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Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative <t>PCR</t> analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic <t>DNA</t> (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.
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1) Product Images from "Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice"

Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014391

Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative PCR analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic DNA (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.
Figure Legend Snippet: Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative PCR analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic DNA (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.

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

Identification of the tgε26 transgenic integration site. (A) Physical map of the genomic region between D17mit26-mit80-1 and D17mit 26-mit80-24. a–q: probes for Southern blot analysis. Details of these probes are listed in Supplementary 2 . 1–7: regions of quantitative genomic PCR analysis. S: SalI, E: EcoRI, A: AseI, K: KpnI, Hp: HpaI, Sc: SacI. (B–D) PFGE Southern blot analysis of SalI-digested WT and tgε26 +/+ genomic DNA using the indicated probes. (E) Sequence of Sstr5 -sequence-containing tgε26 +/+ genomic clones that also contained hCD3ε sequence (left). Physical map of the transgenic integration site at the Sstr5 locus (middle). H: HindIII. i: probe for Southern blot analysis. TS1F, TS1R, and CD3e9130F: primers for genomic PCR analysis. Genomic PCR analysis using TS1F/TS1R primers (lanes 1 and 2) and CDe9130F/TS1R primers (lanes 3 and 4) (right). (F, G) Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (H) Physical map of the transgenic integration site at the Metrn locus (left). N: NcoI. q: probe for Southern blot analysis. m7900, m8100, and CD3e23010F: primers for genomic PCR analysis. i-1R, i-1F, i-2R, and i-2F: primers for inverse and nested PCR amplification. Sequence of tgε26 +/+ genomic clones containing both Metrn and hCD3ε sequences (middle). Genomic PCR analysis using m7900/m8100 primers (lanes 1 and 2) and m7900/CD3e23010F primers (lanes 3 and 4) (right). (I, J) PFGE Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (K) Quantitative genomic PCR analysis of WT (open bars) and tgε26 +/+ (black bars) genomic DNA. Primers 1–7 are shown in (A). ct-1 and ct-2 are the genomic regions from chromosome 6 used as controls. Primer sequences are shown in Supplementary Table S3 . Data were normalized to ct-1 signals. Means and standard errors of 3 independent measurements are shown. (L) Configuration of the tgε26 allele.
Figure Legend Snippet: Identification of the tgε26 transgenic integration site. (A) Physical map of the genomic region between D17mit26-mit80-1 and D17mit 26-mit80-24. a–q: probes for Southern blot analysis. Details of these probes are listed in Supplementary 2 . 1–7: regions of quantitative genomic PCR analysis. S: SalI, E: EcoRI, A: AseI, K: KpnI, Hp: HpaI, Sc: SacI. (B–D) PFGE Southern blot analysis of SalI-digested WT and tgε26 +/+ genomic DNA using the indicated probes. (E) Sequence of Sstr5 -sequence-containing tgε26 +/+ genomic clones that also contained hCD3ε sequence (left). Physical map of the transgenic integration site at the Sstr5 locus (middle). H: HindIII. i: probe for Southern blot analysis. TS1F, TS1R, and CD3e9130F: primers for genomic PCR analysis. Genomic PCR analysis using TS1F/TS1R primers (lanes 1 and 2) and CDe9130F/TS1R primers (lanes 3 and 4) (right). (F, G) Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (H) Physical map of the transgenic integration site at the Metrn locus (left). N: NcoI. q: probe for Southern blot analysis. m7900, m8100, and CD3e23010F: primers for genomic PCR analysis. i-1R, i-1F, i-2R, and i-2F: primers for inverse and nested PCR amplification. Sequence of tgε26 +/+ genomic clones containing both Metrn and hCD3ε sequences (middle). Genomic PCR analysis using m7900/m8100 primers (lanes 1 and 2) and m7900/CD3e23010F primers (lanes 3 and 4) (right). (I, J) PFGE Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (K) Quantitative genomic PCR analysis of WT (open bars) and tgε26 +/+ (black bars) genomic DNA. Primers 1–7 are shown in (A). ct-1 and ct-2 are the genomic regions from chromosome 6 used as controls. Primer sequences are shown in Supplementary Table S3 . Data were normalized to ct-1 signals. Means and standard errors of 3 independent measurements are shown. (L) Configuration of the tgε26 allele.

Techniques Used: Transgenic Assay, Southern Blot, Polymerase Chain Reaction, Sequencing, Clone Assay, Nested PCR, Amplification

2) Product Images from "The FTS-Hook-FHIP (FHF) complex interacts with AP-4 to mediate perinuclear distribution of AP-4 and its cargo ATG9A"

Article Title: The FTS-Hook-FHIP (FHF) complex interacts with AP-4 to mediate perinuclear distribution of AP-4 and its cargo ATG9A

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E19-11-0658

Distribution of AP-4 and Hook2 in HeLa cells. (A) IB analysis of HeLa cells treated with siRNA pools as described in Materials and Methods . Control cells were treated with a nontargeting siRNA. Cells were lysed and cleared extracts were subjected to SDS–PAGE and IB with the indicated antibodies. The position of molecular mass markers (in kDa) is indicated at left of blots. Results are representative of at least three independent experiments carried out with different batches of silenced cells prepared on separate days (usually weeks apart). (B) The efficiency of FHIP-L silencing was analyzed by quantitative real-time RT-PCR because of the unavailability of a specific anti-FHIP-L antibody. Results shown represent absolute cDNA levels arising from reverse transcription of 15 ng of total RNA (mean ± SD of technical triplicates; * P
Figure Legend Snippet: Distribution of AP-4 and Hook2 in HeLa cells. (A) IB analysis of HeLa cells treated with siRNA pools as described in Materials and Methods . Control cells were treated with a nontargeting siRNA. Cells were lysed and cleared extracts were subjected to SDS–PAGE and IB with the indicated antibodies. The position of molecular mass markers (in kDa) is indicated at left of blots. Results are representative of at least three independent experiments carried out with different batches of silenced cells prepared on separate days (usually weeks apart). (B) The efficiency of FHIP-L silencing was analyzed by quantitative real-time RT-PCR because of the unavailability of a specific anti-FHIP-L antibody. Results shown represent absolute cDNA levels arising from reverse transcription of 15 ng of total RNA (mean ± SD of technical triplicates; * P

Techniques Used: SDS Page, Quantitative RT-PCR

3) Product Images from "The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription"

Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription

Journal: Virology Journal

doi: 10.1186/s12985-016-0600-9

The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P
Figure Legend Snippet: The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P

Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Injection

UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P
Figure Legend Snippet: UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P

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

Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box
Figure Legend Snippet: Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box

Techniques Used: Mutagenesis, Labeling, Amplification, Expressing, Infection, Polymerase Chain Reaction

4) Product Images from "Pseudouridine formation in archaeal RNAs: The case of Haloferax volcanii"

Article Title: Pseudouridine formation in archaeal RNAs: The case of Haloferax volcanii

Journal: RNA

doi: 10.1261/rna.2712811

cbf5 -deleted strain of H. volcanii is viable. Deletion of the cbf5 gene was confirmed by three independent methods. ( A ) Schematics to show HVO_2493 (thick lines) in the genomes of wild-type (WT) and cbf5 -deleted (Δ cbf5 ) strains and on plasmid pHCbf5. Eco RI and Nde I (in plasmid) sites are marked. Positions of primers 1–5 (Cbf5_Ext_Fwd, Cbf5_Ext_Rev, Cbf5_Int_Fwd, Cbf5_Int_Rew, and HVCBF5-F, respectively) are shown by arrows. The arrow direction indicates 5′ to 3′ direction of the primers. Segment between broken lines is deleted in the Δ cbf5 strain. The His-tag at the C terminus of the Cbf5 protein is indicated by an asterisk. Approximate lengths of the segments are indicated but are not drawn to a scale. ( B ) PCR products using primers designed to anneal outside the gene (primers 1 and 2 in A ) confirm a genomic rearrangement of correctly predicted sizes in WT and mutant strains. ( C ) PCR products using primers designed to anneal within the target gene (primers 3 and 4 in A ) show the absence of the HVO_2493 segment in the mutant and its presence in the WT strain. ( D ) Southern hybridization is consistent with loss of the gene in mutant strain. Blots of Eco RI-digested DNAs of WT and Δ cbf5 strains, and Δ cbf5 strain transformed with pHCbf5 were probed with [5′- 32 P]-labeled HVCBF5-F primer. The predicted sizes of the hybridized fragments are indicated beside the panel. ( E ) Growth curves of WT, Δ cbf5 , and Δ cbf5 +pHCbf5 strains of H. volcanii are shown. OD 600 was measured using a Bioscreen C apparatus. Each point is the mean of three independent cultures; error bars represent standard error. H26 and VDC2364 were used as WT and Δ cbf5 strains.
Figure Legend Snippet: cbf5 -deleted strain of H. volcanii is viable. Deletion of the cbf5 gene was confirmed by three independent methods. ( A ) Schematics to show HVO_2493 (thick lines) in the genomes of wild-type (WT) and cbf5 -deleted (Δ cbf5 ) strains and on plasmid pHCbf5. Eco RI and Nde I (in plasmid) sites are marked. Positions of primers 1–5 (Cbf5_Ext_Fwd, Cbf5_Ext_Rev, Cbf5_Int_Fwd, Cbf5_Int_Rew, and HVCBF5-F, respectively) are shown by arrows. The arrow direction indicates 5′ to 3′ direction of the primers. Segment between broken lines is deleted in the Δ cbf5 strain. The His-tag at the C terminus of the Cbf5 protein is indicated by an asterisk. Approximate lengths of the segments are indicated but are not drawn to a scale. ( B ) PCR products using primers designed to anneal outside the gene (primers 1 and 2 in A ) confirm a genomic rearrangement of correctly predicted sizes in WT and mutant strains. ( C ) PCR products using primers designed to anneal within the target gene (primers 3 and 4 in A ) show the absence of the HVO_2493 segment in the mutant and its presence in the WT strain. ( D ) Southern hybridization is consistent with loss of the gene in mutant strain. Blots of Eco RI-digested DNAs of WT and Δ cbf5 strains, and Δ cbf5 strain transformed with pHCbf5 were probed with [5′- 32 P]-labeled HVCBF5-F primer. The predicted sizes of the hybridized fragments are indicated beside the panel. ( E ) Growth curves of WT, Δ cbf5 , and Δ cbf5 +pHCbf5 strains of H. volcanii are shown. OD 600 was measured using a Bioscreen C apparatus. Each point is the mean of three independent cultures; error bars represent standard error. H26 and VDC2364 were used as WT and Δ cbf5 strains.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Mutagenesis, Hybridization, Transformation Assay, Labeling

pus10 is essential in H. volcanii . pus10 ( HVO_1979 ) could only be deleted from the chromosome when expressed in trans . ( A ) H26 (WT) and two independent isolates VDC2606 and VDC2607 (Δ HVO_1979 with plasmid-borne copy of HVO_1979 under P tna control) were serially diluted from 10 0 to 10 −4 and spotted onto Hv-Min media plates, plus and minus 500 μM tryptophan. Significant growth of the mutant is only observed in the presence of tryptophan. ( B ) Deletion of the chromosomal copy of HVO_1979 was further verified by PCR using primers Pus10_Ext_Fwd and Pus10_Ext_Rev. Predicted sizes of the amplicon are indicated above each band.
Figure Legend Snippet: pus10 is essential in H. volcanii . pus10 ( HVO_1979 ) could only be deleted from the chromosome when expressed in trans . ( A ) H26 (WT) and two independent isolates VDC2606 and VDC2607 (Δ HVO_1979 with plasmid-borne copy of HVO_1979 under P tna control) were serially diluted from 10 0 to 10 −4 and spotted onto Hv-Min media plates, plus and minus 500 μM tryptophan. Significant growth of the mutant is only observed in the presence of tryptophan. ( B ) Deletion of the chromosomal copy of HVO_1979 was further verified by PCR using primers Pus10_Ext_Fwd and Pus10_Ext_Rev. Predicted sizes of the amplicon are indicated above each band.

Techniques Used: Plasmid Preparation, Mutagenesis, Polymerase Chain Reaction, Amplification

5) Product Images from "Changes in DNA methylation assessed by genomic bisulfite sequencing suggest a role for DNA methylation in cotton fruiting branch development"

Article Title: Changes in DNA methylation assessed by genomic bisulfite sequencing suggest a role for DNA methylation in cotton fruiting branch development

Journal: PeerJ

doi: 10.7717/peerj.4945

The DNA methylation levels validated by bisulfite PCR sequencing and BS-seq. DNA methylation levels of Gh_A10G0138 was analysed by BS-seq. Vertical bar mean eleven methylation sites with different methylation levels. Methylation site was examined by BS-PCR bisulfite PCR sequencing. Six methylation sites were found. The height of vertical bar means the methylation levels.
Figure Legend Snippet: The DNA methylation levels validated by bisulfite PCR sequencing and BS-seq. DNA methylation levels of Gh_A10G0138 was analysed by BS-seq. Vertical bar mean eleven methylation sites with different methylation levels. Methylation site was examined by BS-PCR bisulfite PCR sequencing. Six methylation sites were found. The height of vertical bar means the methylation levels.

Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Sequencing, Methylation

6) Product Images from "A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import [OPEN]"

Article Title: A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import [OPEN]

Journal: The Plant Cell

doi: 10.1105/tpc.18.00357

A Remaining Zinc Binding Motif of FtsH12 Is Dispensable for the Essential Function of FtsH12 in Arabidopsis. (A) The position of T-DNA insertion in the FTSH12 locus on the Arabidopsis genome. Transgene cDNA constructs placed under the 35S promoter (35S pro ) together with the positions of PCR primers used for genotyping were also depicted. (B) Functional complementation of the embryo-lethal homozygous ftsH12 null mutant ( emb1047-1 ) by expressing the FtsH12(H769Y) mutant. Individuals carrying the indicated genotypes were grown on the MS media containing 2% sucrose for 25 d. (C) Protein levels in the transformants and in the wild type were analyzed by SDS-PAGE and immunoblotting. Ten micrograms of total protein extracts of each seedling was analyzed. (D) PCR genotyping confirmed the homozygous T-DNA insertions in the ftsh12 locus of the obtained transformants. The forward (F) and backward (B) primers shown in (A) were used. (E) The genomic PCR fragments obtained from the transformants were directly sequenced. Only the mutated region was shown. (F) The FtsH12(H769Y) mutant showed normal chloroplast protein import. Chloroplasts were isolated from the homozygous T-DNA inserted line expressing the wild-type FtsH12 or the mutant FtsH12(H769Y) and used for the in vitro import experiments with pFd*-TEV-ProtA as a model preprotein. Import was performed in the presence of 3 mM ATP for 0, 10, 20, and 40 min and analyzed by SDS-PAGE followed by immunoblotting using anti-FLAG antibody. After 40 min incubation, another aliquot of chloroplasts was ruptured to separate the soluble supernatant containing stroma (S) from the membrane pellet (P) and analyzed similarly. m, mature form; p, precursor form. (G) ATP-dependent import of pFd*-TEV-ProtA into the chloroplasts expressing the wild-type FtsH12 or FtsH12(H769Y) mutant was analyzed as in (F) .
Figure Legend Snippet: A Remaining Zinc Binding Motif of FtsH12 Is Dispensable for the Essential Function of FtsH12 in Arabidopsis. (A) The position of T-DNA insertion in the FTSH12 locus on the Arabidopsis genome. Transgene cDNA constructs placed under the 35S promoter (35S pro ) together with the positions of PCR primers used for genotyping were also depicted. (B) Functional complementation of the embryo-lethal homozygous ftsH12 null mutant ( emb1047-1 ) by expressing the FtsH12(H769Y) mutant. Individuals carrying the indicated genotypes were grown on the MS media containing 2% sucrose for 25 d. (C) Protein levels in the transformants and in the wild type were analyzed by SDS-PAGE and immunoblotting. Ten micrograms of total protein extracts of each seedling was analyzed. (D) PCR genotyping confirmed the homozygous T-DNA insertions in the ftsh12 locus of the obtained transformants. The forward (F) and backward (B) primers shown in (A) were used. (E) The genomic PCR fragments obtained from the transformants were directly sequenced. Only the mutated region was shown. (F) The FtsH12(H769Y) mutant showed normal chloroplast protein import. Chloroplasts were isolated from the homozygous T-DNA inserted line expressing the wild-type FtsH12 or the mutant FtsH12(H769Y) and used for the in vitro import experiments with pFd*-TEV-ProtA as a model preprotein. Import was performed in the presence of 3 mM ATP for 0, 10, 20, and 40 min and analyzed by SDS-PAGE followed by immunoblotting using anti-FLAG antibody. After 40 min incubation, another aliquot of chloroplasts was ruptured to separate the soluble supernatant containing stroma (S) from the membrane pellet (P) and analyzed similarly. m, mature form; p, precursor form. (G) ATP-dependent import of pFd*-TEV-ProtA into the chloroplasts expressing the wild-type FtsH12 or FtsH12(H769Y) mutant was analyzed as in (F) .

Techniques Used: Binding Assay, Construct, Polymerase Chain Reaction, Functional Assay, Mutagenesis, Expressing, Mass Spectrometry, SDS Page, Isolation, In Vitro, Incubation

7) Product Images from "ARHI is a novel epigenetic silenced tumor suppressor in sporadic pheochromocytoma"

Article Title: ARHI is a novel epigenetic silenced tumor suppressor in sporadic pheochromocytoma

Journal: Oncotarget

doi: 10.18632/oncotarget.21149

ARHI retained a hypermethylation copy in sporadic PCC (A) Schematic representations for the localization of CpG islands. Bottom boxes from left to right indicate CpG islands I, II, and III, respectively, and the red bar in shows the CpG sites. The corresponding numbers indicated the location in the genome from the UCSC database. (B) The ARHI promoter included three CpG islands; the methylation ratios were detected by EpiTYPER methylation analysis. The methylation ratios were significantly higher in PCC tumors than normal adrenal tissues. The methylation ratios of the three CpG islands were obviously higher in the tumors with deleted copy numbers of ARHI when compared with those that carried normal copy numbers of ARHI. (C) Correlation between ARHI promoter CpG islands and their expression in PCC samples (n=38). (D) Summary of bisulfite-treated genomic DNA sequencing of PCC samples dependent on ARHI deletion, where the amplified region includes all three CpG islands; 73 CpG dinucleotides (CpGs), represented by circles located on the region, were analyzed by DNA sequencing. Black and white circles represent the methylated and unmethylated CpG dinucleotides, respectively. Each line represents the DNA sequence of a random clone, of which black and white circles represent unmethylated and methylated CpG sites of these regions, respectively. (E) Fluorescence in situ hybridization studies in Subjects 1 and 2. In Subjects 1, the 1chr.p31.3 (ARHI) labeled with Rhodamine BAC clone showed 2 copies (panel a) while the “control”chr.1q21 labeled with FITC BAC clone showed normal hybridization pattern in nuclei (Green). Subjects 2 showed the ARHI deletion (panel b) detected by the Rhodamine BAC clone (arrow). The FITC BAC clone was the control probe. (F) PHPC with ARHI copy number deletion (without endogenous ARHI expression) and with normal ARHI copy number were used to detect the methylation status using bisulfite-treated genomic DNA sequencing. Western blot and RT-PCR were used to determine whether ARHI was expressed at protein and mRNA levels. (G) Effect of DAC expression on methylation status of the ARHI gene promoter. DNA from control or DAC-treated PHPC with negative ARHI expression were collected at the indicated time points, cloned and sequenced to detect CpG-island methylation of the ARHI promoter. (H) ARHI-negative PHPC were treated with DAC, after 24h, 48h, 72h; ARHI was detected using RT-PCR and western blot, the error bars are represented as mean ± SD. (I) ARHI methylated allele analysis and its maternal imprinting in PCC tumors. SNP rs11209207 of normal DNA from 4 PCC patients (lane 1:N1-N4): one allele is lost in tumor DNA (lane 4:P1-P4); the retained allele is methylated (lane 5:Methylated allele1-4). Genotype of SNP rs11209207 of 4 families was shown. Maternal (lane 2:M1-M4); paternal (lane 3:F1-F4).
Figure Legend Snippet: ARHI retained a hypermethylation copy in sporadic PCC (A) Schematic representations for the localization of CpG islands. Bottom boxes from left to right indicate CpG islands I, II, and III, respectively, and the red bar in shows the CpG sites. The corresponding numbers indicated the location in the genome from the UCSC database. (B) The ARHI promoter included three CpG islands; the methylation ratios were detected by EpiTYPER methylation analysis. The methylation ratios were significantly higher in PCC tumors than normal adrenal tissues. The methylation ratios of the three CpG islands were obviously higher in the tumors with deleted copy numbers of ARHI when compared with those that carried normal copy numbers of ARHI. (C) Correlation between ARHI promoter CpG islands and their expression in PCC samples (n=38). (D) Summary of bisulfite-treated genomic DNA sequencing of PCC samples dependent on ARHI deletion, where the amplified region includes all three CpG islands; 73 CpG dinucleotides (CpGs), represented by circles located on the region, were analyzed by DNA sequencing. Black and white circles represent the methylated and unmethylated CpG dinucleotides, respectively. Each line represents the DNA sequence of a random clone, of which black and white circles represent unmethylated and methylated CpG sites of these regions, respectively. (E) Fluorescence in situ hybridization studies in Subjects 1 and 2. In Subjects 1, the 1chr.p31.3 (ARHI) labeled with Rhodamine BAC clone showed 2 copies (panel a) while the “control”chr.1q21 labeled with FITC BAC clone showed normal hybridization pattern in nuclei (Green). Subjects 2 showed the ARHI deletion (panel b) detected by the Rhodamine BAC clone (arrow). The FITC BAC clone was the control probe. (F) PHPC with ARHI copy number deletion (without endogenous ARHI expression) and with normal ARHI copy number were used to detect the methylation status using bisulfite-treated genomic DNA sequencing. Western blot and RT-PCR were used to determine whether ARHI was expressed at protein and mRNA levels. (G) Effect of DAC expression on methylation status of the ARHI gene promoter. DNA from control or DAC-treated PHPC with negative ARHI expression were collected at the indicated time points, cloned and sequenced to detect CpG-island methylation of the ARHI promoter. (H) ARHI-negative PHPC were treated with DAC, after 24h, 48h, 72h; ARHI was detected using RT-PCR and western blot, the error bars are represented as mean ± SD. (I) ARHI methylated allele analysis and its maternal imprinting in PCC tumors. SNP rs11209207 of normal DNA from 4 PCC patients (lane 1:N1-N4): one allele is lost in tumor DNA (lane 4:P1-P4); the retained allele is methylated (lane 5:Methylated allele1-4). Genotype of SNP rs11209207 of 4 families was shown. Maternal (lane 2:M1-M4); paternal (lane 3:F1-F4).

