binding regions in Tradd (intended target) and two unintended targets Ptprd and Adipor1 . Mismatched bases in the five gapmers T1–T5 are indicated in red. Gray lines indicate gapmer binding region. For gapmer sequences, uppercase bold indicates LNA and lowercase indicates DNA. ( B ) Knockdown of the intended target, Tradd mRNA and two unintended targets, Ptprd and Adipor1 mRNA, in mouse liver following five 15 mg/kg doses of gapmers T1–T5 over 2 weeks as measured by qRT-PCR. ( C ) Levels of ALT in mouse serum following five 15 mg/kg doses of gapmers T1–T5 over 2 weeks. Data represent the average ± one standard deviation relative to saline-treated controls (Ctrl) with n = 5 per treatment group. Significance of differences in average transcript levels between groups were evaluated by Student’s t -test. * P

    Techniques Used: Activity Assay, Binding Assay, Quantitative RT-PCR, Standard Deviation

    Evaluation of bindi ng affinity and potency selectivity ratios for four gapmers of different length but the same core target region. ( A ) Expected binding regions in UBE3C (intended target) and four unintended targets. Mismatched bases in the four gapmers U1–U4 are indicated in red. Gray lines indicate nested gapmer binding regions. For gapmer sequences, uppercase bold indicates LNA and lowercase indicates DNA. ( B ) T m s for gapmers U1–U4 binding to UBE3C and each of the unintended targets. ( C ) For each of the unintended targets, the EC 50 for each gapmer relative to the EC 50 for UBE3C was estimated from an 8-point CRC measured by qRT-PCR ( n = 2). Error bars represent one standard deviation as determined from the nonlinear regression using error propagation.
    Figure Legend Snippet: Evaluation of bindi ng affinity and potency selectivity ratios for four gapmers of different length but the same core target region. ( A ) Expected binding regions in UBE3C (intended target) and four unintended targets. Mismatched bases in the four gapmers U1–U4 are indicated in red. Gray lines indicate nested gapmer binding regions. For gapmer sequences, uppercase bold indicates LNA and lowercase indicates DNA. ( B ) T m s for gapmers U1–U4 binding to UBE3C and each of the unintended targets. ( C ) For each of the unintended targets, the EC 50 for each gapmer relative to the EC 50 for UBE3C was estimated from an 8-point CRC measured by qRT-PCR ( n = 2). Error bars represent one standard deviation as determined from the nonlinear regression using error propagation.

    Techniques Used: Binding Assay, Quantitative RT-PCR, Standard Deviation

    2) Product Images from "An optimized isolation protocol yields high‐quality RNA from cassava tissues (Manihot esculenta Crantz)"

    Article Title: An optimized isolation protocol yields high‐quality RNA from cassava tissues (Manihot esculenta Crantz)

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12561

    Quality analyses of cDNA from total RNA that was extracted using the present modified protocol; leaf samples were taken from the cassava genotypes HMC ‐1 and ESP at indicated time points, and qRT ‐ PCR analyses were performed using primers for 18S. (A) Amplification plot and melting curves for HMC ‐1 genotype; (B) amplification plot and melting curve for ESP genotype. For details of all curves, see Fig. 3 legend.
    Figure Legend Snippet: Quality analyses of cDNA from total RNA that was extracted using the present modified protocol; leaf samples were taken from the cassava genotypes HMC ‐1 and ESP at indicated time points, and qRT ‐ PCR analyses were performed using primers for 18S. (A) Amplification plot and melting curves for HMC ‐1 genotype; (B) amplification plot and melting curve for ESP genotype. For details of all curves, see Fig. 3 legend.

    Techniques Used: Modification, End-sequence Profiling, Quantitative RT-PCR, Amplification

    Conventional PCR using primers for G3pdh and cDNA from total RNA that was extracted from various tissues of the ESP genotype grown in pots and in vitro at 15 °C and 30 °C under LD conditions for 16 h inside growth chambers. Biological repeat 1: left panel; M, 1 Kb ladder; TL 48, buds and young leaves from potted plants at 15 °C; TL 54, stems from potted plants grown at 15 °C; right panel, NTC , negative control, water template; gDNA , cassava genomic DNA ; IL 126, roots from in vitro samples grown at 30 °C; IL 138, leaves from in vitro samples grown at 30 °C; biological repeat 2: IL 141, stems from in vitro samples grown at 30 °C. See Fig. S2 for complete PCR analyses of total RNA from potted and in vitro samples.
    Figure Legend Snippet: Conventional PCR using primers for G3pdh and cDNA from total RNA that was extracted from various tissues of the ESP genotype grown in pots and in vitro at 15 °C and 30 °C under LD conditions for 16 h inside growth chambers. Biological repeat 1: left panel; M, 1 Kb ladder; TL 48, buds and young leaves from potted plants at 15 °C; TL 54, stems from potted plants grown at 15 °C; right panel, NTC , negative control, water template; gDNA , cassava genomic DNA ; IL 126, roots from in vitro samples grown at 30 °C; IL 138, leaves from in vitro samples grown at 30 °C; biological repeat 2: IL 141, stems from in vitro samples grown at 30 °C. See Fig. S2 for complete PCR analyses of total RNA from potted and in vitro samples.

    Techniques Used: Polymerase Chain Reaction, End-sequence Profiling, In Vitro, Negative Control

    Conventional PCR with G3pdh primers was performed using cDNA that was reverse‐transcribed from total RNA after extraction using the present protocol. Tissue samples were taken from HMC ‐1 (A) and Esparrago ( ESP ; (B) genotypes (Manihot esculenta) that were grown under long‐day ( LD ) conditions for indicated times; M: 1 Kb ladder; 85: HMC ‐1, 0 h LD ; 86: HMC ‐1, 4 h LD ; 87: HMC ‐1, 8 h LD ; 88: HMC ‐1, 12 h LD ; 89: HMC ‐1, 16 h LD ; 90: HMC ‐1, 20 h LD ; 91: HMC ‐1, 24 h LD ; 197: ESP , 0 h LD ; 198: ESP , 4 h LD ; 199: ESP , 8 h LD ; 200: ESP , 12 h LD ; 201: ESP , 16 h LD ; 202: ESP , 20 h LD ; 203: ESP , 24 h LD ; NTC : non‐template negative control (water template), and gDNA : cassava genomic DNA.
    Figure Legend Snippet: Conventional PCR with G3pdh primers was performed using cDNA that was reverse‐transcribed from total RNA after extraction using the present protocol. Tissue samples were taken from HMC ‐1 (A) and Esparrago ( ESP ; (B) genotypes (Manihot esculenta) that were grown under long‐day ( LD ) conditions for indicated times; M: 1 Kb ladder; 85: HMC ‐1, 0 h LD ; 86: HMC ‐1, 4 h LD ; 87: HMC ‐1, 8 h LD ; 88: HMC ‐1, 12 h LD ; 89: HMC ‐1, 16 h LD ; 90: HMC ‐1, 20 h LD ; 91: HMC ‐1, 24 h LD ; 197: ESP , 0 h LD ; 198: ESP , 4 h LD ; 199: ESP , 8 h LD ; 200: ESP , 12 h LD ; 201: ESP , 16 h LD ; 202: ESP , 20 h LD ; 203: ESP , 24 h LD ; NTC : non‐template negative control (water template), and gDNA : cassava genomic DNA.

    Techniques Used: Polymerase Chain Reaction, End-sequence Profiling, Negative Control

    3) Product Images from "Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞"

    Article Title: Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞

    Journal:

    doi: 10.1091/mbc.E04-09-0788

    In situ hybridization against Fcr1 stains most centromeres. Fluorescent in situ hybridization of digoxigenin-labeled Fcr1 probe (red) against DAPI-stained metaphase chromosome spreads (blue) from cultured cells. Indirect immunofluorescence against XCENP-A
    Figure Legend Snippet: In situ hybridization against Fcr1 stains most centromeres. Fluorescent in situ hybridization of digoxigenin-labeled Fcr1 probe (red) against DAPI-stained metaphase chromosome spreads (blue) from cultured cells. Indirect immunofluorescence against XCENP-A

    Techniques Used: In Situ Hybridization, Labeling, Staining, Cell Culture, Immunofluorescence

    Identification of Fcr1, a 174-base pair centromere-associated repeat. (A) Consensus sequence of the monomer unit (with ends defined arbitrarily) of the DNA satellite cloned from α-CENP-A chromatin immunoprecipitates. The box indicates the putative
    Figure Legend Snippet: Identification of Fcr1, a 174-base pair centromere-associated repeat. (A) Consensus sequence of the monomer unit (with ends defined arbitrarily) of the DNA satellite cloned from α-CENP-A chromatin immunoprecipitates. The box indicates the putative

    Techniques Used: Sequencing, Clone Assay

    Fcr1 is present in large ordered arrays in the frog genome. Southern blot of Nsi I-digested genomic DNA with radioactive probe for Fcr1. Genomic DNA (1 μg per lane) was digested with 25 U (lane 1), 5 U (lane 2), 1 U (lane 3), 0.2 U (lane 4), or
    Figure Legend Snippet: Fcr1 is present in large ordered arrays in the frog genome. Southern blot of Nsi I-digested genomic DNA with radioactive probe for Fcr1. Genomic DNA (1 μg per lane) was digested with 25 U (lane 1), 5 U (lane 2), 1 U (lane 3), 0.2 U (lane 4), or

    Techniques Used: Southern Blot

    Fcr1 hybridization against lampbrush chromosomes confirms consistent staining of 11 of the 18 frog chromosomes. Each of the 18 X. laevis lampbrush chromosomes (stained with DAPI, blue) can be identified by the distinct patterns of loci stained by antibodies
    Figure Legend Snippet: Fcr1 hybridization against lampbrush chromosomes confirms consistent staining of 11 of the 18 frog chromosomes. Each of the 18 X. laevis lampbrush chromosomes (stained with DAPI, blue) can be identified by the distinct patterns of loci stained by antibodies

    Techniques Used: Hybridization, Staining

    4) Product Images from "A Designed Zinc-finger Transcriptional Repressor of Phospholamban Improves Function of the Failing Heart"

    Article Title: A Designed Zinc-finger Transcriptional Repressor of Phospholamban Improves Function of the Failing Heart

    Journal: Molecular Therapy

    doi: 10.1038/mt.2012.80

    Targeting and in vitro analysis of zinc-finger protein transcription factor (ZFP TF) repressors of the rat phospholamban ( PLN ) gene . ( a ) Nuclei of H9C2(2-1) cells were isolated and titrated with DNase I (Worthington) as described. 3 Isolated genomic DNA was double-digested with Bgl II and Afl II. Aliquots of double-digested DNA were subjected to Hind III or Nco I digest for use as genomic size markers. The location of the DNase I hypersensitive region (DHS) is indicated by a solid bar next to the blot and in the schematic diagram. The transcription start site (+1) is marked by the hooked arrow. The nucleotide locations of the Nco I site and the Hind III site are relative to the transcription start site. The open box indicates the location of the probe for Southern blot. ( b ) The structure of the assembled ZFP repressors is depicted in the diagram. NLS, nuclear localization signal; KOX1, repression domain of KOX1; FLAG, FLAG epitope tag. ( c ) H9C2(2-1) cells were transiently transfected with plasmids expressing the indicated ZFP TFs ( Supplementary Materials and Methods ). After 72 hours mRNA was harvested and the levels of PLN mRNA and GAPDH mRNA were quantified using real-time reverse transcription (RT)-PCR (Taqman). The PLN level was normalized to that of GAPDH. ( d ) Variants of the 6439-KOX repressor protein in which the KOX repression domain was either removed (6439-NF) or replaced with an activator (6439-VP16), were tested for function in H9C2(2-1) cells. PLN mRNA levels were measured as in b . ( e ) H9C2(2-1) cells were transiently transfected as indicated, and 72 hours later whole cell extracts were collected and subjected to western blot analysis. A PLN cDNA was used as a positive control for the anti-PLN antibody.
    Figure Legend Snippet: Targeting and in vitro analysis of zinc-finger protein transcription factor (ZFP TF) repressors of the rat phospholamban ( PLN ) gene . ( a ) Nuclei of H9C2(2-1) cells were isolated and titrated with DNase I (Worthington) as described. 3 Isolated genomic DNA was double-digested with Bgl II and Afl II. Aliquots of double-digested DNA were subjected to Hind III or Nco I digest for use as genomic size markers. The location of the DNase I hypersensitive region (DHS) is indicated by a solid bar next to the blot and in the schematic diagram. The transcription start site (+1) is marked by the hooked arrow. The nucleotide locations of the Nco I site and the Hind III site are relative to the transcription start site. The open box indicates the location of the probe for Southern blot. ( b ) The structure of the assembled ZFP repressors is depicted in the diagram. NLS, nuclear localization signal; KOX1, repression domain of KOX1; FLAG, FLAG epitope tag. ( c ) H9C2(2-1) cells were transiently transfected with plasmids expressing the indicated ZFP TFs ( Supplementary Materials and Methods ). After 72 hours mRNA was harvested and the levels of PLN mRNA and GAPDH mRNA were quantified using real-time reverse transcription (RT)-PCR (Taqman). The PLN level was normalized to that of GAPDH. ( d ) Variants of the 6439-KOX repressor protein in which the KOX repression domain was either removed (6439-NF) or replaced with an activator (6439-VP16), were tested for function in H9C2(2-1) cells. PLN mRNA levels were measured as in b . ( e ) H9C2(2-1) cells were transiently transfected as indicated, and 72 hours later whole cell extracts were collected and subjected to western blot analysis. A PLN cDNA was used as a positive control for the anti-PLN antibody.

    Techniques Used: In Vitro, Isolation, Southern Blot, FLAG-tag, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control

    Zinc-finger protein (ZFP)-driven repression of phospholamban ( PLN ) improves calcium transient and contractility in rat cardiomyocytes . ( a , b ) Cultured neonatal rat cardiomyocytes were transduced with adenoviral vectors encoding either the PLN repressor ZFP (6439-KOX) or the repressor domain alone without the ZFP DNA-binding domain (KOX) at MOI 200. Seventy-two hours later, PLN ( a ) mRNA levels and ( b ) protein levels were measured. ( c , d ) Cultured neonatal rat cardiomyocytes infected with either Ad-6439-KOX or Ad-KOX were loaded with Fluo-3 and calcium transients were assessed during excitation-contraction induced by electrical pacing. The rate of calcium reuptake into the sacroplasmic reticulum was measured as the time required to decrease cytosolic calcium levels to ( c ) 50% or ( d ) 5% of their peak after depolarization. ( n = 25 for KOX-transduced cells, n = 25 for 6439-KOX-transduced cells). ( e , f ) Either Ad-6439-KOX or Ad-KOX, mixed with an equal titer of Ad-FS-Red (an adenoviral vector encoding a Fluorescent marker) were codelivered to rat hearts via direct intramyocardial injections. Cardiomyocytes were isolated 72 hours after injection, and edge-detection-based analysis of single-cell contractility during electrical stimulation was performed on FS-Red-positive cells. The rate of ( e ) cell lengthening (relaxation), ( f ) percent cell shortening and the ( g ) rate of cell shortening (contraction) were determined ( n = 15 cells per group three separate injected hearts per treatment; five cotransduced cells per heart examined; and at least five separate contraction-relaxation cycles were analyzed per cell).
    Figure Legend Snippet: Zinc-finger protein (ZFP)-driven repression of phospholamban ( PLN ) improves calcium transient and contractility in rat cardiomyocytes . ( a , b ) Cultured neonatal rat cardiomyocytes were transduced with adenoviral vectors encoding either the PLN repressor ZFP (6439-KOX) or the repressor domain alone without the ZFP DNA-binding domain (KOX) at MOI 200. Seventy-two hours later, PLN ( a ) mRNA levels and ( b ) protein levels were measured. ( c , d ) Cultured neonatal rat cardiomyocytes infected with either Ad-6439-KOX or Ad-KOX were loaded with Fluo-3 and calcium transients were assessed during excitation-contraction induced by electrical pacing. The rate of calcium reuptake into the sacroplasmic reticulum was measured as the time required to decrease cytosolic calcium levels to ( c ) 50% or ( d ) 5% of their peak after depolarization. ( n = 25 for KOX-transduced cells, n = 25 for 6439-KOX-transduced cells). ( e , f ) Either Ad-6439-KOX or Ad-KOX, mixed with an equal titer of Ad-FS-Red (an adenoviral vector encoding a Fluorescent marker) were codelivered to rat hearts via direct intramyocardial injections. Cardiomyocytes were isolated 72 hours after injection, and edge-detection-based analysis of single-cell contractility during electrical stimulation was performed on FS-Red-positive cells. The rate of ( e ) cell lengthening (relaxation), ( f ) percent cell shortening and the ( g ) rate of cell shortening (contraction) were determined ( n = 15 cells per group three separate injected hearts per treatment; five cotransduced cells per heart examined; and at least five separate contraction-relaxation cycles were analyzed per cell).

    Techniques Used: Cell Culture, Transduction, Binding Assay, Infection, Plasmid Preparation, Marker, Isolation, Injection

    5) Product Images from "The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle"

    Article Title: The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

    Journal: Respiratory Research

    doi: 10.1186/1465-9921-14-89

    Immunoblot analysis of dopamine D 1 -like receptors in human and guinea pig airway smooth muscle. Representative immunoblot analyses using antibodies against the dopamine D 1 receptor (A) and D 5 receptor (B) using 100 μg total protein prepared from freshly dissected native human tracheal airway smooth muscle (SM), cultured HASM cells from all three different sources [#1: cultures of immortalized HASM cells gifted from Dr. William Gerthoffer, #2: primary cultured cells gifted from Dr. Reynold A. Panettieri, Jr., and #3: primary cultured cells obtained from Lonza (Walkersville, MD)], freshly dissected native guinea pig tracheal SM, human kidney, and human or guinea pig brain cerebral cortex. White spaces between the lanes in (B) indicate that these lanes were located on the same immunoblot but were not located in neighboring lanes on the original gel and immunoblot image.
    Figure Legend Snippet: Immunoblot analysis of dopamine D 1 -like receptors in human and guinea pig airway smooth muscle. Representative immunoblot analyses using antibodies against the dopamine D 1 receptor (A) and D 5 receptor (B) using 100 μg total protein prepared from freshly dissected native human tracheal airway smooth muscle (SM), cultured HASM cells from all three different sources [#1: cultures of immortalized HASM cells gifted from Dr. William Gerthoffer, #2: primary cultured cells gifted from Dr. Reynold A. Panettieri, Jr., and #3: primary cultured cells obtained from Lonza (Walkersville, MD)], freshly dissected native guinea pig tracheal SM, human kidney, and human or guinea pig brain cerebral cortex. White spaces between the lanes in (B) indicate that these lanes were located on the same immunoblot but were not located in neighboring lanes on the original gel and immunoblot image.

    Techniques Used: Cell Culture

    Immunohistochemical detection of dopamine D 1 receptor expression in human and guinea pig airway smooth muscle. (A and C) Representative immunohistochemical staining of dopamine D 1 receptor (A) , and dopamine D 5 receptor (C) in paraformaldehyde/glutaraldehyde-fixed human trachea. (B and D) Anti-rabbit IgG isotype negative control in serial section of human tracheal airway smooth muscle. (E and G) Representative immunohistochemical staining of dopamine D 1 receptor (E) , and dopamine D 5 receptor (G) in paraformaldehyde-fixed guinea pig trachea. (F and H) Anti-rabbit IgG isotype negative control in serial section of guinea pig trachea. All sections were counterstained with hematoxylin. Calibration bar: 100 μm. ASM, airway smooth muscle; Epi, airway epithelium. Images are representative of at least 3 independent immunohistochemical analyses from human and guinea pig trachea.
    Figure Legend Snippet: Immunohistochemical detection of dopamine D 1 receptor expression in human and guinea pig airway smooth muscle. (A and C) Representative immunohistochemical staining of dopamine D 1 receptor (A) , and dopamine D 5 receptor (C) in paraformaldehyde/glutaraldehyde-fixed human trachea. (B and D) Anti-rabbit IgG isotype negative control in serial section of human tracheal airway smooth muscle. (E and G) Representative immunohistochemical staining of dopamine D 1 receptor (E) , and dopamine D 5 receptor (G) in paraformaldehyde-fixed guinea pig trachea. (F and H) Anti-rabbit IgG isotype negative control in serial section of guinea pig trachea. All sections were counterstained with hematoxylin. Calibration bar: 100 μm. ASM, airway smooth muscle; Epi, airway epithelium. Images are representative of at least 3 independent immunohistochemical analyses from human and guinea pig trachea.