Techniques Used: Periodic Counter-current Chromatography, Methylation, Expressing, DNA Sequencing, Amplification, Sequencing, Fluorescence, In Situ Hybridization, Labeling, BAC Assay, Hybridization, Western Blot, Reverse Transcription Polymerase Chain Reaction, Clone Assay

ARHI lost a functional allele and downregulated in sporadic PCC (A) ARHI copy number variation in PCC tumors. qRT-PCR was carried out with 10 ng per reaction of the same genomic DNA used in the promoter analysis. All samples were then normalized to C2 and the copy number in blood samples was set to 2. Copy number relative ratio
Figure Legend Snippet: ARHI lost a functional allele and downregulated in sporadic PCC (A) ARHI copy number variation in PCC tumors. qRT-PCR was carried out with 10 ng per reaction of the same genomic DNA used in the promoter analysis. All samples were then normalized to C2 and the copy number in blood samples was set to 2. Copy number relative ratio

Techniques Used: Functional Assay, Periodic Counter-current Chromatography, Quantitative RT-PCR

8) Product Images from "The positive feedback loop between ILF3 and lncRNA ILF3-AS1 promotes melanoma proliferation, migration, and invasion"

Article Title: The positive feedback loop between ILF3 and lncRNA ILF3-AS1 promotes melanoma proliferation, migration, and invasion

Journal: Cancer Management and Research

doi: 10.2147/CMAR.S186777

ILF3-AS1 epigenetically activates ILF3 expression. Notes: ( A ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably overexpressed and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( B ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably depleted and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( C ) ILF3 mRNA levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by qRT-PCR. ( D ) ILF3 mRNA levels in ILF3-AS1 stably depleted and control A375 cells were determined by qRT-PCR. ( E ) ILF3 protein levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by Western blot. ( F ) ILF3 protein levels in ILF3-AS1 stably depleted and control A375 cells were determined by Western blot. Results are presented as mean ± SD based on at least three independent experiments. ** P
Figure Legend Snippet: ILF3-AS1 epigenetically activates ILF3 expression. Notes: ( A ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably overexpressed and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( B ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably depleted and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( C ) ILF3 mRNA levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by qRT-PCR. ( D ) ILF3 mRNA levels in ILF3-AS1 stably depleted and control A375 cells were determined by qRT-PCR. ( E ) ILF3 protein levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by Western blot. ( F ) ILF3 protein levels in ILF3-AS1 stably depleted and control A375 cells were determined by Western blot. Results are presented as mean ± SD based on at least three independent experiments. ** P

Techniques Used: Expressing, Binding Assay, Stable Transfection, Chromatin Immunoprecipitation, Quantitative RT-PCR, Western Blot

ILF3 is increased in melanoma and correlated with poor prognosis. Notes: ( A ) ILF3 expression levels in 37 benign nevi and 60 primary melanoma tissues were determined by qRT-PCR. Results are presented as median with IQR; P
Figure Legend Snippet: ILF3 is increased in melanoma and correlated with poor prognosis. Notes: ( A ) ILF3 expression levels in 37 benign nevi and 60 primary melanoma tissues were determined by qRT-PCR. Results are presented as median with IQR; P

Techniques Used: Expressing, Quantitative RT-PCR

ILF3 physically binds and increases the stability of ILF3-AS1 transcript. Notes: ( A ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1 with ILF3-specific antibody compared with nonspecific IgG. β-Actin mRNA was used as a negative control. LincIN was used as a positive control. ( B ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, ILF3 protein levels were determined by Western blot. ( C ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, ILF3 protein levels were determined by Western blot. ( D ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, the cells were treated with 50 µM α-amanitin to block new RNA synthesis, and then, the stability of ILF3-AS1 transcript over time was determined by qRT-PCR. 18S rRNA, a product of RNA polymerase I that is unchanged by α-amanitin, was used as endogenous control. ( E ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, the cells were treated with 50 µM α-amanitin to block new RNA synthesis and, then, the stability of ILF3-AS1 transcript over time was determined by qRT-PCR. 18S rRNA, a product of RNA polymerase I that is unchanged by α-amanitin, was used as endogenous control. ( F ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, ILF3-AS1 transcript levels were determined by qRT-PCR. ( G ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, ILF3-AS1 transcript levels were determined by qRT-PCR. Results are presented as mean ± SD based on at least three independent experiments. ** P
Figure Legend Snippet: ILF3 physically binds and increases the stability of ILF3-AS1 transcript. Notes: ( A ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1 with ILF3-specific antibody compared with nonspecific IgG. β-Actin mRNA was used as a negative control. LincIN was used as a positive control. ( B ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, ILF3 protein levels were determined by Western blot. ( C ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, ILF3 protein levels were determined by Western blot. ( D ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, the cells were treated with 50 µM α-amanitin to block new RNA synthesis, and then, the stability of ILF3-AS1 transcript over time was determined by qRT-PCR. 18S rRNA, a product of RNA polymerase I that is unchanged by α-amanitin, was used as endogenous control. ( E ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, the cells were treated with 50 µM α-amanitin to block new RNA synthesis and, then, the stability of ILF3-AS1 transcript over time was determined by qRT-PCR. 18S rRNA, a product of RNA polymerase I that is unchanged by α-amanitin, was used as endogenous control. ( F ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, ILF3-AS1 transcript levels were determined by qRT-PCR. ( G ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, ILF3-AS1 transcript levels were determined by qRT-PCR. Results are presented as mean ± SD based on at least three independent experiments. ** P

Techniques Used: Quantitative RT-PCR, Negative Control, Positive Control, Over Expression, Plasmid Preparation, Western Blot, Blocking Assay

9) Product Images from "Overexpression of ThVHAc1 and its potential upstream regulator, ThWRKY7, improved plant tolerance of Cadmium stress"

Article Title: Overexpression of ThVHAc1 and its potential upstream regulator, ThWRKY7, improved plant tolerance of Cadmium stress

Journal: Scientific Reports

doi: 10.1038/srep18752

RT-PCR analysis of stress-related genes in Arabidopsis with heterologous expression of ThVHAc1 and in T. hispida with transient expression of ThVHAc1 . ( a ) Gel electrophoresis of AHA genes (At2g07560, At1g80660, At3g42640), ACA genes (At1g27770, At1g08065, At1g08080) and stress-related genes ( ACT (At3g18780), CSD (At1g08830), APX (At1g07890), RBOHC (At5g51060)). ( b–d ) Relative expression levels according to a. ( e ) Gel electrophoresis of CSB1-3 , CAX2 , ADP , GLH , NADPH . ( f ) Relative expression levels according to (e). All experiments were repeated three times. The data are shown as the means ± SD of three independent experiments.
Figure Legend Snippet: RT-PCR analysis of stress-related genes in Arabidopsis with heterologous expression of ThVHAc1 and in T. hispida with transient expression of ThVHAc1 . ( a ) Gel electrophoresis of AHA genes (At2g07560, At1g80660, At3g42640), ACA genes (At1g27770, At1g08065, At1g08080) and stress-related genes ( ACT (At3g18780), CSD (At1g08830), APX (At1g07890), RBOHC (At5g51060)). ( b–d ) Relative expression levels according to a. ( e ) Gel electrophoresis of CSB1-3 , CAX2 , ADP , GLH , NADPH . ( f ) Relative expression levels according to (e). All experiments were repeated three times. The data are shown as the means ± SD of three independent experiments.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Nucleic Acid Electrophoresis, Activated Clotting Time Assay

qRT-PCR analysis of T. hispida V-ATPase subunits in transient expression ThVHAc1 lines. ( a ) Control conditions. ( b ) CdCl 2 treatment. The relative expression levels were all log 2 transformed. The X-axis shows the name of the V-ATPase subunit. The data are shown as the means ± SD of three independent experiments.
Figure Legend Snippet: qRT-PCR analysis of T. hispida V-ATPase subunits in transient expression ThVHAc1 lines. ( a ) Control conditions. ( b ) CdCl 2 treatment. The relative expression levels were all log 2 transformed. The X-axis shows the name of the V-ATPase subunit. The data are shown as the means ± SD of three independent experiments.

Techniques Used: Quantitative RT-PCR, Expressing, Transformation Assay

qRT-PCR analysis and comparison of five AtVHA-c subunits and ThVHAc1 in c1#10. The relative expression levels were all log 2 transformed. The x-axis shows the stress time point. The data are shown as the means ± SD of three independent experiments.
Figure Legend Snippet: qRT-PCR analysis and comparison of five AtVHA-c subunits and ThVHAc1 in c1#10. The relative expression levels were all log 2 transformed. The x-axis shows the stress time point. The data are shown as the means ± SD of three independent experiments.

Techniques Used: Quantitative RT-PCR, Expressing, Transformation Assay

Analysis of transient expression of ThVHAc1 in T. hispida under 100 μM CdCl 2 treatment for 1 h and 2 h. ( a ) qRT-PCR analysis of ThVHAc1 in T. hispida seedlings transiently transformed with 35S::ThVHAc1, RNAi:: ThVHAc1 compared with T-ck. The relative expression levels were all log 2 transformed. ( b ) DAB staining. ( c ) Evans blue staining. ( d ) MDA content. ( e ) Electrolyte leakage. All data are displayed as the mean ± SD of three independent experiments, and significant differences between transgenic lines and WT (P
Figure Legend Snippet: Analysis of transient expression of ThVHAc1 in T. hispida under 100 μM CdCl 2 treatment for 1 h and 2 h. ( a ) qRT-PCR analysis of ThVHAc1 in T. hispida seedlings transiently transformed with 35S::ThVHAc1, RNAi:: ThVHAc1 compared with T-ck. The relative expression levels were all log 2 transformed. ( b ) DAB staining. ( c ) Evans blue staining. ( d ) MDA content. ( e ) Electrolyte leakage. All data are displayed as the mean ± SD of three independent experiments, and significant differences between transgenic lines and WT (P

Techniques Used: Expressing, Quantitative RT-PCR, Transformation Assay, Staining, Multiple Displacement Amplification, Transgenic Assay

10) Product Images from "Differential Regulation of Mitogen- and Stress-activated Protein Kinase-1 and -2 (MSK1 and MSK2) by CK2 following UV Radiation *"

Article Title: Differential Regulation of Mitogen- and Stress-activated Protein Kinase-1 and -2 (MSK1 and MSK2) by CK2 following UV Radiation *

Journal:

doi: 10.1074/jbc.M109.083808

MSK2 is required for p65 transactivation and cell survival following UV-C radiation. A , quantitative real-time PCR analysis of endogenous A20, Bcl-xL, and X-IAP gene expression (normalized to TATA-box binding protein) in MDA-MB-231 cells 6 h following
Figure Legend Snippet: MSK2 is required for p65 transactivation and cell survival following UV-C radiation. A , quantitative real-time PCR analysis of endogenous A20, Bcl-xL, and X-IAP gene expression (normalized to TATA-box binding protein) in MDA-MB-231 cells 6 h following

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Binding Assay, Multiple Displacement Amplification

11) Product Images from "Down-Regulation of DUSP6 Expression in Lung Cancer --Its Mechanism and Potential Role in Carcinogenesis"

Article Title: Down-Regulation of DUSP6 Expression in Lung Cancer --Its Mechanism and Potential Role in Carcinogenesis

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.080489

Expression of DUSP6 in immortalized and cancer cell lines. Copy numbers of DUSP6 and ACTB mRNA were measured by quantitative reverse transcription-PCR. The means and standard deviations (error bars) of the mRNA expression of DUSP6 normalized to that of
Figure Legend Snippet: Expression of DUSP6 in immortalized and cancer cell lines. Copy numbers of DUSP6 and ACTB mRNA were measured by quantitative reverse transcription-PCR. The means and standard deviations (error bars) of the mRNA expression of DUSP6 normalized to that of

Techniques Used: Expressing, Polymerase Chain Reaction

Methylation status of the DUSP6 promoter and intron 1. Cells treated with vehicle (Vcl), AZA, TSA, or a combination of AZA and TSA were examined for restoration of DUSP6 mRNA expression by quantitative reverse transcription-PCR. The copy number of DUSP6
Figure Legend Snippet: Methylation status of the DUSP6 promoter and intron 1. Cells treated with vehicle (Vcl), AZA, TSA, or a combination of AZA and TSA were examined for restoration of DUSP6 mRNA expression by quantitative reverse transcription-PCR. The copy number of DUSP6

Techniques Used: Methylation, Expressing, Polymerase Chain Reaction

12) Product Images from "Down-Regulation of FXYD3 Expression in Human Lung Cancers "

Article Title: Down-Regulation of FXYD3 Expression in Human Lung Cancers

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.080571

Expression of FXYD3 mRNA in cell lines. Levels of FXYD3 mRNA were examined by RT-PCR. PCR products of FXYD3 and β-actin (ACTB) were resolved by electrophoresis in 1% agar and stained with ethidium bromide ( A ). Copy numbers of DUSP6 and β-actin
Figure Legend Snippet: Expression of FXYD3 mRNA in cell lines. Levels of FXYD3 mRNA were examined by RT-PCR. PCR products of FXYD3 and β-actin (ACTB) were resolved by electrophoresis in 1% agar and stained with ethidium bromide ( A ). Copy numbers of DUSP6 and β-actin

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

13) Product Images from "Induction of Short NFATc1/αA Isoform Interferes with Peripheral B Cell Differentiation"

Article Title: Induction of Short NFATc1/αA Isoform Interferes with Peripheral B Cell Differentiation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00032

NFATc1/αA suppresses cell death and Prdm1/ Blimp-1 expression in chicken DT40 B cells. (A) WT, NFATC1 −/−/− , NFATC1 −/−/ α A , and NFATC1 −/−/ α C DT40 cells were stimulated with 30 µg/ml α-IgM M4 Ab for 28 h. Cells were stained by annexin V-Cy3. Shown is the percentage of apoptotic cells in one typical experiment from more than three. (B) Real-time qPCR assays. RNAs were isolated from either untreated DT40 B cells—or from cells treated with 30 µg/ml αIgM for 6 h (+) using a QIAamp RNA Blood kit (QIAGEN), and quantitative RT-PCR assays were performed using TaqMan probes for detecting Bcl-6 and GAPDH3 RNAs as internal control. (C,D) Human NFATc1/αA affects the gene signature of DT40 cells. RNAs from untreated DT40 B cells—or from cells treated with α-IgM for 4 h (C) or T+I for 1 and 4 h (D) were assayed in semi-quantitative PCR assays. In (C,D) , one typical assay from two reactions is shown.
Figure Legend Snippet: NFATc1/αA suppresses cell death and Prdm1/ Blimp-1 expression in chicken DT40 B cells. (A) WT, NFATC1 −/−/− , NFATC1 −/−/ α A , and NFATC1 −/−/ α C DT40 cells were stimulated with 30 µg/ml α-IgM M4 Ab for 28 h. Cells were stained by annexin V-Cy3. Shown is the percentage of apoptotic cells in one typical experiment from more than three. (B) Real-time qPCR assays. RNAs were isolated from either untreated DT40 B cells—or from cells treated with 30 µg/ml αIgM for 6 h (+) using a QIAamp RNA Blood kit (QIAGEN), and quantitative RT-PCR assays were performed using TaqMan probes for detecting Bcl-6 and GAPDH3 RNAs as internal control. (C,D) Human NFATc1/αA affects the gene signature of DT40 cells. RNAs from untreated DT40 B cells—or from cells treated with α-IgM for 4 h (C) or T+I for 1 and 4 h (D) were assayed in semi-quantitative PCR assays. In (C,D) , one typical assay from two reactions is shown.

Techniques Used: Expressing, Staining, Real-time Polymerase Chain Reaction, Isolation, Quantitative RT-PCR

Effect of NFATc1/αA-bio and NFATc1/βC-bio proteins on cell death and the expression of Aicda and Prdm1 genes in murine WEHI 231 B lymphoma cells. (A) WEHI 231 B cells stably infected with retroviral vectors expressing BirA (WEHI-231), Bir A and NFATc1/αA-bio (NFATc1/αA), or BirA and NFATc1/βC-bio (NFATc1/βC) were stimulated with α-IgM or α-IgM α-IgM + αCD40 for 48 or 96 h. Apoptosis was determined by PI staining. MFI: Mean fluorescence intensity. (B) Wild-type (WT) WEHI cells (Co) or cells expressing NFATc1/αA-bio (blue) or NFATc1/βC-bio (red) were left unstimulated or stimulated for 6, 24, or 96 h with α-IgM. RNA was isolated and converted to cDNA libraries. DNA stretches of 50 bp were sequenced on a Illumina HiSeq2500 platform using the Truseq SBS kit-HS V3. Shown are the RNA reads (RPKM) from the Aicda and Prdm1 genes in the three types of WEHI cells. Results of one from two assays are shown. (C) Chromatin immuno precipitation (ChIP) assays for the binding of NFATc1-bio proteins to the Prdm1 gene in WEHI cells stimulated with T+I for 6 h. In the upper panel semi-quantitative PCR assays are shown for the detection of Prdm1 (and β -Actin ) DNA in chromatin precipitations. In the first three lanes, chromatin from WEHI cells transfected with BirA alone, with NFATc1/αA-bio (+BirA) or NFATc1/βC-bio (+BirA) was precipitated with streptavidin-agarose beads. In the next lanes, chromatin was precipitated with Abs specific for histone H3, NFATc1 (7A6), and immunoglobulin. In the last two lanes, DNA input and H 2 O controls are shown. One typical assay from three assays is shown. In the lower panel the enrichment of β -Actin, Rcan1, Prdm1, Il2 , and Ppp3ca DNAs precipitated with streptavidin beads from WEHI cells expressing either NFATc1/αA-bio or NFATc1/βC-bio is shown. Mean values of three assays are shown. (D) ChIP assays indicating histone modifications at the Prdm1 promoter. ChIP assays were performed with chromatin from WEHI cells overexpressing NFATc1/αA-bio (+BirA) (open bars) or NFATc1/βC-bio (+BirA) proteins (gray bars) using Abs directed the histone modifications H3K9me3 and H3K9ac, respectively. In semi-quantitative PCR assays, primers detecting the Prdm1 ) were used. Mean values of three assays are shown.
Figure Legend Snippet: Effect of NFATc1/αA-bio and NFATc1/βC-bio proteins on cell death and the expression of Aicda and Prdm1 genes in murine WEHI 231 B lymphoma cells. (A) WEHI 231 B cells stably infected with retroviral vectors expressing BirA (WEHI-231), Bir A and NFATc1/αA-bio (NFATc1/αA), or BirA and NFATc1/βC-bio (NFATc1/βC) were stimulated with α-IgM or α-IgM α-IgM + αCD40 for 48 or 96 h. Apoptosis was determined by PI staining. MFI: Mean fluorescence intensity. (B) Wild-type (WT) WEHI cells (Co) or cells expressing NFATc1/αA-bio (blue) or NFATc1/βC-bio (red) were left unstimulated or stimulated for 6, 24, or 96 h with α-IgM. RNA was isolated and converted to cDNA libraries. DNA stretches of 50 bp were sequenced on a Illumina HiSeq2500 platform using the Truseq SBS kit-HS V3. Shown are the RNA reads (RPKM) from the Aicda and Prdm1 genes in the three types of WEHI cells. Results of one from two assays are shown. (C) Chromatin immuno precipitation (ChIP) assays for the binding of NFATc1-bio proteins to the Prdm1 gene in WEHI cells stimulated with T+I for 6 h. In the upper panel semi-quantitative PCR assays are shown for the detection of Prdm1 (and β -Actin ) DNA in chromatin precipitations. In the first three lanes, chromatin from WEHI cells transfected with BirA alone, with NFATc1/αA-bio (+BirA) or NFATc1/βC-bio (+BirA) was precipitated with streptavidin-agarose beads. In the next lanes, chromatin was precipitated with Abs specific for histone H3, NFATc1 (7A6), and immunoglobulin. In the last two lanes, DNA input and H 2 O controls are shown. One typical assay from three assays is shown. In the lower panel the enrichment of β -Actin, Rcan1, Prdm1, Il2 , and Ppp3ca DNAs precipitated with streptavidin beads from WEHI cells expressing either NFATc1/αA-bio or NFATc1/βC-bio is shown. Mean values of three assays are shown. (D) ChIP assays indicating histone modifications at the Prdm1 promoter. ChIP assays were performed with chromatin from WEHI cells overexpressing NFATc1/αA-bio (+BirA) (open bars) or NFATc1/βC-bio (+BirA) proteins (gray bars) using Abs directed the histone modifications H3K9me3 and H3K9ac, respectively. In semi-quantitative PCR assays, primers detecting the Prdm1 ) were used. Mean values of three assays are shown.

Techniques Used: Expressing, Stable Transfection, Infection, Staining, Fluorescence, Isolation, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Transfection

Suppression of plasmablast differentiation and of IgG class switch by caNFATc1/αA. (A) Changes in Prdm1 RNA levels in splenic B cells upon stimulation with α-IgM, α-CD40, or LPS for 24 h. pαIgM: cells were “pulsed” for 30 min at 4°C with α-IgM, washed and then treated with α-CD40 or LPS for 24 h. Shown are mean values of Prdm1 RNA reads (RPKM) from two assays. (B) Splenic B cells from wild-type (WT) mice (open columns) or caNfatc1/ α A × Cd23-cre mice (black) were treated for 24–72 h with LPS. Real-time PCR assays for detecting Prdm1 RNA levels. Mean values of three assays are shown. (C,D) WT and caNfatc1/ α A × Cd23-cre mice were immunized with NP-KLH (C) or NP-Ficoll (D) for 21 days, and the serum levels of immunoglobulins were determined in ELISAs. One dot and square indicates one mouse, respectively.
Figure Legend Snippet: Suppression of plasmablast differentiation and of IgG class switch by caNFATc1/αA. (A) Changes in Prdm1 RNA levels in splenic B cells upon stimulation with α-IgM, α-CD40, or LPS for 24 h. pαIgM: cells were “pulsed” for 30 min at 4°C with α-IgM, washed and then treated with α-CD40 or LPS for 24 h. Shown are mean values of Prdm1 RNA reads (RPKM) from two assays. (B) Splenic B cells from wild-type (WT) mice (open columns) or caNfatc1/ α A × Cd23-cre mice (black) were treated for 24–72 h with LPS. Real-time PCR assays for detecting Prdm1 RNA levels. Mean values of three assays are shown. (C,D) WT and caNfatc1/ α A × Cd23-cre mice were immunized with NP-KLH (C) or NP-Ficoll (D) for 21 days, and the serum levels of immunoglobulins were determined in ELISAs. One dot and square indicates one mouse, respectively.

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

14) Product Images from "ghr-miR5272a-mediated regulation of GhMKK6 gene transcription contributes to the immune response in cotton"

Article Title: ghr-miR5272a-mediated regulation of GhMKK6 gene transcription contributes to the immune response in cotton

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erx373

ghr-miR5272a overexpression sensitizes cotton to F. oxysporum . (A) ghr-miR5272a expression patterns in cotton under MeJA and F. oxysporum treatments. (B) miR5272 expression levels in cotton transfected with pCLCrVA-amiR5272. (C) Pathogen disease index in amiR5272-overexpressing plants at 5 d after F. oxysporum infection. (D) Representative phenotypes of amiR5272-overexpressing plants infected with F. oxysporum (5 d after infection). (E) GhMKK6 mRNA relative expression and protein levels in cotton transfected with pCLCrVA-amiR5272. (F) qRT-PCR analysis of expression of SA- and JA-mediated defence pathway genes in amiR5272-overexpressing plants at 5 d after F. oxysporum infection. CRV::00 served as the empty vector control. Data in (A–C, E, F) are means ± SE of three independent experiments ( n =6). Different letters indicate significant differences ( P
Figure Legend Snippet: ghr-miR5272a overexpression sensitizes cotton to F. oxysporum . (A) ghr-miR5272a expression patterns in cotton under MeJA and F. oxysporum treatments. (B) miR5272 expression levels in cotton transfected with pCLCrVA-amiR5272. (C) Pathogen disease index in amiR5272-overexpressing plants at 5 d after F. oxysporum infection. (D) Representative phenotypes of amiR5272-overexpressing plants infected with F. oxysporum (5 d after infection). (E) GhMKK6 mRNA relative expression and protein levels in cotton transfected with pCLCrVA-amiR5272. (F) qRT-PCR analysis of expression of SA- and JA-mediated defence pathway genes in amiR5272-overexpressing plants at 5 d after F. oxysporum infection. CRV::00 served as the empty vector control. Data in (A–C, E, F) are means ± SE of three independent experiments ( n =6). Different letters indicate significant differences ( P

Techniques Used: Over Expression, Expressing, Transfection, Infection, Quantitative RT-PCR, Plasmid Preparation

GhMKK6 -silencing in cotton reduces resistance to F. oxysporum . (A) GhMKK6 RNA relative expression and protein levels in GhMKK6 -silenced cotton. (B) Representative phenotypes of GhMKK6 -silenced plants 5 d after being infected with F. oxysporum . (C) Pathogen disease index in GhMKK6 -silenced plants 5 d after F. oxysporum infection. (D) qRT-PCR analysis for expression of SA- and JA-mediated defence pathway genes in CRV::00 , CRV::01 , CRV::02 , and CRV::03 at 5 d after infection with F. oxysporum . CRV::00 served as the empty vector control. Data in (A, C, D) are means ± SE of three independent experiments; (A, D) n =15, (C) n =6. Different letters indicate significant differences ( P
Figure Legend Snippet: GhMKK6 -silencing in cotton reduces resistance to F. oxysporum . (A) GhMKK6 RNA relative expression and protein levels in GhMKK6 -silenced cotton. (B) Representative phenotypes of GhMKK6 -silenced plants 5 d after being infected with F. oxysporum . (C) Pathogen disease index in GhMKK6 -silenced plants 5 d after F. oxysporum infection. (D) qRT-PCR analysis for expression of SA- and JA-mediated defence pathway genes in CRV::00 , CRV::01 , CRV::02 , and CRV::03 at 5 d after infection with F. oxysporum . CRV::00 served as the empty vector control. Data in (A, C, D) are means ± SE of three independent experiments; (A, D) n =15, (C) n =6. Different letters indicate significant differences ( P

Techniques Used: Expressing, Infection, Quantitative RT-PCR, Plasmid Preparation

Characterization and sequence analysis of GhMKK6 . (A) Multiple amino acid sequence alignment of GhMKK6, AtMEK6, and NtMEK1. The conserved kinase domain S/TXXXXXS/T is indicated by the box. (B) Dendrogram analysis. The numbers above the branches indicate bootstrap values ( > 50%) from 1000 replicates. A, B, C, and D indicate MAPKK groups. (C) qRT-PCR analysis of GhMKK6 expression under SA, MeJA, or F. oxysporum treatment. Data are means ± SE of three independent experiments ( n =6). Different letters indicate significant differences ( P
Figure Legend Snippet: Characterization and sequence analysis of GhMKK6 . (A) Multiple amino acid sequence alignment of GhMKK6, AtMEK6, and NtMEK1. The conserved kinase domain S/TXXXXXS/T is indicated by the box. (B) Dendrogram analysis. The numbers above the branches indicate bootstrap values ( > 50%) from 1000 replicates. A, B, C, and D indicate MAPKK groups. (C) qRT-PCR analysis of GhMKK6 expression under SA, MeJA, or F. oxysporum treatment. Data are means ± SE of three independent experiments ( n =6). Different letters indicate significant differences ( P

Techniques Used: Sequencing, Quantitative RT-PCR, Expressing

Phenotypes of GhMKK6 -overexpressing plants infected with F. oxysporum . (A) qRT-PCR analysis of expression of SA- and JA-mediated defence pathway genes in GhMKK6 -overexpressing plants at 3 d after F. oxysporum infection. (B) Pathogen disease index in GhMKK6 -overexpressing plants at 3 d after F. oxysporum infection. (C) qRT-PCR analysis of RbohB expression in GhMKK6 -overexpressing plants at 3 d after F. oxysporum infection. Data in (A–C) are means ± SE of three independent experiments ( n =6). Different letters indicate significant differences ( P
Figure Legend Snippet: Phenotypes of GhMKK6 -overexpressing plants infected with F. oxysporum . (A) qRT-PCR analysis of expression of SA- and JA-mediated defence pathway genes in GhMKK6 -overexpressing plants at 3 d after F. oxysporum infection. (B) Pathogen disease index in GhMKK6 -overexpressing plants at 3 d after F. oxysporum infection. (C) qRT-PCR analysis of RbohB expression in GhMKK6 -overexpressing plants at 3 d after F. oxysporum infection. Data in (A–C) are means ± SE of three independent experiments ( n =6). Different letters indicate significant differences ( P

Techniques Used: Infection, Quantitative RT-PCR, Expressing

Excessive GhMKK6 activation leads to lesion-mimicking phenotypes. (A) Representative phenotypes of plants transiently expressing GhMKK6 , GhMKK6AA , and GhMKK6EE at 48 and 120 h post-infiltration (hpi). DAB staining was used to show ROS accumulation. (B) GhMKK6 protein levels in plants transiently expressing GhMKK6 , GhMKK6AA , and GhMKK6EE at 5 d post-infiltration. (C, D) qRT-PCR analysis of expression of (C) rbohB and (D) SA- and JA-mediated defence pathway genes in plants transiently expressing GhMKK6 , GhMKK6AA , and GhMKK6EE at 5 d post-infiltration. Data are means ± SE of three independent experiments ( n =6). Different letters indicate significant differences ( P
Figure Legend Snippet: Excessive GhMKK6 activation leads to lesion-mimicking phenotypes. (A) Representative phenotypes of plants transiently expressing GhMKK6 , GhMKK6AA , and GhMKK6EE at 48 and 120 h post-infiltration (hpi). DAB staining was used to show ROS accumulation. (B) GhMKK6 protein levels in plants transiently expressing GhMKK6 , GhMKK6AA , and GhMKK6EE at 5 d post-infiltration. (C, D) qRT-PCR analysis of expression of (C) rbohB and (D) SA- and JA-mediated defence pathway genes in plants transiently expressing GhMKK6 , GhMKK6AA , and GhMKK6EE at 5 d post-infiltration. Data are means ± SE of three independent experiments ( n =6). Different letters indicate significant differences ( P

Techniques Used: Activation Assay, Expressing, Staining, Quantitative RT-PCR

15) Product Images from "Alternative exon usage creates novel transcript variants of tumor suppressor SHREW-1 gene with differential tissue expression profile"

Article Title: Alternative exon usage creates novel transcript variants of tumor suppressor SHREW-1 gene with differential tissue expression profile

Journal: Biology Open

doi: 10.1242/bio.019463

Detection of shrew-1 transcript variant expression during murine mammary gland differentiation. (A) Tissue digest of whole fat pads from a virgin mouse was performed to separate mammary organoids and fibroblasts. RT-PCR was performed with a primer pair to detect protein isoforms 1 and 3 (E1 to E5, isoform 1: 1269 bp; isoform 3: 463 bp). The presence of shrew-1 cDNA was confirmed with a primer pair to amplify E4, which is common to all known transcript variants (E4 to E5; 323 bp). cDNA of brain tissue (E13.5) served as positive control for both shrew-1 transcript variants encoding shrew-1 protein isoforms 1 and 3 respectively; the housekeeping gene BIP served as positive control for cDNA synthesis. cDNA analysis showed that both shrew-1 transcript variants corresponding to protein isoforms 1 and 3 are present in organoids but only isoform 1 is detectable in fibroblasts. M, bp marker. (B) The lactogenic inducible mammary epithelial cell line HC11 was differentiated in vitro with a hormone cocktail (DIP). RT-PCR with primer pairs to detect shrew-1 transcript variant expression (E1 to E5, isoform 1: 1269 bp hash; isoform 3: 463 bp delta) or the presence of all known shrew-1 transcript variants (E4 to E5; 323 bp) indicate that only the shrew-1 transcript encoding protein isoform 3 is expressed during all differentiation stages (delta). M, bp marker.
Figure Legend Snippet: Detection of shrew-1 transcript variant expression during murine mammary gland differentiation. (A) Tissue digest of whole fat pads from a virgin mouse was performed to separate mammary organoids and fibroblasts. RT-PCR was performed with a primer pair to detect protein isoforms 1 and 3 (E1 to E5, isoform 1: 1269 bp; isoform 3: 463 bp). The presence of shrew-1 cDNA was confirmed with a primer pair to amplify E4, which is common to all known transcript variants (E4 to E5; 323 bp). cDNA of brain tissue (E13.5) served as positive control for both shrew-1 transcript variants encoding shrew-1 protein isoforms 1 and 3 respectively; the housekeeping gene BIP served as positive control for cDNA synthesis. cDNA analysis showed that both shrew-1 transcript variants corresponding to protein isoforms 1 and 3 are present in organoids but only isoform 1 is detectable in fibroblasts. M, bp marker. (B) The lactogenic inducible mammary epithelial cell line HC11 was differentiated in vitro with a hormone cocktail (DIP). RT-PCR with primer pairs to detect shrew-1 transcript variant expression (E1 to E5, isoform 1: 1269 bp hash; isoform 3: 463 bp delta) or the presence of all known shrew-1 transcript variants (E4 to E5; 323 bp) indicate that only the shrew-1 transcript encoding protein isoform 3 is expressed during all differentiation stages (delta). M, bp marker.

Techniques Used: Variant Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Marker, In Vitro

16) Product Images from "Downregulation of miR-141-3p promotes bone metastasis via activating NF-κB signaling in prostate cancer"

Article Title: Downregulation of miR-141-3p promotes bone metastasis via activating NF-κB signaling in prostate cancer

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-017-0645-7

miR-141-3p is downregulated in bone metastatic PCa tissues and cells. a miR-141-3p expression levels was decreased in bone metastatic PCa tissues (PCa/BM) compared with that in non-bone metastatic PCa tissues (PCa/nBM) as assessed by analyzing the TCGA PCa miRNA sequencing dataset (PCa/nBM, n = 11; PCa/BM, n = 9). b Percentages and number of samples showed high or low miR-141-3p expression in bone metastatic and non-bone metastatic PCa tissues in PCa dataset from TCGA. c Real-time PCR analysis of miR-141-3p expression in 89 non-bone metastatic and 52 bone metastatic PCa samples. Transcript levels were normalized to U6 expression. d Percentages and number of samples showed high or low miR-141-3p expression in bone metastatic and non-bone metastatic PCa tissues in our PCa tissues. e Real-time PCR analysis of miR-141-3p expression levels in normal prostate epithelial cell (RWPE-1), primary PCa cell 22RV1, bone metastatic PCa cell lines (PC-3, C4-2B and VCaP) and brain metastatic cell line DU145 and lymph node metastatic cell line LNCaP. Transcript levels were normalized to U6 expression. * P
Figure Legend Snippet: miR-141-3p is downregulated in bone metastatic PCa tissues and cells. a miR-141-3p expression levels was decreased in bone metastatic PCa tissues (PCa/BM) compared with that in non-bone metastatic PCa tissues (PCa/nBM) as assessed by analyzing the TCGA PCa miRNA sequencing dataset (PCa/nBM, n = 11; PCa/BM, n = 9). b Percentages and number of samples showed high or low miR-141-3p expression in bone metastatic and non-bone metastatic PCa tissues in PCa dataset from TCGA. c Real-time PCR analysis of miR-141-3p expression in 89 non-bone metastatic and 52 bone metastatic PCa samples. Transcript levels were normalized to U6 expression. d Percentages and number of samples showed high or low miR-141-3p expression in bone metastatic and non-bone metastatic PCa tissues in our PCa tissues. e Real-time PCR analysis of miR-141-3p expression levels in normal prostate epithelial cell (RWPE-1), primary PCa cell 22RV1, bone metastatic PCa cell lines (PC-3, C4-2B and VCaP) and brain metastatic cell line DU145 and lymph node metastatic cell line LNCaP. Transcript levels were normalized to U6 expression. * P

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

Upregulation of miR-141-3p inhibits EMT in PCa cells. a Gene set enrichment analysis (GSEA) revealed that low expression of miR-141-3p expression significantly and positively correlated with the EMT signatures. b Real-time PCR analysis of miR-141-3p expression inVCaP and C4-2B cells transduced with miR-141-3p or transfected with anti-miR-141-3p compared to controls. Transcript levels were normalized by U6 expression. Error bars represent the mean ± s.d. of three independent experiments. * P
Figure Legend Snippet: Upregulation of miR-141-3p inhibits EMT in PCa cells. a Gene set enrichment analysis (GSEA) revealed that low expression of miR-141-3p expression significantly and positively correlated with the EMT signatures. b Real-time PCR analysis of miR-141-3p expression inVCaP and C4-2B cells transduced with miR-141-3p or transfected with anti-miR-141-3p compared to controls. Transcript levels were normalized by U6 expression. Error bars represent the mean ± s.d. of three independent experiments. * P

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

17) Product Images from "Overexpression of OsCIPK30 Enhances Plant Tolerance to Rice stripe virus"

Article Title: Overexpression of OsCIPK30 Enhances Plant Tolerance to Rice stripe virus

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02322

Effect of OsCIPK30 overexpression on RSV infection in rice. (A) Growth of the WT and Ubi-OsCIPK30 transgenic rice (T 1 generation). (B) WT and Ubi-OsCIPK30 T 1 transgenic rice were inoculated with RSV and photographed at 21 dpi. The lower panel of (B) shows the symptomatic leaves of the representative plants. (C) Temporal changes of RSV symptom development in WT and Ubi-OsCIPK30 T 1 transgenic rice (OE-1, OE-2, OE-3). Thirty plants were used for each treatment in the experiment. (D) The expression level of OsCIPK30 in T 1 transgenic rice (OE-1, OE-2, OE-3) by real-time RT-PCR at 21 dpi with RSV. The relative mRNA levels were calculated with respect to the expression level of the corresponding transcript in the WT rice plant. (E) The expression level of NP in T 1 transgenic rice (OE-1, OE-2, OE-3) by real-time RT-PCR at 21 dpi with RSV. The relative mRNA levels were calculated with respect to the expression level of the corresponding transcript in the WT rice plant. All the experiments were repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. The asterisks above the columns represent significance based on an unpaired, two-tailed Student’s t -test relative to the WT. ∗∗ P
Figure Legend Snippet: Effect of OsCIPK30 overexpression on RSV infection in rice. (A) Growth of the WT and Ubi-OsCIPK30 transgenic rice (T 1 generation). (B) WT and Ubi-OsCIPK30 T 1 transgenic rice were inoculated with RSV and photographed at 21 dpi. The lower panel of (B) shows the symptomatic leaves of the representative plants. (C) Temporal changes of RSV symptom development in WT and Ubi-OsCIPK30 T 1 transgenic rice (OE-1, OE-2, OE-3). Thirty plants were used for each treatment in the experiment. (D) The expression level of OsCIPK30 in T 1 transgenic rice (OE-1, OE-2, OE-3) by real-time RT-PCR at 21 dpi with RSV. The relative mRNA levels were calculated with respect to the expression level of the corresponding transcript in the WT rice plant. (E) The expression level of NP in T 1 transgenic rice (OE-1, OE-2, OE-3) by real-time RT-PCR at 21 dpi with RSV. The relative mRNA levels were calculated with respect to the expression level of the corresponding transcript in the WT rice plant. All the experiments were repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. The asterisks above the columns represent significance based on an unpaired, two-tailed Student’s t -test relative to the WT. ∗∗ P

Techniques Used: Over Expression, Infection, Transgenic Assay, Expressing, Quantitative RT-PCR, Two Tailed Test

18) Product Images from "Histone Methyltransferase SETD3 Regulates Muscle Differentiation"

Article Title: Histone Methyltransferase SETD3 Regulates Muscle Differentiation

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.203307

SETD3 promotes muscle cell differentiation. A , phase-contrast images at indicated time points (days) of C2C12 cells ( upper panel ) and the expression levels of SETD3 detected via RT-PCR ( lower panel ). GAPDH was used as a control. B , H9c2 and C2C12 cells
Figure Legend Snippet: SETD3 promotes muscle cell differentiation. A , phase-contrast images at indicated time points (days) of C2C12 cells ( upper panel ) and the expression levels of SETD3 detected via RT-PCR ( lower panel ). GAPDH was used as a control. B , H9c2 and C2C12 cells

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

19) Product Images from "Jacalin Lectin At5g28520 Is Regulated By ABA and miR846"

Article Title: Jacalin Lectin At5g28520 Is Regulated By ABA and miR846

Journal: Plant Signaling & Behavior

doi: 10.4161/psb.24563

Figure 1. Real-time PCR results showing the ABA-regulated expression of ( A ) AT5G28520 and ( B ) pre-miR846 in 10-d-old seedlings over 24 h. Numbers above the bars indicate fold changes (comparing to no ABA at the same time point). Error bars are
Figure Legend Snippet: Figure 1. Real-time PCR results showing the ABA-regulated expression of ( A ) AT5G28520 and ( B ) pre-miR846 in 10-d-old seedlings over 24 h. Numbers above the bars indicate fold changes (comparing to no ABA at the same time point). Error bars are