    Techniques Used: Immunohistochemistry, Expressing, Staining, Negative Control

    Dopamine D 1 receptor agonist-induced cAMP activity in human airway smooth muscle cells. (A) The effects of dopamine (DA; 1 μM) or the dopamine D 1 -like receptor agonists (A68930 or SKF38393; 1 μM respectively) on cAMP production in cultured HASM cells. Number of experiments was shown in parentheses. (B) The effects of dopamine (1 μM) on cAMP production in the presence or absence of the dopamine D 2 receptor antagonist (L-741626; 1 μM for 30 min pretreatment before dopamine treatment). N = 4. (C) Concentration-dependent effect of dopamine D 1 -like receptor agonist A68930 (1 nM - 100 μM) on cAMP production in cultured HASM cells. The cells were incubated with A68930 for 20 min before cAMP assay. N = 4 - 13. (D) Time-course effect of A68930 (1 μM) on cAMP production in cultured human airway smooth muscle cells. N = 6. (E) The effect of dopamine D 1 -like receptor selective antagonists SCH23390 or SCH39166 on A68930-stimulated cAMP production in HASM cells. Cells were pretreated with SCH23390 (1 μM) or SCH39166 (1 μM) for 30 min pretreatment prior to A68930 (1 μM) treatment for 20 min. Data represent means ± SEM. N = 7 - 9. * P
    Figure Legend Snippet: Dopamine D 1 receptor agonist-induced cAMP activity in human airway smooth muscle cells. (A) The effects of dopamine (DA; 1 μM) or the dopamine D 1 -like receptor agonists (A68930 or SKF38393; 1 μM respectively) on cAMP production in cultured HASM cells. Number of experiments was shown in parentheses. (B) The effects of dopamine (1 μM) on cAMP production in the presence or absence of the dopamine D 2 receptor antagonist (L-741626; 1 μM for 30 min pretreatment before dopamine treatment). N = 4. (C) Concentration-dependent effect of dopamine D 1 -like receptor agonist A68930 (1 nM - 100 μM) on cAMP production in cultured HASM cells. The cells were incubated with A68930 for 20 min before cAMP assay. N = 4 - 13. (D) Time-course effect of A68930 (1 μM) on cAMP production in cultured human airway smooth muscle cells. N = 6. (E) The effect of dopamine D 1 -like receptor selective antagonists SCH23390 or SCH39166 on A68930-stimulated cAMP production in HASM cells. Cells were pretreated with SCH23390 (1 μM) or SCH39166 (1 μM) for 30 min pretreatment prior to A68930 (1 μM) treatment for 20 min. Data represent means ± SEM. N = 7 - 9. * P

    Techniques Used: Activity Assay, Cell Culture, Concentration Assay, Incubation, cAMP Assay

    RT-PCR analysis of dopamine D 1 -like receptors in human airway smooth muscle. Representative gel images of RT-PCR analysis of total RNA using primers specific for human dopamine D 1 receptor (A) and D 5 receptor (B) . Total RNA extracted from freshly dissected human tracheal airway smooth muscle or primary cultures of human airway smooth muscle (HASM) cells was analyzed. Lanes 1: base pair standards; Lanes 2: negative control (no RNA); Lanes 3: total RNA from freshly dissected native human airway smooth muscle tissue; Lanes 4: total RNA from primary cultured HASM cells obtained from Lonza (Walkersville, MD); Lanes 5: total RNA from whole human brain. −RT; cDNA synthesis reactions performed in the absence of reverse transcriptase confirming that PCR products were not arising from contaminating genomic DNA.
    Figure Legend Snippet: RT-PCR analysis of dopamine D 1 -like receptors in human airway smooth muscle. Representative gel images of RT-PCR analysis of total RNA using primers specific for human dopamine D 1 receptor (A) and D 5 receptor (B) . Total RNA extracted from freshly dissected human tracheal airway smooth muscle or primary cultures of human airway smooth muscle (HASM) cells was analyzed. Lanes 1: base pair standards; Lanes 2: negative control (no RNA); Lanes 3: total RNA from freshly dissected native human airway smooth muscle tissue; Lanes 4: total RNA from primary cultured HASM cells obtained from Lonza (Walkersville, MD); Lanes 5: total RNA from whole human brain. −RT; cDNA synthesis reactions performed in the absence of reverse transcriptase confirming that PCR products were not arising from contaminating genomic DNA.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Negative Control, Cell Culture, Polymerase Chain Reaction

    6) Product Images from "Pre-mRNA processing enhancer (PPE) elements from intronless genes play additional roles in mRNA biogenesis than do ones from intron-containing genes"

    Article Title: Pre-mRNA processing enhancer (PPE) elements from intronless genes play additional roles in mRNA biogenesis than do ones from intron-containing genes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki506

    The human c-Jun gene contains elements that enhance stabilization, 3′ end processing and cytoplasmic accumulation of intronless transcripts. ( A ) Autoradiogram of quantitative S1 nuclease mapping analysis of β-globin-like RNAs accumulated in the nucleus (N) and cytoplasm (C) 48 h after co-transfection of CV-1PD cells with the indicated plasmids and pRSV-Tori. The S1 nuclease mapping probes were the 5′ end-labeled ones shown in Figure 1B . The samples were analyzed as described in the legend to Figure 2A . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here. ( B ) Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. Portions of the RNA samples from the experiment shown in (A) were mapped with the 3′ end-labeled probe shown in Figure 1B . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here; they were calculated as described in the legend to Figure 2B . ( C ) The β-globin-like RNAs accumulated in the cytoplasm are unspliced. A portion of the RNA sample from the experiment shown in (A), lane 6, was reverse-transcribed and amplified by PCR as described in Figure 2C . Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. The rightmost lane contains a 1 kilobase pair DNA ladder. ( D ) Autoradiogram of quantitative S1 nuclease mapping analysis showing that a 201 nt region of c-Jun is sufficient to enhance cytoplasmic accumulation of intronless β-globin-like transcripts. COS-M6 cells were transfected with the indicated plasmids and processed as described in (A).
    Figure Legend Snippet: The human c-Jun gene contains elements that enhance stabilization, 3′ end processing and cytoplasmic accumulation of intronless transcripts. ( A ) Autoradiogram of quantitative S1 nuclease mapping analysis of β-globin-like RNAs accumulated in the nucleus (N) and cytoplasm (C) 48 h after co-transfection of CV-1PD cells with the indicated plasmids and pRSV-Tori. The S1 nuclease mapping probes were the 5′ end-labeled ones shown in Figure 1B . The samples were analyzed as described in the legend to Figure 2A . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here. ( B ) Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. Portions of the RNA samples from the experiment shown in (A) were mapped with the 3′ end-labeled probe shown in Figure 1B . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here; they were calculated as described in the legend to Figure 2B . ( C ) The β-globin-like RNAs accumulated in the cytoplasm are unspliced. A portion of the RNA sample from the experiment shown in (A), lane 6, was reverse-transcribed and amplified by PCR as described in Figure 2C . Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. The rightmost lane contains a 1 kilobase pair DNA ladder. ( D ) Autoradiogram of quantitative S1 nuclease mapping analysis showing that a 201 nt region of c-Jun is sufficient to enhance cytoplasmic accumulation of intronless β-globin-like transcripts. COS-M6 cells were transfected with the indicated plasmids and processed as described in (A).

    Techniques Used: Cotransfection, Labeling, Amplification, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Transfection

    Effects of the presence of a PRE, PPE, CTE or RRE in obviating the intron requirement for efficient 3′ end processing and cytoplasmic accumulation of human β-globin-like RNA. ( A ) Autoradiogram of quantitative S1 nuclease mapping analysis of the human β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells transfected with the plasmids shown in Figure 1A . CV-1PD cells were co-transfected with 2 μg of the indicated plasmid along with 1 μg of the SV40 T antigen-encoding plasmid pRSV-Tori. Nuclear (N) and cytoplasmic (C) RNAs were harvested 48 h later and analyzed by concurrent quantitative S1 nuclease mapping with the 5′ end-labeled β-globin and β-actin probes shown in Figure 1B . Transfection and RNA fractionation efficiencies were analyzed as described in Methods. The amount of β-globin-like RNA accumulated in the cytoplasm was internally normalized to the amount of cellular β-actin RNA present in the same sample. The numbers shown below the lanes are the percentages relative to the amount of β-globin-like RNA accumulated in the cytoplasm of cells transfected in parallel with β-β1(+)2(+) RNA, with normalization as well to the relative amounts of replicated β-globin plasmid DNA present in the nuclear fractions of those samples. These numbers are mean ± SEM values of data obtained from five independent experiments similar to the one shown here. ( B ) Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. The RNA samples from the experiment in panel A were analyzed with the 3′ end-labeled probe shown in Figure 1B . The numbers below the pairs of lanes are mean ± SEM values of the cleaved RNA (N + C) divided by the cleaved plus uncleaved RNA (N + C) times 100% of data obtained from three independent experiments similar to the one shown here. ( C ) The β-globin-like RNAs accumulated in the cytoplasm are unspliced. Portions of the cytoplasmic RNA samples from the experiment shown in (A) were reverse-transcribed and then amplified by PCR with the primers described in Methods (+RT, lanes 3, 6, 9, 12 and 15). As controls, PCR amplification reactions were performed on the RNA samples without prior reverse transcription (−RT, lanes 2, 5, 8, 11 and 14) and on the plasmid DNAs used in the transfections (DNA, lanes 4, 7, 10, 13 and 16). Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. Lane 1 contained 1 kilobase pair ladder DNA as size markers.
    Figure Legend Snippet: Effects of the presence of a PRE, PPE, CTE or RRE in obviating the intron requirement for efficient 3′ end processing and cytoplasmic accumulation of human β-globin-like RNA. ( A ) Autoradiogram of quantitative S1 nuclease mapping analysis of the human β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells transfected with the plasmids shown in Figure 1A . CV-1PD cells were co-transfected with 2 μg of the indicated plasmid along with 1 μg of the SV40 T antigen-encoding plasmid pRSV-Tori. Nuclear (N) and cytoplasmic (C) RNAs were harvested 48 h later and analyzed by concurrent quantitative S1 nuclease mapping with the 5′ end-labeled β-globin and β-actin probes shown in Figure 1B . Transfection and RNA fractionation efficiencies were analyzed as described in Methods. The amount of β-globin-like RNA accumulated in the cytoplasm was internally normalized to the amount of cellular β-actin RNA present in the same sample. The numbers shown below the lanes are the percentages relative to the amount of β-globin-like RNA accumulated in the cytoplasm of cells transfected in parallel with β-β1(+)2(+) RNA, with normalization as well to the relative amounts of replicated β-globin plasmid DNA present in the nuclear fractions of those samples. These numbers are mean ± SEM values of data obtained from five independent experiments similar to the one shown here. ( B ) Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. The RNA samples from the experiment in panel A were analyzed with the 3′ end-labeled probe shown in Figure 1B . The numbers below the pairs of lanes are mean ± SEM values of the cleaved RNA (N + C) divided by the cleaved plus uncleaved RNA (N + C) times 100% of data obtained from three independent experiments similar to the one shown here. ( C ) The β-globin-like RNAs accumulated in the cytoplasm are unspliced. Portions of the cytoplasmic RNA samples from the experiment shown in (A) were reverse-transcribed and then amplified by PCR with the primers described in Methods (+RT, lanes 3, 6, 9, 12 and 15). As controls, PCR amplification reactions were performed on the RNA samples without prior reverse transcription (−RT, lanes 2, 5, 8, 11 and 14) and on the plasmid DNAs used in the transfections (DNA, lanes 4, 7, 10, 13 and 16). Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. Lane 1 contained 1 kilobase pair ladder DNA as size markers.

    Techniques Used: Transfection, Plasmid Preparation, Labeling, Fractionation, Amplification, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis

    7) Product Images from "Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1"

    Article Title: Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074424

    Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p
    Figure Legend Snippet: Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation

    TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P
    Figure Legend Snippet: TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P

    Techniques Used: Expressing, Cell Culture, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Standard Deviation

    8) Product Images from "Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation"

    Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075648

    Relative levels of the HCV 3’ X-associated proteins in Huh7.5, Clone B, and 293T cells. HCV 3’ X-associated proteins were fractionated on the 2D gel. The level of each protein (spot) was quantified using the PDQuest software, expressed in parts per million (ppm) and compared among Huh7.5, Clone B, and 293T cells (from left to right in each histogram). The identity of each protein revealed by MASS was given on the top of the histogram along with the SwissProt accession number in parenthesis and the standard spot (SSP) number at the bottom of the histogram.
    Figure Legend Snippet: Relative levels of the HCV 3’ X-associated proteins in Huh7.5, Clone B, and 293T cells. HCV 3’ X-associated proteins were fractionated on the 2D gel. The level of each protein (spot) was quantified using the PDQuest software, expressed in parts per million (ppm) and compared among Huh7.5, Clone B, and 293T cells (from left to right in each histogram). The identity of each protein revealed by MASS was given on the top of the histogram along with the SwissProt accession number in parenthesis and the standard spot (SSP) number at the bottom of the histogram.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Software

    Scheme of the RNA affinity chromatography-2D/MASS strategy. Cell extracts were prepared from 293T, Huh7.5, and Clone B cells and were added to streptavidin agarose beads that were pre-conjugated with biotinylated HCV 3’X RNA. The beads were extensively washed to remove unbound proteins, and the proteins that either directly or indirectly bound to RNA were then eluted and analyzed by 2D/MASS.
    Figure Legend Snippet: Scheme of the RNA affinity chromatography-2D/MASS strategy. Cell extracts were prepared from 293T, Huh7.5, and Clone B cells and were added to streptavidin agarose beads that were pre-conjugated with biotinylated HCV 3’X RNA. The beads were extensively washed to remove unbound proteins, and the proteins that either directly or indirectly bound to RNA were then eluted and analyzed by 2D/MASS.

    Techniques Used: Affinity Chromatography

    9) Product Images from "Rai14 (Retinoic Acid Induced Protein 14) Is Involved in Regulating F-Actin Dynamics at the Ectoplasmic Specialization in the Rat Testis*"

    Article Title: Rai14 (Retinoic Acid Induced Protein 14) Is Involved in Regulating F-Actin Dynamics at the Ectoplasmic Specialization in the Rat Testis*

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060656

    Rai14 is an actin-binding protein in the rat testis. ( A ) A study by RT-PCR to confirm the expression of Rai14 in adult rat testis, Sertoli cells (SC, isolated from 20-day-old rat testes and cultured for 4-day), and germ cells (GC, isolated from adult rat testes and cultured for 16 hr). ( B ) Immunoblotting also confirmed the expression of Rai14 in the rat testis, Sertoli and germ cells, and the relative expression of Rai14 in SC vs. GC was shown in the histogram with n = 3 experiments in which the relative expression level of Rai14 in the testis was arbitrarily set at 1 so that the relative expression level between these samples can be compared. ( C ) The specificity of the anti-Rai14 antibody ( Table 1 ) was assessed by immunoblotting using lysates of GC (20 µg protein). ( D ) Using the specific anti-Rai14 antibody, Rai14 was shown to be an actin-binding protein by co-immunoprecipitation (Co-IP); however, Rai14 did not structurally interact with any of the BTB-associated proteins including several actin-binding and regulatory proteins ( e.g ., Arp3, drebrin E, Eps8) and vimentin (an intermediate filament-based constituent protein). However, Rai14 was found to structurally interact with an actin cross-linking protein palladin which is known to be involved in conferring actin filament bundles in other mammalian cells [48] . ( E ) Rai14 (red) was also shown to be an actin-binding protein by dual-labeled immunofluorescence analysis in which it co-localized with F-actin (green) in Sertoli cells. Cell nuclei (blue) were visualized by DAPI. Scale ba = 20 µm, which applies to all other micrographs.
    Figure Legend Snippet: Rai14 is an actin-binding protein in the rat testis. ( A ) A study by RT-PCR to confirm the expression of Rai14 in adult rat testis, Sertoli cells (SC, isolated from 20-day-old rat testes and cultured for 4-day), and germ cells (GC, isolated from adult rat testes and cultured for 16 hr). ( B ) Immunoblotting also confirmed the expression of Rai14 in the rat testis, Sertoli and germ cells, and the relative expression of Rai14 in SC vs. GC was shown in the histogram with n = 3 experiments in which the relative expression level of Rai14 in the testis was arbitrarily set at 1 so that the relative expression level between these samples can be compared. ( C ) The specificity of the anti-Rai14 antibody ( Table 1 ) was assessed by immunoblotting using lysates of GC (20 µg protein). ( D ) Using the specific anti-Rai14 antibody, Rai14 was shown to be an actin-binding protein by co-immunoprecipitation (Co-IP); however, Rai14 did not structurally interact with any of the BTB-associated proteins including several actin-binding and regulatory proteins ( e.g ., Arp3, drebrin E, Eps8) and vimentin (an intermediate filament-based constituent protein). However, Rai14 was found to structurally interact with an actin cross-linking protein palladin which is known to be involved in conferring actin filament bundles in other mammalian cells [48] . ( E ) Rai14 (red) was also shown to be an actin-binding protein by dual-labeled immunofluorescence analysis in which it co-localized with F-actin (green) in Sertoli cells. Cell nuclei (blue) were visualized by DAPI. Scale ba = 20 µm, which applies to all other micrographs.

    Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Cell Culture, Immunoprecipitation, Co-Immunoprecipitation Assay, Labeling, Immunofluorescence

    Knockdown of Rai14 in the Sertoli cell epithelium with an established TJ-permeability barrier in vitro by RNAi disrupts actin filament organization and the TJ barrier. (A) Sertoli cells cultured alone on Matrigel-coated 12-well dishes for 2-day with an established TJ-permeability barrier were transfected with Rai14 siRNA duplexes (Rai14 RNAi) versus non-targeting control duplexes (Ctrl RNAi) at 100 nM using Ribojuice transfection medium for 24 hr, thereafter, cells were washed twice and cultured in F12/DMEM for 12 hr to allow recovery. Thereafter, cells were transfected again under the same conditions for another 24 hr. Thereafter, cells were rinsed with fresh F12/DMEM and cultured for an additional 12 hr before termination, and used to prepare lysates for immunoblotting using antibodies against several BTB-associated constituent or regulatory proteins. A knockdown of Rai14 by ∼50% was noted in which the control was arbitrarily set at 1 against which statistical comparison was performed (B) without any apparent off-target effects (A). The findings shown herein are the results of 3 independent experiments excluding pilot experiments which were used to establish optimal experimental conditions, such as different concentrations of siRNA duplexes and Ribojuice. It was noted that we achieved only ∼50–60% knockdown of Rai14 in several pilot experiments, unlike other target genes [ e.g ., Scribble, β1-integrin, and P-glycoprotein] wherein we could silence the target gene expression by as much as ∼70–90%. **, P
    Figure Legend Snippet: Knockdown of Rai14 in the Sertoli cell epithelium with an established TJ-permeability barrier in vitro by RNAi disrupts actin filament organization and the TJ barrier. (A) Sertoli cells cultured alone on Matrigel-coated 12-well dishes for 2-day with an established TJ-permeability barrier were transfected with Rai14 siRNA duplexes (Rai14 RNAi) versus non-targeting control duplexes (Ctrl RNAi) at 100 nM using Ribojuice transfection medium for 24 hr, thereafter, cells were washed twice and cultured in F12/DMEM for 12 hr to allow recovery. Thereafter, cells were transfected again under the same conditions for another 24 hr. Thereafter, cells were rinsed with fresh F12/DMEM and cultured for an additional 12 hr before termination, and used to prepare lysates for immunoblotting using antibodies against several BTB-associated constituent or regulatory proteins. A knockdown of Rai14 by ∼50% was noted in which the control was arbitrarily set at 1 against which statistical comparison was performed (B) without any apparent off-target effects (A). The findings shown herein are the results of 3 independent experiments excluding pilot experiments which were used to establish optimal experimental conditions, such as different concentrations of siRNA duplexes and Ribojuice. It was noted that we achieved only ∼50–60% knockdown of Rai14 in several pilot experiments, unlike other target genes [ e.g ., Scribble, β1-integrin, and P-glycoprotein] wherein we could silence the target gene expression by as much as ∼70–90%. **, P

    Techniques Used: Permeability, In Vitro, Cell Culture, Transfection, Expressing

    Stage-specific expression of Rai14 and its association with the F-actin-rich ectoplasmic specialization (ES) in the seminiferous epithelium of adult rat testes. Dual-labeled immunofluorescence analysis was performed using frozen cross-sections of testes from adult rat testes to examine co-localization of Rai14 (red) and F-actin (green) in the seminiferous epithelium. At stage VI, Rai14 was weakly detected at the apical ES at the Sertoli-spermatid interface, and its localization to the BTB was also weakly detectable. However, at stage VII-early stage VIII, the expression of Rai14 was the strongest. Rai14 was intensely localized to the apical ES, most abundantly at the front-end of the spermatid head, and co-localized with F-actin at the site (“yellow” in merged image of the apical ES), and its localization at the BTB remained not clearly visible. At stage VIII, Rai14 expression at the apical ES was still considerably strong, but it no longer restricted to the front-end of the spermatid head, instead, Rai14 was scattered around the spermatid head at the apical ES, but not tightly co-localized with the F-actin when compared to late stage VII tubule. In late stage VIII tubules, Rai14 also considerably expressed near the basement membrane (annotated by “white” broken line), consistent with its localization at the BTB (see “white” arrowheads), but not tightly co-localized with F-actin. At stage XI-XII, Rai14 remained localized to the apical ES, but it also shifted to the front-end of the spermatid head, partially co-localized with F-actin. “Yellow” and “green” boxed areas were magnified and shown in corresponding micrographs to better illustrate the localization and/or co-localization of Rai14 and/or Rai14/F-actin at the apical ES. Scale bar = 50 µm or 10 µm in the micrograph or inset, respectively, which apply to all other micrographs and insets.
    Figure Legend Snippet: Stage-specific expression of Rai14 and its association with the F-actin-rich ectoplasmic specialization (ES) in the seminiferous epithelium of adult rat testes. Dual-labeled immunofluorescence analysis was performed using frozen cross-sections of testes from adult rat testes to examine co-localization of Rai14 (red) and F-actin (green) in the seminiferous epithelium. At stage VI, Rai14 was weakly detected at the apical ES at the Sertoli-spermatid interface, and its localization to the BTB was also weakly detectable. However, at stage VII-early stage VIII, the expression of Rai14 was the strongest. Rai14 was intensely localized to the apical ES, most abundantly at the front-end of the spermatid head, and co-localized with F-actin at the site (“yellow” in merged image of the apical ES), and its localization at the BTB remained not clearly visible. At stage VIII, Rai14 expression at the apical ES was still considerably strong, but it no longer restricted to the front-end of the spermatid head, instead, Rai14 was scattered around the spermatid head at the apical ES, but not tightly co-localized with the F-actin when compared to late stage VII tubule. In late stage VIII tubules, Rai14 also considerably expressed near the basement membrane (annotated by “white” broken line), consistent with its localization at the BTB (see “white” arrowheads), but not tightly co-localized with F-actin. At stage XI-XII, Rai14 remained localized to the apical ES, but it also shifted to the front-end of the spermatid head, partially co-localized with F-actin. “Yellow” and “green” boxed areas were magnified and shown in corresponding micrographs to better illustrate the localization and/or co-localization of Rai14 and/or Rai14/F-actin at the apical ES. Scale bar = 50 µm or 10 µm in the micrograph or inset, respectively, which apply to all other micrographs and insets.

    Techniques Used: Expressing, Labeling, Immunofluorescence

    10) Product Images from "Nodal Promotes the Self-Renewal of Human Colon Cancer Stem Cells via an Autocrine Manner through Smad2/3 Signaling Pathway"

    Article Title: Nodal Promotes the Self-Renewal of Human Colon Cancer Stem Cells via an Autocrine Manner through Smad2/3 Signaling Pathway

    Journal: BioMed Research International

    doi: 10.1155/2014/364134

    Expression of CD24 and CD44, as well as Nodal and its receptors in CD44- and CD24-positive and -negative colon cancer cells. (a-b) Immunofluorescence showed the expression of CD24 (a) and CD44 (b) in the freshly isolated CD24- and CD44-positive HCT116 cells. (c-d) RT-PCR revealed transcripts of Nodal, ALK-4, ALK-7, and Actr-IIb in CD44-negative SW480 cells and CD44-negative LOVO cells (c) as well as in CD24-negative HCT116 cells (d). Gapdh was used as a loading control of total RNA. (e) RT-PCR displayed transcripts of Nodal, ALK-4, ALK-7, and Actr-IIb in CD24-positive HCT116 cells. Gapdh was used as a loading control of total RNA.
    Figure Legend Snippet: Expression of CD24 and CD44, as well as Nodal and its receptors in CD44- and CD24-positive and -negative colon cancer cells. (a-b) Immunofluorescence showed the expression of CD24 (a) and CD44 (b) in the freshly isolated CD24- and CD44-positive HCT116 cells. (c-d) RT-PCR revealed transcripts of Nodal, ALK-4, ALK-7, and Actr-IIb in CD44-negative SW480 cells and CD44-negative LOVO cells (c) as well as in CD24-negative HCT116 cells (d). Gapdh was used as a loading control of total RNA. (e) RT-PCR displayed transcripts of Nodal, ALK-4, ALK-7, and Actr-IIb in CD24-positive HCT116 cells. Gapdh was used as a loading control of total RNA.

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

    Protein expression of Nodal ligand and its receptors in various human colorectal cancer cell lines. (a–c) Immunocytochemistry showed translation of NODAL (a), ALK-4 (b), and ACTR-IIB (c) in human colon cancer cell lines, including SW480 cells, LOVO cells, and HCT116 cells. Scale bars in (a)–(c) = 10 μ m.
    Figure Legend Snippet: Protein expression of Nodal ligand and its receptors in various human colorectal cancer cell lines. (a–c) Immunocytochemistry showed translation of NODAL (a), ALK-4 (b), and ACTR-IIB (c) in human colon cancer cell lines, including SW480 cells, LOVO cells, and HCT116 cells. Scale bars in (a)–(c) = 10 μ m.

    Techniques Used: Expressing, Immunocytochemistry

    mRNA expression of Nodal ligand and its three receptors in various human colorectal cancer cell lines. (a–c) RT-PCR revealed the transcripts of Nodal, ALK-4, ALK-7, and Actr-IIb in human colon cancer cell lines, including SW480 cells (a), LOVO cells (b), and HCT116 cells (c). Gapdh served as a loading control of total RNA.
    Figure Legend Snippet: mRNA expression of Nodal ligand and its three receptors in various human colorectal cancer cell lines. (a–c) RT-PCR revealed the transcripts of Nodal, ALK-4, ALK-7, and Actr-IIb in human colon cancer cell lines, including SW480 cells (a), LOVO cells (b), and HCT116 cells (c). Gapdh served as a loading control of total RNA.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    11) Product Images from "Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells"

    Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2014.00110

    Fzd9 enhances β-catenin accumulation in myotubes. (A) GFP and Fzd9-transfected myotubes were immunostained with an anti β-catenin antibody (red). Fluorescence intensity of transfected myotubes was quantified using Metamorph. Quantification of the data ( right panel ) shows that the expression of Fzd9 induces a significant ~2-fold accumulation of β-catenin in the sarcoplasma, compared to control GFP-expressing myotubes. (B) Total protein samples from GFP- and Fzd9-transfected myotubes were separated by SDS-PAGE and immunoblotted with Fzd9 and β-catenin antibodies. Quantification of the Fzd9 or β-catenin against β-actin band intensity ratios shows that Fzd9-overexpressing myotubes display a ~2-fold increase in β-catenin cytosolic levels, which is equivalent to the ~2-fold increase observed for Fzd9 levels, compared to control myotubes ( right panel ). Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to control GFP cells; *** p
    Figure Legend Snippet: Fzd9 enhances β-catenin accumulation in myotubes. (A) GFP and Fzd9-transfected myotubes were immunostained with an anti β-catenin antibody (red). Fluorescence intensity of transfected myotubes was quantified using Metamorph. Quantification of the data ( right panel ) shows that the expression of Fzd9 induces a significant ~2-fold accumulation of β-catenin in the sarcoplasma, compared to control GFP-expressing myotubes. (B) Total protein samples from GFP- and Fzd9-transfected myotubes were separated by SDS-PAGE and immunoblotted with Fzd9 and β-catenin antibodies. Quantification of the Fzd9 or β-catenin against β-actin band intensity ratios shows that Fzd9-overexpressing myotubes display a ~2-fold increase in β-catenin cytosolic levels, which is equivalent to the ~2-fold increase observed for Fzd9 levels, compared to control myotubes ( right panel ). Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to control GFP cells; *** p

    Techniques Used: Transfection, Fluorescence, Expressing, SDS Page

    Most Fzd receptors are expressed in skeletal muscle cells . Total RNAs were extracted from 6-week old mouse diaphragm and cultured myotubes and further subjected to RT-PCR to detect the mRNA expression of all ten mouse Fzd receptors (+). As a negative control, samples were processed in the absence of reverse transcriptase (−). Fzd receptors 1 to 10 are expressed in the mouse diaphragm ( left panel ). In cultured C2C12 myotubes, almost all Fzd receptors are expressed at the mRNA level, except for Fzd8 and Fzd10, which were not amplified by RT-PCR ( right panel ). GAPDH expression was used as a loading control gene. Gels are representative of at least three experiments performed by triplicate.
    Figure Legend Snippet: Most Fzd receptors are expressed in skeletal muscle cells . Total RNAs were extracted from 6-week old mouse diaphragm and cultured myotubes and further subjected to RT-PCR to detect the mRNA expression of all ten mouse Fzd receptors (+). As a negative control, samples were processed in the absence of reverse transcriptase (−). Fzd receptors 1 to 10 are expressed in the mouse diaphragm ( left panel ). In cultured C2C12 myotubes, almost all Fzd receptors are expressed at the mRNA level, except for Fzd8 and Fzd10, which were not amplified by RT-PCR ( right panel ). GAPDH expression was used as a loading control gene. Gels are representative of at least three experiments performed by triplicate.

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Amplification

    Fzd9 impairs agrin-dependent AChR clustering in myotubes. (A) Fzd9 is expressed in the muscle cell line C2C12 throughout differentiation. C2C12 cells were cultured in vitro and differentiated for 0, 3, or 6 days (d0-d6). Total proteins were subjected to Western blot analyses. An expected 56 kDa band is gradually increased during C2C12 cells differentiation. α-tubulin expression was used as a loading control. (B) C2C12 myotubes differentiated for 6 days were analyzed by immunocytochemistry to detect Fzd9. Fzd9 is localized to the plasma membrane of the myotubes (green, upper panel ), similar to Glut1, which was used as a marker of plasma membrane (red, middle panel ). The merge image ( lower panel ) reveals the co-localization of Fzd9 and Glut1. (C) Differentiated C2C12 myotubes were subjected to a sequential fractionation procedure to isolate samples enriched in cytoplasm (cyt) or plasma membrane (mb) proteins. Western blot analyzes showed that α-tubulin is specifically detected in cytoplasmic fractions, whereas the vitamin C transporter SVCT2 was only present in membrane-enriched protein fractions ( left panel ). C2C12 myoblasts were transfected either with GFP or Fzd9 and differentiated. Sequential protein lysates were analyzed by Western blot. Both endogenous Fzd9 (GFP-transfected cells) or overexpressed Fzd9 (Fzd9-transfected cells) were found predominantly in the plasma membrane and were absent in the cytoplasm. As a loading control, β-actin is found only in the cytoplasm-enriched fraction ( right panel ). (D) Myoblasts transfected with plasmids coding for GFP (control) or Fzd9 were differentiated into myotubes and subsequently incubated with 200 pM neural agrin. αBTX staining allows the visualization of the AChRs (red). Automatized quantification of aggregates shows that Fzd9 overexpression induces a decrease in the number of AChR clusters per myotube, as well as a reduction in the total area and average size of AChR clusters, compared to controls. Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to GFP-transfected myotubes). ( ** p
    Figure Legend Snippet: Fzd9 impairs agrin-dependent AChR clustering in myotubes. (A) Fzd9 is expressed in the muscle cell line C2C12 throughout differentiation. C2C12 cells were cultured in vitro and differentiated for 0, 3, or 6 days (d0-d6). Total proteins were subjected to Western blot analyses. An expected 56 kDa band is gradually increased during C2C12 cells differentiation. α-tubulin expression was used as a loading control. (B) C2C12 myotubes differentiated for 6 days were analyzed by immunocytochemistry to detect Fzd9. Fzd9 is localized to the plasma membrane of the myotubes (green, upper panel ), similar to Glut1, which was used as a marker of plasma membrane (red, middle panel ). The merge image ( lower panel ) reveals the co-localization of Fzd9 and Glut1. (C) Differentiated C2C12 myotubes were subjected to a sequential fractionation procedure to isolate samples enriched in cytoplasm (cyt) or plasma membrane (mb) proteins. Western blot analyzes showed that α-tubulin is specifically detected in cytoplasmic fractions, whereas the vitamin C transporter SVCT2 was only present in membrane-enriched protein fractions ( left panel ). C2C12 myoblasts were transfected either with GFP or Fzd9 and differentiated. Sequential protein lysates were analyzed by Western blot. Both endogenous Fzd9 (GFP-transfected cells) or overexpressed Fzd9 (Fzd9-transfected cells) were found predominantly in the plasma membrane and were absent in the cytoplasm. As a loading control, β-actin is found only in the cytoplasm-enriched fraction ( right panel ). (D) Myoblasts transfected with plasmids coding for GFP (control) or Fzd9 were differentiated into myotubes and subsequently incubated with 200 pM neural agrin. αBTX staining allows the visualization of the AChRs (red). Automatized quantification of aggregates shows that Fzd9 overexpression induces a decrease in the number of AChR clusters per myotube, as well as a reduction in the total area and average size of AChR clusters, compared to controls. Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to GFP-transfected myotubes). ( ** p

    Techniques Used: Cell Culture, In Vitro, Western Blot, Expressing, Immunocytochemistry, Marker, Fractionation, Transfection, Incubation, Staining, Over Expression

    Down-regulation of Fzd9 increases agrin-dependent AChR clustering in myotubes. (A,B) The efficiency of shFzd9 was tested by its ability to impair overexpression/function of the Fzd9 construct. (B) HEK293 cells were transfected with Fzd9HA together with a control shRNA (pFUX) or shFzd9, followed by protein homogenization and Western blot. Whereas a 56 kDa band corresponding to Fzd9 is detected in the membrane-enriched fraction of the control condition, Fzd9 expression is drastically silenced in cells transfected with shFzd9. (B) The efficiency of the shFzd9 to affect the functionality of Fzd9HA was assessed by co-transfecting Fzd9HA and Wnt2 in the presence or absence of the shFzd9 plasmid in HEK293 cells. Activation of the TOPflash luciferase reporter gene was used as a readout of activation of the canonical Wnt pathway. These experiments were performed at least three times by triplicate ( ** p
    Figure Legend Snippet: Down-regulation of Fzd9 increases agrin-dependent AChR clustering in myotubes. (A,B) The efficiency of shFzd9 was tested by its ability to impair overexpression/function of the Fzd9 construct. (B) HEK293 cells were transfected with Fzd9HA together with a control shRNA (pFUX) or shFzd9, followed by protein homogenization and Western blot. Whereas a 56 kDa band corresponding to Fzd9 is detected in the membrane-enriched fraction of the control condition, Fzd9 expression is drastically silenced in cells transfected with shFzd9. (B) The efficiency of the shFzd9 to affect the functionality of Fzd9HA was assessed by co-transfecting Fzd9HA and Wnt2 in the presence or absence of the shFzd9 plasmid in HEK293 cells. Activation of the TOPflash luciferase reporter gene was used as a readout of activation of the canonical Wnt pathway. These experiments were performed at least three times by triplicate ( ** p

    Techniques Used: Over Expression, Construct, Transfection, shRNA, Homogenization, Western Blot, Expressing, Plasmid Preparation, Activation Assay, Luciferase

    12) Product Images from "Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis"

    Article Title: Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-10-77

    (A) - Electrophoretic profile of in vitro synthesized messenger RNAs . Lane M: 0.5-10 kb RNA Ladder (Invitrogen), lane 1: 1 μg of mRNA-Hsp65, 2: 1 μg of EF-1α mRNA. The electrophoresis was performed in 1.5% denaturing agarose gel stained with ethidium bromide. (BP-Base pair, M-Marker). ( B) - Detection of the integrity of the mRNA-Hsp65 by RT-PCR. After transfection of HEK 293 cells with the mRNA-Hsp65 cells were maintained in culture for different periods of time, as showed in the figure, in order to analyze the presence of mRNA-Hsp65. Electrophoresis was run in 1.5% agarose gel and stained with ethidium bromide (NC-Negative PCR control, NT-Not transfected, PC-Positive PCR control). ( C) - After contact mRNA-Hsp65 for different periods of time, the total cell lysate was subjected to polyacrylamide gel electrophoresis (12.5%) and the bands transferred to nitrocellulose membrane and incubated with anti-Hsp65 for 2 hours. The reaction was revealed with secondary antibody anti-mouse IgG in the presence of DAB. (RC-Recombinant protein Hsp65, NT-Not transfected)
    Figure Legend Snippet: (A) - Electrophoretic profile of in vitro synthesized messenger RNAs . Lane M: 0.5-10 kb RNA Ladder (Invitrogen), lane 1: 1 μg of mRNA-Hsp65, 2: 1 μg of EF-1α mRNA. The electrophoresis was performed in 1.5% denaturing agarose gel stained with ethidium bromide. (BP-Base pair, M-Marker). ( B) - Detection of the integrity of the mRNA-Hsp65 by RT-PCR. After transfection of HEK 293 cells with the mRNA-Hsp65 cells were maintained in culture for different periods of time, as showed in the figure, in order to analyze the presence of mRNA-Hsp65. Electrophoresis was run in 1.5% agarose gel and stained with ethidium bromide (NC-Negative PCR control, NT-Not transfected, PC-Positive PCR control). ( C) - After contact mRNA-Hsp65 for different periods of time, the total cell lysate was subjected to polyacrylamide gel electrophoresis (12.5%) and the bands transferred to nitrocellulose membrane and incubated with anti-Hsp65 for 2 hours. The reaction was revealed with secondary antibody anti-mouse IgG in the presence of DAB. (RC-Recombinant protein Hsp65, NT-Not transfected)

    Techniques Used: In Vitro, Synthesized, Electrophoresis, Agarose Gel Electrophoresis, Staining, Marker, Reverse Transcription Polymerase Chain Reaction, Transfection, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Incubation, Recombinant

    13) Product Images from "The PXR is a drug target for chronic inflammatory liver disease"

    Article Title: The PXR is a drug target for chronic inflammatory liver disease

    Journal: The Journal of Steroid Biochemistry and Molecular Biology

    doi: 10.1016/j.jsbmb.2010.04.012

    Effect of PXR activators on NF-κB-dependent gene expression in U397-3XκB-LUC cells. (A) Left panel, RT-PCR for PXR and GAPDH in U937-3XκB-LUC cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Figure Legend Snippet: Effect of PXR activators on NF-κB-dependent gene expression in U397-3XκB-LUC cells. (A) Left panel, RT-PCR for PXR and GAPDH in U937-3XκB-LUC cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Luciferase, Activity Assay, Standard Deviation, Two Tailed Test, Concentration Assay

    CsA is an activator of the human PXR. (A) Hep-G2 cells cultured in 24-well plates were transfected with 0.5 μg (ER6) 3 -pGL3promoter and 0.05 μg RL-TK/well and PXR activation determined [6] . Data are the mean and standard deviation of at least 3 separate transfections from the same experiment expressed as fold normalised luciferase expression versus DMSO vehicle control, typical of at least 3 separate experiments. *Significantly different ( P > 95%) luciferase activity versus DMSO using Student's t -test (two tailed). (B) Human hepatocytes were isolated and cultured as outlined in Methods section and treated with the indicated compounds after the first 24 h of culture ( T 0 ) for a further 48 h prior to isolation and analysis of CYP3A4 and β-actin levels by Western blotting. Medium and treatments were replenished every 24 h. (C) RT-PCR for the indicated transcripts in human liver, two separate human hepatocyte preparations (freshly isolated cells) and two separate LS180 cultures (amplified product from the equivalent 100 ng template total RNA per well, 35 PCR cycles). (D) Expression of CYP3A4 mRNA in LS180 cells. LS180 cells were treated with the indicated compound DMSO, 0.1% (v/v); RIF, 20 μM RIF (for 48 h) and CsA and FK506 at the indicated concentrations for 72 h. Culture media and treatments were changed daily. RNA was isolated and transcript CYP3A4 and 18S rRNA levels determined by quantitative RT-PCR. *Significantly different ( P > 95%) transcript level activity versus DMSO using Student's t -test (two tailed).
    Figure Legend Snippet: CsA is an activator of the human PXR. (A) Hep-G2 cells cultured in 24-well plates were transfected with 0.5 μg (ER6) 3 -pGL3promoter and 0.05 μg RL-TK/well and PXR activation determined [6] . Data are the mean and standard deviation of at least 3 separate transfections from the same experiment expressed as fold normalised luciferase expression versus DMSO vehicle control, typical of at least 3 separate experiments. *Significantly different ( P > 95%) luciferase activity versus DMSO using Student's t -test (two tailed). (B) Human hepatocytes were isolated and cultured as outlined in Methods section and treated with the indicated compounds after the first 24 h of culture ( T 0 ) for a further 48 h prior to isolation and analysis of CYP3A4 and β-actin levels by Western blotting. Medium and treatments were replenished every 24 h. (C) RT-PCR for the indicated transcripts in human liver, two separate human hepatocyte preparations (freshly isolated cells) and two separate LS180 cultures (amplified product from the equivalent 100 ng template total RNA per well, 35 PCR cycles). (D) Expression of CYP3A4 mRNA in LS180 cells. LS180 cells were treated with the indicated compound DMSO, 0.1% (v/v); RIF, 20 μM RIF (for 48 h) and CsA and FK506 at the indicated concentrations for 72 h. Culture media and treatments were changed daily. RNA was isolated and transcript CYP3A4 and 18S rRNA levels determined by quantitative RT-PCR. *Significantly different ( P > 95%) transcript level activity versus DMSO using Student's t -test (two tailed).