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

20) Product Images from "ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis"

Article Title: ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis

Journal: Genes & Development

doi: 10.1101/gad.1077503

Expression of the ICE1 gene and subcellular localization of the ICE1 protein. ( A ) ICE1 promoter-driven GUS expression pattern in a wild-type seedling. ( B ) ICE1 promoter- GUS expression in different plant tissues, and the corresponding ICE1 transcript levels as determined by RT–PCR analysis. The tubulin gene was used as an internal control in the RT–PCR. ( C ) RNA blot analysis of ICE1 expression in wild-type seedlings under various abiotic stresses. Plants with the following treatments are shown: control, MS salt only; NaCl, 300 mM NaCl for 5 h; ABA, 100 μM abscisic acid for 3 h; Cold, 0°C for 2 h; Dehydration, air drying for 30 min. ( D ) Localization of GFP–ICE1 fusion protein in the nucleus. Panels a–c show confocal images of root cells in GFP–ICE1 transgenic plants, and panel d shows the location of nuclei as indicated by propidium stain.
Figure Legend Snippet: Expression of the ICE1 gene and subcellular localization of the ICE1 protein. ( A ) ICE1 promoter-driven GUS expression pattern in a wild-type seedling. ( B ) ICE1 promoter- GUS expression in different plant tissues, and the corresponding ICE1 transcript levels as determined by RT–PCR analysis. The tubulin gene was used as an internal control in the RT–PCR. ( C ) RNA blot analysis of ICE1 expression in wild-type seedlings under various abiotic stresses. Plants with the following treatments are shown: control, MS salt only; NaCl, 300 mM NaCl for 5 h; ABA, 100 μM abscisic acid for 3 h; Cold, 0°C for 2 h; Dehydration, air drying for 30 min. ( D ) Localization of GFP–ICE1 fusion protein in the nucleus. Panels a–c show confocal images of root cells in GFP–ICE1 transgenic plants, and panel d shows the location of nuclei as indicated by propidium stain.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Northern blot, Mass Spectrometry, Transgenic Assay, Staining

21) Product Images from "DOR/Tp53inp2 and Tp53inp1 Constitute a Metazoan Gene Family Encoding Dual Regulators of Autophagy and Transcription"

Article Title: DOR/Tp53inp2 and Tp53inp1 Constitute a Metazoan Gene Family Encoding Dual Regulators of Autophagy and Transcription

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034034

Human TP53INP1 activates autophagy. Panel A. HeLa cells were transiently transfected with TP53INP1 and incubated with DMEM (basal) or HBSS for 1 h (amino acid starvation), or 2 µM rapamycin for 3 h. The intracellular localization of TP53INP1 was analyzed by immunofluorescence and is shown in red. The nuclei are shown in blue. Scale bars, 10 µm. Panel B. Confocal images of HeLa cells transiently transfected with TP53INP1 and GFP-LC3, and incubated whether with DMEM, HBSS for 1 h or 2 µM rapamycin for 3 h. The intracellular localization of TP53INP1 was analyzed as in panel A. Scale bars, 10 µm. Contrast-corrected merged RGB pictures and Z-projection of PDM images are shown for all panels. Color scales of PDM images have different maximal values so care must be taken when comparing conditions. PDM values closer to 1 show reliable co-localized pixels. Panel C. HeLa cells were transiently transfected with either pcDNA3, DOR or TP53INP1a and incubated with DMEM or HBSS for 1 h, alone or in combination with 200 nM bafilomycin A1 for 16 h (cells were incubated with BafA1 for 16 h and for the last hour they were incubated with or without HBSS). Cell lysates were obtained and Western blot assays were performed with specific antibodies against L C3 and beta-actin. Panel D. HeLa cells previously infected with lentiviruses encoding scramble RNA or TP53INP1 shRNA were cultured. Cell extracts and total RNA were obtained and TP53INP1 mRNA levels were assayed by real-time PCR. Data are mean ± SEM of 3 independent experiments. * significant difference compared to the Scramble group, at P
Figure Legend Snippet: Human TP53INP1 activates autophagy. Panel A. HeLa cells were transiently transfected with TP53INP1 and incubated with DMEM (basal) or HBSS for 1 h (amino acid starvation), or 2 µM rapamycin for 3 h. The intracellular localization of TP53INP1 was analyzed by immunofluorescence and is shown in red. The nuclei are shown in blue. Scale bars, 10 µm. Panel B. Confocal images of HeLa cells transiently transfected with TP53INP1 and GFP-LC3, and incubated whether with DMEM, HBSS for 1 h or 2 µM rapamycin for 3 h. The intracellular localization of TP53INP1 was analyzed as in panel A. Scale bars, 10 µm. Contrast-corrected merged RGB pictures and Z-projection of PDM images are shown for all panels. Color scales of PDM images have different maximal values so care must be taken when comparing conditions. PDM values closer to 1 show reliable co-localized pixels. Panel C. HeLa cells were transiently transfected with either pcDNA3, DOR or TP53INP1a and incubated with DMEM or HBSS for 1 h, alone or in combination with 200 nM bafilomycin A1 for 16 h (cells were incubated with BafA1 for 16 h and for the last hour they were incubated with or without HBSS). Cell lysates were obtained and Western blot assays were performed with specific antibodies against L C3 and beta-actin. Panel D. HeLa cells previously infected with lentiviruses encoding scramble RNA or TP53INP1 shRNA were cultured. Cell extracts and total RNA were obtained and TP53INP1 mRNA levels were assayed by real-time PCR. Data are mean ± SEM of 3 independent experiments. * significant difference compared to the Scramble group, at P

Techniques Used: Transfection, Incubation, Immunofluorescence, Western Blot, Infection, shRNA, Cell Culture, Real-time Polymerase Chain Reaction

22) Product Images from "Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma"

Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2010.01124.x

Analysis of methylation status in CD147 promoter and CD147 expression level in HCC and ANT tissues. (A) Representative methylation profiles of CpG island in CD147 promoter in HCC and ANT tissues detected by BGS. Open and closed circles indicate unmethylated and methylated CpG sites, respectively. The percentage of methylated CpG sites in all sequenced CpG sites is shown in parentheses. (B) Representative results of MS-PCR analysis in HCC tumour tissues (T) and ANT tissues (N). M: methylation; U: unmethylation. (C) Immunohistochemical staining analysis for CD147 expression in HCC tissues. Top, positive CD147 immunostaining. Bottom, negative CD147 immunostaining. Scale bars, 50 μm. (D) Analysis for correlation of CD147 expression and its promoter metheylation in HCC tissues by calculating Spearman’s ρ method.
Figure Legend Snippet: Analysis of methylation status in CD147 promoter and CD147 expression level in HCC and ANT tissues. (A) Representative methylation profiles of CpG island in CD147 promoter in HCC and ANT tissues detected by BGS. Open and closed circles indicate unmethylated and methylated CpG sites, respectively. The percentage of methylated CpG sites in all sequenced CpG sites is shown in parentheses. (B) Representative results of MS-PCR analysis in HCC tumour tissues (T) and ANT tissues (N). M: methylation; U: unmethylation. (C) Immunohistochemical staining analysis for CD147 expression in HCC tissues. Top, positive CD147 immunostaining. Bottom, negative CD147 immunostaining. Scale bars, 50 μm. (D) Analysis for correlation of CD147 expression and its promoter metheylation in HCC tissues by calculating Spearman’s ρ method.

Techniques Used: Methylation, Expressing, Mass Spectrometry, Polymerase Chain Reaction, Immunohistochemistry, Staining, Immunostaining

Promoter hypomethylation up-regulated CD147 expression through increasing Sp1 binding in vivo . (A, B and C) The mRNA and protein expression of CD147 and Sp1 detected by regular RT-PCR, real-time quantitative RT-PCR and Western blot in QZG cell treated with different concentration of 5-Aza-dC. (D) Demethylation increased the binding of Sp1 to the CD147 promoter in vivo . ChIP assay using antibody against Sp1 was performed in before and after 5-Aza-dC treatment QZG cell. The normal rabbit IgG was used as a negative control and Input indicates 5% input DNA, a positive amplification control.
Figure Legend Snippet: Promoter hypomethylation up-regulated CD147 expression through increasing Sp1 binding in vivo . (A, B and C) The mRNA and protein expression of CD147 and Sp1 detected by regular RT-PCR, real-time quantitative RT-PCR and Western blot in QZG cell treated with different concentration of 5-Aza-dC. (D) Demethylation increased the binding of Sp1 to the CD147 promoter in vivo . ChIP assay using antibody against Sp1 was performed in before and after 5-Aza-dC treatment QZG cell. The normal rabbit IgG was used as a negative control and Input indicates 5% input DNA, a positive amplification control.

Techniques Used: Expressing, Binding Assay, In Vivo, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Concentration Assay, Chromatin Immunoprecipitation, Negative Control, Amplification

Up-expression of CD147 in HCC cell lines. (A) mRNA expressions of CD147 gene were detected by real-time quantitative RT-PCR in HCC cell lines, normal cell lines and normal liver tissues. GAPDH gene was used as an internal control. (B) CD147 protein expression was determined by Western blot with tubulin as an internal control.
Figure Legend Snippet: Up-expression of CD147 in HCC cell lines. (A) mRNA expressions of CD147 gene were detected by real-time quantitative RT-PCR in HCC cell lines, normal cell lines and normal liver tissues. GAPDH gene was used as an internal control. (B) CD147 protein expression was determined by Western blot with tubulin as an internal control.

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

23) Product Images from "Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ"

Article Title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

Journal: BMC Genomics

doi: 10.1186/s12864-016-3331-9

Generation of gene cassette knock-in mice by the enhanced PITCh system. a Schematic diagram of pronuclear injection of Cas9 protein, Actb and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. b PCR screenings of knock-in newborns. c Summary of Actb -TetO-FLEX-hM3Dq/mCherry knock-in mouse production by the enhanced PITCh system. d Sequences of boundaries between Actb and TetO-FLEX- hM3Dq/mCherry cassette. Blue and red characters indicate microhomologies and partial crRNA( gRNA-s1 ) target sequence, respectively. IF: internal forward primer, IR: internal reverse primer, LF: left forward primer, LR: left reverse primer, RF: right forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, WT: wildtype, KI: knock-in, and KI/+: tail genomic DNA of F1 heterozygous knock-in pup derived from #13 (KI#2) F0 knock-in mouse
Figure Legend Snippet: Generation of gene cassette knock-in mice by the enhanced PITCh system. a Schematic diagram of pronuclear injection of Cas9 protein, Actb and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. b PCR screenings of knock-in newborns. c Summary of Actb -TetO-FLEX-hM3Dq/mCherry knock-in mouse production by the enhanced PITCh system. d Sequences of boundaries between Actb and TetO-FLEX- hM3Dq/mCherry cassette. Blue and red characters indicate microhomologies and partial crRNA( gRNA-s1 ) target sequence, respectively. IF: internal forward primer, IR: internal reverse primer, LF: left forward primer, LR: left reverse primer, RF: right forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, WT: wildtype, KI: knock-in, and KI/+: tail genomic DNA of F1 heterozygous knock-in pup derived from #13 (KI#2) F0 knock-in mouse

Techniques Used: Knock-In, Mouse Assay, Injection, Polymerase Chain Reaction, Sequencing, Marker, Derivative Assay

Generation of knock-in mice carrying a gene cassette by the PITCh system. a Targeting strategy for the generation of Actb -TetO-FLEX-hM3Dq/mCherry knock-in mice by the PITCh system. Purple highlights indicate microhomologies between endogenous Actb locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Actb and gRNA-s1 crRNAs, tracrRNA and PITCh-donor. The red, purple, and blue boxes indicate the insert, Actb microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of knock-in newborns. d Summary of Actb -TetO-FLEX-hM3Dq/mCherry knock-in mouse production by the PITCh system. e Sequences of boundaries between Actb and TetO-FLEX- hM3Dq/mCherry cassette. Blue characters indicate microhomologies. IF: internal forward primer, IR: internal reverse primer, LF: left forward primer, LR: left reverse primer, RF: right forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, WT: wildtype, KI: knock-in, WPRE: woodchuck hepatitis virus posttranscriptional regulatory element, pA: polyA, and KI/+: tail genomic DNA of F1 heterozygous knock-in pup derived from #13 (KI#2) F0 knock-in mouse
Figure Legend Snippet: Generation of knock-in mice carrying a gene cassette by the PITCh system. a Targeting strategy for the generation of Actb -TetO-FLEX-hM3Dq/mCherry knock-in mice by the PITCh system. Purple highlights indicate microhomologies between endogenous Actb locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Actb and gRNA-s1 crRNAs, tracrRNA and PITCh-donor. The red, purple, and blue boxes indicate the insert, Actb microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of knock-in newborns. d Summary of Actb -TetO-FLEX-hM3Dq/mCherry knock-in mouse production by the PITCh system. e Sequences of boundaries between Actb and TetO-FLEX- hM3Dq/mCherry cassette. Blue characters indicate microhomologies. IF: internal forward primer, IR: internal reverse primer, LF: left forward primer, LR: left reverse primer, RF: right forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, WT: wildtype, KI: knock-in, WPRE: woodchuck hepatitis virus posttranscriptional regulatory element, pA: polyA, and KI/+: tail genomic DNA of F1 heterozygous knock-in pup derived from #13 (KI#2) F0 knock-in mouse

Techniques Used: Knock-In, Mouse Assay, CRISPR, Injection, Polymerase Chain Reaction, Marker, Derivative Assay

24) Product Images from "MicroRNA-181 Regulates CARM1 and Histone Aginine Methylation to Promote Differentiation of Human Embryonic Stem Cells"

Article Title: MicroRNA-181 Regulates CARM1 and Histone Aginine Methylation to Promote Differentiation of Human Embryonic Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053146

CARM1 is down-regulated post-transcriptionally during hESC differentiation. (A, B) The kinetic expression levels of CARM1 , Nanog , Sox2 , and Oct4 in differentiated hESCs. Human ESCs differentiation was induced by the addition of BMP4 in the absence of bFGF. The expression levels of CARM1 , Nanog , Sox2 , and Oct4 at the mRNA (A) and protein levels (B) were quantified by qRT-PCR and Western blotting, respectively. *, p
Figure Legend Snippet: CARM1 is down-regulated post-transcriptionally during hESC differentiation. (A, B) The kinetic expression levels of CARM1 , Nanog , Sox2 , and Oct4 in differentiated hESCs. Human ESCs differentiation was induced by the addition of BMP4 in the absence of bFGF. The expression levels of CARM1 , Nanog , Sox2 , and Oct4 at the mRNA (A) and protein levels (B) were quantified by qRT-PCR and Western blotting, respectively. *, p

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

The miR-181 family directly regulates CARM1 expression in hESC. (A) The expression levels of mature miRNAs predicted to target the CARM1 3′UTR were monitored in differentiated ESCs by qRT-PCR and normalized to endogenous U6 expression. *, p
Figure Legend Snippet: The miR-181 family directly regulates CARM1 expression in hESC. (A) The expression levels of mature miRNAs predicted to target the CARM1 3′UTR were monitored in differentiated ESCs by qRT-PCR and normalized to endogenous U6 expression. *, p

Techniques Used: Expressing, Quantitative RT-PCR

miR-181c leads to hESC differentiation through negative regulation of CARM1 and H3R17 methylation. (A) Overexpression of miR-181c down-regulated CARM1 expression in comparison to negative control (NC) RNA-transfected ESCs at both the mRNA and protein levels, as determined by qRT-PCR and Western blotting, respectively. H3R17 methylation level and Oct4 , Nanog and Sox2 mRNA expression were also monitored. Samples were assayed in duplicate (n = 3) and normalized to endogenous β-actin expression. **, p
Figure Legend Snippet: miR-181c leads to hESC differentiation through negative regulation of CARM1 and H3R17 methylation. (A) Overexpression of miR-181c down-regulated CARM1 expression in comparison to negative control (NC) RNA-transfected ESCs at both the mRNA and protein levels, as determined by qRT-PCR and Western blotting, respectively. H3R17 methylation level and Oct4 , Nanog and Sox2 mRNA expression were also monitored. Samples were assayed in duplicate (n = 3) and normalized to endogenous β-actin expression. **, p

Techniques Used: Methylation, Over Expression, Expressing, Negative Control, Transfection, Quantitative RT-PCR, Western Blot

25) Product Images from "Regulation of CEACAM1 Protein Expression by the Transcription Factor ETS-1 in BRAF-Mutant Human Metastatic Melanoma Cells"

Article Title: Regulation of CEACAM1 Protein Expression by the Transcription Factor ETS-1 in BRAF-Mutant Human Metastatic Melanoma Cells

Journal: Neoplasia (New York, N.Y.)

doi: 10.1016/j.neo.2018.01.012

Resistance to inhibitors of the MAPK pathway restores CEACAM1 expression. Vemurafenib-resistant (Vem-Res) and selumetinib-resistant (Sel-Res) sublines of 624mel and 526mel cells were tested. (A) The restored expression of pERK. The graph shows the ratio of each indicated protein as normalized according to actin using densitometry. (B) The restored expression of CEACAM1 using flow cytometry. Shaded histograms represent staining with secondary reagent only. Parental (Parent.) and resistant (Res) histograms are indicated in each panel. (C) The effect of each treatment on CEACAM1 isoform expression (long, short) using. RT-PCR. Results are depicted as fold change (RQ) of the parental cell control. Figure shows a representative experiment out of three performed.
Figure Legend Snippet: Resistance to inhibitors of the MAPK pathway restores CEACAM1 expression. Vemurafenib-resistant (Vem-Res) and selumetinib-resistant (Sel-Res) sublines of 624mel and 526mel cells were tested. (A) The restored expression of pERK. The graph shows the ratio of each indicated protein as normalized according to actin using densitometry. (B) The restored expression of CEACAM1 using flow cytometry. Shaded histograms represent staining with secondary reagent only. Parental (Parent.) and resistant (Res) histograms are indicated in each panel. (C) The effect of each treatment on CEACAM1 isoform expression (long, short) using. RT-PCR. Results are depicted as fold change (RQ) of the parental cell control. Figure shows a representative experiment out of three performed.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction

ETS1 upregulates the expression of CEACAM1. (A) CEACAM1 promoter was cloned upstream to firefly luciferase and co-transfected with a normalizing construct of Renilla luciferase, together with a vector encoding for ETS1 (ETS1), mutated ETS1 (ETS1-T38A), or an empty vector (Mock). Relative promoter activity was calculated relative to the effect of transfection with Mock vector. (B) Wild type (WT) or deletions in the putative ETS1 binding site in the negative strand (delETS1(−)), positive strand (delETS1(+)), or both (double) of the CEACAM1 promoter were cloned upstream to firefly luciferase and co-transfected with a normalizing construct of Renilla luciferase, together with a vector encoding for ETS1 (ETS1) or an empty vector (Mock). Data shown are normalized to Mock. (C) The effect of ETS1 (ETS1) compared to an empty vector (Mock) on CEACAM1 isoform (long, short) expression following transfected into melanoma was tested at the mRNA level using RT-PCR. (D) CEACAM1 expression was tested at the protein level using flow cytometry. The histograms of each of the transfectants are indicated in the figure. (A-C) The average results of three independent experiments. Significance was tested with Student’s t test, ** depicts P value of
Figure Legend Snippet: ETS1 upregulates the expression of CEACAM1. (A) CEACAM1 promoter was cloned upstream to firefly luciferase and co-transfected with a normalizing construct of Renilla luciferase, together with a vector encoding for ETS1 (ETS1), mutated ETS1 (ETS1-T38A), or an empty vector (Mock). Relative promoter activity was calculated relative to the effect of transfection with Mock vector. (B) Wild type (WT) or deletions in the putative ETS1 binding site in the negative strand (delETS1(−)), positive strand (delETS1(+)), or both (double) of the CEACAM1 promoter were cloned upstream to firefly luciferase and co-transfected with a normalizing construct of Renilla luciferase, together with a vector encoding for ETS1 (ETS1) or an empty vector (Mock). Data shown are normalized to Mock. (C) The effect of ETS1 (ETS1) compared to an empty vector (Mock) on CEACAM1 isoform (long, short) expression following transfected into melanoma was tested at the mRNA level using RT-PCR. (D) CEACAM1 expression was tested at the protein level using flow cytometry. The histograms of each of the transfectants are indicated in the figure. (A-C) The average results of three independent experiments. Significance was tested with Student’s t test, ** depicts P value of

Techniques Used: Expressing, Clone Assay, Luciferase, Transfection, Construct, Plasmid Preparation, Activity Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry

Inhibition of MAPK pathway downregulates CEACAM1 expression. The indicated BRAF mutant or wild-type (WT) melanoma cells were incubated with vemurafenib (VEM), selumetinib (SEL), or control (DMSO). (A) The effect of each treatment on pERK. (B) The effect of different doses of each treatment on CEACAM1 expression, as tested by flow cytometry, in each of the melanoma cell lines, in the indicated time points. Shaded histograms represent staining with secondary reagent only. Black histograms represent treatment with DMSO. Gray and dotted histograms represent treatment with 0.1 μM or 1 μM, respectively, of VEM or SEL. (C) The effect of each treatment on CEACAM1 isoform expression (long, short) using RT-PCR. Results are depicted as fold change (RQ) of the DMSO control. Figure shows a representative experiment out of three performed.
Figure Legend Snippet: Inhibition of MAPK pathway downregulates CEACAM1 expression. The indicated BRAF mutant or wild-type (WT) melanoma cells were incubated with vemurafenib (VEM), selumetinib (SEL), or control (DMSO). (A) The effect of each treatment on pERK. (B) The effect of different doses of each treatment on CEACAM1 expression, as tested by flow cytometry, in each of the melanoma cell lines, in the indicated time points. Shaded histograms represent staining with secondary reagent only. Black histograms represent treatment with DMSO. Gray and dotted histograms represent treatment with 0.1 μM or 1 μM, respectively, of VEM or SEL. (C) The effect of each treatment on CEACAM1 isoform expression (long, short) using RT-PCR. Results are depicted as fold change (RQ) of the DMSO control. Figure shows a representative experiment out of three performed.