    Techniques Used: Cell Culture, Transfection, Activation Assay, Standard Deviation, Luciferase, Expressing, Activity Assay, Two Tailed Test, Isolation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Quantitative RT-PCR

    Effect of PXR activators or NF-κB inhibitors on TNFα-promoter reporter gene expression. (A and C) Cells were transfected with either pXP1 (EMPTY) or TNFα-Prom, both with renilla plasmid RL-TK (10:1 ratio). After 24 h, cells were treated as indicated (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) and after a further 24 h, cells were harvested and reporter gene expression determined. Data are the mean and standard deviation of 3 separate experiments, significantly different ( P > 95%) reporter gene expression versus *untreated or DMSO vehicle control; $ versus TNFα-induced DMSO control or ¶ LPS-induced DMSO control using Student's t -test (two tailed). (B) RT-PCR for TLR4 and TNFR1 in the various cell types (amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (D) RT-PCR for human PXR and glyceraldehyde phosphate dehydrogenase (GAPDH) transcripts in the indicated cell types (amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (E) percentage secretion of TNFα over 24 h by human Kupffer cells versus LPS treatment alone (100% arb). Cells were incubated with the indicated PXR activator (RIF or HYP) added from a 1000-fold molar concentrated stock in dimethyl sulfoxide (DMSO). DMSO, 0.1% (v/v) as control. Data are the mean and standard deviation of results from 3 separate patient cell preparations. *Significantly different secretion ( P > 95%) versus LPS + DMSO using Student's t -test (two tailed). (F) TNFα secretion by Kupffer cells isolated from PXR +/+ or PXR −/− mice expressed as the mean and standard deviation levels from 3 separate incubations from a single pool of cells versus DMSO treated cells. DMSO, 0.5% (v/v); PCN, 20 μM from a 4 mM stock in DMSO, LPS, 100 ng/ml. Results are typical of 3 separate experiments. Significantly different ( P > 95%) compared to *DMSO or ¶ LPS-treated cells using Student's t -test (two tailed).
    Figure Legend Snippet: Effect of PXR activators or NF-κB inhibitors on TNFα-promoter reporter gene expression. (A and C) Cells were transfected with either pXP1 (EMPTY) or TNFα-Prom, both with renilla plasmid RL-TK (10:1 ratio). After 24 h, cells were treated as indicated (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) and after a further 24 h, cells were harvested and reporter gene expression determined. Data are the mean and standard deviation of 3 separate experiments, significantly different ( P > 95%) reporter gene expression versus *untreated or DMSO vehicle control; $ versus TNFα-induced DMSO control or ¶ LPS-induced DMSO control using Student's t -test (two tailed). (B) RT-PCR for TLR4 and TNFR1 in the various cell types (amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (D) RT-PCR for human PXR and glyceraldehyde phosphate dehydrogenase (GAPDH) transcripts in the indicated cell types (amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (E) percentage secretion of TNFα over 24 h by human Kupffer cells versus LPS treatment alone (100% arb). Cells were incubated with the indicated PXR activator (RIF or HYP) added from a 1000-fold molar concentrated stock in dimethyl sulfoxide (DMSO). DMSO, 0.1% (v/v) as control. Data are the mean and standard deviation of results from 3 separate patient cell preparations. *Significantly different secretion ( P > 95%) versus LPS + DMSO using Student's t -test (two tailed). (F) TNFα secretion by Kupffer cells isolated from PXR +/+ or PXR −/− mice expressed as the mean and standard deviation levels from 3 separate incubations from a single pool of cells versus DMSO treated cells. DMSO, 0.5% (v/v); PCN, 20 μM from a 4 mM stock in DMSO, LPS, 100 ng/ml. Results are typical of 3 separate experiments. Significantly different ( P > 95%) compared to *DMSO or ¶ LPS-treated cells using Student's t -test (two tailed).

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Standard Deviation, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Incubation, Isolation, Mouse Assay

    14) Product Images from "Regenerative Response in Ischemic Brain Restricted by p21cip1/waf1"

    Article Title: Regenerative Response in Ischemic Brain Restricted by p21cip1/waf1

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20031385

    Increased cell proliferation in p21 −/− brain tissues after MCAO. BrdU was injected into the littermate or age-matched p21 −/− or p21 +/+ mice on day 7 and 8 or day 14 and 15; and animals were killed 9 d or 16 d after MCAO for 20 min. Immunohistochemistry was performed on free-floating 40-μm coronal sections pretreated by denaturing DNA, and a specific stereological analysis ( 18 ) was applied to enumerate the labeled cells per volume as detailed in Materials and Methods. A few BrdU-labeled cells were detected in the noninjured p21 −/− and p21 +/+ mice. There was an increased BrdU labeling (black cells) in the region corresponding to the SGZ of dentate gyrus in hippocampus (A) or the SVZ in lateral ventricle (B) after MCAO. Mean values from multiple experiments are summarized in the graphs under each histological picture. The noninjured samples are marked as control in the graph. Black and white bars indicate mean values ± SD from p21 −/− and p21 +/+ genotype, respectively. A significant increase of BrdU-labeled cells in both genotypes and a larger fraction of BrdU-positive cells in p21 −/− genotype was observed 9 d after MCAO (P
    Figure Legend Snippet: Increased cell proliferation in p21 −/− brain tissues after MCAO. BrdU was injected into the littermate or age-matched p21 −/− or p21 +/+ mice on day 7 and 8 or day 14 and 15; and animals were killed 9 d or 16 d after MCAO for 20 min. Immunohistochemistry was performed on free-floating 40-μm coronal sections pretreated by denaturing DNA, and a specific stereological analysis ( 18 ) was applied to enumerate the labeled cells per volume as detailed in Materials and Methods. A few BrdU-labeled cells were detected in the noninjured p21 −/− and p21 +/+ mice. There was an increased BrdU labeling (black cells) in the region corresponding to the SGZ of dentate gyrus in hippocampus (A) or the SVZ in lateral ventricle (B) after MCAO. Mean values from multiple experiments are summarized in the graphs under each histological picture. The noninjured samples are marked as control in the graph. Black and white bars indicate mean values ± SD from p21 −/− and p21 +/+ genotype, respectively. A significant increase of BrdU-labeled cells in both genotypes and a larger fraction of BrdU-positive cells in p21 −/− genotype was observed 9 d after MCAO (P

    Techniques Used: Injection, Mouse Assay, Immunohistochemistry, Labeling

    15) Product Images from "Loss of Parp-1 affects gene expression profile in a genome-wide manner in ES cells and liver cells"

    Article Title: Loss of Parp-1 affects gene expression profile in a genome-wide manner in ES cells and liver cells

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-8-41

    Effect of Parp-1 deficiency on gene expression . Gene expression data from microarray analyses are plotted for Parp-1 -/- versus wild-type ( Parp-1 +/+ ) ES cell lines (A-C) or the livers (D-F). Horizontal and vertical axes represent expression levels normalized for an individual gene. Each point represents normalized expression data for an individual gene. The genes that showed standard deviations greater than 2.0 in the normalized data of both genotypes (A and D) were excluded and gene lists were constructed with p
    Figure Legend Snippet: Effect of Parp-1 deficiency on gene expression . Gene expression data from microarray analyses are plotted for Parp-1 -/- versus wild-type ( Parp-1 +/+ ) ES cell lines (A-C) or the livers (D-F). Horizontal and vertical axes represent expression levels normalized for an individual gene. Each point represents normalized expression data for an individual gene. The genes that showed standard deviations greater than 2.0 in the normalized data of both genotypes (A and D) were excluded and gene lists were constructed with p

    Techniques Used: Expressing, Microarray, Construct

    Confirmation of differentially expressed genes in microarray analysis by northern blot analysis (A), and functional categorization of up- and down-regulated genes (B) . Ten micrograms of total RNA were used for northern blot analysis in (A). Copy numbers were calculated from the radioactivities of the probe control.
    Figure Legend Snippet: Confirmation of differentially expressed genes in microarray analysis by northern blot analysis (A), and functional categorization of up- and down-regulated genes (B) . Ten micrograms of total RNA were used for northern blot analysis in (A). Copy numbers were calculated from the radioactivities of the probe control.

    Techniques Used: Microarray, Northern Blot, Functional Assay

    16) Product Images from "Differential regulation of repeated histone genes during the fission yeast cell cycle"

    Article Title: Differential regulation of repeated histone genes during the fission yeast cell cycle

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm213

    Identification of UTR regions for individual histone H3 and H4 gene pairs. ( A ) 3′- and 5′-RACE were performed using total RNA prepared from exponentially growing wild-type cells. The amplified PCR products were cloned and sequenced ( > 5 clones per RACE reaction). Arrows and lollipops indicate the locations of the 5′-ends of cDNAs obtained by 5′-RACE and the 3′-ends of cDNAs obtained by 3′-RACE, respectively. Ellipses indicate the locations of the AACCCT-box. Short gray and white bars indicate the allele-specific probes used for northern blotting ( Figures 4 A and 5 A) and quantitative RT-PCR ( Figures 4 B, 5 B, 6 and 7 ) analyses, respectively. ( B ) To determine whether the different-sized transcripts are actually produced from the histone alleles as predicted by the above RACE methods, the total RNA was separated on a denaturing polyacrylamide gel and blotted onto a Nylon membrane. The mixed H3- and H4-ORF probes that hybridized to the three histone H3 genes and three histone H4 genes, respectively, were used for northern analyses. The location of the transcript corresponding to the bands indicated by an asterisk could not be mapped on the hht1 + locus by the RACE methods in the present study.
    Figure Legend Snippet: Identification of UTR regions for individual histone H3 and H4 gene pairs. ( A ) 3′- and 5′-RACE were performed using total RNA prepared from exponentially growing wild-type cells. The amplified PCR products were cloned and sequenced ( > 5 clones per RACE reaction). Arrows and lollipops indicate the locations of the 5′-ends of cDNAs obtained by 5′-RACE and the 3′-ends of cDNAs obtained by 3′-RACE, respectively. Ellipses indicate the locations of the AACCCT-box. Short gray and white bars indicate the allele-specific probes used for northern blotting ( Figures 4 A and 5 A) and quantitative RT-PCR ( Figures 4 B, 5 B, 6 and 7 ) analyses, respectively. ( B ) To determine whether the different-sized transcripts are actually produced from the histone alleles as predicted by the above RACE methods, the total RNA was separated on a denaturing polyacrylamide gel and blotted onto a Nylon membrane. The mixed H3- and H4-ORF probes that hybridized to the three histone H3 genes and three histone H4 genes, respectively, were used for northern analyses. The location of the transcript corresponding to the bands indicated by an asterisk could not be mapped on the hht1 + locus by the RACE methods in the present study.

    Techniques Used: Amplification, Polymerase Chain Reaction, Clone Assay, Northern Blot, Quantitative RT-PCR, Produced

    17) Product Images from "The non-dosage compensated Lsp1? gene of Drosophila melanogaster escapes acetylation by MOF in larval fat body nuclei, but is flanked by two dosage compensated genes"

    Article Title: The non-dosage compensated Lsp1? gene of Drosophila melanogaster escapes acetylation by MOF in larval fat body nuclei, but is flanked by two dosage compensated genes

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-35

    The genes flanking Lsp1 α are dosage compensated, and CG2556 is expressed in the same tissue as Lsp1 α (A) The predicted genes flanking Lsp1 α (exons in black). (B) Northern RNA hybridization analysis of 10 μg of total RNA from embryos 0 – 2 h after laying (E0), embryos 12 h after laying (E12), first instar larvae (L1), second instar larvae (L2), third instar larvae (L3), pupae (P), adult males (M), and adult females (F). All embryo, larval and pupae samples consist of mixed male and female RNA. Northerns were probed with cDNAs for Lsp1 α (a) and rp49 (b). (C) Northern hybridization analysis of 2 μg of poly(A) mRNA from the developmental stages described in (B). Northerns were probed with cDNAs for CG15926 (a), CG2560 (b), CG2556 (c) and rp49 (d). (D) Real-time RT-PCR of Lsp1 α, Lsd-2 , Gpdh , rp49 , CG2556 and CG2560 in male fat body and whole third instar male larvae cDNA. The fold enrichment of each transcript in fat body compared to whole larvae cDNA is shown. (E) CG2560 and Pgd mRNA was measured by RNase protection relative to rp49 in male and female first instar larvae. Mean female/male transcript ratios and 95% confidence intervals are indicated for 3 experiments. (F) Lsp1 α, CG2556, Pgd and rp49 mRNA was measured by RNase protection in male and female y w staged-third instar larvae. Mean female/male transcript ratios and 95% confidence intervals are indicated for 3 experiments.
    Figure Legend Snippet: The genes flanking Lsp1 α are dosage compensated, and CG2556 is expressed in the same tissue as Lsp1 α (A) The predicted genes flanking Lsp1 α (exons in black). (B) Northern RNA hybridization analysis of 10 μg of total RNA from embryos 0 – 2 h after laying (E0), embryos 12 h after laying (E12), first instar larvae (L1), second instar larvae (L2), third instar larvae (L3), pupae (P), adult males (M), and adult females (F). All embryo, larval and pupae samples consist of mixed male and female RNA. Northerns were probed with cDNAs for Lsp1 α (a) and rp49 (b). (C) Northern hybridization analysis of 2 μg of poly(A) mRNA from the developmental stages described in (B). Northerns were probed with cDNAs for CG15926 (a), CG2560 (b), CG2556 (c) and rp49 (d). (D) Real-time RT-PCR of Lsp1 α, Lsd-2 , Gpdh , rp49 , CG2556 and CG2560 in male fat body and whole third instar male larvae cDNA. The fold enrichment of each transcript in fat body compared to whole larvae cDNA is shown. (E) CG2560 and Pgd mRNA was measured by RNase protection relative to rp49 in male and female first instar larvae. Mean female/male transcript ratios and 95% confidence intervals are indicated for 3 experiments. (F) Lsp1 α, CG2556, Pgd and rp49 mRNA was measured by RNase protection in male and female y w staged-third instar larvae. Mean female/male transcript ratios and 95% confidence intervals are indicated for 3 experiments.

    Techniques Used: Northern Blot, Hybridization, Quantitative RT-PCR

    18) Product Images from "Ectopic Lymphoid Structures Support Ongoing Production of Class-Switched Autoantibodies in Rheumatoid SynoviumAutoimmune Responses in the Rheumatoid Synovium"

    Article Title: Ectopic Lymphoid Structures Support Ongoing Production of Class-Switched Autoantibodies in Rheumatoid SynoviumAutoimmune Responses in the Rheumatoid Synovium

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0060001

    Rheumatoid Synovial Grafts in SCID Mice Maintain AID Expression and the Up-regulation of Genes Determining Lymphoneogenesis (A) Rheumatoid synovial tissue ( n = 56) was transplanted into SCID mice (arrows depict site of transplants). (B) Transplanted RA synovial tissue in SCID mouse. (C–F) Montage image of a paraffin embedded haematoxylin and eosin stained section of transplanted tissue depicting the whole graft (original magnification 4×, area outlined in black equates to aggregate seen at higher magnification in [D] and [E]). Sequential sections of transplants stained using conventional immunohistochemistry for CD21 (D), AID (E), and double immunofluorescence for Ki67 (green) and CD20 (red) (F) (inset at 40× magnification, with arrowheads indicating the cells staining positively for Ki67 and CD20) demonstrated persistent FDCs, associated expression of AID (original magnification 40×) and continued proliferation of B cells. Four weeks post-transplantation, total RNA from the grafts was reverse transcribed and used for QT-PCR to determine the mRNA expression levels of AID and CD21L and a number of genes known to correlate with lymphoid tissue organization. (G) A significant correlation between levels of transcripts for AID and CD21L was found and AID was detected only in the presence of CD21L. (H–L) Tissues were stratified according to the presence or absence of AID as determined by QT-PCR, and analysed for the expression of the selected cytokines/chemokines genes between the groups. A significant difference is seen in levels of APRIL (H), CXCL13 (L), TNFα (J), and LTβ (K). Significant p -values are depicted, Mann-Whitney U test. No significant difference is seen between levels of BAFF (I). The lower and upper margins of the box represent the 25th and 75th percentiles, with the extended arms representing the 10th and 90th percentiles, respectively. The median is shown as a horizontal line within the box. Scale bars: 6 mm (B) 2 mm (C), 50 μm (D, E, F).
    Figure Legend Snippet: Rheumatoid Synovial Grafts in SCID Mice Maintain AID Expression and the Up-regulation of Genes Determining Lymphoneogenesis (A) Rheumatoid synovial tissue ( n = 56) was transplanted into SCID mice (arrows depict site of transplants). (B) Transplanted RA synovial tissue in SCID mouse. (C–F) Montage image of a paraffin embedded haematoxylin and eosin stained section of transplanted tissue depicting the whole graft (original magnification 4×, area outlined in black equates to aggregate seen at higher magnification in [D] and [E]). Sequential sections of transplants stained using conventional immunohistochemistry for CD21 (D), AID (E), and double immunofluorescence for Ki67 (green) and CD20 (red) (F) (inset at 40× magnification, with arrowheads indicating the cells staining positively for Ki67 and CD20) demonstrated persistent FDCs, associated expression of AID (original magnification 40×) and continued proliferation of B cells. Four weeks post-transplantation, total RNA from the grafts was reverse transcribed and used for QT-PCR to determine the mRNA expression levels of AID and CD21L and a number of genes known to correlate with lymphoid tissue organization. (G) A significant correlation between levels of transcripts for AID and CD21L was found and AID was detected only in the presence of CD21L. (H–L) Tissues were stratified according to the presence or absence of AID as determined by QT-PCR, and analysed for the expression of the selected cytokines/chemokines genes between the groups. A significant difference is seen in levels of APRIL (H), CXCL13 (L), TNFα (J), and LTβ (K). Significant p -values are depicted, Mann-Whitney U test. No significant difference is seen between levels of BAFF (I). The lower and upper margins of the box represent the 25th and 75th percentiles, with the extended arms representing the 10th and 90th percentiles, respectively. The median is shown as a horizontal line within the box. Scale bars: 6 mm (B) 2 mm (C), 50 μm (D, E, F).

    Techniques Used: Mouse Assay, Expressing, Staining, Immunohistochemistry, Immunofluorescence, Transplantation Assay, Polymerase Chain Reaction, MANN-WHITNEY

    19) Product Images from "C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle"

    Article Title: C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle

    Journal: Nature Communications

    doi: 10.1038/ncomms7894

    Characterization of truncated CEP250 transcripts and C-Nap1 mislocalization in SHGC primary fibroblasts. ( a ) Structure of the 5′ region of the bovine CEP250 gene. The 5′ untranslated region is depicted in white. Exons are numbered from the first coding exon. Two transcripts (a and a′) are detected in wild-type cells including one lacking exon 3 and retaining the last five nucleotides of intron 3 (a′). Mutation in exon 4 generates two shorter transcripts: transcript (b) starts at an AG dinucleotide likely located at the end of intron 4 and includes exon 5 (depicted as *); transcript (c) starts in exon 6 at nucleotide 656 of the open reading frame. ( b ) Agarose gel showing the 5′ RACE products amplified using RT primer GSP1 and GSP2 located in exon 9 and PCR primers GSP3 or GSP4 located in exon 8 (black and white triangles, respectively), with or without formamide. The longest products were purified and sequenced (boxes). Original gel is presented in Supplementary Fig. 6 . ( c ) Relative CEP250 mRNA levels estimated by RT–qPCR targeting the indicated exons. Values are provided relative to wild type. Mutant, n =3 and wild-type, n =2. ( d ) C-Nap1 subcellular localization in wild-type and SHGC mutant fibroblasts using C-Nap1 C-terminus labelling (R63 serum). In 92% of wild-type cells ( n =113), centrosomes (white arrows) are labelled with an antibody against γ-tubulin (red) and with R63 (green). In 91% of mutant cells ( n =97), only γ-tubulin labelling is detected on split centrosomes (double arrows). The anti-C-Nap1 antibody only produced a heterogeneous labelling (green colour on the merge) but no labelling of split centrosomes. Scale bar, 10 μm.
    Figure Legend Snippet: Characterization of truncated CEP250 transcripts and C-Nap1 mislocalization in SHGC primary fibroblasts. ( a ) Structure of the 5′ region of the bovine CEP250 gene. The 5′ untranslated region is depicted in white. Exons are numbered from the first coding exon. Two transcripts (a and a′) are detected in wild-type cells including one lacking exon 3 and retaining the last five nucleotides of intron 3 (a′). Mutation in exon 4 generates two shorter transcripts: transcript (b) starts at an AG dinucleotide likely located at the end of intron 4 and includes exon 5 (depicted as *); transcript (c) starts in exon 6 at nucleotide 656 of the open reading frame. ( b ) Agarose gel showing the 5′ RACE products amplified using RT primer GSP1 and GSP2 located in exon 9 and PCR primers GSP3 or GSP4 located in exon 8 (black and white triangles, respectively), with or without formamide. The longest products were purified and sequenced (boxes). Original gel is presented in Supplementary Fig. 6 . ( c ) Relative CEP250 mRNA levels estimated by RT–qPCR targeting the indicated exons. Values are provided relative to wild type. Mutant, n =3 and wild-type, n =2. ( d ) C-Nap1 subcellular localization in wild-type and SHGC mutant fibroblasts using C-Nap1 C-terminus labelling (R63 serum). In 92% of wild-type cells ( n =113), centrosomes (white arrows) are labelled with an antibody against γ-tubulin (red) and with R63 (green). In 91% of mutant cells ( n =97), only γ-tubulin labelling is detected on split centrosomes (double arrows). The anti-C-Nap1 antibody only produced a heterogeneous labelling (green colour on the merge) but no labelling of split centrosomes. Scale bar, 10 μm.