Techniques Used: Inhibition, Expressing, Mutagenesis, Incubation, Flow Cytometry, Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction

26) Product Images from "Crystal Structure of the N-terminal Domain of the Yeast General Corepressor Tup1p and Its Functional Implications *"

Article Title: Crystal Structure of the N-terminal Domain of the Yeast General Corepressor Tup1p and Its Functional Implications *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.369652

Quantitative RT-PCR complementation assays. A , Tup1p point mutants in which one, two, or four acidic residues had been replaced. B , Tup1p point mutants in which a hydrophobic residue in a coiled coil had been replaced. Samples of the S. cerevisiae strain YMH427, which harbors a deletion in TUP1 (Δ tup1 ), were transformed with one of the plasmids that harbored a mutated TUP1 . The ability of the expressed, mutated Tup1ps to repress ANB1 ( blue bars ), STE2 ( yellow bars ), and SUC2 ( red bars ) expression was measured by quantitative RT-PCR.
Figure Legend Snippet: Quantitative RT-PCR complementation assays. A , Tup1p point mutants in which one, two, or four acidic residues had been replaced. B , Tup1p point mutants in which a hydrophobic residue in a coiled coil had been replaced. Samples of the S. cerevisiae strain YMH427, which harbors a deletion in TUP1 (Δ tup1 ), were transformed with one of the plasmids that harbored a mutated TUP1 . The ability of the expressed, mutated Tup1ps to repress ANB1 ( blue bars ), STE2 ( yellow bars ), and SUC2 ( red bars ) expression was measured by quantitative RT-PCR.

Techniques Used: Quantitative RT-PCR, Transformation Assay, Expressing

27) Product Images from "P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression"

Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.201

The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. qRT-PCR was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P
Figure Legend Snippet: The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. qRT-PCR was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P

Techniques Used: In Vivo, Transfection, Injection, Mouse Assay, Quantitative RT-PCR, Expressing, Histopathology, Staining, Immunohistochemistry

TUG1 could epigenetically regulate HOXB7 by binding to PRC2. ( a ) qRT-PCR was performed to detect the expression of HOX genes in transfected cells, and western blot assays were used to detect the level of HOXB7 after the transfection of si-TUG1. IHC assays were used to detect the HOXB7 in tumor sections from shTUG1-transfected cells. ( b ) RIP experiments were performed in SPC-A1 and the coprecipitated RNA was subjected to qRT-PCR for TUG1. HOTAIR was used as a positive control. The fold enrichment of TUG1 in EZH2 RIP is relative to its matching IgG control RIP. TUG1 nuclear localization, as identified using qRT-PCR in fractionated SPC-A1 and A549 cells. ( c ) ChIP of H3K27me3 and EZH2 of the promoter region of HOXB7 locus after siRNA treatment targeting si-NC or si-TUG1; qPCR were performed to determine the quantitation of ChIP assays. The levels of qPCR products are expressed as a percentage of the input DNA. Error bars indicate means±S.E.M. * P
Figure Legend Snippet: TUG1 could epigenetically regulate HOXB7 by binding to PRC2. ( a ) qRT-PCR was performed to detect the expression of HOX genes in transfected cells, and western blot assays were used to detect the level of HOXB7 after the transfection of si-TUG1. IHC assays were used to detect the HOXB7 in tumor sections from shTUG1-transfected cells. ( b ) RIP experiments were performed in SPC-A1 and the coprecipitated RNA was subjected to qRT-PCR for TUG1. HOTAIR was used as a positive control. The fold enrichment of TUG1 in EZH2 RIP is relative to its matching IgG control RIP. TUG1 nuclear localization, as identified using qRT-PCR in fractionated SPC-A1 and A549 cells. ( c ) ChIP of H3K27me3 and EZH2 of the promoter region of HOXB7 locus after siRNA treatment targeting si-NC or si-TUG1; qPCR were performed to determine the quantitation of ChIP assays. The levels of qPCR products are expressed as a percentage of the input DNA. Error bars indicate means±S.E.M. * P

Techniques Used: Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Immunohistochemistry, Positive Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitation Assay

p53 induces TUG1 through interacting with the promoter region of TUG1. ( a ) Analysis of TUG1 expression levels in NSCLC cell lines (A549, SPC-A1, SK-MES-1, NCI-H1299 and NCI-H1650) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( b ) Description of p53RE and mutant p53RE in promoter region of TUG1. The position of ChIP primers ( e ) was indicated by arrows. ( c ) Western blotting was used to detect the p53 induction by doxo. qRT-PCR was used to detect the effect of doxo on TUG1 expression in p53-WT and p53-null cells. ( d ) Induction of TUG1 by ectopically expressed p53 (wild-type p53 or mutant p53). Overexpression was confirmed by western blotting. ( e ) Induction of TUG1 promoter activity by p53, but not mutant p53 in NCI-H1299 cell lines. ( f ) Deletion and mutation analysis of the promoter activity to determine the role of the p53RE in p53-mediated regulation of TUG1. ( g ) The p53 binding at the promoter regions of TUG1 was assessed by ChIP analysis. ChIP primers were detailed in Materials and Methods section. Shown are representative images of three independent experiments. Error bars indicate means±S.E.M. * P
Figure Legend Snippet: p53 induces TUG1 through interacting with the promoter region of TUG1. ( a ) Analysis of TUG1 expression levels in NSCLC cell lines (A549, SPC-A1, SK-MES-1, NCI-H1299 and NCI-H1650) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( b ) Description of p53RE and mutant p53RE in promoter region of TUG1. The position of ChIP primers ( e ) was indicated by arrows. ( c ) Western blotting was used to detect the p53 induction by doxo. qRT-PCR was used to detect the effect of doxo on TUG1 expression in p53-WT and p53-null cells. ( d ) Induction of TUG1 by ectopically expressed p53 (wild-type p53 or mutant p53). Overexpression was confirmed by western blotting. ( e ) Induction of TUG1 promoter activity by p53, but not mutant p53 in NCI-H1299 cell lines. ( f ) Deletion and mutation analysis of the promoter activity to determine the role of the p53RE in p53-mediated regulation of TUG1. ( g ) The p53 binding at the promoter regions of TUG1 was assessed by ChIP analysis. ChIP primers were detailed in Materials and Methods section. Shown are representative images of three independent experiments. Error bars indicate means±S.E.M. * P

Techniques Used: Expressing, Quantitative RT-PCR, Mutagenesis, Chromatin Immunoprecipitation, Western Blot, Over Expression, Activity Assay, Binding Assay

Analysis of TUG1 expression in NSCLC tissues and clinical parameters. ( a ) TUG1 was detected in 192 pairs of NSCLC tissues by qRT-PCR. The levels of TUG1 in NSCLC tissues are significantly lower than those in non-tumorous tissues. The ΔCt value was determined by subtracting the GAPDH Ct value from the TUG1 Ct value (relative to a single reference value). Smaller ΔCt value indicates higher expression. ( b and c ) Data are presented as relative expression level in tumor tissues (shown as ΔCt). TUG1 expression was significantly lower in patients with a higher pathological stage and big tumor size. ( d ) Patients with low levels of TUG1 expression showed reduced survival times compared with patients with high levels of TUG1 expression ( P
Figure Legend Snippet: Analysis of TUG1 expression in NSCLC tissues and clinical parameters. ( a ) TUG1 was detected in 192 pairs of NSCLC tissues by qRT-PCR. The levels of TUG1 in NSCLC tissues are significantly lower than those in non-tumorous tissues. The ΔCt value was determined by subtracting the GAPDH Ct value from the TUG1 Ct value (relative to a single reference value). Smaller ΔCt value indicates higher expression. ( b and c ) Data are presented as relative expression level in tumor tissues (shown as ΔCt). TUG1 expression was significantly lower in patients with a higher pathological stage and big tumor size. ( d ) Patients with low levels of TUG1 expression showed reduced survival times compared with patients with high levels of TUG1 expression ( P

Techniques Used: Expressing, Quantitative RT-PCR

TUG1 could participate in AKT and MAPK pathway through modulating HOXB7. ( A ) MTT analysis of cell proliferation by co-transfection (si-NC, si-HOXB7, si-HOXB7+si-TUG1). At 48 h after transfection of si-HOXB7, the cell cycle and apoptosis were analyzed by flow cytometry. LR, early apoptotic cells. UR, terminal apoptotic cells. ( B ) qRT-PCR were performed to detect the expression of HOXB7 after overexpression of p53 and transfected with p53 followed by transfection with si-TUG1. ( C ) Western blotting analysis of the expression of p-ERK, total ERK, p-AKT, total AKT, p-GSK-3 β , total GSK-3 β proteins in indicated si-NC-transfected, si-HOXB7 and si-TUG1-transfected SPC-A1 cell lines. ( D ) Immunostaining of HOXB7 was negatively or very weakly positive in corresponding non-tumor lung tissues (a and b), but was strongly positive in squamous cell carcinoma tissues (c and d) and lung adenocarcinoma (e and f). Bar, 100 μ m. ( E ) The immunoreactivity of HOXB7 protein in NSCLC tissues showed a statistically significant inverse correlation with the relative level of TUG1 expression. Error bars indicate means±S.E.M. * P
Figure Legend Snippet: TUG1 could participate in AKT and MAPK pathway through modulating HOXB7. ( A ) MTT analysis of cell proliferation by co-transfection (si-NC, si-HOXB7, si-HOXB7+si-TUG1). At 48 h after transfection of si-HOXB7, the cell cycle and apoptosis were analyzed by flow cytometry. LR, early apoptotic cells. UR, terminal apoptotic cells. ( B ) qRT-PCR were performed to detect the expression of HOXB7 after overexpression of p53 and transfected with p53 followed by transfection with si-TUG1. ( C ) Western blotting analysis of the expression of p-ERK, total ERK, p-AKT, total AKT, p-GSK-3 β , total GSK-3 β proteins in indicated si-NC-transfected, si-HOXB7 and si-TUG1-transfected SPC-A1 cell lines. ( D ) Immunostaining of HOXB7 was negatively or very weakly positive in corresponding non-tumor lung tissues (a and b), but was strongly positive in squamous cell carcinoma tissues (c and d) and lung adenocarcinoma (e and f). Bar, 100 μ m. ( E ) The immunoreactivity of HOXB7 protein in NSCLC tissues showed a statistically significant inverse correlation with the relative level of TUG1 expression. Error bars indicate means±S.E.M. * P

Techniques Used: MTT Assay, Cotransfection, Transfection, Flow Cytometry, Cytometry, Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Immunostaining

28) Product Images from "A single nucleotide polymorphism in the Bax gene promoter affects transcription and influences retinal ganglion cell death"

Article Title: A single nucleotide polymorphism in the Bax gene promoter affects transcription and influences retinal ganglion cell death

Journal: ASN NEURO

doi: 10.1042/AN20100003

DBA/2J and 129B6 mice differentially express Bax ( A ) Quantitative PCR analysis of latent Bax transcript levels (means±SEM) in 129B6 and DBA/2J mice. Values shown are the number of Bax mRNA molecules [per 2.5 pg of poly(A) RNA input] normalized to the number of S16 molecules in each sample. Reducing Bax gene dosage to one gene caused a 50% reduction of Bax transcripts (for 129B6, P = 0.004, for DBA/2J, P = 0.002, control retinas of Bax +/− mice compared with control retinas of Bax +/+ mice) and no transcripts were detected in knock-out mice. Similarly, 129B6 mice had approximately twice the amount of Bax mRNA in the retina (*129B6 Bax +/+ compared with DBA/2J Bax +/+ mice, P = 0.008; **129B6 Bax +/− compared with DBA/2J Bax +/− mice, P = 0.002). ( B ) Representative immunoblot showing relative Bax protein levels in DBA/2J and 129B6 neurons. Bax protein levels were 1.2- to 1.8-fold higher in 129B6 Bax+/+ mice compared with DBA/2J Bax +/+ mice (when normalized to actin in each lane). Similarly, 129B6 Bax +/− mice expressed 1.4- to 2.0-fold as much Bax as DBA/2J Bax +/− mice. No Bax protein was detected in Bax −/− mice. ( C ) Histogram of BclX transcript levels in the retinas of Bax heterozygous mice from each strain. Unlike Bax mRNA, no difference in BclX mRNA was detected between strains ( P = 0.26), or between wild-type and knockout mice (data not shown). ( D ) Blot strips taken from the same lanes of retina homogenates of wild-type DBA/2J or 129B6 mice. To accurately assess the relative levels of BAX and BCL-X, the strips were developed equally, until BCL-X staining began to saturate the colorimetric reaction. BCL-X shows up as two prominent bands, probably reflecting modified and deamidated polypeptides ( Johnstone, 2002 ). BAX levels, under these conditions, are just detectable. Chemiluminescence detection of BAX staining clearly shows, however, that this antibody specifically interacted with BAX protein in similar samples (see B ). Actin was also evaluated as a loading control. ( E ) Histogram showing changes in Bax transcript abundance (means±S.E.M.), 4 days after ONC. Retinal transcript levels were examined at 4 days after injury when others have reported moderate increases in Bax mRNA after optic nerve axotomy ( Näpänkangas et al., 2003 ). ONC caused a 42.0±13.2% decrease in Bax mRNA in DBA/2J mice relative to control eyes (* P = 0.003) and a 79.0±3.8% decrease in 129B6 mice relative to control eyes (** P = 4.6×10 −5 ). The control retinas of 129B6 Bax +/+ mice contained 2.2-fold the amount of Bax mRNA compared with control DBA/2J Bax +/+ retinas ( P = 2.0×10 −4 ). Thus Bax expression was not differentially increased between strains, and instead showed a relative decrease after injury.
Figure Legend Snippet: DBA/2J and 129B6 mice differentially express Bax ( A ) Quantitative PCR analysis of latent Bax transcript levels (means±SEM) in 129B6 and DBA/2J mice. Values shown are the number of Bax mRNA molecules [per 2.5 pg of poly(A) RNA input] normalized to the number of S16 molecules in each sample. Reducing Bax gene dosage to one gene caused a 50% reduction of Bax transcripts (for 129B6, P = 0.004, for DBA/2J, P = 0.002, control retinas of Bax +/− mice compared with control retinas of Bax +/+ mice) and no transcripts were detected in knock-out mice. Similarly, 129B6 mice had approximately twice the amount of Bax mRNA in the retina (*129B6 Bax +/+ compared with DBA/2J Bax +/+ mice, P = 0.008; **129B6 Bax +/− compared with DBA/2J Bax +/− mice, P = 0.002). ( B ) Representative immunoblot showing relative Bax protein levels in DBA/2J and 129B6 neurons. Bax protein levels were 1.2- to 1.8-fold higher in 129B6 Bax+/+ mice compared with DBA/2J Bax +/+ mice (when normalized to actin in each lane). Similarly, 129B6 Bax +/− mice expressed 1.4- to 2.0-fold as much Bax as DBA/2J Bax +/− mice. No Bax protein was detected in Bax −/− mice. ( C ) Histogram of BclX transcript levels in the retinas of Bax heterozygous mice from each strain. Unlike Bax mRNA, no difference in BclX mRNA was detected between strains ( P = 0.26), or between wild-type and knockout mice (data not shown). ( D ) Blot strips taken from the same lanes of retina homogenates of wild-type DBA/2J or 129B6 mice. To accurately assess the relative levels of BAX and BCL-X, the strips were developed equally, until BCL-X staining began to saturate the colorimetric reaction. BCL-X shows up as two prominent bands, probably reflecting modified and deamidated polypeptides ( Johnstone, 2002 ). BAX levels, under these conditions, are just detectable. Chemiluminescence detection of BAX staining clearly shows, however, that this antibody specifically interacted with BAX protein in similar samples (see B ). Actin was also evaluated as a loading control. ( E ) Histogram showing changes in Bax transcript abundance (means±S.E.M.), 4 days after ONC. Retinal transcript levels were examined at 4 days after injury when others have reported moderate increases in Bax mRNA after optic nerve axotomy ( Näpänkangas et al., 2003 ). ONC caused a 42.0±13.2% decrease in Bax mRNA in DBA/2J mice relative to control eyes (* P = 0.003) and a 79.0±3.8% decrease in 129B6 mice relative to control eyes (** P = 4.6×10 −5 ). The control retinas of 129B6 Bax +/+ mice contained 2.2-fold the amount of Bax mRNA compared with control DBA/2J Bax +/+ retinas ( P = 2.0×10 −4 ). Thus Bax expression was not differentially increased between strains, and instead showed a relative decrease after injury.

Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Knock-Out, Staining, Modification, Expressing

29) Product Images from "Identification of target genes for wild type and truncated HMGA2 in mesenchymal stem-like cells"

Article Title: Identification of target genes for wild type and truncated HMGA2 in mesenchymal stem-like cells

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-329

Characterization of hMSC-TERT20 cells stably over-expressing eGFP-HMGA2 WT and eGFP-HMGA2 TRUNC . a ) Nuclear localization of eGFP-tagged HMGA2 proteins was observed in hMSC-TERT20 over-expressing eGFP-HMGA2 WT or eGFP-HMGA2 TRUNC . Endogenous HMGA2 was visualized by immunofluorescent staining with an anti-HMGA2 antibody (The intensity in parental cells was adjusted to make observation of nucleolar fluorescence possible). The nucleolus was detected with an anti-B23 antibody, while heterochromatin was stained by DAPI, showing co-localization of HMGA2 with both nucleoli and heterochromatin. Arrowheads show concentration of endogenous or eGFP-tagged HMGA2 at sites that correspond with nucleoli, while an arrow indicates discrete foci of HMGA2 within nucleoli. b ) Expression levels of endogenous and exogenous HMGA2 transcripts; expected size of the endogenous transcript is 4468 bp, while the exogenous is only 327 bp due to the lack of the 3' UTR. Top panel, northern blot of HMGA2 mRNAs. Lower panel, quantitation of total HMGA2 mRNA levels by real-time PCR, normalized for TBP expression and represented as fold induction over parental cells. c ) Expression levels of endogenous and exogenous HMGA2 proteins, as detected on western blot. d ) Accumulation of fat in cell cultures grown in basal medium supplemented with MDI and Rosiglitazone for 14 days. e ) Relative accumulation of fat-bound Oil Red O after 14 days of differentiation of hMSC-TERT20 cells. Data from two independent experiments are shown. Par, Parental cells; eGFP, eGFP-transduced; WT, transduced with eGFP-HMGA2 WT ; Trunc, transduced with eGFP-HMGA2 TRUNC f) Expression of CD24 and HLA-DRA on each hMSC TERT20-derived cell lines as measured by flow cytometric mean fluorescence intensity (MFI).
Figure Legend Snippet: Characterization of hMSC-TERT20 cells stably over-expressing eGFP-HMGA2 WT and eGFP-HMGA2 TRUNC . a ) Nuclear localization of eGFP-tagged HMGA2 proteins was observed in hMSC-TERT20 over-expressing eGFP-HMGA2 WT or eGFP-HMGA2 TRUNC . Endogenous HMGA2 was visualized by immunofluorescent staining with an anti-HMGA2 antibody (The intensity in parental cells was adjusted to make observation of nucleolar fluorescence possible). The nucleolus was detected with an anti-B23 antibody, while heterochromatin was stained by DAPI, showing co-localization of HMGA2 with both nucleoli and heterochromatin. Arrowheads show concentration of endogenous or eGFP-tagged HMGA2 at sites that correspond with nucleoli, while an arrow indicates discrete foci of HMGA2 within nucleoli. b ) Expression levels of endogenous and exogenous HMGA2 transcripts; expected size of the endogenous transcript is 4468 bp, while the exogenous is only 327 bp due to the lack of the 3' UTR. Top panel, northern blot of HMGA2 mRNAs. Lower panel, quantitation of total HMGA2 mRNA levels by real-time PCR, normalized for TBP expression and represented as fold induction over parental cells. c ) Expression levels of endogenous and exogenous HMGA2 proteins, as detected on western blot. d ) Accumulation of fat in cell cultures grown in basal medium supplemented with MDI and Rosiglitazone for 14 days. e ) Relative accumulation of fat-bound Oil Red O after 14 days of differentiation of hMSC-TERT20 cells. Data from two independent experiments are shown. Par, Parental cells; eGFP, eGFP-transduced; WT, transduced with eGFP-HMGA2 WT ; Trunc, transduced with eGFP-HMGA2 TRUNC f) Expression of CD24 and HLA-DRA on each hMSC TERT20-derived cell lines as measured by flow cytometric mean fluorescence intensity (MFI).