    Techniques Used: Mutagenesis, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Purification, Quantitative RT-PCR, Produced

    20) Product Images from "Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor"

    Article Title: Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor

    Journal: Nuclear Receptor

    doi: 10.1186/1478-1336-2-1

    CAR expression and alternative splicing of exon 9 during enterocytic differentiation of Caco-2 TC7 cells . (A) Northern Blot analysis with polyadenylated RNA of the indicated cell lines. Caco-2 TC7 cells were analyzed from subconfluent (sub), confluent (confl) and 15 days post-confluent (15d pc) cultures. The blot was sequentially hybridized with probes for the genes indicated. The arrow marks the major CAR mRNA species of 1.4 to 1.7 kb. (B) Analysis of the expression of transcripts with alternatively spliced exon 9 by qualitative RT-PCR with random hexamer primed cDNA of polyadenylated RNA of Caco-2 TC7 cells cultured until confluence (confl) and for 15 days post-confluent (15d pc). PCR was performed with primers F2/R2 (Table 1 ). SV1 and SV5 denote control reactions performed with DNA of the corresponding CAR isoform expression plasmids. The lane on the left shows a 50 bp ladder size marker. By mixing DNA of SV1 and SV5 CAR isoform expression plasmids in different molar ratios, we confirmed that both fragments were amplified with equal efficiency (data not shown).
    Figure Legend Snippet: CAR expression and alternative splicing of exon 9 during enterocytic differentiation of Caco-2 TC7 cells . (A) Northern Blot analysis with polyadenylated RNA of the indicated cell lines. Caco-2 TC7 cells were analyzed from subconfluent (sub), confluent (confl) and 15 days post-confluent (15d pc) cultures. The blot was sequentially hybridized with probes for the genes indicated. The arrow marks the major CAR mRNA species of 1.4 to 1.7 kb. (B) Analysis of the expression of transcripts with alternatively spliced exon 9 by qualitative RT-PCR with random hexamer primed cDNA of polyadenylated RNA of Caco-2 TC7 cells cultured until confluence (confl) and for 15 days post-confluent (15d pc). PCR was performed with primers F2/R2 (Table 1 ). SV1 and SV5 denote control reactions performed with DNA of the corresponding CAR isoform expression plasmids. The lane on the left shows a 50 bp ladder size marker. By mixing DNA of SV1 and SV5 CAR isoform expression plasmids in different molar ratios, we confirmed that both fragments were amplified with equal efficiency (data not shown).

    Techniques Used: Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Random Hexamer Labeling, Cell Culture, Polymerase Chain Reaction, Marker, Amplification

    21) Product Images from "Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells"

    Article Title: Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

    Journal: Jundishapur Journal of Microbiology

    doi: 10.5812/jjm.22570

    Agarose Gel Electrophoresis Analysis the Expression of SAG1 in CHO Cells by RT-PCR Assay Line M, 1 kb DNA ladder; Line 1, Transfected CHO cells with empty pVAX1 plasmid; Line 2, Transfected CHO cells with pVAX-SAG1.
    Figure Legend Snippet: Agarose Gel Electrophoresis Analysis the Expression of SAG1 in CHO Cells by RT-PCR Assay Line M, 1 kb DNA ladder; Line 1, Transfected CHO cells with empty pVAX1 plasmid; Line 2, Transfected CHO cells with pVAX-SAG1.

    Techniques Used: Agarose Gel Electrophoresis, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation

    22) Product Images from "Expression of Perforin Gene for Early Development of Nephrons in Olive Flounder (Paralichthys olivaceus)"

    Article Title: Expression of Perforin Gene for Early Development of Nephrons in Olive Flounder (Paralichthys olivaceus)

    Journal: Development & Reproduction

    doi: 10.12717/DR.2013.17.4.321

    Expression of perforin gene induces in the development stage. (A) The experiment was flounder fertilized eggs from the tank, oxygen supply and maintain a temperature of 20°C. Embryo and larva were harvested during hours post fertilization (HPF) and days after hatching (DAH) for the indicated periods. The RNA extracted and perforin was analyzed by RT-PCR method. (B) The mRNA expression of perforin was analyzed by real-time PCR. Different letters over each bar with the standard deviation represent significant differences one group according to unpaired matched comparisons ( p
    Figure Legend Snippet: Expression of perforin gene induces in the development stage. (A) The experiment was flounder fertilized eggs from the tank, oxygen supply and maintain a temperature of 20°C. Embryo and larva were harvested during hours post fertilization (HPF) and days after hatching (DAH) for the indicated periods. The RNA extracted and perforin was analyzed by RT-PCR method. (B) The mRNA expression of perforin was analyzed by real-time PCR. Different letters over each bar with the standard deviation represent significant differences one group according to unpaired matched comparisons ( p

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

    23) Product Images from "TgpA, a Protein with a Eukaryotic-Like Transglutaminase Domain, Plays a Critical Role in the Viability of Pseudomonas aeruginosa"

    Article Title: TgpA, a Protein with a Eukaryotic-Like Transglutaminase Domain, Plays a Critical Role in the Viability of Pseudomonas aeruginosa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050323

    Genetic organization and transcription analysis of the genomic region including PA2873 locus ( tgpA ). (A) PA2875-2874-2873-2872 gene cluster is represented according to visualization by GBrowse in Pseudomonas Genome Database. Locations of fragments amplified by the oligo pairs (Roman numbers) used in RT-PCR-based transcription analysis (B) are shown along the region map. The position of plasmid pDM4 cointegration in PAO1 PA2875::pDM4 strain is indicated. (B) Total RNA was extracted from PAO1 and PAO1 PA2875::pDM4 cells in both exponential (E) and stationary (S) phases, and analyzed by RT-PCR. RT untreated samples (RT−) as controls of genomic DNA contamination were included in the analysis.
    Figure Legend Snippet: Genetic organization and transcription analysis of the genomic region including PA2873 locus ( tgpA ). (A) PA2875-2874-2873-2872 gene cluster is represented according to visualization by GBrowse in Pseudomonas Genome Database. Locations of fragments amplified by the oligo pairs (Roman numbers) used in RT-PCR-based transcription analysis (B) are shown along the region map. The position of plasmid pDM4 cointegration in PAO1 PA2875::pDM4 strain is indicated. (B) Total RNA was extracted from PAO1 and PAO1 PA2875::pDM4 cells in both exponential (E) and stationary (S) phases, and analyzed by RT-PCR. RT untreated samples (RT−) as controls of genomic DNA contamination were included in the analysis.

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    24) Product Images from "Nuclear Import and DNA Binding of the ZHD5 Transcription Factor Is Modulated by a Competitive Peptide Inhibitor in Arabidopsis *"

    Article Title: Nuclear Import and DNA Binding of the ZHD5 Transcription Factor Is Modulated by a Competitive Peptide Inhibitor in Arabidopsis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.167692

    Phenotypic recovery of transgenic plants overexpressing ZHD5 by MIF1 coexpression. A , phenotypes of transgenic plants. The MIF1 or ZHD5 gene was overexpressed under the control of the CaMV 35 S promoter. Five-week-old transgenic plants grown in soil were photographed. The MIF1 x ZHD5 plants were produced by genetic cross of the 35S: MIF1 and 35S: ZHD5 transgenic plants. The inlet shows an enlarged view of the 35S: MIF1 transgenic plant. B , expression of the MIF1 and ZHD5 genes in the transgenic plants. Transcript levels were determined by qRT-PCR using total RNA samples extracted from 2-week-old, whole plants grown on MS-agar plates. Biological triplicates were averaged. Error bars , S.E. Statistical significance was determined using Student's t test (*, p
    Figure Legend Snippet: Phenotypic recovery of transgenic plants overexpressing ZHD5 by MIF1 coexpression. A , phenotypes of transgenic plants. The MIF1 or ZHD5 gene was overexpressed under the control of the CaMV 35 S promoter. Five-week-old transgenic plants grown in soil were photographed. The MIF1 x ZHD5 plants were produced by genetic cross of the 35S: MIF1 and 35S: ZHD5 transgenic plants. The inlet shows an enlarged view of the 35S: MIF1 transgenic plant. B , expression of the MIF1 and ZHD5 genes in the transgenic plants. Transcript levels were determined by qRT-PCR using total RNA samples extracted from 2-week-old, whole plants grown on MS-agar plates. Biological triplicates were averaged. Error bars , S.E. Statistical significance was determined using Student's t test (*, p

    Techniques Used: Transgenic Assay, Produced, Expressing, Quantitative RT-PCR, Mass Spectrometry

    25) Product Images from "Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro"

    Article Title: Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0337479100

    RT-PCR of total RNA isolated from rabbit ileal loops. PCR was performed as described in Materials and Methods by using primers specific for selected V. cholerae genes and either reverse-transcribed RNA (cDNA) or chromosomal DNA as template.
    Figure Legend Snippet: RT-PCR of total RNA isolated from rabbit ileal loops. PCR was performed as described in Materials and Methods by using primers specific for selected V. cholerae genes and either reverse-transcribed RNA (cDNA) or chromosomal DNA as template.

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

    26) Product Images from "Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity"

    Article Title: Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity

    Journal: British Journal of Haematology

    doi: 10.1111/bjh.12622

    CRBN mRNA and CRBN protein levels do not correlate in MM cell lines with intrinsic sensitivity or resistance to the IMiD agent lenalidomide. CRBN mRNA and CRBN protein levels were measured in 12 human MM cell lines by TaqMan polymerase chain reaction and Western blot, respectively. mRNA and protein were normalized to HRPT1 and β-tubulin, respectively. Graph represents the average of 3–4 experiments. CRBN, cereblon; LEN, lenalidomide; MM, multiple myeloma; mRNA, messenger RNA.
    Figure Legend Snippet: CRBN mRNA and CRBN protein levels do not correlate in MM cell lines with intrinsic sensitivity or resistance to the IMiD agent lenalidomide. CRBN mRNA and CRBN protein levels were measured in 12 human MM cell lines by TaqMan polymerase chain reaction and Western blot, respectively. mRNA and protein were normalized to HRPT1 and β-tubulin, respectively. Graph represents the average of 3–4 experiments. CRBN, cereblon; LEN, lenalidomide; MM, multiple myeloma; mRNA, messenger RNA.

    Techniques Used: Polymerase Chain Reaction, Western Blot

    27) Product Images from "Modulation of α-Enolase Post-Translational Modifications by Dengue Virus: Increased Secretion of the Basic Isoforms in Infected Hepatic Cells"

    Article Title: Modulation of α-Enolase Post-Translational Modifications by Dengue Virus: Increased Secretion of the Basic Isoforms in Infected Hepatic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088314

    α-enolase in the conditioned medium of DENV-infected cells is not due to cell death. HepG2 cells were either mock-infected or infected with DENV (MOI = 1, 2 and 4) for 24 h. (A) Cell viability was measured by trypan blue exclusion assay. Graph shows mean ± SEM from 5 independent experiments. (B) Cell viability was assessed by MTT reduction assay. Graph shows mean ± SEM from 6 independent experiments. (C) Cell lysis was assessed by LDH release assay. Graph shows mean ± SEM from 5 independent experiments. Differences between samples from mock-infected and DENV-infected cells were assessed using repeated measures ANOVA test.
    Figure Legend Snippet: α-enolase in the conditioned medium of DENV-infected cells is not due to cell death. HepG2 cells were either mock-infected or infected with DENV (MOI = 1, 2 and 4) for 24 h. (A) Cell viability was measured by trypan blue exclusion assay. Graph shows mean ± SEM from 5 independent experiments. (B) Cell viability was assessed by MTT reduction assay. Graph shows mean ± SEM from 6 independent experiments. (C) Cell lysis was assessed by LDH release assay. Graph shows mean ± SEM from 5 independent experiments. Differences between samples from mock-infected and DENV-infected cells were assessed using repeated measures ANOVA test.

    Techniques Used: Infection, Trypan Blue Exclusion Assay, MTT Assay, Lysis, Lactate Dehydrogenase Assay

    DENV infection does not affect the distribution of intracellular α-enolase isoforms. HepG2 cells were either mock-infected or infected with DENV (MOI = 4) for 24 h. Cell lysates proteins (80 µg) were analyzed by two-dimensional gel electrophoresis (2-DE). (A) Gels stained with colloidal Coomassie blue. The circles indicate the region for α-enolase separation which was selected accordingly to the theoretical molecular mass and isoeletric point of the protein. (B) Immunodetection of α-enolase isoforms on the 2-DE Western blot. α-enolase spots were detected in the region correspondent to the circles in Figure 5A . The figure shows a representative blot of 4 independent experiments. (C) Densitometric analysis of α-enolase spots. Graph shows mean ± SEM from 4 independent experiments. Differences between samples from mock and DENV-infected cells were assessed using repeated measures two-way ANOVA.
    Figure Legend Snippet: DENV infection does not affect the distribution of intracellular α-enolase isoforms. HepG2 cells were either mock-infected or infected with DENV (MOI = 4) for 24 h. Cell lysates proteins (80 µg) were analyzed by two-dimensional gel electrophoresis (2-DE). (A) Gels stained with colloidal Coomassie blue. The circles indicate the region for α-enolase separation which was selected accordingly to the theoretical molecular mass and isoeletric point of the protein. (B) Immunodetection of α-enolase isoforms on the 2-DE Western blot. α-enolase spots were detected in the region correspondent to the circles in Figure 5A . The figure shows a representative blot of 4 independent experiments. (C) Densitometric analysis of α-enolase spots. Graph shows mean ± SEM from 4 independent experiments. Differences between samples from mock and DENV-infected cells were assessed using repeated measures two-way ANOVA.

    Techniques Used: Infection, Two-Dimensional Gel Electrophoresis, Electrophoresis, Staining, Immunodetection, Western Blot

    DENV infection does not modulate α-enolase gene expression and intracellular content in HepG2 cells. HepG2 cells were either mock-infected or infected with DENV (MOI = 1, 2 and 4) for 24 h. (A) ENO1 expression was measured by quantitative RT-PCR. Graph shows mean ± SEM from 5 independent experiments. (B) Western blot analysis of cell lysates proteins. Upper panel shows a representative blot and bottom panel shows the densitometric analysis of α-enolase bands normalized by the content of GAPDH. Graph shows mean ± SEM from 3 independent experiments. Comparison between mock and DENV-infected cells was analyzed by repeated measures ANOVA. (C) α-enolase content in cell lysates was measured by ELISA. Graph shows mean ± SEM from 3 independent experiments. Differences between samples from mock and DENV-infected cells were assessed by paired t test.
    Figure Legend Snippet: DENV infection does not modulate α-enolase gene expression and intracellular content in HepG2 cells. HepG2 cells were either mock-infected or infected with DENV (MOI = 1, 2 and 4) for 24 h. (A) ENO1 expression was measured by quantitative RT-PCR. Graph shows mean ± SEM from 5 independent experiments. (B) Western blot analysis of cell lysates proteins. Upper panel shows a representative blot and bottom panel shows the densitometric analysis of α-enolase bands normalized by the content of GAPDH. Graph shows mean ± SEM from 3 independent experiments. Comparison between mock and DENV-infected cells was analyzed by repeated measures ANOVA. (C) α-enolase content in cell lysates was measured by ELISA. Graph shows mean ± SEM from 3 independent experiments. Differences between samples from mock and DENV-infected cells were assessed by paired t test.

    Techniques Used: Infection, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

    DENV infection induces α-enolase secretion in HepG2 cells. HepG2 cells were either mock-infected or infected with DENV (MOI = 1, 2 and 4) for 24 h. (A) DENV+ cells were measured by flow cytometry using an anti-DENV monoclonal antibody. Representative flow cytometry analysis of mock and DENV-infected HepG2 cells. Shaded histogram indicates DENV-infected HepG2 cells (MOI = 4) stained with an isotype control antibody, open histograms in red, blue, yellow and black show mock, DENV-infected cells with MOI = 1, MOI = 2 and MOI = 4, respectively. The inset graph shows the frequency of DENV+ cells and data represents mean ± SEM from 8 independent experiments. (B) α-enolase content in the conditioned medium was measured by ELISA. Graph shows mean ± SEM from 6 independent experiments. Differences between mock and DENV-infected cells were assessed using repeated measures ANOVA and Dunnet post-hoc test. Statistical significance compared with the control is indicated by asterisks (* p
    Figure Legend Snippet: DENV infection induces α-enolase secretion in HepG2 cells. HepG2 cells were either mock-infected or infected with DENV (MOI = 1, 2 and 4) for 24 h. (A) DENV+ cells were measured by flow cytometry using an anti-DENV monoclonal antibody. Representative flow cytometry analysis of mock and DENV-infected HepG2 cells. Shaded histogram indicates DENV-infected HepG2 cells (MOI = 4) stained with an isotype control antibody, open histograms in red, blue, yellow and black show mock, DENV-infected cells with MOI = 1, MOI = 2 and MOI = 4, respectively. The inset graph shows the frequency of DENV+ cells and data represents mean ± SEM from 8 independent experiments. (B) α-enolase content in the conditioned medium was measured by ELISA. Graph shows mean ± SEM from 6 independent experiments. Differences between mock and DENV-infected cells were assessed using repeated measures ANOVA and Dunnet post-hoc test. Statistical significance compared with the control is indicated by asterisks (* p

    Techniques Used: Infection, Flow Cytometry, Cytometry, Staining, Enzyme-linked Immunosorbent Assay

    DENV infection induces changes in the distribution of secreted α-enolase isoforms. HepG2 cells were either mock-infected or infected with DENV (MOI = 4) for 24 h. Conditioned medium was collected and concentrated and the proteins were analyzed by two-dimensional gel electrophoresis (2-DE). (A) Gels stained with colloidal Coomassie blue. The circles indicate the region for α-enolase separation, which was selected accordingly to the theoretical molecular mass and isoeletric point of the protein. (B) Immunodetection of α-enolase isoforms on the 2-DE Western blot. α-enolase spots were detected in the region correspondent to the circles in Figure 3A . The figure shows a representative blot of 4 independent experiments (C) Densitometric analysis of α-enolase spots. Graph shows mean ± SEM from 4 independent experiments. Differences between spots from mock and DENV-infected cells were assessed using repeated measures two-way ANOVA followed by Bonferroni post hoc test. Statistical significance compared with the control is indicated by asterisks (*** p
    Figure Legend Snippet: DENV infection induces changes in the distribution of secreted α-enolase isoforms. HepG2 cells were either mock-infected or infected with DENV (MOI = 4) for 24 h. Conditioned medium was collected and concentrated and the proteins were analyzed by two-dimensional gel electrophoresis (2-DE). (A) Gels stained with colloidal Coomassie blue. The circles indicate the region for α-enolase separation, which was selected accordingly to the theoretical molecular mass and isoeletric point of the protein. (B) Immunodetection of α-enolase isoforms on the 2-DE Western blot. α-enolase spots were detected in the region correspondent to the circles in Figure 3A . The figure shows a representative blot of 4 independent experiments (C) Densitometric analysis of α-enolase spots. Graph shows mean ± SEM from 4 independent experiments. Differences between spots from mock and DENV-infected cells were assessed using repeated measures two-way ANOVA followed by Bonferroni post hoc test. Statistical significance compared with the control is indicated by asterisks (*** p

    Techniques Used: Infection, Two-Dimensional Gel Electrophoresis, Electrophoresis, Staining, Immunodetection, Western Blot

    28) Product Images from "Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis"

    Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis

    Journal: Nature Communications

    doi: 10.1038/ncomms10893

    The DGKA DMR shows gene enhancer characteristics. ( a ) DGKA mRNA expression in primary human dermal fibroblasts on exposure to increasing doses of gamma irradiation. Data depict mean±s.e.m. from duplicate experiments in three different normal human dermal fibroblast strains derived from healthy donors. ( b ) Radiation-inducible and constitutive DGKA mRNA expression correlated with DNA methylation at the intragenic DGKA DMR in dermal fibroblasts ( n =16). ( c ) Chromatin immunoprecipitation in differentially methylated patient-derived fibroblasts ( n =8) and hypermethylated control cell lines (BJ, IMR90, HCT116) for enhancer histone marks (H3K4me1 and H3K27ac), as well as promoter marks (H3K4me3) at the DGKA DMR. Data depict mean±s.e.m. of four independent immunoprecipitations (triplicates for mRNA and DNA methylation measurements) per fibroblast strain. ( d ) Map of the interrogated DGKA 5′ UTR with CpG islands (green), DGKA transcripts (blue), ChIP-quantitative PCR amplicons (red) and luciferase reporter inserts (black). ( e ) Luciferase assay in pGL 4.10-based reporters measuring promoter activity in three different upstream elements P1, P2 and P3 (for their location, see d ) and the DGKA DMR region. ( f ) Influence of sequence orientation (arrow to the left/right: sense/antisense orientation) and DNA CpG methylation (grey/black bars: unmethylated/methylated insert) on the DGKA DMR gene enhancer activity in a luciferase reporter plasmid carrying a CpG-free EF1alpha minimal promoter (EF1a). Graphs ( e , f ) depict mean±s.e.m. of four independent replicates in HEK293T cells. Vector maps ( e , f ) indicate DMR inserts (blue), minimal promoter (red) and luciferase (grey), hatched boxes indicate lack of vector insert. ( g ) Chromatin conformation capture depicting the interaction of the DGKA DMR with the surrounding DGKA locus in two patient fibroblasts with low- or high-DMR methylation. Data show mean±s.e.m. of three independent experiments. Insert shows gene regions tested with DGKA transcripts depicted in blue and CpG islands in green. **** P
    Figure Legend Snippet: The DGKA DMR shows gene enhancer characteristics. ( a ) DGKA mRNA expression in primary human dermal fibroblasts on exposure to increasing doses of gamma irradiation. Data depict mean±s.e.m. from duplicate experiments in three different normal human dermal fibroblast strains derived from healthy donors. ( b ) Radiation-inducible and constitutive DGKA mRNA expression correlated with DNA methylation at the intragenic DGKA DMR in dermal fibroblasts ( n =16). ( c ) Chromatin immunoprecipitation in differentially methylated patient-derived fibroblasts ( n =8) and hypermethylated control cell lines (BJ, IMR90, HCT116) for enhancer histone marks (H3K4me1 and H3K27ac), as well as promoter marks (H3K4me3) at the DGKA DMR. Data depict mean±s.e.m. of four independent immunoprecipitations (triplicates for mRNA and DNA methylation measurements) per fibroblast strain. ( d ) Map of the interrogated DGKA 5′ UTR with CpG islands (green), DGKA transcripts (blue), ChIP-quantitative PCR amplicons (red) and luciferase reporter inserts (black). ( e ) Luciferase assay in pGL 4.10-based reporters measuring promoter activity in three different upstream elements P1, P2 and P3 (for their location, see d ) and the DGKA DMR region. ( f ) Influence of sequence orientation (arrow to the left/right: sense/antisense orientation) and DNA CpG methylation (grey/black bars: unmethylated/methylated insert) on the DGKA DMR gene enhancer activity in a luciferase reporter plasmid carrying a CpG-free EF1alpha minimal promoter (EF1a). Graphs ( e , f ) depict mean±s.e.m. of four independent replicates in HEK293T cells. Vector maps ( e , f ) indicate DMR inserts (blue), minimal promoter (red) and luciferase (grey), hatched boxes indicate lack of vector insert. ( g ) Chromatin conformation capture depicting the interaction of the DGKA DMR with the surrounding DGKA locus in two patient fibroblasts with low- or high-DMR methylation. Data show mean±s.e.m. of three independent experiments. Insert shows gene regions tested with DGKA transcripts depicted in blue and CpG islands in green. **** P

    Techniques Used: Expressing, Irradiation, Derivative Assay, DNA Methylation Assay, Chromatin Immunoprecipitation, Methylation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Sequencing, CpG Methylation Assay, Plasmid Preparation

    29) Product Images from "Genome-wide characterization of soybean P1B-ATPases gene family provides functional implications in cadmium responses"

    Article Title: Genome-wide characterization of soybean P1B-ATPases gene family provides functional implications in cadmium responses

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2730-2

    Expression patterns of HMA genes in six tissues in two soybean genotypes by qRT-PCR. Samples are color coded: HX3 without Cd treatment (HX3-CK) in green, HX3 with Cd treatment (HX3-Cd) in gray, ZH24 without Cd treatment (ZH24-CK) in red, ZH24 with Cd treatment (ZH24-Cd) in blue. For each gene, the expression levels obtained by normalization to ACT3 are presented on relative scales. Data are average values ± SD from three experiments, each carried out in triplicate. The significance of the changes between HX3 and ZH24 with or without Cd was assessed using the Student’s t -test at the level of P ≤ 0.05 (“*”) and P ≤ 0.01 (“**”)
    Figure Legend Snippet: Expression patterns of HMA genes in six tissues in two soybean genotypes by qRT-PCR. Samples are color coded: HX3 without Cd treatment (HX3-CK) in green, HX3 with Cd treatment (HX3-Cd) in gray, ZH24 without Cd treatment (ZH24-CK) in red, ZH24 with Cd treatment (ZH24-Cd) in blue. For each gene, the expression levels obtained by normalization to ACT3 are presented on relative scales. Data are average values ± SD from three experiments, each carried out in triplicate. The significance of the changes between HX3 and ZH24 with or without Cd was assessed using the Student’s t -test at the level of P ≤ 0.05 (“*”) and P ≤ 0.01 (“**”)

    Techniques Used: Expressing, Quantitative RT-PCR

    30) Product Images from "A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant Binding immunoglobulin Proteins"

    Article Title: A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant Binding immunoglobulin Proteins

    Journal: Nature Communications

    doi: 10.1038/ncomms11685

    PsAvh262 is an essential virulence factor of Phytophthora sojae . ( a ) Expression profile of PsAvh262 during P. sojae strain P6497 infection of soybean hypocotyls. The susceptible soybean cultivar Williams was used as the host. Total RNA was extracted from mycelia (MY) or infected soybean leaves at 1.5, 3, 6, 12 and 24 h post inoculation (h.p.i.). Transcript levels of PsAvh262 were determined by qRT–PCR. The P. sojae actin gene (VMD GeneID: 108986) was used as the pathogen internal control gene, ( b ) Silencing of PsAvh262 in P. sojae greatly impaired the virulence in soybean hypocotyls. Relative transcript levels of PsAvh262 (upper panel) in the P. sojae transformants were determined by qRT–PCR. Disease symptoms (lower panel) in etiolated hypocotyls were observed. Pictures were taken at 7 days post inoculation (d.p.i.). S12 and S141 were non-silenced transformants carrying the same silencing construct. ( c ) Expression of PsAvh262 in soybean hairy roots enhanced P. sojae infection. Hairy roots expressing GFP-PsAvh262 or GFP were inoculated with mycelia plugs of RFP-labelled wild-type P. sojae strain P6497 (P6497-RFP). Oospore production in the infected hair roots was observed under a confocal microscope (left panel), and lesion length was determined (middle panel) at 48 h.p.i. Expression of GFP or GFP-PsAvh262 was confirmed by western blotting using an anti-GFP antibody (right panel). ( d ) Expression of PsAvh262 in N. benthamiana enhanced infection of Phytophthora capsici . Leaf regions transiently expressing PsAvh262 or GFP, were inoculated with mycelia plugs of P. capsici . Infected leaves were stained with Trypan blue at 36 h.p.i. to visualize disease lesions (left panel) and the sizes of the lesions were determined (middle panel). Expression of GFP or PsAvh262-GFP was confirmed by western blotting using an anti-GFP antibody (right panel). Δ, non-specific band when using anti-GFP, which are common contaminants of western blots present in many published articles detecting GFP-fused proteins expressed in the plant cells 13 64 ; #, PsAvh262 derived band; this may due to some unknown modification or degradation. Error bars represent the mean±s.d.( n =3) and asterisks ( ** or *** ) denote significant differences ( P
    Figure Legend Snippet: PsAvh262 is an essential virulence factor of Phytophthora sojae . ( a ) Expression profile of PsAvh262 during P. sojae strain P6497 infection of soybean hypocotyls. The susceptible soybean cultivar Williams was used as the host. Total RNA was extracted from mycelia (MY) or infected soybean leaves at 1.5, 3, 6, 12 and 24 h post inoculation (h.p.i.). Transcript levels of PsAvh262 were determined by qRT–PCR. The P. sojae actin gene (VMD GeneID: 108986) was used as the pathogen internal control gene, ( b ) Silencing of PsAvh262 in P. sojae greatly impaired the virulence in soybean hypocotyls. Relative transcript levels of PsAvh262 (upper panel) in the P. sojae transformants were determined by qRT–PCR. Disease symptoms (lower panel) in etiolated hypocotyls were observed. Pictures were taken at 7 days post inoculation (d.p.i.). S12 and S141 were non-silenced transformants carrying the same silencing construct. ( c ) Expression of PsAvh262 in soybean hairy roots enhanced P. sojae infection. Hairy roots expressing GFP-PsAvh262 or GFP were inoculated with mycelia plugs of RFP-labelled wild-type P. sojae strain P6497 (P6497-RFP). Oospore production in the infected hair roots was observed under a confocal microscope (left panel), and lesion length was determined (middle panel) at 48 h.p.i. Expression of GFP or GFP-PsAvh262 was confirmed by western blotting using an anti-GFP antibody (right panel). ( d ) Expression of PsAvh262 in N. benthamiana enhanced infection of Phytophthora capsici . Leaf regions transiently expressing PsAvh262 or GFP, were inoculated with mycelia plugs of P. capsici . Infected leaves were stained with Trypan blue at 36 h.p.i. to visualize disease lesions (left panel) and the sizes of the lesions were determined (middle panel). Expression of GFP or PsAvh262-GFP was confirmed by western blotting using an anti-GFP antibody (right panel). Δ, non-specific band when using anti-GFP, which are common contaminants of western blots present in many published articles detecting GFP-fused proteins expressed in the plant cells 13 64 ; #, PsAvh262 derived band; this may due to some unknown modification or degradation. Error bars represent the mean±s.d.( n =3) and asterisks ( ** or *** ) denote significant differences ( P

    Techniques Used: Expressing, Infection, Quantitative RT-PCR, Construct, Microscopy, Western Blot, Staining, Derivative Assay, Modification

    31) Product Images from "Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis"

    Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7833

    Features of intronic location of ultraconserved RNA (uc). 8+ in CASZ1 A. Schematic representation of the transcript including uc.8+ with respect to CASZ1 . J82 RNA was retrotranscribed by using the SMARTer Rapid Amplification of cDNA Ends (RACE) cDNA Amplification kit (Clontech). Primers used for the 5′ RACE were as follows: Universal Primer Mix (UPM) that recognized the SMARTer oligonucleotide added at the 5′ end, gene specific primers 1 (GSP1) that recognized the sense transcript, and GSP2 primers that recognized the antisense transcript. The arrows represent the direction of amplification from the gene-specific primers that successfully amplified the unknown regions of the TUC8 gene. B. 5′- and 3′-RACE polymerase chain reaction (PCR) performed to amplify the uc.8+ cDNA. C. Sequence of the complete uc.8+ transcript (2435 bases) as determined using RACE. The yellow sequence was reported by Bejerano et al , 2004 [v1].
    Figure Legend Snippet: Features of intronic location of ultraconserved RNA (uc). 8+ in CASZ1 A. Schematic representation of the transcript including uc.8+ with respect to CASZ1 . J82 RNA was retrotranscribed by using the SMARTer Rapid Amplification of cDNA Ends (RACE) cDNA Amplification kit (Clontech). Primers used for the 5′ RACE were as follows: Universal Primer Mix (UPM) that recognized the SMARTer oligonucleotide added at the 5′ end, gene specific primers 1 (GSP1) that recognized the sense transcript, and GSP2 primers that recognized the antisense transcript. The arrows represent the direction of amplification from the gene-specific primers that successfully amplified the unknown regions of the TUC8 gene. B. 5′- and 3′-RACE polymerase chain reaction (PCR) performed to amplify the uc.8+ cDNA. C. Sequence of the complete uc.8+ transcript (2435 bases) as determined using RACE. The yellow sequence was reported by Bejerano et al , 2004 [v1].

    Techniques Used: Rapid Amplification of cDNA Ends, Amplification, Polymerase Chain Reaction, Sequencing

    Cellular localization of ultraconserved RNA (uc). 8+ A. Images acquired using inverted fluorescence microscope (magnification, 20×) of J82 control cells (Mock) after transfection with PNA-TO scramble-R8 (PNA-scramble) or with TO-PNA1-R8, the PNA complementary to uc.8+ (PNA-uc.8+). Images were recorded with excitation wavelength (lex)=450–490 nm (DAPI) or lex=510–540 nm (PNA-TO); the superimposition of the images recorded is also reported (Merge). All images were taken with the same confocal microscopy settings. Scale bar, 100 mm. Nuclei of J82 cells were stained with DAPI (blue). B. Inverse correlation between the expression of microRNA (miR)-596 and MMP9 in BlCa samples from 20 patients measured using qRT-PCR. C. Relative expression of MMP9 in siRNA-3 anti-uc.8+ –transfected J82 cells. Endogenous uc.8+ levels in the control cells are shown in grey. Data are expressed as the means ± standard deviation of triplicate values. P values were obtained using the Student t test for independent samples. **P
    Figure Legend Snippet: Cellular localization of ultraconserved RNA (uc). 8+ A. Images acquired using inverted fluorescence microscope (magnification, 20×) of J82 control cells (Mock) after transfection with PNA-TO scramble-R8 (PNA-scramble) or with TO-PNA1-R8, the PNA complementary to uc.8+ (PNA-uc.8+). Images were recorded with excitation wavelength (lex)=450–490 nm (DAPI) or lex=510–540 nm (PNA-TO); the superimposition of the images recorded is also reported (Merge). All images were taken with the same confocal microscopy settings. Scale bar, 100 mm. Nuclei of J82 cells were stained with DAPI (blue). B. Inverse correlation between the expression of microRNA (miR)-596 and MMP9 in BlCa samples from 20 patients measured using qRT-PCR. C. Relative expression of MMP9 in siRNA-3 anti-uc.8+ –transfected J82 cells. Endogenous uc.8+ levels in the control cells are shown in grey. Data are expressed as the means ± standard deviation of triplicate values. P values were obtained using the Student t test for independent samples. **P

    Techniques Used: Fluorescence, Microscopy, Transfection, Confocal Microscopy, Staining, Expressing, Quantitative RT-PCR, Standard Deviation

    Ultraconserved RNA (uc). 8+ and microRNA (miR)-596 interaction and target regulation in bladder cancer (BlCa) cells A. Representative positive correlation between the expression of uc.8+ and that of miR-596 in BlCa samples from 20 patients (Table 1 , dataset 4) measured using qRT-PCR. Correlation was computed using the Spearman correlation coefficient. B. Expression of miR-596 in J82 cell extracts after retrieval of endogenous uc.8+ with a peptide nucleic acid (PNA)/uc.8+ probe. Data are expressed as the means ± standard deviation (SD) of triplicate values. P values were obtained using the Student t test for independent samples. ***P
    Figure Legend Snippet: Ultraconserved RNA (uc). 8+ and microRNA (miR)-596 interaction and target regulation in bladder cancer (BlCa) cells A. Representative positive correlation between the expression of uc.8+ and that of miR-596 in BlCa samples from 20 patients (Table 1 , dataset 4) measured using qRT-PCR. Correlation was computed using the Spearman correlation coefficient. B. Expression of miR-596 in J82 cell extracts after retrieval of endogenous uc.8+ with a peptide nucleic acid (PNA)/uc.8+ probe. Data are expressed as the means ± standard deviation (SD) of triplicate values. P values were obtained using the Student t test for independent samples. ***P

    Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation

    Independent regulation of ultraconserved RNA (uc). 8+ and CASZ1 in bladder cancer (BlCa) tissues A. Schematic representation of the intronic localization of uc.8+ within CASZ1 . CASZ1 exons are indicated by black boxes. The locations of the uc.8+ forward (F) and reverse (R) primers used for qRT-PCR and the probe used for in situ hybridization are shown. Of the siRNAs targeting CASZ1 , siRNA-1 is located at the 5′ untranslated region (UTR), siRNA-2 is located in exon 6, and siRNA-3 is located at the 3′ UTR. B. RNA levels of CASZ1 and uc.8+ were determined by qRT-PCR in BlCa (n=19, black dots) and control normal bladder epithelium (NBE) samples (n=11, empty circles). Results are presented as means ± standard deviation (SD). Spearman correlation coefficient and P values are indicated. C. CASZ1 expression after silencing of uc.8+. The expression of CASZ1 was not affected in J82 cells transfected with three different siRNAs anti-uc.8+ or siRNA control. D. J82 cells were transfected with siRNA anti- CASZ1 or siRNA control. The CASZ1 level was determined by qRT-PCR. uc.8+ expression was not affected by any of the siRNAs anti- CASZ1 used. Data are expressed as the mean ± SD of triplicate values.
    Figure Legend Snippet: Independent regulation of ultraconserved RNA (uc). 8+ and CASZ1 in bladder cancer (BlCa) tissues A. Schematic representation of the intronic localization of uc.8+ within CASZ1 . CASZ1 exons are indicated by black boxes. The locations of the uc.8+ forward (F) and reverse (R) primers used for qRT-PCR and the probe used for in situ hybridization are shown. Of the siRNAs targeting CASZ1 , siRNA-1 is located at the 5′ untranslated region (UTR), siRNA-2 is located in exon 6, and siRNA-3 is located at the 3′ UTR. B. RNA levels of CASZ1 and uc.8+ were determined by qRT-PCR in BlCa (n=19, black dots) and control normal bladder epithelium (NBE) samples (n=11, empty circles). Results are presented as means ± standard deviation (SD). Spearman correlation coefficient and P values are indicated. C. CASZ1 expression after silencing of uc.8+. The expression of CASZ1 was not affected in J82 cells transfected with three different siRNAs anti-uc.8+ or siRNA control. D. J82 cells were transfected with siRNA anti- CASZ1 or siRNA control. The CASZ1 level was determined by qRT-PCR. uc.8+ expression was not affected by any of the siRNAs anti- CASZ1 used. Data are expressed as the mean ± SD of triplicate values.

    Techniques Used: Quantitative RT-PCR, In Situ Hybridization, Standard Deviation, Expressing, Transfection

    Effect of ultraconserved RNA (uc). 8+ silencing on bladder cancer (BlCa) cell proliferation, migration, and invasion A. J82 cells were transfected with siRNA anti-uc.8+ or siRNA control and were seeded in 96-well plates. Cell proliferation was determined at the indicated time points. The number of cells per well was measured by the absorbance at 595 nm. The results show data from at least three independent experiments. Cell growth after transfection with siRNA-3 anti-uc.8+ was not significantly different from that of cells transfected with siRNA-2 anti-uc.8+. P values were obtained using the Student t test for independent samples. *P
    Figure Legend Snippet: Effect of ultraconserved RNA (uc). 8+ silencing on bladder cancer (BlCa) cell proliferation, migration, and invasion A. J82 cells were transfected with siRNA anti-uc.8+ or siRNA control and were seeded in 96-well plates. Cell proliferation was determined at the indicated time points. The number of cells per well was measured by the absorbance at 595 nm. The results show data from at least three independent experiments. Cell growth after transfection with siRNA-3 anti-uc.8+ was not significantly different from that of cells transfected with siRNA-2 anti-uc.8+. P values were obtained using the Student t test for independent samples. *P

    Techniques Used: Migration, Transfection

    32) Product Images from "Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo"

    Article Title: Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2010.67

    Tube formation experiment with HUVECs. (A) Representative images of tube formation. The HUVECs of AAV-VEGF/BMP group displayed stronger migration, proliferation, and tube formation ability than other three groups (magnification×200). (B) The data is expressed as the mean±SD from three independent experiments. b P
    Figure Legend Snippet: Tube formation experiment with HUVECs. (A) Representative images of tube formation. The HUVECs of AAV-VEGF/BMP group displayed stronger migration, proliferation, and tube formation ability than other three groups (magnification×200). (B) The data is expressed as the mean±SD from three independent experiments. b P

    Techniques Used: Migration

    Representative images of orthotopic ossification in the rabbit hind limb. Orthotopic ossification was radiographically evident in AAV-VEGF/BMP group eight weeks post injection (indicated as arrows). However, no radiographic evidence of bone formation was observed in AAV-GFP group and AAV-VEGF group.
    Figure Legend Snippet: Representative images of orthotopic ossification in the rabbit hind limb. Orthotopic ossification was radiographically evident in AAV-VEGF/BMP group eight weeks post injection (indicated as arrows). However, no radiographic evidence of bone formation was observed in AAV-GFP group and AAV-VEGF group.