Techniques Used: Stable Transfection, Expressing, Staining, Fluorescence, Concentration Assay, Northern Blot, Quantitation Assay, Real-time Polymerase Chain Reaction, Western Blot, Transduction, Derivative Assay, Flow Cytometry

30) Product Images from "Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη -mediated phosphorylation in Nicotiana benthamiana"

Article Title: Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη -mediated phosphorylation in Nicotiana benthamiana

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006789

NbSKη- silenced N . benthamiana plants display the phenotype similar to N . benthamiana plants infected by PVX-C4. (A and B) qRT-PCR (A) and western blot (B) assays of NbSKη gene expression in wild-type (WT) and NbSKη -silenced plants. Relative accumulation level of NbSKη transcripts is normalized against the amount of actin transcript. Error bar denotes the standard deviation of three biological replicates. (C) Growth of the wild-type (WT), mock (TRV-GFP) and NbSKη -silenced (TRV- NbSKη ) N . benthamiana plants at 35 dpi. (D) The leaf petioles of NbSKη- silenced N . benthamiana leave mimics that of N . benthamiana leave inoculated with PVX-C4. The phenotype of 1st-5th leaves of plants under different treatments was shown. (E) The petiole length of the 3rd leaf of plants under different treatments. Each treatment had fifteen plants at the same developmental stage. ** represent significant difference (P value
Figure Legend Snippet: NbSKη- silenced N . benthamiana plants display the phenotype similar to N . benthamiana plants infected by PVX-C4. (A and B) qRT-PCR (A) and western blot (B) assays of NbSKη gene expression in wild-type (WT) and NbSKη -silenced plants. Relative accumulation level of NbSKη transcripts is normalized against the amount of actin transcript. Error bar denotes the standard deviation of three biological replicates. (C) Growth of the wild-type (WT), mock (TRV-GFP) and NbSKη -silenced (TRV- NbSKη ) N . benthamiana plants at 35 dpi. (D) The leaf petioles of NbSKη- silenced N . benthamiana leave mimics that of N . benthamiana leave inoculated with PVX-C4. The phenotype of 1st-5th leaves of plants under different treatments was shown. (E) The petiole length of the 3rd leaf of plants under different treatments. Each treatment had fifteen plants at the same developmental stage. ** represent significant difference (P value

Techniques Used: Infection, Quantitative RT-PCR, Western Blot, Expressing, Standard Deviation, Significance Assay

31) Product Images from "The feedback loop of LITAF and BCL6 is involved in regulating apoptosis in B cell non-Hodgkin's-lymphoma"

Article Title: The feedback loop of LITAF and BCL6 is involved in regulating apoptosis in B cell non-Hodgkin's-lymphoma

Journal: Oncotarget

doi: 10.18632/oncotarget.12680

Transcriptional regulation of BCL6 by LITAF A. qRT-PCR showing temporal regulation of BCL6 and its target genes by LITAF in Ramos and OCI-Ly6 cells 48 h post-infection. Control, cells with pLVX virus; LITAF-myc, cells with over-expressed LITAF. B. Effect of silencing LITAF on BCL6 , PRDM1 and c-Myc mRNA in OCI-Ly3 and Namalwa cells. LITAF-shR, shRNA against LITAF; CTRL-shR, scrambled shRNA. C. Graphic representation of the BCL6 gene highlighting the three potential LITAF binding sites. The sequences of A (−87 to +65), B (+66 to +206) and C (+358 to +488) of the BCL6 gene are shown containing the LITAF consensus sites (CTCCC) underlined. Sequence numbers are in reference to the + 1 nucleotide identified by previous study [ 43 ]. D. ChIP assay in OCI-Ly3 cells. PCR was performed with primers specific for three potential regulating regions (A, B and C) in the promoter of BCL6 , respectively. The PCR product was analyzed by agarose gel electrophoresis (left), and qRT-PCR results are expressed as fold enrichment calculated as the percentage of input for the specific antibody (LITAF) with respect to IgG control of three replicates (right). Marker, 100-bp ladder. E. Schematic representation of the wild-type and three mutant (CTCCC to CTAAA) BCL6 reporters used in the experiments. F. The luciferase activity of BCL6 reporter plasmid in cell lines including 293T, Ramos and OCI-Ly6 after over-expression of LITAF (upper); and OCI-Ly3 cells infected with shRNA for LITAF or control virus (lower). G. Luciferase activity of wild-type (WT) or mutant (Mut-A, Mut-B and Mut-C) BCL6 reporters in OCI-Ly6 cells transfected with LITAF. Mean±s.d. of three technical replicates were plotted. * P
Figure Legend Snippet: Transcriptional regulation of BCL6 by LITAF A. qRT-PCR showing temporal regulation of BCL6 and its target genes by LITAF in Ramos and OCI-Ly6 cells 48 h post-infection. Control, cells with pLVX virus; LITAF-myc, cells with over-expressed LITAF. B. Effect of silencing LITAF on BCL6 , PRDM1 and c-Myc mRNA in OCI-Ly3 and Namalwa cells. LITAF-shR, shRNA against LITAF; CTRL-shR, scrambled shRNA. C. Graphic representation of the BCL6 gene highlighting the three potential LITAF binding sites. The sequences of A (−87 to +65), B (+66 to +206) and C (+358 to +488) of the BCL6 gene are shown containing the LITAF consensus sites (CTCCC) underlined. Sequence numbers are in reference to the + 1 nucleotide identified by previous study [ 43 ]. D. ChIP assay in OCI-Ly3 cells. PCR was performed with primers specific for three potential regulating regions (A, B and C) in the promoter of BCL6 , respectively. The PCR product was analyzed by agarose gel electrophoresis (left), and qRT-PCR results are expressed as fold enrichment calculated as the percentage of input for the specific antibody (LITAF) with respect to IgG control of three replicates (right). Marker, 100-bp ladder. E. Schematic representation of the wild-type and three mutant (CTCCC to CTAAA) BCL6 reporters used in the experiments. F. The luciferase activity of BCL6 reporter plasmid in cell lines including 293T, Ramos and OCI-Ly6 after over-expression of LITAF (upper); and OCI-Ly3 cells infected with shRNA for LITAF or control virus (lower). G. Luciferase activity of wild-type (WT) or mutant (Mut-A, Mut-B and Mut-C) BCL6 reporters in OCI-Ly6 cells transfected with LITAF. Mean±s.d. of three technical replicates were plotted. * P

Techniques Used: Quantitative RT-PCR, Infection, shRNA, Binding Assay, Sequencing, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Mutagenesis, Luciferase, Activity Assay, Plasmid Preparation, Over Expression, Transfection

32) Product Images from "Down-Regulation of FXYD3 Expression in Human Lung Cancers "

Article Title: Down-Regulation of FXYD3 Expression in Human Lung Cancers

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.080571

Expression of FXYD3 mRNA in cell lines. Levels of FXYD3 mRNA were examined by RT-PCR. PCR products of FXYD3 and β-actin (ACTB) were resolved by electrophoresis in 1% agar and stained with ethidium bromide ( A ). Copy numbers of DUSP6 and β-actin
Figure Legend Snippet: Expression of FXYD3 mRNA in cell lines. Levels of FXYD3 mRNA were examined by RT-PCR. PCR products of FXYD3 and β-actin (ACTB) were resolved by electrophoresis in 1% agar and stained with ethidium bromide ( A ). Copy numbers of DUSP6 and β-actin

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

33) Product Images from "Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2"

Article Title: Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2

Journal: Malaria Journal

doi: 10.1186/s12936-016-1377-4

Complementation of Plasmodium berghei N-acylated Rab5b with conventional Rab5 and Rab5b from other apicomplexan parasites. a Schematic representation of PbRab5b genomic-locus replacement strategy. The targeting construct, consisting of the 5′ and 3′ regions ( two black boxes ) and PbRab5b open reading frame (ORF) ( orange ) fused to mAG ( dark green ), P. berghei DHFR 3′ untranslated region (PbDT) ( blue ), and the selectable marker TgDHFR expression cassette ( white ), was integrated into the PbRab5b genomic locus ( orange ) by double-crossover homologous recombination. The positions of the four PCR primers used to confirm the plasmid integration ( right panel ) are indicated. b Growth curve of the wild-type parasites ( blue ) and transgenic parasites for which the PbRab5b open reading frame replaced with PbRab5b-mAG ( red ). Bars standard deviation (n = 5). c Multiple amino acid sequence alignment of Plasmodium Rab5 isoforms. The GTP-binding consensus sequences and lipid modification sites are indicated red and blue boxes , respectively. The effector regions are shown as grey , black or magenta boxes . The conserved glutamine residue which was mutated to create a constitutively active mutant is shown in a red arrow . d Complementation test of the PbRab5b knockout. The coding region of PbRab5b was replaced with each gene-of-interest (GOI). The result of the complementation analysis is shown at the right ; +, complemented or −, uncomplemented (n = 3)
Figure Legend Snippet: Complementation of Plasmodium berghei N-acylated Rab5b with conventional Rab5 and Rab5b from other apicomplexan parasites. a Schematic representation of PbRab5b genomic-locus replacement strategy. The targeting construct, consisting of the 5′ and 3′ regions ( two black boxes ) and PbRab5b open reading frame (ORF) ( orange ) fused to mAG ( dark green ), P. berghei DHFR 3′ untranslated region (PbDT) ( blue ), and the selectable marker TgDHFR expression cassette ( white ), was integrated into the PbRab5b genomic locus ( orange ) by double-crossover homologous recombination. The positions of the four PCR primers used to confirm the plasmid integration ( right panel ) are indicated. b Growth curve of the wild-type parasites ( blue ) and transgenic parasites for which the PbRab5b open reading frame replaced with PbRab5b-mAG ( red ). Bars standard deviation (n = 5). c Multiple amino acid sequence alignment of Plasmodium Rab5 isoforms. The GTP-binding consensus sequences and lipid modification sites are indicated red and blue boxes , respectively. The effector regions are shown as grey , black or magenta boxes . The conserved glutamine residue which was mutated to create a constitutively active mutant is shown in a red arrow . d Complementation test of the PbRab5b knockout. The coding region of PbRab5b was replaced with each gene-of-interest (GOI). The result of the complementation analysis is shown at the right ; +, complemented or −, uncomplemented (n = 3)

Techniques Used: Construct, Marker, Expressing, Homologous Recombination, Polymerase Chain Reaction, Plasmid Preparation, Transgenic Assay, Standard Deviation, Sequencing, Binding Assay, Modification, Mutagenesis, Knock-Out

34) Product Images from "Non-canonical TAF complexes regulate active promoters in human embryonic stem cells"

Article Title: Non-canonical TAF complexes regulate active promoters in human embryonic stem cells

Journal: eLife

doi: 10.7554/eLife.00068

Validation of results presented in Figure 8C using a second, unrelated siRNA. qRT-PCR analysis monitoring expression of NANOG in H9 cells treated with a TAF siRNA. Values are given relative to that obtained with a control luciferase siRNA, which was set to 1. Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.00068.023
Figure Legend Snippet: Validation of results presented in Figure 8C using a second, unrelated siRNA. qRT-PCR analysis monitoring expression of NANOG in H9 cells treated with a TAF siRNA. Values are given relative to that obtained with a control luciferase siRNA, which was set to 1. Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.00068.023

Techniques Used: Quantitative RT-PCR, Expressing, Luciferase

shRNA-mediated knockdown efficiency of TAFs in H9 hESCs. qRT-PCR analysis monitoring TAF expression in H9 cells treated with two independent shRNAs directed against the indicated TAF. TAF expression is specified relative to that obtained with a control non-silencing shRNA, which was set to 1. Data are represented as mean ± SD. DOI: http://dx.doi.org/10.7554/eLife.00068.020
Figure Legend Snippet: shRNA-mediated knockdown efficiency of TAFs in H9 hESCs. qRT-PCR analysis monitoring TAF expression in H9 cells treated with two independent shRNAs directed against the indicated TAF. TAF expression is specified relative to that obtained with a control non-silencing shRNA, which was set to 1. Data are represented as mean ± SD. DOI: http://dx.doi.org/10.7554/eLife.00068.020

Techniques Used: shRNA, Quantitative RT-PCR, Expressing

Validation of results presented in Figure 8B using a second, unrelated siRNA. qRT-PCR analysis monitoring expression of differentiation markers in H9 cells treated with a TAF siRNA. Values are given relative to that obtained with a control luciferase siRNA, which was set to 1. Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.00068.022
Figure Legend Snippet: Validation of results presented in Figure 8B using a second, unrelated siRNA. qRT-PCR analysis monitoring expression of differentiation markers in H9 cells treated with a TAF siRNA. Values are given relative to that obtained with a control luciferase siRNA, which was set to 1. Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.00068.022

Techniques Used: Quantitative RT-PCR, Expressing, Luciferase

Confirmation of TAF requirement for transcriptional activity using a second, unrelated siRNA. qRT-PCR analysis monitoring expression of class I and class II genes in H9 cells treated with a TAF3, TAF5, TAF2, TAF6, TAF7 or TAF11 siRNA. Expression of each gene is specified relative to that obtained with a control luciferase siRNA, which was set to 1. Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.00068.012
Figure Legend Snippet: Confirmation of TAF requirement for transcriptional activity using a second, unrelated siRNA. qRT-PCR analysis monitoring expression of class I and class II genes in H9 cells treated with a TAF3, TAF5, TAF2, TAF6, TAF7 or TAF11 siRNA. Expression of each gene is specified relative to that obtained with a control luciferase siRNA, which was set to 1. Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.00068.012

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

siRNA-mediated knockdown efficiency of TAFs in H9 hESCs. qRT-PCR analysis monitoring TAF expression in H9 cells treated with two independent siRNAs ( A ) directed against the indicated TAF. TAF expression is specified relative to that obtained with a control luciferase siRNA, which was set to 1. Data are represented as mean ± SD. DOI: http://dx.doi.org/10.7554/eLife.00068.011
Figure Legend Snippet: siRNA-mediated knockdown efficiency of TAFs in H9 hESCs. qRT-PCR analysis monitoring TAF expression in H9 cells treated with two independent siRNAs ( A ) directed against the indicated TAF. TAF expression is specified relative to that obtained with a control luciferase siRNA, which was set to 1. Data are represented as mean ± SD. DOI: http://dx.doi.org/10.7554/eLife.00068.011

Techniques Used: Quantitative RT-PCR, Expressing, Luciferase

35) Product Images from "MicroRNA-340-mediated Degradation of Microphthalmia-associated Transcription Factor mRNA Is Inhibited by the Coding Region Determinant-binding Protein *"

Article Title: MicroRNA-340-mediated Degradation of Microphthalmia-associated Transcription Factor mRNA Is Inhibited by the Coding Region Determinant-binding Protein *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.109298

MITF mRNA with short 3′-UTR is more abundant in melanoma cell lines. A , graphical representation of different MITF mRNA isoforms with varying lengths of 3′-UTRs. The approximate positions of the reported miRNAs are shown on long and medium 3′-UTRs. B , real time quantitative PCR with primers specific for long, medium, and short 3′-UTRs (see “Experimental Procedures” for primer sequence) using RNA extracted from NHMs and the indicated melanoma cell lines.
Figure Legend Snippet: MITF mRNA with short 3′-UTR is more abundant in melanoma cell lines. A , graphical representation of different MITF mRNA isoforms with varying lengths of 3′-UTRs. The approximate positions of the reported miRNAs are shown on long and medium 3′-UTRs. B , real time quantitative PCR with primers specific for long, medium, and short 3′-UTRs (see “Experimental Procedures” for primer sequence) using RNA extracted from NHMs and the indicated melanoma cell lines.

Techniques Used: Real-time Polymerase Chain Reaction, Sequencing

CRD-BP is a positive regulator of MITF expression. A , 451Lu cells were transfected with either control shRNA or shRNA against CRD-BP. 48 h after transfection, the cells were collected and assayed for levels of MITF mRNA by quantitative RT-PCR, normalized to GAPDH expression, and presented as percentages of control (scrambled shRNA). B , 1241 Mel and Mel IM cells were co-transfected with the indicated shRNA-expressing plasmids, β-galactosidase-expressing plasmid, and pHTRPL4, where the luciferase gene is expressed under the MITF-dependent promoter. The values corresponding to luciferase activity normalized to β-galactosidase expression are presented. C , NHMs were electroporated using AMAXA TM with either CRD-BP-overexpressing plasmid or empty vector. 72 h after transfection, the cells were collected, assayed for levels of MITF mRNA by quantitative RT-PCR normalized to GAPDH expression, and presented as percentages of control (pBABE). D , NHM cells were electroporated using AMAXA TM with indicated plasmids. 48 h after electroporation, the cells were stained for senescence-associated β-galactosidase, and the percentages of β-galactosidase-positive cells were calculated. E , 451Lu cells were co-transfected with Tet-off plasmid, p-BIG-MITF, and either pcDNA control or CRD-BP-expressing plasmid. The turnover of MITF transcript was analyzed by real time RT-PCR after stopping transcription by doxycycline treatment for the indicated time points. Normalization was done with respect to GAPDH expression. All of the results are representative of three separate experiments and are expressed as the mean values ± S.D. ( error bars . F , FLAG immunoprecipitation of UV cross-linked ribonuclear protein complexes. Protein extracts from 293T cells, transfected with FLAG-CRDBP, were incubated with internally 32 P-labeled RNA transcripts of three fragments of the MITF mRNA coding region, MITF full-length mRNA, and the short 3′-UTR. Fragment 1 contains nucleotides 1–421 of the coding region, fragment 2 consists of nucleotides 422–841, and fragment 3 consists of nucleotides 842–1260 of the MITF coding region. Ribonuclear protein complexes were precipitated with anti-FLAG antibodies, analyzed on PAGE, and autoradiographed.
Figure Legend Snippet: CRD-BP is a positive regulator of MITF expression. A , 451Lu cells were transfected with either control shRNA or shRNA against CRD-BP. 48 h after transfection, the cells were collected and assayed for levels of MITF mRNA by quantitative RT-PCR, normalized to GAPDH expression, and presented as percentages of control (scrambled shRNA). B , 1241 Mel and Mel IM cells were co-transfected with the indicated shRNA-expressing plasmids, β-galactosidase-expressing plasmid, and pHTRPL4, where the luciferase gene is expressed under the MITF-dependent promoter. The values corresponding to luciferase activity normalized to β-galactosidase expression are presented. C , NHMs were electroporated using AMAXA TM with either CRD-BP-overexpressing plasmid or empty vector. 72 h after transfection, the cells were collected, assayed for levels of MITF mRNA by quantitative RT-PCR normalized to GAPDH expression, and presented as percentages of control (pBABE). D , NHM cells were electroporated using AMAXA TM with indicated plasmids. 48 h after electroporation, the cells were stained for senescence-associated β-galactosidase, and the percentages of β-galactosidase-positive cells were calculated. E , 451Lu cells were co-transfected with Tet-off plasmid, p-BIG-MITF, and either pcDNA control or CRD-BP-expressing plasmid. The turnover of MITF transcript was analyzed by real time RT-PCR after stopping transcription by doxycycline treatment for the indicated time points. Normalization was done with respect to GAPDH expression. All of the results are representative of three separate experiments and are expressed as the mean values ± S.D. ( error bars . F , FLAG immunoprecipitation of UV cross-linked ribonuclear protein complexes. Protein extracts from 293T cells, transfected with FLAG-CRDBP, were incubated with internally 32 P-labeled RNA transcripts of three fragments of the MITF mRNA coding region, MITF full-length mRNA, and the short 3′-UTR. Fragment 1 contains nucleotides 1–421 of the coding region, fragment 2 consists of nucleotides 422–841, and fragment 3 consists of nucleotides 842–1260 of the MITF coding region. Ribonuclear protein complexes were precipitated with anti-FLAG antibodies, analyzed on PAGE, and autoradiographed.

Techniques Used: Expressing, Transfection, shRNA, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Activity Assay, Electroporation, Staining, Immunoprecipitation, Incubation, Labeling, Polyacrylamide Gel Electrophoresis

CRD-BP inhibition reduces mitfa expression and pigmentation in zebrafish embryos. A , zebrafish embryos were injected with CRD-BP morpholino (5 or 10 ng) or standard control morpholino. Expression levels of the zebrafish MITF ortholog, mitfa , were assayed by real time quantitative PCR at 24 h post-fertilization and normalized to zebrafish α- tubulin . Morphant expression levels are presented relative to levels of expression in control embryos. B , representative zebrafish embryo at 48 h post-fertilization, injected with control or CRD-BP morpholino. CRD-BP morphants have less pigmentation and reduced numbers of pigment cells (melanophores) in 33 of 33 morphants (from seven independent experiments).
Figure Legend Snippet: CRD-BP inhibition reduces mitfa expression and pigmentation in zebrafish embryos. A , zebrafish embryos were injected with CRD-BP morpholino (5 or 10 ng) or standard control morpholino. Expression levels of the zebrafish MITF ortholog, mitfa , were assayed by real time quantitative PCR at 24 h post-fertilization and normalized to zebrafish α- tubulin . Morphant expression levels are presented relative to levels of expression in control embryos. B , representative zebrafish embryo at 48 h post-fertilization, injected with control or CRD-BP morpholino. CRD-BP morphants have less pigmentation and reduced numbers of pigment cells (melanophores) in 33 of 33 morphants (from seven independent experiments).

Techniques Used: Inhibition, Expressing, Injection, Real-time Polymerase Chain Reaction

The abundant MITF mRNA with short 3′-UTR is also regulated by miRNA. A , Dicer wt and Dicer Ex5/Ex5 HCT116 cells were co-transfected with Tet-off plasmid and p-BIG-MITF plasmid with short 3′-UTR. Transcription was stopped by treatment with doxycycline for the indicated durations. The stability of MITF transcripts was analyzed by measuring MITF mRNA levels with real time RT-PCR (normalized to GAPDH expression). B , p-BIG-MITF plasmid with short 3′-UTR was expressed in DLD1 cells, Dicer wt ( wt ), and Dicer Ex5/Ex5 ( ex5/ex5 ) under the control of the Tet-off system. The stability of MITF mRNA was analyzed as in A. C , stability of MITF transcript with short 3′-UTR expressed in RKO wt and RKO Dicer Ex5/Ex5 cells was analyzed as in A . All of the results are representative of three separate experiments and are expressed as the mean values ± S.D. ( error bars . D , sequence of the short 3′-UTR of MITF showing binding sites for miR-340 (in bold type ) and miR-548c-3p ( underlined ). E , the levels of endogenous MITF mRNA in 451Lu cells, transfected with the indicated miR-Sponge constructs, were estimated by real time RT-PCR after normalization with respect to GAPDH expression. The results are representative of three separate experiments and are expressed as percentages of control (SP-CXCR4) as the mean values ± S.D. ( error bars ). F , immunoblot analysis of MITF expression in the 451Lu cells transfected with the indicated miR-Sponge constructs ( upper panel ). The lower panel .
Figure Legend Snippet: The abundant MITF mRNA with short 3′-UTR is also regulated by miRNA. A , Dicer wt and Dicer Ex5/Ex5 HCT116 cells were co-transfected with Tet-off plasmid and p-BIG-MITF plasmid with short 3′-UTR. Transcription was stopped by treatment with doxycycline for the indicated durations. The stability of MITF transcripts was analyzed by measuring MITF mRNA levels with real time RT-PCR (normalized to GAPDH expression). B , p-BIG-MITF plasmid with short 3′-UTR was expressed in DLD1 cells, Dicer wt ( wt ), and Dicer Ex5/Ex5 ( ex5/ex5 ) under the control of the Tet-off system. The stability of MITF mRNA was analyzed as in A. C , stability of MITF transcript with short 3′-UTR expressed in RKO wt and RKO Dicer Ex5/Ex5 cells was analyzed as in A . All of the results are representative of three separate experiments and are expressed as the mean values ± S.D. ( error bars . D , sequence of the short 3′-UTR of MITF showing binding sites for miR-340 (in bold type ) and miR-548c-3p ( underlined ). E , the levels of endogenous MITF mRNA in 451Lu cells, transfected with the indicated miR-Sponge constructs, were estimated by real time RT-PCR after normalization with respect to GAPDH expression. The results are representative of three separate experiments and are expressed as percentages of control (SP-CXCR4) as the mean values ± S.D. ( error bars ). F , immunoblot analysis of MITF expression in the 451Lu cells transfected with the indicated miR-Sponge constructs ( upper panel ). The lower panel .

Techniques Used: Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Sequencing, Binding Assay, Construct

MITF mRNA is a target of miR-340. A , 451Lu cells were co-transfected with Tet-off plasmid, p-BIG-MITF plasmid with short 3′-UTR, and the indicated miR-Sponge construct. Transcription was stopped by treatment with doxycycline for the indicated durations. The stability of MITF transcripts was analyzed by measuring MITF mRNA levels with real time RT-PCR (normalized to GAPDH expression). B , 451Lu cells were co-transfected with Tet-off plasmid, p-BIGL-MITF-Short MITF 3′-UTR, and the indicated miR-Sponge construct. The turnover of chimeric Luciferase-MITF-3′-UTR transcripts was analyzed as in A. C , 451Lu cells were co-transfected with β-galactosidase-expressing plasmid, Tet-off plasmid, p-BIGL-MITF wt , or p-BIGL-MITF δmiR-340 and the indicated miR-Sponge constructs. After 24 h the luciferase activity was measured. The values represent luciferase activity normalized to β-galactosidase. D , 451Lu cells were co-transfected with Tet-off plasmid, p-BIG-MITF wt , or p-BIG-MITF δmiR-340 . The stability of MITF transcripts was analyzed as in A . All of the results are representative of three separate experiments and are expressed as the mean values ± S.D. ( error bars .
Figure Legend Snippet: MITF mRNA is a target of miR-340. A , 451Lu cells were co-transfected with Tet-off plasmid, p-BIG-MITF plasmid with short 3′-UTR, and the indicated miR-Sponge construct. Transcription was stopped by treatment with doxycycline for the indicated durations. The stability of MITF transcripts was analyzed by measuring MITF mRNA levels with real time RT-PCR (normalized to GAPDH expression). B , 451Lu cells were co-transfected with Tet-off plasmid, p-BIGL-MITF-Short MITF 3′-UTR, and the indicated miR-Sponge construct. The turnover of chimeric Luciferase-MITF-3′-UTR transcripts was analyzed as in A. C , 451Lu cells were co-transfected with β-galactosidase-expressing plasmid, Tet-off plasmid, p-BIGL-MITF wt , or p-BIGL-MITF δmiR-340 and the indicated miR-Sponge constructs. After 24 h the luciferase activity was measured. The values represent luciferase activity normalized to β-galactosidase. D , 451Lu cells were co-transfected with Tet-off plasmid, p-BIG-MITF wt , or p-BIG-MITF δmiR-340 . The stability of MITF transcripts was analyzed as in A . All of the results are representative of three separate experiments and are expressed as the mean values ± S.D. ( error bars .

Techniques Used: Transfection, Plasmid Preparation, Construct, Quantitative RT-PCR, Expressing, Luciferase, Activity Assay

36) Product Images from "HetR homodimer is a DNA-binding protein required for heterocyst differentiation, and the DNA-binding activity is inhibited by PatS"

Article Title: HetR homodimer is a DNA-binding protein required for heterocyst differentiation, and the DNA-binding activity is inhibited by PatS

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

doi: 10.1073/pnas.0400429101

Real-time PCR analysis of the expression of hetR . The wild-type cells were subjected to a nitrogen step-down with (squares) or without (circles) 1 μM PatS pentapeptide, and total RNA was isolated at the times indicated for real-time PCR. The expression of hetRc48a in C48 after nitrogen step-down is also shown (triangles). Each point is an average of four independent values, and all points are normalized against the maximum value of the hetR mRNA at 24 h after nitrogen step-down.
Figure Legend Snippet: Real-time PCR analysis of the expression of hetR . The wild-type cells were subjected to a nitrogen step-down with (squares) or without (circles) 1 μM PatS pentapeptide, and total RNA was isolated at the times indicated for real-time PCR. The expression of hetRc48a in C48 after nitrogen step-down is also shown (triangles). Each point is an average of four independent values, and all points are normalized against the maximum value of the hetR mRNA at 24 h after nitrogen step-down.

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

EMSA for DNA binding by HetR proteins. ( A ) Homodimerization of HetR is required for its DNA-binding activity. A DNA fragment from the hetR promoter (F hetR -1, lanes 1–11) and a fragment from the coding region of hetR (F hetR -3, lanes 12 and 13) were PCR-amplified and end-labeled with 32 P for EMSA. Lanes 1–4, HetR concentration-dependent effect on gel mobility. The concentrations (in μM) are shown above the gels. Lane 5, same as in lane 4 except that 2 mM DTT was included in the reaction solution. Lanes 6–8, concentration-dependent DNA binding by HetR S179N . Lanes 9–11, no mobility shift was detected with HetR C48A . Lanes 12 and 13, no gel shift was observed at HetR concentrations as shown when F hetR -3 was used for EMSA. ( B ) HetR binding to the fragment F hetR -2 from the hetR promoter. Lane 1, no HetR added; lanes 2–4, HetR, HetR S179N , and HetR C48A were added, respectively; lane 5, F hetR -3 was used with the addition of HetR. ( C ) HetR binding to the fragment from the hepA promoter. Lanes 1 and 2, F hepA -1; lanes 3 and 4, F hepA -2. Lanes 1 and 3, no addition of HetR; lane 2 and 4, with addition of HetR. ( D ) HetR binding to the fragments from the patS promoter. Lanes 1 and 2, F patS -1; lanes 3 and 4, F patS -2. Lanes 1 and 3, no addition of HetR; lanes 2 and 4, with addition of HetR. The concentrations of HetR proteins in B – D were all 1 μM.
Figure Legend Snippet: EMSA for DNA binding by HetR proteins. ( A ) Homodimerization of HetR is required for its DNA-binding activity. A DNA fragment from the hetR promoter (F hetR -1, lanes 1–11) and a fragment from the coding region of hetR (F hetR -3, lanes 12 and 13) were PCR-amplified and end-labeled with 32 P for EMSA. Lanes 1–4, HetR concentration-dependent effect on gel mobility. The concentrations (in μM) are shown above the gels. Lane 5, same as in lane 4 except that 2 mM DTT was included in the reaction solution. Lanes 6–8, concentration-dependent DNA binding by HetR S179N . Lanes 9–11, no mobility shift was detected with HetR C48A . Lanes 12 and 13, no gel shift was observed at HetR concentrations as shown when F hetR -3 was used for EMSA. ( B ) HetR binding to the fragment F hetR -2 from the hetR promoter. Lane 1, no HetR added; lanes 2–4, HetR, HetR S179N , and HetR C48A were added, respectively; lane 5, F hetR -3 was used with the addition of HetR. ( C ) HetR binding to the fragment from the hepA promoter. Lanes 1 and 2, F hepA -1; lanes 3 and 4, F hepA -2. Lanes 1 and 3, no addition of HetR; lane 2 and 4, with addition of HetR. ( D ) HetR binding to the fragments from the patS promoter. Lanes 1 and 2, F patS -1; lanes 3 and 4, F patS -2. Lanes 1 and 3, no addition of HetR; lanes 2 and 4, with addition of HetR. The concentrations of HetR proteins in B – D were all 1 μM.

Techniques Used: Binding Assay, Activity Assay, Polymerase Chain Reaction, Amplification, Labeling, Concentration Assay, Mobility Shift, Electrophoretic Mobility Shift Assay

The expression of patS depends on the HetR dimer. Total RNA was isolated from the cells of the wild-type strain (circles) and C48 (squares) at the times indicated after nitrogen step-down and used as templates for reverse transcription. The relative amount of the patS mRNA was analyzed by real-time PCR. Each point is an average of four independent values and is normalized against the maximum values of the patS mRNA in the wild-type strain at 24 h.
Figure Legend Snippet: The expression of patS depends on the HetR dimer. Total RNA was isolated from the cells of the wild-type strain (circles) and C48 (squares) at the times indicated after nitrogen step-down and used as templates for reverse transcription. The relative amount of the patS mRNA was analyzed by real-time PCR. Each point is an average of four independent values and is normalized against the maximum values of the patS mRNA in the wild-type strain at 24 h.

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

37) Product Images from "Attenuated phenotypes and analysis of a herpes simplex virus 1 strain with partial deletion of the UL7, UL41 and LAT genes"

Article Title: Attenuated phenotypes and analysis of a herpes simplex virus 1 strain with partial deletion of the UL7, UL41 and LAT genes

Journal: Virologica Sinica

doi: 10.1007/s12250-017-3947-1

Design of the HSV-1 M1, M2 and M3 strains and verification of their mutant genes. (A) Flowchart of the design and construction of the M1, M2 and M3 viruses. (B) Identification of the mutated UL7 gene in the purified M1 virus. The UL7 mutation (225–254, 30-bp deletion) was identified via PCR using UL7-sense and UL7-antisense primers. (C) Identification of the mutated UL41 gene in the purified M2 virus. The UL41 mutation (375–433, 59-bp deletion) was identified via PCR using the UL41-F and UL41-R primers. (D) Identification of the mutated LAT gene (937–1074, 138-bp deletion) in the purified M3 virus. The LAT mutation was identified via PCR using the LAT-F and LAT-R primers.
Figure Legend Snippet: Design of the HSV-1 M1, M2 and M3 strains and verification of their mutant genes. (A) Flowchart of the design and construction of the M1, M2 and M3 viruses. (B) Identification of the mutated UL7 gene in the purified M1 virus. The UL7 mutation (225–254, 30-bp deletion) was identified via PCR using UL7-sense and UL7-antisense primers. (C) Identification of the mutated UL41 gene in the purified M2 virus. The UL41 mutation (375–433, 59-bp deletion) was identified via PCR using the UL41-F and UL41-R primers. (D) Identification of the mutated LAT gene (937–1074, 138-bp deletion) in the purified M3 virus. The LAT mutation was identified via PCR using the LAT-F and LAT-R primers.

Techniques Used: Mutagenesis, Purification, Polymerase Chain Reaction

The three mutants showed a lower capacity for latent infection of the nervous system of mice compared with the WT strain. Levels of (A) RS1 and (B) LAT expression in the trigeminal nerve of mice challenged with WT, M1, M2, M3 or PBS at 14 or 28 d.p.i., as determined by relative RT-PCR. The graphic indicates the fold change in the RNA levels detected in virus-infected mice compared with PBS-injected mice. The RNA levels of the housekeeping gene GAPDH were used to normalize the target RNA levels detected in the mouse tissues. Relative quantification was performed using the comparative Ct method (∆∆Ct), with the RNA from the PBS-infected mice being used for calibration. (C) Positive LAT RNA expression, as determined using chromogenic in situ hybridization (× 200 magnification), is highlighted with black arrows. Brown-dot positivity can be observed for the WT-, M1- and M2-infected mice, with no positive signal for the M3- or PBS-infected mice.
Figure Legend Snippet: The three mutants showed a lower capacity for latent infection of the nervous system of mice compared with the WT strain. Levels of (A) RS1 and (B) LAT expression in the trigeminal nerve of mice challenged with WT, M1, M2, M3 or PBS at 14 or 28 d.p.i., as determined by relative RT-PCR. The graphic indicates the fold change in the RNA levels detected in virus-infected mice compared with PBS-injected mice. The RNA levels of the housekeeping gene GAPDH were used to normalize the target RNA levels detected in the mouse tissues. Relative quantification was performed using the comparative Ct method (∆∆Ct), with the RNA from the PBS-infected mice being used for calibration. (C) Positive LAT RNA expression, as determined using chromogenic in situ hybridization (× 200 magnification), is highlighted with black arrows. Brown-dot positivity can be observed for the WT-, M1- and M2-infected mice, with no positive signal for the M3- or PBS-infected mice.

Techniques Used: Infection, Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, RNA Expression, Chromogenic In Situ Hybridization

38) Product Images from "Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts"

Article Title: Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

Journal: Virology

doi: 10.1016/j.virol.2011.12.009

Establishment of Dulac-CD163 porcine kidney cells as a cell line stably expressing porcine CD163. (A) RT-PCR for porcine CD163 mRNA in Dulac-CD163. Total cellular RNA was extracted and treated with DNase I (1 unit/μg of RNA) and PCR was performed after RT (lanes 4 and 6) or without RT (lanes 3 and 5) reactions. Dulac, parental cells; Dulac-CD163, porcine CD163 gene transformed cells. Lane 2, CD163 positive control. (B) Western blot for porcine CD163. Cell lysates were prepared from Dulac-CD163 and resolved by 7.5% SDS-PAGE, followed by blotting to the PVDF membrane and probing with porcine CD163-specific MAb. (C) Immunofluorescence. (D) Permissiveness of Dulac-CD163 cells for PRRSV. Expression of porcine CD163 rendered non-permissive Dulac cells permissive for PRRSV infection. Cells were infected with PRRSV-GFP and the GFP expression was observed by fluorescent microscopy (panel d). PRRSV-specific cytopathic effect was also evident in Dulac-CD163 cells (panel c).
Figure Legend Snippet: Establishment of Dulac-CD163 porcine kidney cells as a cell line stably expressing porcine CD163. (A) RT-PCR for porcine CD163 mRNA in Dulac-CD163. Total cellular RNA was extracted and treated with DNase I (1 unit/μg of RNA) and PCR was performed after RT (lanes 4 and 6) or without RT (lanes 3 and 5) reactions. Dulac, parental cells; Dulac-CD163, porcine CD163 gene transformed cells. Lane 2, CD163 positive control. (B) Western blot for porcine CD163. Cell lysates were prepared from Dulac-CD163 and resolved by 7.5% SDS-PAGE, followed by blotting to the PVDF membrane and probing with porcine CD163-specific MAb. (C) Immunofluorescence. (D) Permissiveness of Dulac-CD163 cells for PRRSV. Expression of porcine CD163 rendered non-permissive Dulac cells permissive for PRRSV infection. Cells were infected with PRRSV-GFP and the GFP expression was observed by fluorescent microscopy (panel d). PRRSV-specific cytopathic effect was also evident in Dulac-CD163 cells (panel c).

Techniques Used: Stable Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Transformation Assay, Positive Control, Western Blot, SDS Page, Immunofluorescence, Infection, Microscopy

39) Product Images from "Toward the development of a single-round infection assay based on EGFP reporting for anti-HIV-1 drug discovery"

Article Title: Toward the development of a single-round infection assay based on EGFP reporting for anti-HIV-1 drug discovery

Journal: Reports of Biochemistry & Molecular Biology

doi:

. pmzNL4-3/GFP plasmid construction and analysis. Agarose gel electrophoresis of three PCR products resulting from sequential amplification of gfp gene that led to the construction of the 809 bp final product (A). The scheme of the amplified gfp fragment
Figure Legend Snippet: . pmzNL4-3/GFP plasmid construction and analysis. Agarose gel electrophoresis of three PCR products resulting from sequential amplification of gfp gene that led to the construction of the 809 bp final product (A). The scheme of the amplified gfp fragment

Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification

40) Product Images from "A Conserved E2F6-Binding Element in Murine Meiosis-Specific Gene Promoters 1"

Article Title: A Conserved E2F6-Binding Element in Murine Meiosis-Specific Gene Promoters 1

Journal: Biology of Reproduction

doi: 10.1095/biolreprod.108.067645

E2F6 binds to meiosis-specific genes. ChIP of endogenous E2F6 binding activity in wt R1 ES cells. ChIP was performed using mouse-anti-E2F6 antibody and analyzed by semiquantitative PCR. Input and IgG are shown as controls. E2F6 binding activity on: (
Figure Legend Snippet: E2F6 binds to meiosis-specific genes. ChIP of endogenous E2F6 binding activity in wt R1 ES cells. ChIP was performed using mouse-anti-E2F6 antibody and analyzed by semiquantitative PCR. Input and IgG are shown as controls. E2F6 binding activity on: (

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Activity Assay, Polymerase Chain Reaction

Meiosis-specific genes are repressed by E2F6. Semiquantitative RT-PCR analysis of meiosis-specific gene expression in R1, HA-E2F6, and HA-ΔC-E2F6 ES cells. A ) Genes derepressed in E2f6−/− MEFs. B ) Other meiosis-specific genes.
Figure Legend Snippet: Meiosis-specific genes are repressed by E2F6. Semiquantitative RT-PCR analysis of meiosis-specific gene expression in R1, HA-E2F6, and HA-ΔC-E2F6 ES cells. A ) Genes derepressed in E2f6−/− MEFs. B ) Other meiosis-specific genes.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

Slc25a31 transcription is repressed by stable E2F6 overexpression in ES cells and is derepressed in E2f6 −/− MEFs. A ) Semiquantitative RT-PCR analysis of Slc25a31 and Actb expression in parental R1, HA-E2F6 (HA), and HA-ΔC-E2F6
Figure Legend Snippet: Slc25a31 transcription is repressed by stable E2F6 overexpression in ES cells and is derepressed in E2f6 −/− MEFs. A ) Semiquantitative RT-PCR analysis of Slc25a31 and Actb expression in parental R1, HA-E2F6 (HA), and HA-ΔC-E2F6

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

Limited meiosis-specific genes are derepressed in E2f6−/− MEFs. Semiquantitative RT-PCR analysis of meiosis-specific gene expression in wt testis from 6-wk-old mice, R1 ES cells, wt MEFs, and E2f6−/− MEFs. A ) E2f6 and genes
Figure Legend Snippet: Limited meiosis-specific genes are derepressed in E2f6−/− MEFs. Semiquantitative RT-PCR analysis of meiosis-specific gene expression in wt testis from 6-wk-old mice, R1 ES cells, wt MEFs, and E2f6−/− MEFs. A ) E2f6 and genes

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

Related Articles

Real-time Polymerase Chain Reaction:

Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice
Article Snippet: .. For quantitative PCR analysis, genomic DNA was PCR-amplified and analyzed using SYBR Green PCR Master Mix (Takara) and a 7900HT Sequence Detection System (Applied Biosystems). ..

Transfection:

Article Title: The positive feedback loop between ILF3 and lncRNA ILF3-AS1 promotes melanoma proliferation, migration, and invasion
Article Snippet: .. Plasmids’ construction and transfection The complementary DNA encoding ILF3 was PCR-amplified by the Ex Taq DNA polymerase hot-start version (Takara) and subcloned into the Nhe I and Kpn I sites of pcDNA3.1(+) plasmid (Thermo Fisher Scientific). ..

Clone Assay:

Article Title: A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import [OPEN]
Article Snippet: .. For expression of the long form (amino acids 417–946) (FtsHi1C_L) and the short form (amino acids 417–684) (FtsHi1C_S) of the C-terminal domain of Arabidopsis FtsHi1 as a fusion protein with GST (from Schistosoma japonicum ), the corresponding cDNA fragments were PCR-amplified and cloned into pCold-GST vector (Takara) and modified by KOD One PCR enzyme (Toyobo). ..

SYBR Green Assay:

Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice
Article Snippet: .. For quantitative PCR analysis, genomic DNA was PCR-amplified and analyzed using SYBR Green PCR Master Mix (Takara) and a 7900HT Sequence Detection System (Applied Biosystems). ..

Sequencing:

Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice
Article Snippet: .. For quantitative PCR analysis, genomic DNA was PCR-amplified and analyzed using SYBR Green PCR Master Mix (Takara) and a 7900HT Sequence Detection System (Applied Biosystems). ..

Polymerase Chain Reaction:

Article Title: ARHI is a novel epigenetic silenced tumor suppressor in sporadic pheochromocytoma
Article Snippet: .. For each reaction, 30 ng of tumor DNA was PCR-amplified for 35 cycles using HotStar Taq Polymerase (Takara). .. Sequences were analyzed by alignment to the UCSC sequence using the DNAMAN6.0 tool (LynnonBiosoft).

Article Title: The positive feedback loop between ILF3 and lncRNA ILF3-AS1 promotes melanoma proliferation, migration, and invasion
Article Snippet: .. Plasmids’ construction and transfection The complementary DNA encoding ILF3 was PCR-amplified by the Ex Taq DNA polymerase hot-start version (Takara) and subcloned into the Nhe I and Kpn I sites of pcDNA3.1(+) plasmid (Thermo Fisher Scientific). ..

Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice
Article Snippet: .. For quantitative PCR analysis, genomic DNA was PCR-amplified and analyzed using SYBR Green PCR Master Mix (Takara) and a 7900HT Sequence Detection System (Applied Biosystems). ..

Article Title: A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import [OPEN]
Article Snippet: .. For expression of the long form (amino acids 417–946) (FtsHi1C_L) and the short form (amino acids 417–684) (FtsHi1C_S) of the C-terminal domain of Arabidopsis FtsHi1 as a fusion protein with GST (from Schistosoma japonicum ), the corresponding cDNA fragments were PCR-amplified and cloned into pCold-GST vector (Takara) and modified by KOD One PCR enzyme (Toyobo). ..

Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription
Article Snippet: .. The genomic region surrounding the CRISPR target site of the UL7 gene was PCR-amplified using PrimeSTAR DNA polymerase (TAKARA, Dalian, Liaoning, China) with the primers UL7-sense and UL7-antisense. .. The PCR products were purified using the Universal DNA Purification Kit (Tiangen, Beijing, China).

Article Title: Changes in DNA methylation assessed by genomic bisulfite sequencing suggest a role for DNA methylation in cotton fruiting branch development
Article Snippet: .. The bisulfite-converted DNA was PCR-amplified with ExTaq DNA Polymerase (Takara, Dalian, Shandong, China). ..

Article Title: The FTS-Hook-FHIP (FHF) complex interacts with AP-4 to mediate perinuclear distribution of AP-4 and its cargo ATG9A
Article Snippet: .. The human Hook1, Hook2, Hook3, and FTS cDNAs were PCR-amplified and subcloned into the pGBT9 and pGADT7 vectors (Clontech). ..

Article Title: Pseudouridine formation in archaeal RNAs: The case of Haloferax volcanii
Article Snippet: .. Briefly, regions of ∼600 bp upstream of and downstream from the gene to be deleted, along with a small portion of the gene at both ends to avoid affecting neighboring genes, were PCR-amplified from H. volcanii genomic DNA and recombined between the Xho I and EcoR I sites of pTA131 using InFusion (Clontech); 0.2 μL of the reaction mixture was transformed to TOP10 chemically competent cells (Invitrogen) and plated onto LB plus ampicillin (100 μg/mL), isopropyl β-D-1-thiogalactopyranoside (0.2 mM), and bromo-chloro-indolyl-galactopyranoside (40 μg/mL). .. Clones were screened by PCR using M13 primers (which anneal on either side of the multiple cloning site of pTA131) using 5′ Master Mix (Eppendorf) as per the directions, and confirmed by sequencing using the UF Sequencing facility.

Expressing:

Article Title: A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import [OPEN]
Article Snippet: .. For expression of the long form (amino acids 417–946) (FtsHi1C_L) and the short form (amino acids 417–684) (FtsHi1C_S) of the C-terminal domain of Arabidopsis FtsHi1 as a fusion protein with GST (from Schistosoma japonicum ), the corresponding cDNA fragments were PCR-amplified and cloned into pCold-GST vector (Takara) and modified by KOD One PCR enzyme (Toyobo). ..

CRISPR:

Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription
Article Snippet: .. The genomic region surrounding the CRISPR target site of the UL7 gene was PCR-amplified using PrimeSTAR DNA polymerase (TAKARA, Dalian, Liaoning, China) with the primers UL7-sense and UL7-antisense. .. The PCR products were purified using the Universal DNA Purification Kit (Tiangen, Beijing, China).

Modification:

Article Title: A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import [OPEN]
Article Snippet: .. For expression of the long form (amino acids 417–946) (FtsHi1C_L) and the short form (amino acids 417–684) (FtsHi1C_S) of the C-terminal domain of Arabidopsis FtsHi1 as a fusion protein with GST (from Schistosoma japonicum ), the corresponding cDNA fragments were PCR-amplified and cloned into pCold-GST vector (Takara) and modified by KOD One PCR enzyme (Toyobo). ..

Transformation Assay:

Article Title: Pseudouridine formation in archaeal RNAs: The case of Haloferax volcanii
Article Snippet: .. Briefly, regions of ∼600 bp upstream of and downstream from the gene to be deleted, along with a small portion of the gene at both ends to avoid affecting neighboring genes, were PCR-amplified from H. volcanii genomic DNA and recombined between the Xho I and EcoR I sites of pTA131 using InFusion (Clontech); 0.2 μL of the reaction mixture was transformed to TOP10 chemically competent cells (Invitrogen) and plated onto LB plus ampicillin (100 μg/mL), isopropyl β-D-1-thiogalactopyranoside (0.2 mM), and bromo-chloro-indolyl-galactopyranoside (40 μg/mL). .. Clones were screened by PCR using M13 primers (which anneal on either side of the multiple cloning site of pTA131) using 5′ Master Mix (Eppendorf) as per the directions, and confirmed by sequencing using the UF Sequencing facility.

Plasmid Preparation:

Article Title: The positive feedback loop between ILF3 and lncRNA ILF3-AS1 promotes melanoma proliferation, migration, and invasion
Article Snippet: .. Plasmids’ construction and transfection The complementary DNA encoding ILF3 was PCR-amplified by the Ex Taq DNA polymerase hot-start version (Takara) and subcloned into the Nhe I and Kpn I sites of pcDNA3.1(+) plasmid (Thermo Fisher Scientific). ..

Article Title: A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import [OPEN]
Article Snippet: .. For expression of the long form (amino acids 417–946) (FtsHi1C_L) and the short form (amino acids 417–684) (FtsHi1C_S) of the C-terminal domain of Arabidopsis FtsHi1 as a fusion protein with GST (from Schistosoma japonicum ), the corresponding cDNA fragments were PCR-amplified and cloned into pCold-GST vector (Takara) and modified by KOD One PCR enzyme (Toyobo). ..

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    TaKaRa pcr amplified fragments
    Colocalization of endogenous proteins with the H/ACA RNA transcription site. (A) Double fluorescence of a fixed and permeabilized <t>E3</t> cell after induction of the transgene and immunostained for endogenous NAP57 (panel 1) and for the H/ACA RNA transcription site by FISH with the probe to intron 1 (panel 2). Panel 3 shows a merge of the two in pseudocolor. Panel 4 depicts the corresponding phase-contrast image outlining, with a box indicating the area containing the transcription site, which is enlarged in the insets. (B–D) Same as in A, except immunostained for the endogenous proteins indicated on the left, and the transcription site was identified by the fluorescence of transfected MS2-RFP (panel 2). (E) Bar diagram of the percentage of cells, with the indicated proteins observed at the site of H/ACA RNA transcription. The following numbers of cells with a transcription site (in parentheses) were counted for each protein: NAP57 (150); NOP10 (101); NHP2 (101); GAR1 (104); and NAF1 (159). Questionable colocalizations were discounted. (F) ChIPs of the GFP-fusion constructs indicated on top after their transient transfection into E3 cells and induction of the transgene. Immunoprecipitations were performed with GFP antibodies. Ethidium bromide stain of DNA amplified by <t>PCR,</t> with primers to the regions of the transgene indicated on the right and separated on agarose gels. Amplifications of the input (I; odd lanes) and the ChIPs (even lanes) are shown. (G) Same as in B and C, except stained for the Cajal body marker protein coilin.
    Pcr Amplified Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pcr amplified myc
    <t>MYC</t> regulates the invasion of JEG-3 cells. JEG-3 cells were transfected with MYC overexpression construct, MYC siRNA, or the corresponding empty vectors. 48 h later, the levels of a MYC mRNA and b MYC protein in the cells were analyzed by real-time <t>PCR</t> and Western blot analysis respectively, using β-actin as the internal control. c Matrigel-based Transwell invasion assay was set up 24 h after transfection, and the number of invading cells was counted 24 h later at 200× magnification (scale bars = 100 μm). This figure shows the representative images from three independent experiments, and the values are expressed as the mean ± standard deviation. * p
    Pcr Amplified Myc, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Colocalization of endogenous proteins with the H/ACA RNA transcription site. (A) Double fluorescence of a fixed and permeabilized E3 cell after induction of the transgene and immunostained for endogenous NAP57 (panel 1) and for the H/ACA RNA transcription site by FISH with the probe to intron 1 (panel 2). Panel 3 shows a merge of the two in pseudocolor. Panel 4 depicts the corresponding phase-contrast image outlining, with a box indicating the area containing the transcription site, which is enlarged in the insets. (B–D) Same as in A, except immunostained for the endogenous proteins indicated on the left, and the transcription site was identified by the fluorescence of transfected MS2-RFP (panel 2). (E) Bar diagram of the percentage of cells, with the indicated proteins observed at the site of H/ACA RNA transcription. The following numbers of cells with a transcription site (in parentheses) were counted for each protein: NAP57 (150); NOP10 (101); NHP2 (101); GAR1 (104); and NAF1 (159). Questionable colocalizations were discounted. (F) ChIPs of the GFP-fusion constructs indicated on top after their transient transfection into E3 cells and induction of the transgene. Immunoprecipitations were performed with GFP antibodies. Ethidium bromide stain of DNA amplified by PCR, with primers to the regions of the transgene indicated on the right and separated on agarose gels. Amplifications of the input (I; odd lanes) and the ChIPs (even lanes) are shown. (G) Same as in B and C, except stained for the Cajal body marker protein coilin.

    Journal: The Journal of Cell Biology

    Article Title: Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells

    doi: 10.1083/jcb.200601105

    Figure Lengend Snippet: Colocalization of endogenous proteins with the H/ACA RNA transcription site. (A) Double fluorescence of a fixed and permeabilized E3 cell after induction of the transgene and immunostained for endogenous NAP57 (panel 1) and for the H/ACA RNA transcription site by FISH with the probe to intron 1 (panel 2). Panel 3 shows a merge of the two in pseudocolor. Panel 4 depicts the corresponding phase-contrast image outlining, with a box indicating the area containing the transcription site, which is enlarged in the insets. (B–D) Same as in A, except immunostained for the endogenous proteins indicated on the left, and the transcription site was identified by the fluorescence of transfected MS2-RFP (panel 2). (E) Bar diagram of the percentage of cells, with the indicated proteins observed at the site of H/ACA RNA transcription. The following numbers of cells with a transcription site (in parentheses) were counted for each protein: NAP57 (150); NOP10 (101); NHP2 (101); GAR1 (104); and NAF1 (159). Questionable colocalizations were discounted. (F) ChIPs of the GFP-fusion constructs indicated on top after their transient transfection into E3 cells and induction of the transgene. Immunoprecipitations were performed with GFP antibodies. Ethidium bromide stain of DNA amplified by PCR, with primers to the regions of the transgene indicated on the right and separated on agarose gels. Amplifications of the input (I; odd lanes) and the ChIPs (even lanes) are shown. (G) Same as in B and C, except stained for the Cajal body marker protein coilin.

    Article Snippet: The pTet-globin-snoE3-CFP-18MS2-2 (for E3 cells) and pTet-globin-CFP-18MS2-2 (for E3-minus cells) were generated by sequentially inserting the following, mostly PCR-amplified fragments, into pCMV-globin ( ): CFP with a COOH-terminal SKL tripeptide from pECFP-N1 (CLONTECH Laboratories, Inc.); 18 MS2 repeats from pSL-24X ( ; six repeats were lost because of recombination in Escherichia coli ); Tet promoter from pTRE-2 (CLONTECH Laboratories, Inc.) replacing the CMV promoter; and, after excision of the neomycin-resistance gene, rat snoRNA E3 from pTM105 ( ).

    Techniques: Fluorescence, Fluorescence In Situ Hybridization, Transfection, Construct, Staining, Amplification, Polymerase Chain Reaction, Marker

    Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative PCR analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic DNA (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.

    Journal: PLoS ONE

    Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice

    doi: 10.1371/journal.pone.0014391

    Figure Lengend Snippet: Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative PCR analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic DNA (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.

    Article Snippet: For quantitative PCR analysis, genomic DNA was PCR-amplified and analyzed using SYBR Green PCR Master Mix (Takara) and a 7900HT Sequence Detection System (Applied Biosystems).

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

    Identification of the tgε26 transgenic integration site. (A) Physical map of the genomic region between D17mit26-mit80-1 and D17mit 26-mit80-24. a–q: probes for Southern blot analysis. Details of these probes are listed in Supplementary 2 . 1–7: regions of quantitative genomic PCR analysis. S: SalI, E: EcoRI, A: AseI, K: KpnI, Hp: HpaI, Sc: SacI. (B–D) PFGE Southern blot analysis of SalI-digested WT and tgε26 +/+ genomic DNA using the indicated probes. (E) Sequence of Sstr5 -sequence-containing tgε26 +/+ genomic clones that also contained hCD3ε sequence (left). Physical map of the transgenic integration site at the Sstr5 locus (middle). H: HindIII. i: probe for Southern blot analysis. TS1F, TS1R, and CD3e9130F: primers for genomic PCR analysis. Genomic PCR analysis using TS1F/TS1R primers (lanes 1 and 2) and CDe9130F/TS1R primers (lanes 3 and 4) (right). (F, G) Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (H) Physical map of the transgenic integration site at the Metrn locus (left). N: NcoI. q: probe for Southern blot analysis. m7900, m8100, and CD3e23010F: primers for genomic PCR analysis. i-1R, i-1F, i-2R, and i-2F: primers for inverse and nested PCR amplification. Sequence of tgε26 +/+ genomic clones containing both Metrn and hCD3ε sequences (middle). Genomic PCR analysis using m7900/m8100 primers (lanes 1 and 2) and m7900/CD3e23010F primers (lanes 3 and 4) (right). (I, J) PFGE Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (K) Quantitative genomic PCR analysis of WT (open bars) and tgε26 +/+ (black bars) genomic DNA. Primers 1–7 are shown in (A). ct-1 and ct-2 are the genomic regions from chromosome 6 used as controls. Primer sequences are shown in Supplementary Table S3 . Data were normalized to ct-1 signals. Means and standard errors of 3 independent measurements are shown. (L) Configuration of the tgε26 allele.

    Journal: PLoS ONE

    Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice

    doi: 10.1371/journal.pone.0014391

    Figure Lengend Snippet: Identification of the tgε26 transgenic integration site. (A) Physical map of the genomic region between D17mit26-mit80-1 and D17mit 26-mit80-24. a–q: probes for Southern blot analysis. Details of these probes are listed in Supplementary 2 . 1–7: regions of quantitative genomic PCR analysis. S: SalI, E: EcoRI, A: AseI, K: KpnI, Hp: HpaI, Sc: SacI. (B–D) PFGE Southern blot analysis of SalI-digested WT and tgε26 +/+ genomic DNA using the indicated probes. (E) Sequence of Sstr5 -sequence-containing tgε26 +/+ genomic clones that also contained hCD3ε sequence (left). Physical map of the transgenic integration site at the Sstr5 locus (middle). H: HindIII. i: probe for Southern blot analysis. TS1F, TS1R, and CD3e9130F: primers for genomic PCR analysis. Genomic PCR analysis using TS1F/TS1R primers (lanes 1 and 2) and CDe9130F/TS1R primers (lanes 3 and 4) (right). (F, G) Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (H) Physical map of the transgenic integration site at the Metrn locus (left). N: NcoI. q: probe for Southern blot analysis. m7900, m8100, and CD3e23010F: primers for genomic PCR analysis. i-1R, i-1F, i-2R, and i-2F: primers for inverse and nested PCR amplification. Sequence of tgε26 +/+ genomic clones containing both Metrn and hCD3ε sequences (middle). Genomic PCR analysis using m7900/m8100 primers (lanes 1 and 2) and m7900/CD3e23010F primers (lanes 3 and 4) (right). (I, J) PFGE Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (K) Quantitative genomic PCR analysis of WT (open bars) and tgε26 +/+ (black bars) genomic DNA. Primers 1–7 are shown in (A). ct-1 and ct-2 are the genomic regions from chromosome 6 used as controls. Primer sequences are shown in Supplementary Table S3 . Data were normalized to ct-1 signals. Means and standard errors of 3 independent measurements are shown. (L) Configuration of the tgε26 allele.

    Article Snippet: For quantitative PCR analysis, genomic DNA was PCR-amplified and analyzed using SYBR Green PCR Master Mix (Takara) and a 7900HT Sequence Detection System (Applied Biosystems).

    Techniques: Transgenic Assay, Southern Blot, Polymerase Chain Reaction, Sequencing, Clone Assay, Nested PCR, Amplification

    The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P

    Journal: Virology Journal

    Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription

    doi: 10.1186/s12985-016-0600-9

    Figure Lengend Snippet: The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P

    Article Snippet: The genomic region surrounding the CRISPR target site of the UL7 gene was PCR-amplified using PrimeSTAR DNA polymerase (TAKARA, Dalian, Liaoning, China) with the primers UL7-sense and UL7-antisense.

    Techniques: Infection, Mouse Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Injection

    UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P

    Journal: Virology Journal

    Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription

    doi: 10.1186/s12985-016-0600-9

    Figure Lengend Snippet: UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P

    Article Snippet: The genomic region surrounding the CRISPR target site of the UL7 gene was PCR-amplified using PrimeSTAR DNA polymerase (TAKARA, Dalian, Liaoning, China) with the primers UL7-sense and UL7-antisense.

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

    Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box

    Journal: Virology Journal

    Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription

    doi: 10.1186/s12985-016-0600-9

    Figure Lengend Snippet: Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box

    Article Snippet: The genomic region surrounding the CRISPR target site of the UL7 gene was PCR-amplified using PrimeSTAR DNA polymerase (TAKARA, Dalian, Liaoning, China) with the primers UL7-sense and UL7-antisense.

    Techniques: Mutagenesis, Labeling, Amplification, Expressing, Infection, Polymerase Chain Reaction

    MYC regulates the invasion of JEG-3 cells. JEG-3 cells were transfected with MYC overexpression construct, MYC siRNA, or the corresponding empty vectors. 48 h later, the levels of a MYC mRNA and b MYC protein in the cells were analyzed by real-time PCR and Western blot analysis respectively, using β-actin as the internal control. c Matrigel-based Transwell invasion assay was set up 24 h after transfection, and the number of invading cells was counted 24 h later at 200× magnification (scale bars = 100 μm). This figure shows the representative images from three independent experiments, and the values are expressed as the mean ± standard deviation. * p

    Journal: BMC Cell Biology

    Article Title: MicroRNA-34a inhibits human trophoblast cell invasion by targeting MYC

    doi: 10.1186/s12860-015-0068-2

    Figure Lengend Snippet: MYC regulates the invasion of JEG-3 cells. JEG-3 cells were transfected with MYC overexpression construct, MYC siRNA, or the corresponding empty vectors. 48 h later, the levels of a MYC mRNA and b MYC protein in the cells were analyzed by real-time PCR and Western blot analysis respectively, using β-actin as the internal control. c Matrigel-based Transwell invasion assay was set up 24 h after transfection, and the number of invading cells was counted 24 h later at 200× magnification (scale bars = 100 μm). This figure shows the representative images from three independent experiments, and the values are expressed as the mean ± standard deviation. * p

    Article Snippet: PCR-amplified MYC -coding sequences were cloned into pcDNA3.1 vectors (TaKaRa Clontech) to generate pcDNA3.1-MYC .

    Techniques: Transfection, Over Expression, Construct, Real-time Polymerase Chain Reaction, Western Blot, Transwell Invasion Assay, Standard Deviation