    Techniques Used: Injection

    Expression of hVEGF 165 and hBMP-7. (A–D) Representative images of RT-PCR assay of AAV-GFP group (A), AAV-VEGF group (B), AAV-BMP group (C) and AAV-VEGF/BMP group (D). The size of the PCR products for hVEGF 165 , hBMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the brightness of the VEGF 165 or BMP-7 bands increased in AAV-VEGF/BMP group. No hVEGF 165 or hBMP-7 band could be detected in AAV-GFP group. Track 1–7 stands for the 1st, 2nd, 3rd, 7th, 14th, 21st, and 28th days post-transfection. (E–H) Representative images of Western blotting assay of AAV-GFP group (E), AAV-VEGF group (F), AAV-BMP group (G) and AAV-VEGF/BMP group (H). The molecular weights of hVEGF 165 , hBMP-7, and β-actin were 23 kDa, 55 kDa and 43 kDa respectively. Strong staining with the expected molecular weight was observed in AAV-VEGF/BMP group, and no hVEGF 165 or hBMP-7 band was observed in AAV-GFP group. (I) ELISA assay for VEGF protein expression. The data is expressed as the mean±SD from three independent experiments. b P
    Figure Legend Snippet: Expression of hVEGF 165 and hBMP-7. (A–D) Representative images of RT-PCR assay of AAV-GFP group (A), AAV-VEGF group (B), AAV-BMP group (C) and AAV-VEGF/BMP group (D). The size of the PCR products for hVEGF 165 , hBMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the brightness of the VEGF 165 or BMP-7 bands increased in AAV-VEGF/BMP group. No hVEGF 165 or hBMP-7 band could be detected in AAV-GFP group. Track 1–7 stands for the 1st, 2nd, 3rd, 7th, 14th, 21st, and 28th days post-transfection. (E–H) Representative images of Western blotting assay of AAV-GFP group (E), AAV-VEGF group (F), AAV-BMP group (G) and AAV-VEGF/BMP group (H). The molecular weights of hVEGF 165 , hBMP-7, and β-actin were 23 kDa, 55 kDa and 43 kDa respectively. Strong staining with the expected molecular weight was observed in AAV-VEGF/BMP group, and no hVEGF 165 or hBMP-7 band was observed in AAV-GFP group. (I) ELISA assay for VEGF protein expression. The data is expressed as the mean±SD from three independent experiments. b P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Infection, Transfection, Western Blot, Staining, Molecular Weight, Enzyme-linked Immunosorbent Assay

    Blood flow in the rabbit hind limb. Blood flow in the anterior tibial artery of ischemic and normal hind limbs was measured at rest with an Aspen Advanced Doppler ultrasound device using a perivascular flow probe 8 weeks post-injection. (A) Representative record images of blood flow in AAV-GFP, AAV-VEGF, and AAV-BMP and AAV-VEGF/BMP group. (B) Mean blood flow in the ischemic limbs 8 weeks post-injection. Values are expressed as a percentage of contralateral limbs and are shown as mean values±SEM from three independent experiments. b P
    Figure Legend Snippet: Blood flow in the rabbit hind limb. Blood flow in the anterior tibial artery of ischemic and normal hind limbs was measured at rest with an Aspen Advanced Doppler ultrasound device using a perivascular flow probe 8 weeks post-injection. (A) Representative record images of blood flow in AAV-GFP, AAV-VEGF, and AAV-BMP and AAV-VEGF/BMP group. (B) Mean blood flow in the ischemic limbs 8 weeks post-injection. Values are expressed as a percentage of contralateral limbs and are shown as mean values±SEM from three independent experiments. b P

    Techniques Used: Flow Cytometry, Injection

    33) Product Images from "Characterization and tissue-specific expression patterns of the Plasmodium chabaudi cir multigene family"

    Article Title: Characterization and tissue-specific expression patterns of the Plasmodium chabaudi cir multigene family

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-10-272

    Transcriptional changes of cir gene expression during the course of infection . For RFLP analyses of expression changes of the cir genes during the course of infection, blood of female NMRI mice infected with 100 pRBCs were passaged on days 7, 14 and 21 days post infections (d.p.i.) into naïve female NMRI mice. Blood of these passaged mice was again collected at 30% parasitaemia. After amplification using the subfamily-specific primers for both subfamilies, 150 ng of purified RT-PCR products (approx. 600 bp) were digested with Alu I and 30 ng were then analysed using the DNA 1000 kit for the Agilent 2100 bioanalyzer. The RT-PCR RFLP profiles for four mice of subfamily 1 are presented in (A). The restriction digest of subfamily 2 are shown in the upper panel of (B) where only at day 7 p.i. and day 14 p.i. of mouse 1 a few smaller restriction fragments could be detected. Therefore a second digest with Xap I for 3 h at 37°C was performed (B, lower panel). DNA 1000 bp ladder was used and in each lane an upper (1000 bp, purple) and a lower (25 bp, green) marker are indicated.
    Figure Legend Snippet: Transcriptional changes of cir gene expression during the course of infection . For RFLP analyses of expression changes of the cir genes during the course of infection, blood of female NMRI mice infected with 100 pRBCs were passaged on days 7, 14 and 21 days post infections (d.p.i.) into naïve female NMRI mice. Blood of these passaged mice was again collected at 30% parasitaemia. After amplification using the subfamily-specific primers for both subfamilies, 150 ng of purified RT-PCR products (approx. 600 bp) were digested with Alu I and 30 ng were then analysed using the DNA 1000 kit for the Agilent 2100 bioanalyzer. The RT-PCR RFLP profiles for four mice of subfamily 1 are presented in (A). The restriction digest of subfamily 2 are shown in the upper panel of (B) where only at day 7 p.i. and day 14 p.i. of mouse 1 a few smaller restriction fragments could be detected. Therefore a second digest with Xap I for 3 h at 37°C was performed (B, lower panel). DNA 1000 bp ladder was used and in each lane an upper (1000 bp, purple) and a lower (25 bp, green) marker are indicated.

    Techniques Used: Expressing, Infection, Mouse Assay, Amplification, Purification, Reverse Transcription Polymerase Chain Reaction, Marker

    Expression profile analyses of cir genes in different tissues of infected mice by RT-PCR RFLP . For RFLP analyses, organs and blood were collected at a parasitaemia of 30% from female NMRI mice infected with 100 pRBCs. After RT-PCR amplification for the six host tissues with the subfamily-specific primers, 150 ng of purified RT-PCR products (approx. 600 bp) were digested with Alu I. The DNA fragments (30 ng) were analysed using the DNA 1000 kit for the Agilent 2100 bioanalyzer for accurate sizing. Compared are RT-PCR RFLP profiles of blood, liver, spleen, kidney, lung and brain for four mice for cir subfamily 1 (A) and cir subfamily 2 (B). DNA 1000 bp ladder was used and in each lane an upper (1000 bp, purple) and a lower (25 bp, green) marker are indicated.
    Figure Legend Snippet: Expression profile analyses of cir genes in different tissues of infected mice by RT-PCR RFLP . For RFLP analyses, organs and blood were collected at a parasitaemia of 30% from female NMRI mice infected with 100 pRBCs. After RT-PCR amplification for the six host tissues with the subfamily-specific primers, 150 ng of purified RT-PCR products (approx. 600 bp) were digested with Alu I. The DNA fragments (30 ng) were analysed using the DNA 1000 kit for the Agilent 2100 bioanalyzer for accurate sizing. Compared are RT-PCR RFLP profiles of blood, liver, spleen, kidney, lung and brain for four mice for cir subfamily 1 (A) and cir subfamily 2 (B). DNA 1000 bp ladder was used and in each lane an upper (1000 bp, purple) and a lower (25 bp, green) marker are indicated.

    Techniques Used: Expressing, Infection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Purification, Marker

    Transcriptional changes of cir genes during intraerythrocytic development . For expression profiling of the cir genes at three different time points in the life cycle, 30 μl tail vein blood of female NMRI mice infected with 100 pRBCs were collected 3 h (early trophozoites), 10 h (late trophozoites) and 17 h (mature trophozoites and early schizonts) after beginning of the light cycle on day 13 p.i. (parasitaemia about 30%). Amplification was performed using the subfamily-specific primers for both subfamilies and 150 ng of purified RT-PCR products (approx. 600 bp) were digested with Alu I and 30 ng were then analysed using the DNA 1000 kit for the Agilent 2100 bioanalyzer. The RFLP profiles of subfamily 1 are shown for four mice in (A). The restriction digests of subfamily 2 are shown in the upper panel of (B). Only a few restriction fragments were detected in all samples, therefore RT-PCR products were also restricted with Xap I (B, lower panel). DNA 1000 bp ladder was used and in each lane an upper (1000 bp, purple) and a lower (25 bp, green) marker were indicated.
    Figure Legend Snippet: Transcriptional changes of cir genes during intraerythrocytic development . For expression profiling of the cir genes at three different time points in the life cycle, 30 μl tail vein blood of female NMRI mice infected with 100 pRBCs were collected 3 h (early trophozoites), 10 h (late trophozoites) and 17 h (mature trophozoites and early schizonts) after beginning of the light cycle on day 13 p.i. (parasitaemia about 30%). Amplification was performed using the subfamily-specific primers for both subfamilies and 150 ng of purified RT-PCR products (approx. 600 bp) were digested with Alu I and 30 ng were then analysed using the DNA 1000 kit for the Agilent 2100 bioanalyzer. The RFLP profiles of subfamily 1 are shown for four mice in (A). The restriction digests of subfamily 2 are shown in the upper panel of (B). Only a few restriction fragments were detected in all samples, therefore RT-PCR products were also restricted with Xap I (B, lower panel). DNA 1000 bp ladder was used and in each lane an upper (1000 bp, purple) and a lower (25 bp, green) marker were indicated.

    Techniques Used: Expressing, Mouse Assay, Infection, Amplification, Purification, Reverse Transcription Polymerase Chain Reaction, Marker

    34) Product Images from "Over-expression of Grhl2 causes spina bifida in the Axial defects mutant mouse"

    Article Title: Over-expression of Grhl2 causes spina bifida in the Axial defects mutant mouse

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddr031

    Spinal neural tube closure in Axd and Grhl2 GT compound heterozygotes. ( A ) The length of the PNP (the open region of spinal neural folds) was assessed in relation to developmental stage (as determined by the number of somites) in E10.5 embryos from crosses between Axd/+ and Grhl2 GT /+ mice. Mean PNP length was significantly greater in Axd/+ embryos than all other genotypes at the 30–34 somite stages (* P
    Figure Legend Snippet: Spinal neural tube closure in Axd and Grhl2 GT compound heterozygotes. ( A ) The length of the PNP (the open region of spinal neural folds) was assessed in relation to developmental stage (as determined by the number of somites) in E10.5 embryos from crosses between Axd/+ and Grhl2 GT /+ mice. Mean PNP length was significantly greater in Axd/+ embryos than all other genotypes at the 30–34 somite stages (* P

    Techniques Used: Mouse Assay

    Increased ventral curvature and diminished hindgut cell proliferation in Axd mutant embryos. ( A – C ) In the caudal region of E10.5 Grhl2 GT/+ (A and B) and Grhl2 GT/GT (C) embryos, X-Gal staining is detected in the hindgut underlying the PNP region (sections in B and C at the level of the dotted line in A). Note staining also in surface ectoderm. The open neural folds in the Grhl2 GT/GT embryo (C) correspond to failure of spinal neurulation in this genotype. ( D – F ) Ventral curvature of the caudal region appears greater in Axd/Axd embryos (E) than in wild-type littermates (D). Quantitation of ventral curvature (angle between lines a and b in A) in a series of 27–33 somite stage embryos at E10.5. Axd/Axd embryos ( n = 10) have significantly greater caudal curvature than either +/+ ( n = 10) or Axd/+ ( n = 18) littermates (* P
    Figure Legend Snippet: Increased ventral curvature and diminished hindgut cell proliferation in Axd mutant embryos. ( A – C ) In the caudal region of E10.5 Grhl2 GT/+ (A and B) and Grhl2 GT/GT (C) embryos, X-Gal staining is detected in the hindgut underlying the PNP region (sections in B and C at the level of the dotted line in A). Note staining also in surface ectoderm. The open neural folds in the Grhl2 GT/GT embryo (C) correspond to failure of spinal neurulation in this genotype. ( D – F ) Ventral curvature of the caudal region appears greater in Axd/Axd embryos (E) than in wild-type littermates (D). Quantitation of ventral curvature (angle between lines a and b in A) in a series of 27–33 somite stage embryos at E10.5. Axd/Axd embryos ( n = 10) have significantly greater caudal curvature than either +/+ ( n = 10) or Axd/+ ( n = 18) littermates (* P

    Techniques Used: Mutagenesis, Staining, Quantitation Assay

    Expression of Grhl2 is elevated in Axd mutant embryos. ( A and B ) Expression analysis by qRT–PCR using mRNA extracted from whole embryos (A) and isolated caudal regions (B), at the 30–31 somite stage. There is significant up-regulation of Grhl2 expression in Axd/Axd embryos compared with wild-type littermates, particularly from the 25 somite stage onwards (A). Heterozygous embryos exhibit an intermediate level of Grhl2 expression in the caudal region (B), whereas no difference in expression was observed for the remaining genes in the Axd critical region. ( C – F ) Whole-mount in situ hybridization for Grhl2 consistently shows more intense staining in intact Axd/Axd embryos (D) compared with wild-type controls (C). Sites of expression include the otic vesicle (white arrowhead in C), pharyngeal region (white arrowhead in D) and forebrain (black arrowhead in D). Sections through the caudal region (at the level of the dotted lines in C and D) reveal expression of Grhl2 in the hindgut endoderm, whereas the neural folds are negative (E and F). Note the more intense hindgut expression in the Axd/Axd embryo (F) with apparent ectopic expression in mesoderm lateral to the hindgut, which is not observed in wild-type (E). Scale bars: 1 mm (C and D) and 0.1 mm (E and F).
    Figure Legend Snippet: Expression of Grhl2 is elevated in Axd mutant embryos. ( A and B ) Expression analysis by qRT–PCR using mRNA extracted from whole embryos (A) and isolated caudal regions (B), at the 30–31 somite stage. There is significant up-regulation of Grhl2 expression in Axd/Axd embryos compared with wild-type littermates, particularly from the 25 somite stage onwards (A). Heterozygous embryos exhibit an intermediate level of Grhl2 expression in the caudal region (B), whereas no difference in expression was observed for the remaining genes in the Axd critical region. ( C – F ) Whole-mount in situ hybridization for Grhl2 consistently shows more intense staining in intact Axd/Axd embryos (D) compared with wild-type controls (C). Sites of expression include the otic vesicle (white arrowhead in C), pharyngeal region (white arrowhead in D) and forebrain (black arrowhead in D). Sections through the caudal region (at the level of the dotted lines in C and D) reveal expression of Grhl2 in the hindgut endoderm, whereas the neural folds are negative (E and F). Note the more intense hindgut expression in the Axd/Axd embryo (F) with apparent ectopic expression in mesoderm lateral to the hindgut, which is not observed in wild-type (E). Scale bars: 1 mm (C and D) and 0.1 mm (E and F).

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Isolation, In Situ Hybridization, Staining

    The Axd mutation maps to a critical region on mouse chromosome 15. ( A ) In DNA from embryos with spina bifida from an intercross of Axd and C57BL/6 mice, typing of polymorphic markers showed a peak of BALB/c and Axd alleles (non-C57BL/6) between D15Mit252 and D15Mit171 (22.6–79.4 Mb). Within this region, a peak of Axd alleles (non-BALB/c or C57BL/6) was present at 38.5–43.6 Mb. ( B ) Using additional novel polymorphic markers, a critical region of 1.1 Mb was defined between markers CA15–16 and CA15-05 (36.4–37.5 Mb). This region contains 10 known genes (black boxes), as well as 3 RNA genes and various putative processed transcripts (as of Ensembl release 60). The marker D15Mit250 showed 100% association of homozygosity for Axd alleles with spina bifida. ( C ) In embryos that were genotyped using D15Mit250, the PNP length was compared at stages during and after spinal neural tube closure. The PNP fails to shorten and close in homozygous Axd/Axd embryos (black circles), leading to spina bifida. In contrast, most wild-type embryos exhibit closed PNPs by the 29–31 somite stage (white circles) and Axd/+ embryos complete closure by the 35–37 somite stage in most cases (grey circles).
    Figure Legend Snippet: The Axd mutation maps to a critical region on mouse chromosome 15. ( A ) In DNA from embryos with spina bifida from an intercross of Axd and C57BL/6 mice, typing of polymorphic markers showed a peak of BALB/c and Axd alleles (non-C57BL/6) between D15Mit252 and D15Mit171 (22.6–79.4 Mb). Within this region, a peak of Axd alleles (non-BALB/c or C57BL/6) was present at 38.5–43.6 Mb. ( B ) Using additional novel polymorphic markers, a critical region of 1.1 Mb was defined between markers CA15–16 and CA15-05 (36.4–37.5 Mb). This region contains 10 known genes (black boxes), as well as 3 RNA genes and various putative processed transcripts (as of Ensembl release 60). The marker D15Mit250 showed 100% association of homozygosity for Axd alleles with spina bifida. ( C ) In embryos that were genotyped using D15Mit250, the PNP length was compared at stages during and after spinal neural tube closure. The PNP fails to shorten and close in homozygous Axd/Axd embryos (black circles), leading to spina bifida. In contrast, most wild-type embryos exhibit closed PNPs by the 29–31 somite stage (white circles) and Axd/+ embryos complete closure by the 35–37 somite stage in most cases (grey circles).

    Techniques Used: Mutagenesis, Mouse Assay, Marker

    35) Product Images from "Over-expression of Grhl2 causes spina bifida in the Axial defects mutant mouse"

    Article Title: Over-expression of Grhl2 causes spina bifida in the Axial defects mutant mouse

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddr031

    The Axd mutation maps to a critical region on mouse chromosome 15. ( A ) In DNA from embryos with spina bifida from an intercross of Axd and C57BL/6 mice, typing of polymorphic markers showed a peak of BALB/c and Axd alleles (non-C57BL/6) between D15Mit252 and D15Mit171 (22.6–79.4 Mb). Within this region, a peak of Axd alleles (non-BALB/c or C57BL/6) was present at 38.5–43.6 Mb. ( B ) Using additional novel polymorphic markers, a critical region of 1.1 Mb was defined between markers CA15–16 and CA15-05 (36.4–37.5 Mb). This region contains 10 known genes (black boxes), as well as 3 RNA genes and various putative processed transcripts (as of Ensembl release 60). The marker D15Mit250 showed 100% association of homozygosity for Axd alleles with spina bifida. ( C ) In embryos that were genotyped using D15Mit250, the PNP length was compared at stages during and after spinal neural tube closure. The PNP fails to shorten and close in homozygous Axd/Axd embryos (black circles), leading to spina bifida. In contrast, most wild-type embryos exhibit closed PNPs by the 29–31 somite stage (white circles) and Axd/+ embryos complete closure by the 35–37 somite stage in most cases (grey circles).
    Figure Legend Snippet: The Axd mutation maps to a critical region on mouse chromosome 15. ( A ) In DNA from embryos with spina bifida from an intercross of Axd and C57BL/6 mice, typing of polymorphic markers showed a peak of BALB/c and Axd alleles (non-C57BL/6) between D15Mit252 and D15Mit171 (22.6–79.4 Mb). Within this region, a peak of Axd alleles (non-BALB/c or C57BL/6) was present at 38.5–43.6 Mb. ( B ) Using additional novel polymorphic markers, a critical region of 1.1 Mb was defined between markers CA15–16 and CA15-05 (36.4–37.5 Mb). This region contains 10 known genes (black boxes), as well as 3 RNA genes and various putative processed transcripts (as of Ensembl release 60). The marker D15Mit250 showed 100% association of homozygosity for Axd alleles with spina bifida. ( C ) In embryos that were genotyped using D15Mit250, the PNP length was compared at stages during and after spinal neural tube closure. The PNP fails to shorten and close in homozygous Axd/Axd embryos (black circles), leading to spina bifida. In contrast, most wild-type embryos exhibit closed PNPs by the 29–31 somite stage (white circles) and Axd/+ embryos complete closure by the 35–37 somite stage in most cases (grey circles).

    Techniques Used: Mutagenesis, Mouse Assay, Marker

    36) Product Images from "Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with X-linked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes"

    Article Title: Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with X-linked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes

    Journal: Neurogenetics

    doi: 10.1007/s10048-010-0253-6

    Identified single nucleotide polymorphisms (SNPs) of ABCD2 , ABCD3 , and ABCD4 ( upper panel ). Comprehensive resequencing of ABCD2 , ABCD3 , and ABCD4 genes of the 40 patients with adrenoleukodystrophy (ALD) revealed two novel SNPs, nine SNPs (six known and three novel SNPs), and 13 SNPs (seven known and six novel SNPs), respectively. Red characters indicate the novel SNPs, blue characters indicate the SNPs identified in the coding region, and black characters indicate the SNPs identified in the noncoding region. Linkage disequilibrium (LD) map of SNPs of ABCD4 in Japanese patients with ALD and the controls using the Haploview version 4.1 ( lower panel ). The five known SNPs (rs17782508, rs2301345, rs4148077, rs4148078, and rs3742801) were in complete disequilibrium in Japanese patients with ALD and the controls (LOD = 43.97, r 2 = 1.0, D′ = 1.0). Novel SNP7 and the five known SNPs (rs17782508, rs2301345, rs4148077, rs4148078, and rs3742801) were not in strong disequilibrium in Japanese patients with ALD and the controls (LOD = 1.15, r 2 = 0.037, D′ = 0.706), although novel SNP7 and the five known SNPs (rs17782508, rs2301345, rs4148077, rs4148078, and rs3742801) were strong disequilibrium only in Japanese patients with ALD (LOD = 2.02, r 2 = 0.221, D′ = 1.0). The number in the box indicates the data of D′. The color of the box is determined from the LOD score and D′. The block was determined using a confidence interval algorithm [ 33 ]
    Figure Legend Snippet: Identified single nucleotide polymorphisms (SNPs) of ABCD2 , ABCD3 , and ABCD4 ( upper panel ). Comprehensive resequencing of ABCD2 , ABCD3 , and ABCD4 genes of the 40 patients with adrenoleukodystrophy (ALD) revealed two novel SNPs, nine SNPs (six known and three novel SNPs), and 13 SNPs (seven known and six novel SNPs), respectively. Red characters indicate the novel SNPs, blue characters indicate the SNPs identified in the coding region, and black characters indicate the SNPs identified in the noncoding region. Linkage disequilibrium (LD) map of SNPs of ABCD4 in Japanese patients with ALD and the controls using the Haploview version 4.1 ( lower panel ). The five known SNPs (rs17782508, rs2301345, rs4148077, rs4148078, and rs3742801) were in complete disequilibrium in Japanese patients with ALD and the controls (LOD = 43.97, r 2 = 1.0, D′ = 1.0). Novel SNP7 and the five known SNPs (rs17782508, rs2301345, rs4148077, rs4148078, and rs3742801) were not in strong disequilibrium in Japanese patients with ALD and the controls (LOD = 1.15, r 2 = 0.037, D′ = 0.706), although novel SNP7 and the five known SNPs (rs17782508, rs2301345, rs4148077, rs4148078, and rs3742801) were strong disequilibrium only in Japanese patients with ALD (LOD = 2.02, r 2 = 0.221, D′ = 1.0). The number in the box indicates the data of D′. The color of the box is determined from the LOD score and D′. The block was determined using a confidence interval algorithm [ 33 ]

    Techniques Used: Blocking Assay

    37) Product Images from "Selective Decrease of Components of the Creatine Kinase System and ATP Synthase Complex in Chronic Chagas Disease Cardiomyopathy"

    Article Title: Selective Decrease of Components of the Creatine Kinase System and ATP Synthase Complex in Chronic Chagas Disease Cardiomyopathy

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001205

    mRNA expression of energy metabolism enzymes by real time RT-PCR. (A) ATPα: ATP synthase alpha subunit, (B) ATPβ: ATP synthase beta subunit, (C) CKM: creatine kinase M and (D) CKMit: mitochondrial creatine kinase. The horizontal lines show statistically significant changes: *p
    Figure Legend Snippet: mRNA expression of energy metabolism enzymes by real time RT-PCR. (A) ATPα: ATP synthase alpha subunit, (B) ATPβ: ATP synthase beta subunit, (C) CKM: creatine kinase M and (D) CKMit: mitochondrial creatine kinase. The horizontal lines show statistically significant changes: *p

    Techniques Used: Expressing, Quantitative RT-PCR

    38) Product Images from "Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae"

    Article Title: Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq583

    CPA-sRNA isolation and size distribution. ( A ) Strategy for CPA-sRNA preparation from mycelial total RNA. The protocol ensures capture of RNA species that possess both a 5′-cap and a 3′-polyadenylated tail. The first treatment with BAP prevents RNA containing a 5′-free phosphate from being able to ligate to the 5′-linker. The use of (dT) 20 VN oligo for single-strand cDNA priming allows cDNA to be synthesized exclusively from RNA containing polyA. Following amplification by PCR, small cDNAs (
    Figure Legend Snippet: CPA-sRNA isolation and size distribution. ( A ) Strategy for CPA-sRNA preparation from mycelial total RNA. The protocol ensures capture of RNA species that possess both a 5′-cap and a 3′-polyadenylated tail. The first treatment with BAP prevents RNA containing a 5′-free phosphate from being able to ligate to the 5′-linker. The use of (dT) 20 VN oligo for single-strand cDNA priming allows cDNA to be synthesized exclusively from RNA containing polyA. Following amplification by PCR, small cDNAs (

    Techniques Used: Isolation, Synthesized, Amplification, Polymerase Chain Reaction

    39) Product Images from "Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici"

    Article Title: Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici

    Journal: Fungal Genetics and Biology

    doi: 10.1016/j.fgb.2015.03.023

    Agarose gels showing the outcome of control PCR experiments. (A) DNA fragments of 5′ end of the myosin chitin synthase 1 ( mcs1 ) were amplified using primers CC-125 and CC-117 (see Table 1 ). In all three preparations, no PCR fragment was found in the absence of template (control), whereas strong bands of 707 bp appeared after PCR on total RNA preparations (lanes 3 and 4). These bands were not present when RNA which had been pre-treated with DNase I to remove contaminating genomic DNA (lanes 5 and 6). After transcribing this purified RNA into cDNA, PCR product of 585 bp appeared confirming the splicing of 122 bp predicted intron. The absence of 122 bp intron on the cDNA product was further confirmed by cDNA sequencing. Note that (1/10) and (1/50) indicate dilutions (1/10: 1 part RNA, 9 parts water; 1/50: 1 part RNA, 49 parts water). (B) Random amplification of yeast colonies with match maker PCR mix generated products with maximum sizes of 2000 bp in all three cDNA libraries (only IPO323_Yeasts shown). This suggests that entire open reading frames of proteins, up to ∼600–700 aa long, are represented in the library. Note that PCRs designed to amplify shorter fragments (585 bp and 1544 bp) of the chitin synthase gene mcs1 (5568 bp without introns) still produced positive bands (see main text). This suggests that fragments of larger genes are also represented in the libraries. (C) Primers were designed to amplify the entire open reading frame of the small GTPases rab7 (815 bp) and rab11 (807 bp) (see Table 1 , rab7 : primers SK-Sep-63 and SK-Sep-64; rab11 : primers SK-Sep-65 and SK-Sep-66). Both open reading frames were amplified from genomic DNA of IPO323. Smaller fragments (615 bp and 633 bp) were found after PCR reactions using cDNA from all three preparations (IPO323_Yeasts, IPO323_Hyphae, K4418_mixed). This corresponds with the predicted presence of introns in both genes ( rab7 : 815 bp; rab11 : 807 bp; see main text for more details) and further confirmed by DNA sequencing.
    Figure Legend Snippet: Agarose gels showing the outcome of control PCR experiments. (A) DNA fragments of 5′ end of the myosin chitin synthase 1 ( mcs1 ) were amplified using primers CC-125 and CC-117 (see Table 1 ). In all three preparations, no PCR fragment was found in the absence of template (control), whereas strong bands of 707 bp appeared after PCR on total RNA preparations (lanes 3 and 4). These bands were not present when RNA which had been pre-treated with DNase I to remove contaminating genomic DNA (lanes 5 and 6). After transcribing this purified RNA into cDNA, PCR product of 585 bp appeared confirming the splicing of 122 bp predicted intron. The absence of 122 bp intron on the cDNA product was further confirmed by cDNA sequencing. Note that (1/10) and (1/50) indicate dilutions (1/10: 1 part RNA, 9 parts water; 1/50: 1 part RNA, 49 parts water). (B) Random amplification of yeast colonies with match maker PCR mix generated products with maximum sizes of 2000 bp in all three cDNA libraries (only IPO323_Yeasts shown). This suggests that entire open reading frames of proteins, up to ∼600–700 aa long, are represented in the library. Note that PCRs designed to amplify shorter fragments (585 bp and 1544 bp) of the chitin synthase gene mcs1 (5568 bp without introns) still produced positive bands (see main text). This suggests that fragments of larger genes are also represented in the libraries. (C) Primers were designed to amplify the entire open reading frame of the small GTPases rab7 (815 bp) and rab11 (807 bp) (see Table 1 , rab7 : primers SK-Sep-63 and SK-Sep-64; rab11 : primers SK-Sep-65 and SK-Sep-66). Both open reading frames were amplified from genomic DNA of IPO323. Smaller fragments (615 bp and 633 bp) were found after PCR reactions using cDNA from all three preparations (IPO323_Yeasts, IPO323_Hyphae, K4418_mixed). This corresponds with the predicted presence of introns in both genes ( rab7 : 815 bp; rab11 : 807 bp; see main text for more details) and further confirmed by DNA sequencing.

    Techniques Used: Polymerase Chain Reaction, Amplification, Purification, Sequencing, Generated, Produced, DNA Sequencing

    40) Product Images from "Differential transcription profiles of long non-coding RNAs in primary human brain microvascular endothelial cells in response to meningitic Escherichia coli"

    Article Title: Differential transcription profiles of long non-coding RNAs in primary human brain microvascular endothelial cells in response to meningitic Escherichia coli

    Journal: Scientific Reports

    doi: 10.1038/srep38903

    Transcriptional levels of lncRNAs in U251 and HUVEC cells in response to meningitic E. coli challenge. The relative expression levels of the 30 most differentially expressed lncRNAs as indicated were examined using qPCR. Lnc-CXCL3-1 and lnc-C5-1 was the most significantly upregulated lncRNAs in U251 cells. Lnc-ITGA11-1 and lnc-DIRC1-1 almost maintained their expression levels in U251 cells. In contrast, lnc-ANKRD37-1 and lnc-RAB11B-1 were the lncRNAs that increased the most in HUVECs, but lnc-CXCL3-1 and lnc-PTTG1-1 were almost unchanged in these cells. Similarly, all the downregulated lncRNAs in primary hBMECs were significantly decreased in HUVECs upon infection. The lnc-SMNDC1-1 was undetectable in both U251 and HUVEC cells, which was marked by the empty bar graphs with # symbol. Data are expressed as mean ± SEM from three separate experiments.
    Figure Legend Snippet: Transcriptional levels of lncRNAs in U251 and HUVEC cells in response to meningitic E. coli challenge. The relative expression levels of the 30 most differentially expressed lncRNAs as indicated were examined using qPCR. Lnc-CXCL3-1 and lnc-C5-1 was the most significantly upregulated lncRNAs in U251 cells. Lnc-ITGA11-1 and lnc-DIRC1-1 almost maintained their expression levels in U251 cells. In contrast, lnc-ANKRD37-1 and lnc-RAB11B-1 were the lncRNAs that increased the most in HUVECs, but lnc-CXCL3-1 and lnc-PTTG1-1 were almost unchanged in these cells. Similarly, all the downregulated lncRNAs in primary hBMECs were significantly decreased in HUVECs upon infection. The lnc-SMNDC1-1 was undetectable in both U251 and HUVEC cells, which was marked by the empty bar graphs with # symbol. Data are expressed as mean ± SEM from three separate experiments.

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

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    Real-time Polymerase Chain Reaction:

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    Transfection:

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    Polymerase Chain Reaction:

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    Mutagenesis:

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    Isolation:

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    Quantitative RT-PCR:

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    SYBR Green Assay:

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    High Throughput Screening Assay:

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    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.

    Random Hexamer Labeling:

    Article Title: Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability
    Article Snippet: .. RT-qPCR quantification of RNA abundance 2–50 ng of mRNA (depending on sample) was used for reverse transcription using Superscript II [Life technologies] with random hexamer primers. cDNA was quantified on a StepOnePlus Real-Time PCR system using a SYBR green PCR mix [ThermoFisher] with gene specific primers ( ). .. High-throughput sequencing and RNAseq quantification 50–100 ng mRNA was used as input material to generate strand-specific sequencing libraries using the NEXTflex Rapid Directional Illumina RNA-Seq Library Prep Kit [BioO] according to the manufacturer’s instructions.

    Sequencing:

    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.

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    Thermo Fisher dna digestion mix
    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary <t>DNA</t> (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease <t>(DNase</t> I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Dna Digestion Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnase r
    Subcellular localization of viral circRNAs. ( A ) Nuclear vs. cytoplasmic localization of viral circRNAs. RNA extracted from nuclear (Nuc) and cytoplasmic (Cyto) fractions of the KSHV and EBV coinfected BC1 cell line was either treated (+) or untreated (−) with RNase R. BSJs spanning PCR products from intron-retaining circBART_1.1 and circBART_2.1 were detected mainly in the nuclear fraction. Exonic circBART_1.2 and circBART_2.2 were found in both fractions. The circvIRF4 junction-spanning PCR products were detected in both fractions. Protein immunoblotting for lamin A/C (nuclear) and LAMP1 (cytoplasmic) was used to confirm fractionation quality. ( B ) Polysome profile for viral circRNAs. RNA polysome sucrose gradient profile (254-nm absorbance) of BC1 cell lysates after treatment with CHX ( Top ). Fraction 1 is at the top of the gradient (free mRNAs) and 12 corresponds to the bottom of the gradient. Polysome RNA fractions are represented by fractions 9 to 12. Quantitative PCR revealed that circvIRF4, circBART_1, and circBART_2 RNAs were not detected in polysomal fractions, whereas mRNAs for translated v-cyclin, LMP2, and GAPDH proteins preferentially fractionated with polysomes ( Bottom ).
    Rnase R, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i reaction buffer
    Overview of RT-RamDA and single-cell RamDA-seq.  a  Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I.  b  Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog ,  Pou5f1 ,  Zfp42 , and  Sox2 ) and three housekeeping ( Gnb2l1 ,  Atp5a1 , and  Tubb5 ) genes using a conventional method (−) as a standard.  c  Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section.  d  Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in  b  and  d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile.  e  Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in  d  and  e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Dnase I Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: To eliminate genomic DNA contamination, 1 μL of genomic DNA digestion mix (0.5× PrimeScript Buffer, 0.2 U of DNase I Amplification Grade, 1: 5 000 000 ERCC RNA Spike-In Mix I (Thermo Fisher) in RNase-free water) was added to 1 μL of the denatured sample.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    Subcellular localization of viral circRNAs. ( A ) Nuclear vs. cytoplasmic localization of viral circRNAs. RNA extracted from nuclear (Nuc) and cytoplasmic (Cyto) fractions of the KSHV and EBV coinfected BC1 cell line was either treated (+) or untreated (−) with RNase R. BSJs spanning PCR products from intron-retaining circBART_1.1 and circBART_2.1 were detected mainly in the nuclear fraction. Exonic circBART_1.2 and circBART_2.2 were found in both fractions. The circvIRF4 junction-spanning PCR products were detected in both fractions. Protein immunoblotting for lamin A/C (nuclear) and LAMP1 (cytoplasmic) was used to confirm fractionation quality. ( B ) Polysome profile for viral circRNAs. RNA polysome sucrose gradient profile (254-nm absorbance) of BC1 cell lysates after treatment with CHX ( Top ). Fraction 1 is at the top of the gradient (free mRNAs) and 12 corresponds to the bottom of the gradient. Polysome RNA fractions are represented by fractions 9 to 12. Quantitative PCR revealed that circvIRF4, circBART_1, and circBART_2 RNAs were not detected in polysomal fractions, whereas mRNAs for translated v-cyclin, LMP2, and GAPDH proteins preferentially fractionated with polysomes ( Bottom ).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Circular DNA tumor viruses make circular RNAs

    doi: 10.1073/pnas.1811728115

    Figure Lengend Snippet: Subcellular localization of viral circRNAs. ( A ) Nuclear vs. cytoplasmic localization of viral circRNAs. RNA extracted from nuclear (Nuc) and cytoplasmic (Cyto) fractions of the KSHV and EBV coinfected BC1 cell line was either treated (+) or untreated (−) with RNase R. BSJs spanning PCR products from intron-retaining circBART_1.1 and circBART_2.1 were detected mainly in the nuclear fraction. Exonic circBART_1.2 and circBART_2.2 were found in both fractions. The circvIRF4 junction-spanning PCR products were detected in both fractions. Protein immunoblotting for lamin A/C (nuclear) and LAMP1 (cytoplasmic) was used to confirm fractionation quality. ( B ) Polysome profile for viral circRNAs. RNA polysome sucrose gradient profile (254-nm absorbance) of BC1 cell lysates after treatment with CHX ( Top ). Fraction 1 is at the top of the gradient (free mRNAs) and 12 corresponds to the bottom of the gradient. Polysome RNA fractions are represented by fractions 9 to 12. Quantitative PCR revealed that circvIRF4, circBART_1, and circBART_2 RNAs were not detected in polysomal fractions, whereas mRNAs for translated v-cyclin, LMP2, and GAPDH proteins preferentially fractionated with polysomes ( Bottom ).

    Article Snippet: One microgram of DNase digested RNA was either treated or untreated with Rnase R and reverse-transcribed using SuperScript IV (Thermo Fisher) with random hexamers in a total volume of 20 µL, according to the manufacturer’s protocol.

    Techniques: Polymerase Chain Reaction, Fractionation, Real-time Polymerase Chain Reaction

    Genetic cargo of mRPC derived extracellular vesicles. ( A ) A 1.5% denaturing agarose gel loaded with total RNA from mRPCs and EVs. Total RNA from EVs consisted primarily of species below 800 nucleotides (nt) lacking 28S and 18S rRNA. EVs were treated with RNase and no difference was detected when compared with non-treated EVs, indicating the RNA of EVs was enclosed within the vesicle membrane. ( B ) Transcription factors, a cell-cycle regulator and intermediate filaments were identified in both mRPCs and EVs included Pax6, Hes1, Sox2, Ki67, GFAP and Nestin. The transcription factors identified are collectively involved in facilitating mRPC multipotency, cell-cycle and fate specification during retinogenesis. GFP, GAPDH and β-actin mRNAs were also detected in mRPCs and EVs. Next, the presence of miRNAs with established expression and function during retinogensis were chosen for analysis. ( C ) Selected miRNA species analyzed included Let7d, miR-9, miR-182 and miR-204. U6 snRNA was used as control. Data presented were combined from four independent replicates.

    Journal: Scientific Reports

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins

    doi: 10.1038/s41598-018-20421-1

    Figure Lengend Snippet: Genetic cargo of mRPC derived extracellular vesicles. ( A ) A 1.5% denaturing agarose gel loaded with total RNA from mRPCs and EVs. Total RNA from EVs consisted primarily of species below 800 nucleotides (nt) lacking 28S and 18S rRNA. EVs were treated with RNase and no difference was detected when compared with non-treated EVs, indicating the RNA of EVs was enclosed within the vesicle membrane. ( B ) Transcription factors, a cell-cycle regulator and intermediate filaments were identified in both mRPCs and EVs included Pax6, Hes1, Sox2, Ki67, GFAP and Nestin. The transcription factors identified are collectively involved in facilitating mRPC multipotency, cell-cycle and fate specification during retinogenesis. GFP, GAPDH and β-actin mRNAs were also detected in mRPCs and EVs. Next, the presence of miRNAs with established expression and function during retinogensis were chosen for analysis. ( C ) Selected miRNA species analyzed included Let7d, miR-9, miR-182 and miR-204. U6 snRNA was used as control. Data presented were combined from four independent replicates.

    Article Snippet: Prior to RNA and miRNA extraction, EVs were treated with 100 ug/ml RNAse (Thermoscientific) for 30 min at 37 °C according to manufacturer’s instruction.

    Techniques: Derivative Assay, Agarose Gel Electrophoresis, Expressing

    Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.

    Journal: Scientific Reports

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins

    doi: 10.1038/s41598-018-20421-1

    Figure Lengend Snippet: Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.

    Article Snippet: Prior to RNA and miRNA extraction, EVs were treated with 100 ug/ml RNAse (Thermoscientific) for 30 min at 37 °C according to manufacturer’s instruction.

    Techniques: Incubation, Labeling, Reverse Transcription Polymerase Chain Reaction, Negative Control, Software

    Overview of RT-RamDA and single-cell RamDA-seq.  a  Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I.  b  Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog ,  Pou5f1 ,  Zfp42 , and  Sox2 ) and three housekeeping ( Gnb2l1 ,  Atp5a1 , and  Tubb5 ) genes using a conventional method (−) as a standard.  c  Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section.  d  Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in  b  and  d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile.  e  Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in  d  and  e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay