kpni  (Roche)


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    Structured Review

    Roche kpni
    Creation of two PF11_0394/GFP clonal parasite populations for protein trafficking studies . A ) Transfection schematic demonstrating how the PF11_0394/GFP construct was created using the pPM2GT vector (obtained from MR4) and successfully incorporated into the genome of the parasite via homologous recombination technology [ 19 ]. Expression of GFP is driven by the endogenous promoter of PF11_0394 . Generation of two PF11_0394/GFP clonal parasite populations were verified by both PCR analysis ( B ) and Southern blot analysis using digoxigenin (DIG) technology coupled with autoradiography ( C ). Arrows indicate the predicted integration products of 10,454 base pairs and 6,602 base pairs using a PF11_0394 specific probe after restriction digestion with SapI and <t>KpnI.</t> The product indicated by an asterisk is of unknown origin and is likely a rearrangement of the plasmid that occurred after transfection. The drug cassette within the vector used for positive selection is human dihydrofolate reductase (hDHFR). GFP = Green fluorescent protein, Wt = Wild-type parasite genomic <t>DNA,</t> Cl = Clonal PF11_0494/GFP parasite genomic DNA, and Ep = PF11_0394/GFP plasmid DNA representing the episome. The arrows on the transfection schematic represent primers used for PCR analysis.
    Kpni, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites"

    Article Title: Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-11-80

    Creation of two PF11_0394/GFP clonal parasite populations for protein trafficking studies . A ) Transfection schematic demonstrating how the PF11_0394/GFP construct was created using the pPM2GT vector (obtained from MR4) and successfully incorporated into the genome of the parasite via homologous recombination technology [ 19 ]. Expression of GFP is driven by the endogenous promoter of PF11_0394 . Generation of two PF11_0394/GFP clonal parasite populations were verified by both PCR analysis ( B ) and Southern blot analysis using digoxigenin (DIG) technology coupled with autoradiography ( C ). Arrows indicate the predicted integration products of 10,454 base pairs and 6,602 base pairs using a PF11_0394 specific probe after restriction digestion with SapI and KpnI. The product indicated by an asterisk is of unknown origin and is likely a rearrangement of the plasmid that occurred after transfection. The drug cassette within the vector used for positive selection is human dihydrofolate reductase (hDHFR). GFP = Green fluorescent protein, Wt = Wild-type parasite genomic DNA, Cl = Clonal PF11_0494/GFP parasite genomic DNA, and Ep = PF11_0394/GFP plasmid DNA representing the episome. The arrows on the transfection schematic represent primers used for PCR analysis.
    Figure Legend Snippet: Creation of two PF11_0394/GFP clonal parasite populations for protein trafficking studies . A ) Transfection schematic demonstrating how the PF11_0394/GFP construct was created using the pPM2GT vector (obtained from MR4) and successfully incorporated into the genome of the parasite via homologous recombination technology [ 19 ]. Expression of GFP is driven by the endogenous promoter of PF11_0394 . Generation of two PF11_0394/GFP clonal parasite populations were verified by both PCR analysis ( B ) and Southern blot analysis using digoxigenin (DIG) technology coupled with autoradiography ( C ). Arrows indicate the predicted integration products of 10,454 base pairs and 6,602 base pairs using a PF11_0394 specific probe after restriction digestion with SapI and KpnI. The product indicated by an asterisk is of unknown origin and is likely a rearrangement of the plasmid that occurred after transfection. The drug cassette within the vector used for positive selection is human dihydrofolate reductase (hDHFR). GFP = Green fluorescent protein, Wt = Wild-type parasite genomic DNA, Cl = Clonal PF11_0494/GFP parasite genomic DNA, and Ep = PF11_0394/GFP plasmid DNA representing the episome. The arrows on the transfection schematic represent primers used for PCR analysis.

    Techniques Used: Transfection, Construct, Plasmid Preparation, Homologous Recombination, Expressing, Polymerase Chain Reaction, Southern Blot, Autoradiography, Selection

    2) Product Images from "Dominance of HIV-1 Subtype CRF01_AE in Sexually Acquired Cases Leads to a New Epidemic in Yunnan Province of China"

    Article Title: Dominance of HIV-1 Subtype CRF01_AE in Sexually Acquired Cases Leads to a New Epidemic in Yunnan Province of China

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0030443

    Phylogenetic Neighbor-Joining Tree for HIV-1 p17 Sequences Obtained from All 16 Prefectures of Yunnan Individual sequences are color coded, with the colors corresponding to those of original geographic sites on the map of Yunnan ( Figure 1 ). The horizontal branch was drawn in accordance with their relative genetic distances. The vertical lines are present purely for clarity of the tree presentation. The bootstrap values of 1,000 replicates are labeled on the major branches. The reference sequences for classifying HIV-1 genotypes were included and were originally obtained from the NIH/NIAID–funded HIV Databases.
    Figure Legend Snippet: Phylogenetic Neighbor-Joining Tree for HIV-1 p17 Sequences Obtained from All 16 Prefectures of Yunnan Individual sequences are color coded, with the colors corresponding to those of original geographic sites on the map of Yunnan ( Figure 1 ). The horizontal branch was drawn in accordance with their relative genetic distances. The vertical lines are present purely for clarity of the tree presentation. The bootstrap values of 1,000 replicates are labeled on the major branches. The reference sequences for classifying HIV-1 genotypes were included and were originally obtained from the NIH/NIAID–funded HIV Databases.

    Techniques Used: Labeling

    3) Product Images from "Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes"

    Article Title: Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029806

    Contribution of TLR ligands to B cell activation. A: Stimulation of human B cells with TLR ligands. CFSE-labelled CD19+ human B cells were stimulated with or without TLR ligands (TLR2 ligand MALP-2, TLR4 ligand LPS (0.1 and 1 µg/ml) or TLR9 ligand CpG ODN 2006 (CpG)) and/or BCR stimuli (5 µg/ml SpA or anti-Ig (aIg)). After 5 days cells were harvested, stained with anti-CD20 and analyzed by flow cytometry. The graphs depict cell survival (percentage of live gated cells; upper right angle) and cell proliferation (CFSE dilution; % proliferating cells of live gated cells (left) where indicated). The experiment shown is representative of n≥3 experiments. B–D: Assessment of TLR2 activity in PWM preparations. HEK293 cells were transfected with pTLR2 or lipofectamine alone (Lf). B: non-transfected and pTLR2-transfected HEK293 cells were stained for TLR2 expression with anti-TLR2 mAb or the respective isotype control as indicated. C: HEK293 cells transfected with pTLR2 or Lf only were stimulated with Pam 3 CSK 4 (P3) or PWM (10 µg/ml). After 24 hours cellular supernatants were collected and analyzed for IL-8 secretion. One representative experiment of n≥3 experiments is shown. D: HEK293 cells transfected with pTLR2 were pretreated with anti-TLR2 mAb or the isotype control before stimulation with Pam 3 CSK 4 (P3) or PWM (10 µg/ml). IL-8 was quantified in the 24 hour supernatants. E: 16S rDNA PCR. DNA isolation and PCR amplification of bacterial 16S ribosomal DNA from DNA from E. coli (EC), a negative clinical specimen (NC), a positive clinical sample (CS) and the PWM preparation (PW) or the water control (H 2 O), with an expected PCR fragment size of approximately 900 bp.
    Figure Legend Snippet: Contribution of TLR ligands to B cell activation. A: Stimulation of human B cells with TLR ligands. CFSE-labelled CD19+ human B cells were stimulated with or without TLR ligands (TLR2 ligand MALP-2, TLR4 ligand LPS (0.1 and 1 µg/ml) or TLR9 ligand CpG ODN 2006 (CpG)) and/or BCR stimuli (5 µg/ml SpA or anti-Ig (aIg)). After 5 days cells were harvested, stained with anti-CD20 and analyzed by flow cytometry. The graphs depict cell survival (percentage of live gated cells; upper right angle) and cell proliferation (CFSE dilution; % proliferating cells of live gated cells (left) where indicated). The experiment shown is representative of n≥3 experiments. B–D: Assessment of TLR2 activity in PWM preparations. HEK293 cells were transfected with pTLR2 or lipofectamine alone (Lf). B: non-transfected and pTLR2-transfected HEK293 cells were stained for TLR2 expression with anti-TLR2 mAb or the respective isotype control as indicated. C: HEK293 cells transfected with pTLR2 or Lf only were stimulated with Pam 3 CSK 4 (P3) or PWM (10 µg/ml). After 24 hours cellular supernatants were collected and analyzed for IL-8 secretion. One representative experiment of n≥3 experiments is shown. D: HEK293 cells transfected with pTLR2 were pretreated with anti-TLR2 mAb or the isotype control before stimulation with Pam 3 CSK 4 (P3) or PWM (10 µg/ml). IL-8 was quantified in the 24 hour supernatants. E: 16S rDNA PCR. DNA isolation and PCR amplification of bacterial 16S ribosomal DNA from DNA from E. coli (EC), a negative clinical specimen (NC), a positive clinical sample (CS) and the PWM preparation (PW) or the water control (H 2 O), with an expected PCR fragment size of approximately 900 bp.

    Techniques Used: Activation Assay, Staining, Flow Cytometry, Cytometry, Activity Assay, Transfection, Expressing, Polymerase Chain Reaction, DNA Extraction, Amplification

    4) Product Images from "The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells"

    Article Title: The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018072

    Detection of GFP-coding sequences within genomic DNA prepared from terminally differentiated PC12 cells transduced with RT43.2GFP vectors. A . Representative gel picture showing genomic DNA fragment with size > 10 Kb was used for PCR analysis. Differentiated PC12 cells (5 days with NGF treatment) were exposed to RT43.2GFP vectors for 3 days, and then genomic DNA was isolated and run on a 0.8% agarose gel to purify DNA fragment with size > 10 Kb. (lane 2). Non-transduced PC12 cells were used as a negative control as represented by lane 1. B . PCR analysis. The primer pair used for lane 1 (H 2 O), lane 2 (control PC12 genomic DNA) and lane 3 (differentiated gel purified transduced PC12 genomic DNA) generated a GFP fragment of 636 bp in lane 3. Control primers specific for GAPDH were used for samples in lanes 5–7. Lanes 5 is an H 2 O control, lane 6 contains DNA purified from control PC12 cells and lane 7 contains gel purified DNA from transduced differentiated PC12 cells; lane 4 is an empty spacer lane.
    Figure Legend Snippet: Detection of GFP-coding sequences within genomic DNA prepared from terminally differentiated PC12 cells transduced with RT43.2GFP vectors. A . Representative gel picture showing genomic DNA fragment with size > 10 Kb was used for PCR analysis. Differentiated PC12 cells (5 days with NGF treatment) were exposed to RT43.2GFP vectors for 3 days, and then genomic DNA was isolated and run on a 0.8% agarose gel to purify DNA fragment with size > 10 Kb. (lane 2). Non-transduced PC12 cells were used as a negative control as represented by lane 1. B . PCR analysis. The primer pair used for lane 1 (H 2 O), lane 2 (control PC12 genomic DNA) and lane 3 (differentiated gel purified transduced PC12 genomic DNA) generated a GFP fragment of 636 bp in lane 3. Control primers specific for GAPDH were used for samples in lanes 5–7. Lanes 5 is an H 2 O control, lane 6 contains DNA purified from control PC12 cells and lane 7 contains gel purified DNA from transduced differentiated PC12 cells; lane 4 is an empty spacer lane.

    Techniques Used: Transduction, Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Negative Control, Purification, Generated

    5) Product Images from "Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome"

    Article Title: Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr451

    ( a ) Schematic representation of the intramolecular ligation technique to obtain the preCore and YMDD Pol motif close to each other. ( 1 ) The circle in a continuous line represents the complete HBV genome (3200 bp). The semicircles in discontinuous lines represent the first (first PCR, 2700 bp) and the nested (2194 bp for genotype A2 sequences and 2155 bp for genotype D sequences) PCR products. ( 2 ) The product obtained after intramolecular ligation and primers used to sequence the circular molecule ( Table 2 ) are indicated in the figure. ( b ) Agarose gel obtained after intramolecular digestion. Two markers (M-I and M-II) were loaded in the gel; band lengths are shown at the left side of the image. Two samples (S1 and S2) were loaded and four bands were detected; band lengths are indicated at the right side of the image. A band with an apparent size of 1700 bp was the circular molecule, with the preCore and YMDD Pol motif close to each other. Band B was the nested PCR product without ligation, and band D were dimers of nested PCR products without ligation. Band C corresponded to the ligated dimers. B , C and D bands were not used, because the main interest was to analyze regions from the same genome. ( c ) Sequence of the circular construct including the preCore region, YMDD polymerase motif (in box), and HindIII sequence. PreCore (1, 2, 14, 15, 28 and 29) and Core ( 1 ) codons analyzed in the text are also enclosed in boxes.
    Figure Legend Snippet: ( a ) Schematic representation of the intramolecular ligation technique to obtain the preCore and YMDD Pol motif close to each other. ( 1 ) The circle in a continuous line represents the complete HBV genome (3200 bp). The semicircles in discontinuous lines represent the first (first PCR, 2700 bp) and the nested (2194 bp for genotype A2 sequences and 2155 bp for genotype D sequences) PCR products. ( 2 ) The product obtained after intramolecular ligation and primers used to sequence the circular molecule ( Table 2 ) are indicated in the figure. ( b ) Agarose gel obtained after intramolecular digestion. Two markers (M-I and M-II) were loaded in the gel; band lengths are shown at the left side of the image. Two samples (S1 and S2) were loaded and four bands were detected; band lengths are indicated at the right side of the image. A band with an apparent size of 1700 bp was the circular molecule, with the preCore and YMDD Pol motif close to each other. Band B was the nested PCR product without ligation, and band D were dimers of nested PCR products without ligation. Band C corresponded to the ligated dimers. B , C and D bands were not used, because the main interest was to analyze regions from the same genome. ( c ) Sequence of the circular construct including the preCore region, YMDD polymerase motif (in box), and HindIII sequence. PreCore (1, 2, 14, 15, 28 and 29) and Core ( 1 ) codons analyzed in the text are also enclosed in boxes.

    Techniques Used: Ligation, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Nested PCR, Construct

    6) Product Images from "Transient transformation of Podosphaera xanthii by electroporation of conidia"

    Article Title: Transient transformation of Podosphaera xanthii by electroporation of conidia

    Journal: BMC Microbiology

    doi: 10.1186/s12866-014-0338-8

    Molecular analysis of P. xanthii transformants obtained after electro-transformation with plasmid pCPXHY1eGFP. Genomic DNA from potential transformants was subjected to PCR amplification of the egfp gene using the primer pair GFP-F and GFP-R. The size of the expected PCR product is indicated on the left. Lanes are: 1, the plasmid pCPXHY1eGFP; 2, DNA from an untransformed colony; 3–8, potential hygromycin B-resistant and fluorescent transformants. M, molecular size marker 1 kb ladder.
    Figure Legend Snippet: Molecular analysis of P. xanthii transformants obtained after electro-transformation with plasmid pCPXHY1eGFP. Genomic DNA from potential transformants was subjected to PCR amplification of the egfp gene using the primer pair GFP-F and GFP-R. The size of the expected PCR product is indicated on the left. Lanes are: 1, the plasmid pCPXHY1eGFP; 2, DNA from an untransformed colony; 3–8, potential hygromycin B-resistant and fluorescent transformants. M, molecular size marker 1 kb ladder.

    Techniques Used: Transformation Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Marker

    7) Product Images from "A WASp-VASP complex regulates actin polymerization at the plasma membrane"

    Article Title: A WASp-VASP complex regulates actin polymerization at the plasma membrane

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.20.5603

    Fig. 3. Overexpressed FRB–WASp418–502 induces F-actin clusters in BHK-21 cells. ( A ) Lysates of RBL-2H3 cell line expressing FRB–WASp418–502, or BHK-21 cells overexpressing FRB–WASp418–502 or Scar-WA were run on 12% SDS–PAGE (9 µg total protein per lane) and blotted on to PVDF. Blots were revealed using mouse anti-myc tag antibody (clone 9E10) and the ECL procedure. The signal in RBL-2H3 cells is ∼5-fold less than in BHK-21 cells (compare lanes 1 and 3). Taking into consideration that ∼10% of BHK-21 cells expressed the FRB–WASp418–502 protein, we estimate that the level of expression of FRB–WASp418–502 is ∼50-fold lower in the RBL-2H3 stable cell line compared with that in BHK-21 cells. ( B – D ) BHK-21 cells expressing the Scar-WA domain. ( E – G ) BHK-21 cells expressing the FRB–WASp418–502 construct. (B and E) Localization of myc-tagged constructs as revealed using anti-myc tag antibody. (C and F) Distribution of F-actin. Arrows point at clusters of WASp or Scar C-terminal domains and F-actin. Arrowheads show non-transfected cells. (D and G) Superimposition of anti-myc and F-actin images. Bar: 10 µm.
    Figure Legend Snippet: Fig. 3. Overexpressed FRB–WASp418–502 induces F-actin clusters in BHK-21 cells. ( A ) Lysates of RBL-2H3 cell line expressing FRB–WASp418–502, or BHK-21 cells overexpressing FRB–WASp418–502 or Scar-WA were run on 12% SDS–PAGE (9 µg total protein per lane) and blotted on to PVDF. Blots were revealed using mouse anti-myc tag antibody (clone 9E10) and the ECL procedure. The signal in RBL-2H3 cells is ∼5-fold less than in BHK-21 cells (compare lanes 1 and 3). Taking into consideration that ∼10% of BHK-21 cells expressed the FRB–WASp418–502 protein, we estimate that the level of expression of FRB–WASp418–502 is ∼50-fold lower in the RBL-2H3 stable cell line compared with that in BHK-21 cells. ( B – D ) BHK-21 cells expressing the Scar-WA domain. ( E – G ) BHK-21 cells expressing the FRB–WASp418–502 construct. (B and E) Localization of myc-tagged constructs as revealed using anti-myc tag antibody. (C and F) Distribution of F-actin. Arrows point at clusters of WASp or Scar C-terminal domains and F-actin. Arrowheads show non-transfected cells. (D and G) Superimposition of anti-myc and F-actin images. Bar: 10 µm.

    Techniques Used: Expressing, SDS Page, Stable Transfection, Construct, Transfection

    Fig. 4. Immunofluorescence localization of the Arp2/3 complex in cell lines expressing the isolated VCA domain (WASp418–502) or WASp105–502. RBL-2H3 cell lines expressing FRB–WASp418–502 ( A – D ) or FRB–WASp105–502 ( E – H ) were plated on to glass coverslips coated with anti-CD25 antibodies and treated with DMSO as control (A, B, E and F) or rapamycin (C, D, G and H), fixed, and stained with phalloidin (A, C, E and G) and anti-p41Arc, a subunit of the Arp2/3 complex (B, D, F and H). Confocal sections were recorded at the level of the ventral surface. Bar: 5 µm.
    Figure Legend Snippet: Fig. 4. Immunofluorescence localization of the Arp2/3 complex in cell lines expressing the isolated VCA domain (WASp418–502) or WASp105–502. RBL-2H3 cell lines expressing FRB–WASp418–502 ( A – D ) or FRB–WASp105–502 ( E – H ) were plated on to glass coverslips coated with anti-CD25 antibodies and treated with DMSO as control (A, B, E and F) or rapamycin (C, D, G and H), fixed, and stained with phalloidin (A, C, E and G) and anti-p41Arc, a subunit of the Arp2/3 complex (B, D, F and H). Confocal sections were recorded at the level of the ventral surface. Bar: 5 µm.

    Techniques Used: Immunofluorescence, Expressing, Isolation, Staining

    Fig. 2. Requirement for N-terminal regions of WASp in actin assembly. F-actin distribution in stable cell lines expressing the surface receptor CD25–FKBP 2 in combination with various FRB–WASp constructs. Control cells cultured in the absence of rapamycin ( A , C , E and G ). Membrane translocation of FRB–WASp constructs to the cytoplasmic region of CD25–FKBP 2 receptors was induced by rapamycin treatment ( B , D , F and H ). Images shown are confocal sections of the ventral cell surface. (A and B) Wasp105–502, (C and D) WASp418–502, (E and F) WASp238–502, (G and H) WASp196–502. Bar: 10 µm.
    Figure Legend Snippet: Fig. 2. Requirement for N-terminal regions of WASp in actin assembly. F-actin distribution in stable cell lines expressing the surface receptor CD25–FKBP 2 in combination with various FRB–WASp constructs. Control cells cultured in the absence of rapamycin ( A , C , E and G ). Membrane translocation of FRB–WASp constructs to the cytoplasmic region of CD25–FKBP 2 receptors was induced by rapamycin treatment ( B , D , F and H ). Images shown are confocal sections of the ventral cell surface. (A and B) Wasp105–502, (C and D) WASp418–502, (E and F) WASp238–502, (G and H) WASp196–502. Bar: 10 µm.

    Techniques Used: Stable Transfection, Expressing, Construct, Cell Culture, Translocation Assay

    Fig. 6. The PRD of WASp mediates direct interaction with VASP. ( A ) VASP pulldown from RBL-2H3 cell lysates using the GST–WASp fusion proteins indicated. GST, GST–WASp155–502, –WASp155–429, –WASp418–502 (VCA domain) or –WASpΔPRD (WASp155–502 deleted of the PRD, position 310–390) immobilized on glutathione–Sepharose beads was incubated with cell lysates prepared from unstimulated RBL-2H3 cells, or cells stimulated by cross-linking of FcεRI (x Fc). Bound material was subjected to western blotting with anti-VASP antibodies. Lanes 1–3: total cell lysates; lanes 4–8: GST pulldown from RBL-2H3 cell lysate; lanes 9–13: GST pulldown from lysate of RBL-2H3 cells activated by FcεRI cross-linking. ( B ) The central PRD of WASp mediates direct binding to VASP. GST or GST–WASp fusion proteins (1 µg) bound to glutathione–Sepharose beads were incubated with recombinant His 6 –VASP (250 nM) and bound material was subjected to western blotting with anti-VASP antibodies. Lanes 1–4: the indicated amount of His 6 –VASP; lanes 5–9: unbound material (2% of total); lanes 10–14: bound material (50% of total). ( C ) Estimation of the equilibrium binding constant of VASP with WASp155–390. His 6 –VASP or GST–WASp(155–390)His 6 (1 µg each) was visualized by Coomassie Blue staining of the gel (lanes 1 and 2). VASP (50 nM) was incubated in the presence of increasing concentrations of WASp155–390 as indicated, in the presence of a fixed amount of glutathione–Sepharose beads. Bound and unbound proteins were subjected to SDS–PAGE followed by western blotting with anti-VASP antibody (lanes 3–8). A value of ∼1 µM was derived for the equilibrium binding constant for VASP with WASp155–390. The results shown are representative of four independent experiments with two different preparations of recombinant proteins.
    Figure Legend Snippet: Fig. 6. The PRD of WASp mediates direct interaction with VASP. ( A ) VASP pulldown from RBL-2H3 cell lysates using the GST–WASp fusion proteins indicated. GST, GST–WASp155–502, –WASp155–429, –WASp418–502 (VCA domain) or –WASpΔPRD (WASp155–502 deleted of the PRD, position 310–390) immobilized on glutathione–Sepharose beads was incubated with cell lysates prepared from unstimulated RBL-2H3 cells, or cells stimulated by cross-linking of FcεRI (x Fc). Bound material was subjected to western blotting with anti-VASP antibodies. Lanes 1–3: total cell lysates; lanes 4–8: GST pulldown from RBL-2H3 cell lysate; lanes 9–13: GST pulldown from lysate of RBL-2H3 cells activated by FcεRI cross-linking. ( B ) The central PRD of WASp mediates direct binding to VASP. GST or GST–WASp fusion proteins (1 µg) bound to glutathione–Sepharose beads were incubated with recombinant His 6 –VASP (250 nM) and bound material was subjected to western blotting with anti-VASP antibodies. Lanes 1–4: the indicated amount of His 6 –VASP; lanes 5–9: unbound material (2% of total); lanes 10–14: bound material (50% of total). ( C ) Estimation of the equilibrium binding constant of VASP with WASp155–390. His 6 –VASP or GST–WASp(155–390)His 6 (1 µg each) was visualized by Coomassie Blue staining of the gel (lanes 1 and 2). VASP (50 nM) was incubated in the presence of increasing concentrations of WASp155–390 as indicated, in the presence of a fixed amount of glutathione–Sepharose beads. Bound and unbound proteins were subjected to SDS–PAGE followed by western blotting with anti-VASP antibody (lanes 3–8). A value of ∼1 µM was derived for the equilibrium binding constant for VASP with WASp155–390. The results shown are representative of four independent experiments with two different preparations of recombinant proteins.

    Techniques Used: Incubation, Western Blot, Binding Assay, Recombinant, Staining, SDS Page, Derivative Assay

    8) Product Images from "Long-term treatment outcomes of clevudine in antiviral-naive patients with chronic hepatitis B"

    Article Title: Long-term treatment outcomes of clevudine in antiviral-naive patients with chronic hepatitis B

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v18.i47.6943

    Decrement changes in serum hepatitis B virus DNA titer and cumulative rates of hepatitis B virus DNA non-detection during the on-treatment period. A: Hepatitis B virus (HBV) DNA decrement changes; B: HBV DNA non-detection rate.
    Figure Legend Snippet: Decrement changes in serum hepatitis B virus DNA titer and cumulative rates of hepatitis B virus DNA non-detection during the on-treatment period. A: Hepatitis B virus (HBV) DNA decrement changes; B: HBV DNA non-detection rate.

    Techniques Used:

    9) Product Images from "Prevalence of the BRAF p.v600e variant in patients with colorectal cancer from Mexico and its estimated frequency in Latin American and Caribbean populations"

    Article Title: Prevalence of the BRAF p.v600e variant in patients with colorectal cancer from Mexico and its estimated frequency in Latin American and Caribbean populations

    Journal: Journal of Investigative Medicine

    doi: 10.1136/jim-2020-001301

    Partial electropherogram of DNA from a patient with colorectal cancer from Western Mexico showing heterozygosity for the BRAF p.V600E variant.
    Figure Legend Snippet: Partial electropherogram of DNA from a patient with colorectal cancer from Western Mexico showing heterozygosity for the BRAF p.V600E variant.

    Techniques Used: Western Blot, Variant Assay

    10) Product Images from "Mouse chromocenters DNA content: sequencing and in silico analysis"

    Article Title: Mouse chromocenters DNA content: sequencing and in silico analysis

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4534-z

    FISH and fiber-FISH with IAP probe on M.musculus nucleus, metaphase chromosomes and extended chromatin. a – IAP probe (red) on interphase nucleus from L929 cell culture; b - IAP probe (red) on metaphase plate from L929 cell culture; c – IAP probe (red) and MiSat (green) on bone marrow metaphase plate; d – fiber-FISH on L929 chromatin with IAP probe (red) and MiSat (green). DAPI counterstain is blue. Bar – 10 μm
    Figure Legend Snippet: FISH and fiber-FISH with IAP probe on M.musculus nucleus, metaphase chromosomes and extended chromatin. a – IAP probe (red) on interphase nucleus from L929 cell culture; b - IAP probe (red) on metaphase plate from L929 cell culture; c – IAP probe (red) and MiSat (green) on bone marrow metaphase plate; d – fiber-FISH on L929 chromatin with IAP probe (red) and MiSat (green). DAPI counterstain is blue. Bar – 10 μm

    Techniques Used: Fluorescence In Situ Hybridization, Cell Culture

    11) Product Images from "Oral Immunogenicity of Human Papillomavirus-Like Particles Expressed in Potato"

    Article Title: Oral Immunogenicity of Human Papillomavirus-Like Particles Expressed in Potato

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.16.8702-8711.2003

    Southern and Northern blot analyses of selected HPV11 L1st potato transformants. (A) Plant genomic DNA was prepared and Southern blotted as described in Materials and Methods. DNAs extracted from several HPV11 L1st lines (i.e., lines 8, 10, 15, 22, and 23) contained bands that varied in molecular weight, consistent with A. tumefaciens -mediated random insertions that were reactive with an HPV11 L1-specific nucleic acid probe. wt, wild type. (B) Total RNAs extracted from the same lines were Northern blotted and probed as for panel A. Top, methylene blue-stained blot to verify loading (rRNA bands); bottom, blot hybridized with HPV11 L1 probe.
    Figure Legend Snippet: Southern and Northern blot analyses of selected HPV11 L1st potato transformants. (A) Plant genomic DNA was prepared and Southern blotted as described in Materials and Methods. DNAs extracted from several HPV11 L1st lines (i.e., lines 8, 10, 15, 22, and 23) contained bands that varied in molecular weight, consistent with A. tumefaciens -mediated random insertions that were reactive with an HPV11 L1-specific nucleic acid probe. wt, wild type. (B) Total RNAs extracted from the same lines were Northern blotted and probed as for panel A. Top, methylene blue-stained blot to verify loading (rRNA bands); bottom, blot hybridized with HPV11 L1 probe.

    Techniques Used: Northern Blot, Molecular Weight, Staining

    12) Product Images from "The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)"

    Article Title: The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

    Journal: Molecular Biology of the Cell

    doi:

    (A) Yrb1p is a major Xpo1p-binding protein. Xpo1p-ZZ was purified from yeast extracts in the presence of recombinant Gsp1pQ71L-GTP as described in Materials and Methods, and proteins bound to immobilized Xpo1p were eluted with 500 mM KCl. The input (I), flow-through (FT), and eluate (E) were analyzed by SDS PAGE and Coomassie blue staining. The two major proteins in the eluate were identified by MALDI and Western blotting and correspond to Gsp1p and Yrb1p. Molecular weight markers (M) are in kDa. (B-E) Yrb1p shuttles between the cytoplasm and the nucleus. Wild-type cells (B) or xpo1–1 cells (C-E) expressing GFP-YRB1 were grown in liquid medium at 25°C. The cultures were kept at 25°C (C), shifted to 37°C for 2 h (D), or shifted to 37°C for 2 h and then shifted back to 25°C for 2 h (E). To inhibit protein synthesis, cycloheximide (final concentration 0.1 mg/ml) was added to the cultures before the temperature shift. The cells were viewed by fluorescence microscopy to visualize GFP-Yrb1p. The perinuclear staining in wild-type cells is indicated by arrows. (F-K) Yrb1p has two nuclear targeting sequences. Wild-type cells (F, H, and J) or xpo1–1 mutants (G, I, and K) were transformed with plasmids encoding GFP-Yrb1p, GFP-Yrb1ΔN62p, or GFP-Yrb1p 1–40, as indicated. The cultures were incubated at 30°C ( XPO1 cells) or 25°C ( xpo1–1 cells) in raffinose-containing medium. Expression of the GFP fusions was induced by 2% galactose and repressed by addition of 2% glucose after 1 h. Cells were kept at 30°C ( XPO1 ) or shifted to 37°C for 2 h ( xpo1–1 ) and viewed by fluorescence microscopy.
    Figure Legend Snippet: (A) Yrb1p is a major Xpo1p-binding protein. Xpo1p-ZZ was purified from yeast extracts in the presence of recombinant Gsp1pQ71L-GTP as described in Materials and Methods, and proteins bound to immobilized Xpo1p were eluted with 500 mM KCl. The input (I), flow-through (FT), and eluate (E) were analyzed by SDS PAGE and Coomassie blue staining. The two major proteins in the eluate were identified by MALDI and Western blotting and correspond to Gsp1p and Yrb1p. Molecular weight markers (M) are in kDa. (B-E) Yrb1p shuttles between the cytoplasm and the nucleus. Wild-type cells (B) or xpo1–1 cells (C-E) expressing GFP-YRB1 were grown in liquid medium at 25°C. The cultures were kept at 25°C (C), shifted to 37°C for 2 h (D), or shifted to 37°C for 2 h and then shifted back to 25°C for 2 h (E). To inhibit protein synthesis, cycloheximide (final concentration 0.1 mg/ml) was added to the cultures before the temperature shift. The cells were viewed by fluorescence microscopy to visualize GFP-Yrb1p. The perinuclear staining in wild-type cells is indicated by arrows. (F-K) Yrb1p has two nuclear targeting sequences. Wild-type cells (F, H, and J) or xpo1–1 mutants (G, I, and K) were transformed with plasmids encoding GFP-Yrb1p, GFP-Yrb1ΔN62p, or GFP-Yrb1p 1–40, as indicated. The cultures were incubated at 30°C ( XPO1 cells) or 25°C ( xpo1–1 cells) in raffinose-containing medium. Expression of the GFP fusions was induced by 2% galactose and repressed by addition of 2% glucose after 1 h. Cells were kept at 30°C ( XPO1 ) or shifted to 37°C for 2 h ( xpo1–1 ) and viewed by fluorescence microscopy.

    Techniques Used: Binding Assay, Purification, Recombinant, Flow Cytometry, SDS Page, Staining, Western Blot, Molecular Weight, Expressing, Concentration Assay, Fluorescence, Microscopy, Transformation Assay, Incubation

    13) Product Images from "Tissue-Specific Sequence Alterations in the Human Immunodeficiency Virus Type 1 Envelope Favoring CCR5 Usage Contribute to Persistence of Dual-Tropic Virus in the Brain ▿"

    Article Title: Tissue-Specific Sequence Alterations in the Human Immunodeficiency Virus Type 1 Envelope Favoring CCR5 Usage Contribute to Persistence of Dual-Tropic Virus in the Brain ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02648-08

    Coreceptor usage for HIV-1 entry into PBMC and MDM. PBMC (A and B) or MDM (C and D) were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped luciferase
    Figure Legend Snippet: Coreceptor usage for HIV-1 entry into PBMC and MDM. PBMC (A and B) or MDM (C and D) were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped luciferase

    Techniques Used: Infection, Luciferase

    Coreceptor preference for HIV-1 entry into JC53 cells. JC53 cells were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped luciferase reporter virus,
    Figure Legend Snippet: Coreceptor preference for HIV-1 entry into JC53 cells. JC53 cells were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped luciferase reporter virus,

    Techniques Used: Infection, Luciferase

    Env V3 determinants influencing coreceptor usage for HIV-1 entry into PBMC and MDM. PBMC (A) or MDM (B) were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped
    Figure Legend Snippet: Env V3 determinants influencing coreceptor usage for HIV-1 entry into PBMC and MDM. PBMC (A) or MDM (B) were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped

    Techniques Used: Infection

    14) Product Images from "Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans"

    Article Title: Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002395

    The Hemotin peptide is required for proper endosomal maturation in hemocytes. (A–A”) Distribution of acidic organelles in hemo A4 mutant ex vivo hemocytes revealed by the expression of LAMP1-GFP lysosomal marker. The intracellular vacuoles that disrupt the beta-tubulin cytoskeleton (A, A”; red) accumulate LAMP1-GFP positive compartments (A, A’; green). Compare with wild-type in S2A–S2A” Fig . Scale bar (5 μm). (B–B”) Distribution of the endosomal marker FYVE (PI(3)P binding zinc finger domain, early endosomal marker, named after being found in Fab1, YOTP, Vac1, EEA1) (green) (B, B’) and Lysotracker (red) (B, B”) organelles in a hemo A4 mutant ex vivo hemocyte showing enlarged intracellular compartments coexpressing FYVE and Lysotracker. Scale bar (5 μm). (C–C”) Wild-type ex vivo hemocyte labelled as in (B), showing little overlap between early endosome-FYVE positive (green) (C,C’) and lysosomal (red) (C,C”) compartments. (D) Quantification of the FYVE OAI in ex vivo hemocytes (see Materials and Methods ). hemo A4 mutants display a significantly larger FYVE area than wild-type. This phenotype is rescued by the expression of the hemo full length transcript ( UAS-hemoFL ) and is specific to hemo -ORF function, as it is also rescued by the expression of the hemo -ORF mini gene ( UAS-hemo-ORF ) or C-terminal-tagged hemo -GFP (UAS-hemo-GFP ). No rescue was observed by a CG7691 genomic fragment ( CG7691-GF ), or with a hemo full-length transcript containing a frameshift in the hemo -ORF ( UAS-hemoFS ), or with the ORF2 mini gene ( UAS-ORF2 ). All UAS constructs were driven with He-Gal4. Error bars represent SEM. One-way ANOVA analysis shows that there is a statistically significant difference between these groups [F(7,286) = 27.12, p
    Figure Legend Snippet: The Hemotin peptide is required for proper endosomal maturation in hemocytes. (A–A”) Distribution of acidic organelles in hemo A4 mutant ex vivo hemocytes revealed by the expression of LAMP1-GFP lysosomal marker. The intracellular vacuoles that disrupt the beta-tubulin cytoskeleton (A, A”; red) accumulate LAMP1-GFP positive compartments (A, A’; green). Compare with wild-type in S2A–S2A” Fig . Scale bar (5 μm). (B–B”) Distribution of the endosomal marker FYVE (PI(3)P binding zinc finger domain, early endosomal marker, named after being found in Fab1, YOTP, Vac1, EEA1) (green) (B, B’) and Lysotracker (red) (B, B”) organelles in a hemo A4 mutant ex vivo hemocyte showing enlarged intracellular compartments coexpressing FYVE and Lysotracker. Scale bar (5 μm). (C–C”) Wild-type ex vivo hemocyte labelled as in (B), showing little overlap between early endosome-FYVE positive (green) (C,C’) and lysosomal (red) (C,C”) compartments. (D) Quantification of the FYVE OAI in ex vivo hemocytes (see Materials and Methods ). hemo A4 mutants display a significantly larger FYVE area than wild-type. This phenotype is rescued by the expression of the hemo full length transcript ( UAS-hemoFL ) and is specific to hemo -ORF function, as it is also rescued by the expression of the hemo -ORF mini gene ( UAS-hemo-ORF ) or C-terminal-tagged hemo -GFP (UAS-hemo-GFP ). No rescue was observed by a CG7691 genomic fragment ( CG7691-GF ), or with a hemo full-length transcript containing a frameshift in the hemo -ORF ( UAS-hemoFS ), or with the ORF2 mini gene ( UAS-ORF2 ). All UAS constructs were driven with He-Gal4. Error bars represent SEM. One-way ANOVA analysis shows that there is a statistically significant difference between these groups [F(7,286) = 27.12, p

    Techniques Used: Mutagenesis, Ex Vivo, Expressing, Marker, Binding Assay, Construct

    Identification and phenotypical characterisation of the hemotin gene. (A) hemo genomic locus including the hemo , CG7691 , fray , and fruitless genes (blue arrows). The hemo A4 deletion (red bar) was generated by FRT-mediated recombination using the P{RS3}fray CB-0706-3 and the P-Bac{WH}fru f02684 transposable elements (blue triangles). Transcript models are represented under their respective genes, orange boxes represent coding exons, whereas gray boxes indicate noncoding exons (untranslated regions, UTRs). hemo A4 completely removes hemo and CG7691 plus the first noncoding exons of fray and fruitless . The P{PZ}fray 07551 insertion is a lethal fray allele [ 25 ] and was used for genetic complementation experiments between hemo A4 and fray . (B) Top: Ribosomal profiling reads obtained from polyribosomes from S2 cells (Poly-Riboseq;(3)) mapped to the hemo full-length transcript ( hemoFL ). hemo -ORF is translated more efficiently than ORF2 ( hemo -ORF RPKM: 29.4, coverage: 0.9 ORF; ORF2 RPKM: 6.6, coverage: 0.7. Note that the reads per kilobase of transcript per million mapped reads [RPKM] value of ORF2 is below the 11.8 cut-off to be considered translated [ 3 ]). Bottom: schematic representation of other constructs used in this manuscript. hemo -ORF is a minigene consisting of an mRNA fragment truncated immediately after the hemo -ORF stop codon, ORF2 consists of a mini-gene construct carrying the ORF2 sequence only, including 6 nt upstream of its start codon (to conserve its endogenous Kozak sequence). hemo -GFP (green fluorescent protein) is a hemo -ORF-GFP fusion construct in which the GFP sequence (devoid of a start codon) was cloned into the hemoFL construct, immediately downstream and in frame with hemo -ORF (devoid of a stop codon) (see Materials and Methods ). (C) Pattern of expression of hemo in germ band-retracted embryos revealed by in situ hybridisation. hemo is specifically expressed in embryonic hemocytes (arrows; compare with D) in the head, amnioserosa, and dispersed along the body. (D) Spatial distribution of embryonic hemocytes at germ band retraction stage revealed by in situ hybridisation of hemocyte-specific croquemort ( crq ) gene, showing similar distribution in the head, amnioserosa, and along the body (arrows). (E) Cluster of early embryonic hemocytes of the cephalic region expressing the hemo transcript revealed by FISH (fluorescent in situ hybridisation). Some hemocytes show drop-shape morphologies (asterisk) and membrane projections such as filopodia (arrows). (F) Embryonic hemocytes labelled with crq-Gal4;UAS-GFP expression from the head region displaying similar cellular morphologies (arrows and asterisk) as those in E. (G–H) White prepupal thoracic hemocytes revealed by crq-Gal4;UAS-GFP expression in wild-type (G) and hemo A4 mutants (H). In hemo A4 mutants, hemocytes display enlarged vacuoles within the cytoplasm (arrowheads), with larger occupied area index (OAI). Scale bar (50 μm). (I–N) hemocytes observed ex vivo [ 15 ] showing Tubulin (green) and Actin (red) cytoskeletons and nuclei (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, DAPI) with its corresponding orthogonal projection of confocal microscopy z-stacks (above inset) showing only tubulin cytoskeleton (green) and DAPI (blue) staining in the nucleus (n). Scale bar (5 μm). (I) Wild-type hemocyte. (J) hemo A4 mutant hemocyte shows large disruptions of the tubulin cytoskeleton that appear as rounded vacuoles (arrows; arrowhead in inset). (K) Knocking down the expression of hemo with a UAS-hemo-RNAi construct phenocopies the vacuolation phenotype (arrows and arrowhead in inset). (L) Expression of hemo full length transcript ( UAS-hemoFL ) rescues the vacuolated hemo A4 phenotype. Expression of hemo -ORF only (M) also rescues the hemo A4 mutant vacuolation. (N) Expression of ORF2 does not rescue the hemo A4 mutant vacuolated phenotype (arrows and arrowhead in inset). (O) Vacuolation measurements in ex vivo primary pre pupal hemocytes. hemo A4 mutant hemocytes show significantly higher occupied volume index (OVI) (see Materials and Methods ) than wild-type. Rescue experiments show that the vacuolation phenotype is specific to the peptide encoded by hemo -ORF. All upstream activating sequence (UAS) constructs were driven by crq-Gal4 . Error bars represent standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA test indicating that samples were significantly different [F(9,486) = 9.5, p
    Figure Legend Snippet: Identification and phenotypical characterisation of the hemotin gene. (A) hemo genomic locus including the hemo , CG7691 , fray , and fruitless genes (blue arrows). The hemo A4 deletion (red bar) was generated by FRT-mediated recombination using the P{RS3}fray CB-0706-3 and the P-Bac{WH}fru f02684 transposable elements (blue triangles). Transcript models are represented under their respective genes, orange boxes represent coding exons, whereas gray boxes indicate noncoding exons (untranslated regions, UTRs). hemo A4 completely removes hemo and CG7691 plus the first noncoding exons of fray and fruitless . The P{PZ}fray 07551 insertion is a lethal fray allele [ 25 ] and was used for genetic complementation experiments between hemo A4 and fray . (B) Top: Ribosomal profiling reads obtained from polyribosomes from S2 cells (Poly-Riboseq;(3)) mapped to the hemo full-length transcript ( hemoFL ). hemo -ORF is translated more efficiently than ORF2 ( hemo -ORF RPKM: 29.4, coverage: 0.9 ORF; ORF2 RPKM: 6.6, coverage: 0.7. Note that the reads per kilobase of transcript per million mapped reads [RPKM] value of ORF2 is below the 11.8 cut-off to be considered translated [ 3 ]). Bottom: schematic representation of other constructs used in this manuscript. hemo -ORF is a minigene consisting of an mRNA fragment truncated immediately after the hemo -ORF stop codon, ORF2 consists of a mini-gene construct carrying the ORF2 sequence only, including 6 nt upstream of its start codon (to conserve its endogenous Kozak sequence). hemo -GFP (green fluorescent protein) is a hemo -ORF-GFP fusion construct in which the GFP sequence (devoid of a start codon) was cloned into the hemoFL construct, immediately downstream and in frame with hemo -ORF (devoid of a stop codon) (see Materials and Methods ). (C) Pattern of expression of hemo in germ band-retracted embryos revealed by in situ hybridisation. hemo is specifically expressed in embryonic hemocytes (arrows; compare with D) in the head, amnioserosa, and dispersed along the body. (D) Spatial distribution of embryonic hemocytes at germ band retraction stage revealed by in situ hybridisation of hemocyte-specific croquemort ( crq ) gene, showing similar distribution in the head, amnioserosa, and along the body (arrows). (E) Cluster of early embryonic hemocytes of the cephalic region expressing the hemo transcript revealed by FISH (fluorescent in situ hybridisation). Some hemocytes show drop-shape morphologies (asterisk) and membrane projections such as filopodia (arrows). (F) Embryonic hemocytes labelled with crq-Gal4;UAS-GFP expression from the head region displaying similar cellular morphologies (arrows and asterisk) as those in E. (G–H) White prepupal thoracic hemocytes revealed by crq-Gal4;UAS-GFP expression in wild-type (G) and hemo A4 mutants (H). In hemo A4 mutants, hemocytes display enlarged vacuoles within the cytoplasm (arrowheads), with larger occupied area index (OAI). Scale bar (50 μm). (I–N) hemocytes observed ex vivo [ 15 ] showing Tubulin (green) and Actin (red) cytoskeletons and nuclei (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, DAPI) with its corresponding orthogonal projection of confocal microscopy z-stacks (above inset) showing only tubulin cytoskeleton (green) and DAPI (blue) staining in the nucleus (n). Scale bar (5 μm). (I) Wild-type hemocyte. (J) hemo A4 mutant hemocyte shows large disruptions of the tubulin cytoskeleton that appear as rounded vacuoles (arrows; arrowhead in inset). (K) Knocking down the expression of hemo with a UAS-hemo-RNAi construct phenocopies the vacuolation phenotype (arrows and arrowhead in inset). (L) Expression of hemo full length transcript ( UAS-hemoFL ) rescues the vacuolated hemo A4 phenotype. Expression of hemo -ORF only (M) also rescues the hemo A4 mutant vacuolation. (N) Expression of ORF2 does not rescue the hemo A4 mutant vacuolated phenotype (arrows and arrowhead in inset). (O) Vacuolation measurements in ex vivo primary pre pupal hemocytes. hemo A4 mutant hemocytes show significantly higher occupied volume index (OVI) (see Materials and Methods ) than wild-type. Rescue experiments show that the vacuolation phenotype is specific to the peptide encoded by hemo -ORF. All upstream activating sequence (UAS) constructs were driven by crq-Gal4 . Error bars represent standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA test indicating that samples were significantly different [F(9,486) = 9.5, p

    Techniques Used: Generated, BAC Assay, Construct, Sequencing, Clone Assay, Expressing, In Situ, Hybridization, Fluorescence In Situ Hybridization, Ex Vivo, Confocal Microscopy, Staining, Mutagenesis

    15) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    Detection of rifampin-resistant mutations in TB using the modified PR-PCR and next-generation sequencing. (a) Primer and template sequences. TB-NC is a negative control plasmid containing the rpoB gene fragment without any rifampin-sensitive mutations. TB-H526Y and TB-S531L are positive control plasmids containing the rpoB gene fragment with the H526Y(CAC→TAC) and S531L(TCG→TTG) mutations, respectively. (b) Gel electrophoresis of the amplicons for the two mutations in the TB rpoB gene using the modified PR-PCR. (c) Detection of two rifampin-resistant TB mutants using 454 high-throughput sequencing.
    Figure Legend Snippet: Detection of rifampin-resistant mutations in TB using the modified PR-PCR and next-generation sequencing. (a) Primer and template sequences. TB-NC is a negative control plasmid containing the rpoB gene fragment without any rifampin-sensitive mutations. TB-H526Y and TB-S531L are positive control plasmids containing the rpoB gene fragment with the H526Y(CAC→TAC) and S531L(TCG→TTG) mutations, respectively. (b) Gel electrophoresis of the amplicons for the two mutations in the TB rpoB gene using the modified PR-PCR. (c) Detection of two rifampin-resistant TB mutants using 454 high-throughput sequencing.

    Techniques Used: Modification, Polymerase Chain Reaction, Next-Generation Sequencing, Negative Control, Plasmid Preparation, Positive Control, Nucleic Acid Electrophoresis

    16) Product Images from "Expression of the inactivating deiodinase, Deiodinase 3, in the pre-metamorphic tadpole retina"

    Article Title: Expression of the inactivating deiodinase, Deiodinase 3, in the pre-metamorphic tadpole retina

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0195374

    Expression of dio3 contributes to modulate T 3 transcriptional response in the developing retina. Fig 4A. Real-time q-PCR analysis of eGFP , dio3 , klf9 , thibz and thrb for their T 3 transcriptional response in NF48 eye from reporter transgenic line p dio3 -GFP. Gene expression was normalized against odc . mRNA levels from vehicle controls (CTL) were used as reference values. Results pooled from two to three independent experiments are represented as scatter dot plots mean with SD. 14≥n≥6 per group. Non-parametric ANOVA, Kruskall Wallis with uncorrected Dunn’s test (PRISM7) was used to assess statistical significance. *, p
    Figure Legend Snippet: Expression of dio3 contributes to modulate T 3 transcriptional response in the developing retina. Fig 4A. Real-time q-PCR analysis of eGFP , dio3 , klf9 , thibz and thrb for their T 3 transcriptional response in NF48 eye from reporter transgenic line p dio3 -GFP. Gene expression was normalized against odc . mRNA levels from vehicle controls (CTL) were used as reference values. Results pooled from two to three independent experiments are represented as scatter dot plots mean with SD. 14≥n≥6 per group. Non-parametric ANOVA, Kruskall Wallis with uncorrected Dunn’s test (PRISM7) was used to assess statistical significance. *, p

    Techniques Used: Expressing, Polymerase Chain Reaction, Transgenic Assay, CTL Assay

    17) Product Images from "Waterpipe smoking induces epigenetic changes in the small airway epithelium"

    Article Title: Waterpipe smoking induces epigenetic changes in the small airway epithelium

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171112

    Genome-wide methylation differences of Small Airway Epithelium (SAE) DNA of waterpipe smokers vs nonsmokers. The data is derived from analysis of n = 7 nonsmokers and n = 7 waterpipe smokers. A . Principal component analysis using all HELP assay probesets corrected for covariates as input dataset. Shown are the first 3 principal components representing the largest variability among the groups. Each circle represents an individual subject (green = nonsmokers, orange = waterpipe smokers). B . Volcano plot. Assessment of differential DNA methylation of SAE for all probesets comparing waterpipe smokers vs nonsmokers; y-axis, negative log 10 of p value; x-axis, log 2 -transformed fold-change; red dots are probesets with differential DNA methylation, gray dots are probesets without differential methylation. Differentially methylated probesets with p
    Figure Legend Snippet: Genome-wide methylation differences of Small Airway Epithelium (SAE) DNA of waterpipe smokers vs nonsmokers. The data is derived from analysis of n = 7 nonsmokers and n = 7 waterpipe smokers. A . Principal component analysis using all HELP assay probesets corrected for covariates as input dataset. Shown are the first 3 principal components representing the largest variability among the groups. Each circle represents an individual subject (green = nonsmokers, orange = waterpipe smokers). B . Volcano plot. Assessment of differential DNA methylation of SAE for all probesets comparing waterpipe smokers vs nonsmokers; y-axis, negative log 10 of p value; x-axis, log 2 -transformed fold-change; red dots are probesets with differential DNA methylation, gray dots are probesets without differential methylation. Differentially methylated probesets with p

    Techniques Used: Genome Wide, Methylation, Derivative Assay, HELP Assay, DNA Methylation Assay, Transformation Assay

    Correlation of Small Airway Epithelium (SAE) DNA methylation and gene expression. Unique genes that show differences in DNA methylation between waterpipe smokers and nonsmokers (p
    Figure Legend Snippet: Correlation of Small Airway Epithelium (SAE) DNA methylation and gene expression. Unique genes that show differences in DNA methylation between waterpipe smokers and nonsmokers (p

    Techniques Used: DNA Methylation Assay, Expressing

    18) Product Images from "Cloning and functional analysis of a fructosyltransferase cDNA for synthesis of highly polymerized levans in timothy (Phleum pratense L.)"

    Article Title: Cloning and functional analysis of a fructosyltransferase cDNA for synthesis of highly polymerized levans in timothy (Phleum pratense L.)

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ern337

    Phylogenetic tree of FTs and vacuolar invertases (vINV) of Poaceae species based on predicted amino acid sequences. Scale bar indicates branch length. Genbank accession numbers of genes used in this analysis are: Hordeum vulgare 6-SFT, X83233; Triticum aestivum 6-SFT, AB029887; Agropyron cristatum 6-SFT, AF211253; Lolium perenne 6-FT, AF494041; Lolium temulentum 6-FT, AJ532550; Poa secunda 6-SFT, AF192394; Triticum aestivum 1-SST, AB029888; Lolium perenne 1-SST, AY245431; Festuca arundinacea 1-SST, AJ297369; Triticum aestivum 1-FFT, AB088409; Lolium perenne 6G-FFT, AF492836; Hordeum vulgare vINV, AJ623275; Triticum aestivum vINV, AJ635225; Lolium perenne vINV, AY082350; Lolium temulentum vINV, AJ532549; Saccharum officinarum vINV, AY302083; Zea mays vINV, AJ563384; Oryza sativa vINV, AF276704.
    Figure Legend Snippet: Phylogenetic tree of FTs and vacuolar invertases (vINV) of Poaceae species based on predicted amino acid sequences. Scale bar indicates branch length. Genbank accession numbers of genes used in this analysis are: Hordeum vulgare 6-SFT, X83233; Triticum aestivum 6-SFT, AB029887; Agropyron cristatum 6-SFT, AF211253; Lolium perenne 6-FT, AF494041; Lolium temulentum 6-FT, AJ532550; Poa secunda 6-SFT, AF192394; Triticum aestivum 1-SST, AB029888; Lolium perenne 1-SST, AY245431; Festuca arundinacea 1-SST, AJ297369; Triticum aestivum 1-FFT, AB088409; Lolium perenne 6G-FFT, AF492836; Hordeum vulgare vINV, AJ623275; Triticum aestivum vINV, AJ635225; Lolium perenne vINV, AY082350; Lolium temulentum vINV, AJ532549; Saccharum officinarum vINV, AY302083; Zea mays vINV, AJ563384; Oryza sativa vINV, AF276704.

    Techniques Used:

    Alignment of the amino acid sequences of 6-SFTs and putative 6-FTs from Poaceae plants. Bars indicate the three conserved motifs that are crucial for β-fructosidase catalysis. The arrow indicates the predicted N-terminus of the mature PpFT1 protein.
    Figure Legend Snippet: Alignment of the amino acid sequences of 6-SFTs and putative 6-FTs from Poaceae plants. Bars indicate the three conserved motifs that are crucial for β-fructosidase catalysis. The arrow indicates the predicted N-terminus of the mature PpFT1 protein.

    Techniques Used:

    19) Product Images from "Forebrain‐Specific Transgene Rescue of 11β‐HSD1 Associates with Impaired Spatial Memory and Reduced Hippocampal Brain‐Derived Neurotrophic Factor mRNA Levels in Aged 11β‐HSD1 Deficient Mice"

    Article Title: Forebrain‐Specific Transgene Rescue of 11β‐HSD1 Associates with Impaired Spatial Memory and Reduced Hippocampal Brain‐Derived Neurotrophic Factor mRNA Levels in Aged 11β‐HSD1 Deficient Mice

    Journal: Journal of Neuroendocrinology

    doi: 10.1111/jne.12447

    Effect of forebrain‐specific ‘rescue’ of 11β‐hydroxysteroid dehydrogenase type 1 (11β‐ HSD 1) in aged hsd11b1 −/− mice on brain‐derived neurotrophic factor (BDNF) mRNA levels in the hippocampus and cortex. ( a ) Autoradiograph showing distribution of BDNF mRNA in coronal section at the level of the dorsal hippocampus from a control adult C57 BL 6/J brain. ( b ) BDNF mRNA levels in hippocampal subregions and cortex of young 6‐month‐old and aged 24‐month‐old C57 BL 6/J control mice. ( c ) BDNF mRNA levels in hippocampal subregions and cortex of aged 24‐month‐old wild type ( WT ), hsd11b1 −/− ( HSD 1 KO ) and Tg+ HSD 1 KO mice (n = 6–7 per genotype). Data are the mean ± SEM . ( d ) Representative dark‐field photomicrographs of in situ hybridisation showing BDNF mRNA in the CA 3 subregion of the hippocampus from wild type ( WT ; young 6‐month‐old and aged 24‐month‐old) aged HSD 1 KO and aged Tg+ HSD 1 KO mice. Autoradiographic silver grains appear white. Post‐hoc Bonferroni's multiple comparisons test adjusted P‐values: ***P
    Figure Legend Snippet: Effect of forebrain‐specific ‘rescue’ of 11β‐hydroxysteroid dehydrogenase type 1 (11β‐ HSD 1) in aged hsd11b1 −/− mice on brain‐derived neurotrophic factor (BDNF) mRNA levels in the hippocampus and cortex. ( a ) Autoradiograph showing distribution of BDNF mRNA in coronal section at the level of the dorsal hippocampus from a control adult C57 BL 6/J brain. ( b ) BDNF mRNA levels in hippocampal subregions and cortex of young 6‐month‐old and aged 24‐month‐old C57 BL 6/J control mice. ( c ) BDNF mRNA levels in hippocampal subregions and cortex of aged 24‐month‐old wild type ( WT ), hsd11b1 −/− ( HSD 1 KO ) and Tg+ HSD 1 KO mice (n = 6–7 per genotype). Data are the mean ± SEM . ( d ) Representative dark‐field photomicrographs of in situ hybridisation showing BDNF mRNA in the CA 3 subregion of the hippocampus from wild type ( WT ; young 6‐month‐old and aged 24‐month‐old) aged HSD 1 KO and aged Tg+ HSD 1 KO mice. Autoradiographic silver grains appear white. Post‐hoc Bonferroni's multiple comparisons test adjusted P‐values: ***P

    Techniques Used: Mouse Assay, Derivative Assay, Autoradiography, In Situ, Hybridization

    20) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    Blocking efficiency of three kinds of blocked primers in PCR amplifications mediated by Taq DNA polymerase and high-fidelity DNA polymerase. (a) Primer and template sets. WT and Mu indicate wild-type and mutant types, respectively. The 3'-blocked forward primer completely matches the WT sequence but forms a mismatch with the Mu sequence (Mu*1) at the 3'-end. The reverse primer matches both the WT and Mu sequences. The 3'-terminal nucleotide of the blocked primer is blocked with-Pi or-Amino C6, or replaced with ddCTP. (b) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by Taq DNA polymerase. (c) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by high-fidelity DNA polymerase (KOD FX). (d) Discrimination efficiency of the typical proofreading-PCR medicated by various amounts (0.5, 1, 1.5 and 2 U) of KOD FX DNA polymerase using the ddC-blocked primer. All PCR amplifications were performed in a total volume of 20 μL containing 1 U of Taq or KOD FX DNA polymerase (Panel b and c) or various amounts of KOD FX DNA polymerase (Panel d). The cycling conditions consisted of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. NTC: no-template control.
    Figure Legend Snippet: Blocking efficiency of three kinds of blocked primers in PCR amplifications mediated by Taq DNA polymerase and high-fidelity DNA polymerase. (a) Primer and template sets. WT and Mu indicate wild-type and mutant types, respectively. The 3'-blocked forward primer completely matches the WT sequence but forms a mismatch with the Mu sequence (Mu*1) at the 3'-end. The reverse primer matches both the WT and Mu sequences. The 3'-terminal nucleotide of the blocked primer is blocked with-Pi or-Amino C6, or replaced with ddCTP. (b) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by Taq DNA polymerase. (c) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by high-fidelity DNA polymerase (KOD FX). (d) Discrimination efficiency of the typical proofreading-PCR medicated by various amounts (0.5, 1, 1.5 and 2 U) of KOD FX DNA polymerase using the ddC-blocked primer. All PCR amplifications were performed in a total volume of 20 μL containing 1 U of Taq or KOD FX DNA polymerase (Panel b and c) or various amounts of KOD FX DNA polymerase (Panel d). The cycling conditions consisted of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. NTC: no-template control.

    Techniques Used: Blocking Assay, Polymerase Chain Reaction, Mutagenesis, Sequencing

    Detection of rifampin-resistant mutations in TB using the modified PR-PCR and next-generation sequencing. (a) Primer and template sequences. TB-NC is a negative control plasmid containing the rpoB gene fragment without any rifampin-sensitive mutations. TB-H526Y and TB-S531L are positive control plasmids containing the rpoB gene fragment with the H526Y(CAC→TAC) and S531L(TCG→TTG) mutations, respectively. (b) Gel electrophoresis of the amplicons for the two mutations in the TB rpoB gene using the modified PR-PCR. (c) Detection of two rifampin-resistant TB mutants using 454 high-throughput sequencing.
    Figure Legend Snippet: Detection of rifampin-resistant mutations in TB using the modified PR-PCR and next-generation sequencing. (a) Primer and template sequences. TB-NC is a negative control plasmid containing the rpoB gene fragment without any rifampin-sensitive mutations. TB-H526Y and TB-S531L are positive control plasmids containing the rpoB gene fragment with the H526Y(CAC→TAC) and S531L(TCG→TTG) mutations, respectively. (b) Gel electrophoresis of the amplicons for the two mutations in the TB rpoB gene using the modified PR-PCR. (c) Detection of two rifampin-resistant TB mutants using 454 high-throughput sequencing.

    Techniques Used: Modification, Polymerase Chain Reaction, Next-Generation Sequencing, Negative Control, Plasmid Preparation, Positive Control, Nucleic Acid Electrophoresis

    Optimization of annealing temperature and amount of high-fidelity DNA polymerase in the modified PR-PCR. (a) Discrimination efficiency of the modified PR-PCR for known mutations using a mixture of 1 U of Taq DNA polymerase and various amount (0.05, 0.1 and 0.3 U) of KOD FX DNA polymerase in the reaction. The PCR amplifications were performed in a total volume of 20 μL under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. (b) Optimization of annealing temperature for the modified PR-PCR using a mixture of 1 U of Taq DNA polymerase and 0.1 U of KOD FX DNA polymerase. As the optimal discrimination efficiency was obtained when the annealing temperature was within a scope of 61.8°C–62.6°C, the results from 50°C to 61.2°C were not shown. The temperature 62.2°C was selected for subsequent experiments. (c) Optimization of amount of high-fidelity DNA polymerase for the modified PR-PCR with an input of 1 U of Taq DNA polymerase at an annealing temperature of 62.2°C.
    Figure Legend Snippet: Optimization of annealing temperature and amount of high-fidelity DNA polymerase in the modified PR-PCR. (a) Discrimination efficiency of the modified PR-PCR for known mutations using a mixture of 1 U of Taq DNA polymerase and various amount (0.05, 0.1 and 0.3 U) of KOD FX DNA polymerase in the reaction. The PCR amplifications were performed in a total volume of 20 μL under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. (b) Optimization of annealing temperature for the modified PR-PCR using a mixture of 1 U of Taq DNA polymerase and 0.1 U of KOD FX DNA polymerase. As the optimal discrimination efficiency was obtained when the annealing temperature was within a scope of 61.8°C–62.6°C, the results from 50°C to 61.2°C were not shown. The temperature 62.2°C was selected for subsequent experiments. (c) Optimization of amount of high-fidelity DNA polymerase for the modified PR-PCR with an input of 1 U of Taq DNA polymerase at an annealing temperature of 62.2°C.

    Techniques Used: Modification, Polymerase Chain Reaction

    Sensitivity and selectivity of the modified PR-PCR for mutation detection using a fusion-blocked primer and adaptor. (a) Diagram of the fusion-blocked primer and adaptor used in the reactions. (b) Primer and adaptor sequences. (c) Sensitivity of the modified PR-PCR. (d) Selectivity of the modified PR-PCR. All reactions were performed in a total volume of 25 μL containing a mixture of 1 μL of gDNA template at 50 ng/μL, 2.5 μL of (10×) Taq PCR buffer, 0.2 mM dNTPs, 0.15 U of PrimeSTAR HS DNA polymerase, 0.75 U of Taq DNA polymerase, 1 μL of DMSO and 0.3 μM each of the reverse primer, fusion-blocked forward primer and adaptor. The DNA templates were prepared by mixing different amounts of MCF-7 (WT) gDNA (from 0 to 50 ng) among HCC1937 (mutant type, MT) gDNA (from 50 to 0 ng) with concentrations from 0 to 100%. The reactions were performed under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 98°C for 10 s, annealing at 59°C for 30 s and extension at 72°C for 20 s.
    Figure Legend Snippet: Sensitivity and selectivity of the modified PR-PCR for mutation detection using a fusion-blocked primer and adaptor. (a) Diagram of the fusion-blocked primer and adaptor used in the reactions. (b) Primer and adaptor sequences. (c) Sensitivity of the modified PR-PCR. (d) Selectivity of the modified PR-PCR. All reactions were performed in a total volume of 25 μL containing a mixture of 1 μL of gDNA template at 50 ng/μL, 2.5 μL of (10×) Taq PCR buffer, 0.2 mM dNTPs, 0.15 U of PrimeSTAR HS DNA polymerase, 0.75 U of Taq DNA polymerase, 1 μL of DMSO and 0.3 μM each of the reverse primer, fusion-blocked forward primer and adaptor. The DNA templates were prepared by mixing different amounts of MCF-7 (WT) gDNA (from 0 to 50 ng) among HCC1937 (mutant type, MT) gDNA (from 50 to 0 ng) with concentrations from 0 to 100%. The reactions were performed under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 98°C for 10 s, annealing at 59°C for 30 s and extension at 72°C for 20 s.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    21) Product Images from "ToxR Recognizes a Direct Repeat Element in the toxT, ompU, ompT, and ctxA Promoters of Vibrio cholerae To Regulate Transcription"

    Article Title: ToxR Recognizes a Direct Repeat Element in the toxT, ompU, ompT, and ctxA Promoters of Vibrio cholerae To Regulate Transcription

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00889-12

    Models for the role of ToxR in TcpP-mediated toxT activation. (A) As previously described, the toxT ). (B) In the “hand-holding” model, ToxR and TcpP interact in the inner membrane of V. cholerae ), and then ToxR escorts TcpP to the toxT promoter where ToxR relieves H-NS repression and maintains interaction with TcpP while TcpP stimulates transcription. (C) In the “catch and release” model, ToxR also interacts with TcpP and recruits TcpP to the toxT ). (D) In the “promoter alteration” model, interaction between ToxR and TcpP is not required for toxT activation; rather, ToxR binding to the toxT promoter displaces H-NS and alters the toxT promoter architecture such that a normally weak TcpP-binding site is altered in some way to facilitate enhanced TcpP binding, thus allowing TcpP-mediated activation of the toxT promoter. (E) In the “membrane recruitment” model, again interaction between ToxR and TcpP is not required, but the role of ToxR is to simply recruit the toxT promoter to the membrane where TcpP has easier access to its DNA-binding site. This model takes into account the fact that TcpP binding to the toxT promoter requires higher concentrations of V. cholerae ) and the fact that membrane localization was previously shown to be required for ToxR to facilitate TcpP-mediated toxT ).
    Figure Legend Snippet: Models for the role of ToxR in TcpP-mediated toxT activation. (A) As previously described, the toxT ). (B) In the “hand-holding” model, ToxR and TcpP interact in the inner membrane of V. cholerae ), and then ToxR escorts TcpP to the toxT promoter where ToxR relieves H-NS repression and maintains interaction with TcpP while TcpP stimulates transcription. (C) In the “catch and release” model, ToxR also interacts with TcpP and recruits TcpP to the toxT ). (D) In the “promoter alteration” model, interaction between ToxR and TcpP is not required for toxT activation; rather, ToxR binding to the toxT promoter displaces H-NS and alters the toxT promoter architecture such that a normally weak TcpP-binding site is altered in some way to facilitate enhanced TcpP binding, thus allowing TcpP-mediated activation of the toxT promoter. (E) In the “membrane recruitment” model, again interaction between ToxR and TcpP is not required, but the role of ToxR is to simply recruit the toxT promoter to the membrane where TcpP has easier access to its DNA-binding site. This model takes into account the fact that TcpP binding to the toxT promoter requires higher concentrations of V. cholerae ) and the fact that membrane localization was previously shown to be required for ToxR to facilitate TcpP-mediated toxT ).

    Techniques Used: Activation Assay, Binding Assay

    ToxR fails to bind or activate a toxT-lacZ derivative containing the degenerate ToxR-binding site from −69 to −56. (A) toxT promoter derivatives driving lacZ expression were tested for activation in wild-type V. cholerae (O395) or the toxR mutant strain EK307 with or without overexpression of ToxRS from plasmid pVJ21. n = 6. (B) Electrophoretic mobility shift analysis of full-length (−172 to +45), −100 to +45, −81 to +45, and −46 to + 45 toxT derivatives with increasing concentrations of ToxR-containing membranes shows the degenerate ToxR-binding site from −69 to −56 has weak ToxR binding capacity. Negative-control gel shifting with membranes lacking ToxR (ToxR − ) was also tested and showed minimal background. DNA bound by membrane-localized ToxR is retained in the well of the gel. The percentage of shifting by membranes is indicated under each lane as determined by ImageJ.
    Figure Legend Snippet: ToxR fails to bind or activate a toxT-lacZ derivative containing the degenerate ToxR-binding site from −69 to −56. (A) toxT promoter derivatives driving lacZ expression were tested for activation in wild-type V. cholerae (O395) or the toxR mutant strain EK307 with or without overexpression of ToxRS from plasmid pVJ21. n = 6. (B) Electrophoretic mobility shift analysis of full-length (−172 to +45), −100 to +45, −81 to +45, and −46 to + 45 toxT derivatives with increasing concentrations of ToxR-containing membranes shows the degenerate ToxR-binding site from −69 to −56 has weak ToxR binding capacity. Negative-control gel shifting with membranes lacking ToxR (ToxR − ) was also tested and showed minimal background. DNA bound by membrane-localized ToxR is retained in the well of the gel. The percentage of shifting by membranes is indicated under each lane as determined by ImageJ.

    Techniques Used: Binding Assay, Expressing, Activation Assay, Mutagenesis, Over Expression, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Negative Control

    DNA sequence of the V. cholerae classical strain O395 promoter-proximal region of the toxT promoter and ToxR-dependent activation of single-base-pair substitutions. (A) Nucleotides are numbered relative to the toxT ). The solid gray arrows above the sequence indicate the position of the putative 5′-TNAAA-N 5 ). Single-nucleotide substitutions generated within the toxT promoter region from −100 to −57 are indicated on the bottom line in italics. (B) Effects of ToxR-binding site mutations on toxT-lacZ activity in wild-type V. cholerae strain O395. Strains carrying a plasmid-borne wild-type toxT-lacZ fusion (−172 to +45), single-base-pair substitution toxT promoter mutants, promoter deletion derivatives, or empty vector (promoterless lacZ vector, pTG24) were assessed for β-galactosidase activity. The positions of substitutions and endpoints are indicated relative to the toxT transcription start site. Error bars represent the standard deviations for each data set. *, P
    Figure Legend Snippet: DNA sequence of the V. cholerae classical strain O395 promoter-proximal region of the toxT promoter and ToxR-dependent activation of single-base-pair substitutions. (A) Nucleotides are numbered relative to the toxT ). The solid gray arrows above the sequence indicate the position of the putative 5′-TNAAA-N 5 ). Single-nucleotide substitutions generated within the toxT promoter region from −100 to −57 are indicated on the bottom line in italics. (B) Effects of ToxR-binding site mutations on toxT-lacZ activity in wild-type V. cholerae strain O395. Strains carrying a plasmid-borne wild-type toxT-lacZ fusion (−172 to +45), single-base-pair substitution toxT promoter mutants, promoter deletion derivatives, or empty vector (promoterless lacZ vector, pTG24) were assessed for β-galactosidase activity. The positions of substitutions and endpoints are indicated relative to the toxT transcription start site. Error bars represent the standard deviations for each data set. *, P

    Techniques Used: Sequencing, Activation Assay, Generated, Binding Assay, Activity Assay, Plasmid Preparation

    The ToxR consensus-binding site is required for ToxR-mediated activation of the ompU and ctxA promoters and repression of the ompT promoter. (A) Location of consensus ToxR-binding sites in the ompU , ctxA , and ompT promoters. Nucleotides comprising potential ToxR-binding sites are in bold, while the opposite strand sequences, matching the toxT promoter consensus ToxR-binding site, are shown in gray. Those nucleotides targeted for mutagenesis are highlighted in gray and underlined. (B) Effects of transversion mutations on ToxR-mediated activation of the ompU promoter in wild-type V. cholerae or the toxR mutant strain, EK307. (C) Effect of mutations in the consensus ToxR-binding site within the promoter-proximal heptad repeat of the ctxA promoter. ctxA-lacZ expression was measured in a Δ toxT strain (ToxR dependent) or wild-type V. cholerae O395 (ToxT dependent). (D) Effects of ompT transversion mutations on ToxR-mediated repression of the ompT promoter in wild-type V. cholerae or the toxR mutant strain, EK307. *, P
    Figure Legend Snippet: The ToxR consensus-binding site is required for ToxR-mediated activation of the ompU and ctxA promoters and repression of the ompT promoter. (A) Location of consensus ToxR-binding sites in the ompU , ctxA , and ompT promoters. Nucleotides comprising potential ToxR-binding sites are in bold, while the opposite strand sequences, matching the toxT promoter consensus ToxR-binding site, are shown in gray. Those nucleotides targeted for mutagenesis are highlighted in gray and underlined. (B) Effects of transversion mutations on ToxR-mediated activation of the ompU promoter in wild-type V. cholerae or the toxR mutant strain, EK307. (C) Effect of mutations in the consensus ToxR-binding site within the promoter-proximal heptad repeat of the ctxA promoter. ctxA-lacZ expression was measured in a Δ toxT strain (ToxR dependent) or wild-type V. cholerae O395 (ToxT dependent). (D) Effects of ompT transversion mutations on ToxR-mediated repression of the ompT promoter in wild-type V. cholerae or the toxR mutant strain, EK307. *, P

    Techniques Used: Binding Assay, Activation Assay, Mutagenesis, Expressing

    22) Product Images from "Effect of Leflunomide, Cidofovir and Ciprofloxacin on Replication of BKPyV in a Salivary Gland In Vitro Culture System"

    Article Title: Effect of Leflunomide, Cidofovir and Ciprofloxacin on Replication of BKPyV in a Salivary Gland In Vitro Culture System

    Journal: Antiviral research

    doi: 10.1016/j.antiviral.2015.02.002

    Effect of drug treatment on lab-strain and patient-derived BKPy virus progeny release from human salivary gland cells HSG cells were infected with ( A ) lab-strain BKPyV (VR837) or ( B ) BKPyV isolated from the saliva of two HIVSGD patients and a lab-adapted virus strain (MM), or (C) BKPyV isolated from urine of a lung transplant patient, and treated with drug as described in the materials and methods. At stated times post infection supernatant was collected, Dnase-treated and qPCR performed for TAg and VP1 (data not shown) DNA copy no. A standard curve (data not shown) was constructed using a plasmid coding for BKPyV whole genome. The error bars represent the SD and p-value calculated using the t-test.
    Figure Legend Snippet: Effect of drug treatment on lab-strain and patient-derived BKPy virus progeny release from human salivary gland cells HSG cells were infected with ( A ) lab-strain BKPyV (VR837) or ( B ) BKPyV isolated from the saliva of two HIVSGD patients and a lab-adapted virus strain (MM), or (C) BKPyV isolated from urine of a lung transplant patient, and treated with drug as described in the materials and methods. At stated times post infection supernatant was collected, Dnase-treated and qPCR performed for TAg and VP1 (data not shown) DNA copy no. A standard curve (data not shown) was constructed using a plasmid coding for BKPyV whole genome. The error bars represent the SD and p-value calculated using the t-test.

    Techniques Used: Derivative Assay, Infection, Isolation, Real-time Polymerase Chain Reaction, Construct, Plasmid Preparation

    23) 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:

    Production of the first generation GFP-mzNL4-3 virions. In contrast to the cells transfected with pUC19 control plasmid (A1-A3), cells co-transfected with pmzNL4-3/GFP and helper plasmids (pSPAX2, pMD2G) produced large round syncytia (B1, shown with arrows)
    Figure Legend Snippet: Production of the first generation GFP-mzNL4-3 virions. In contrast to the cells transfected with pUC19 control plasmid (A1-A3), cells co-transfected with pmzNL4-3/GFP and helper plasmids (pSPAX2, pMD2G) produced large round syncytia (B1, shown with arrows)

    Techniques Used: Transfection, Plasmid Preparation, Produced

    . 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

    24) Product Images from "Crosstalk between Immune Cells and Adipocytes Requires Both Paracrine Factors and Cell Contact to Modify Cytokine Secretion"

    Article Title: Crosstalk between Immune Cells and Adipocytes Requires Both Paracrine Factors and Cell Contact to Modify Cytokine Secretion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077306

    Rates of cytokine secretion and overall levels are affected by direct contact between adipocytes and splenocytes. Differentiated 3T3-L1 adipocytes and isolated murine splenocytes were co-cultured with no contact (separated by a 0.4 µm transwell filter system) (dashed lines) or with direct contact (solid lines) and incubated with LPS (1 µg/mL) for 0, 8, 24 and 48 h. Cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were measured in culture media following these incubation times by capture ELISA. All experimental points were performed in triplicate.
    Figure Legend Snippet: Rates of cytokine secretion and overall levels are affected by direct contact between adipocytes and splenocytes. Differentiated 3T3-L1 adipocytes and isolated murine splenocytes were co-cultured with no contact (separated by a 0.4 µm transwell filter system) (dashed lines) or with direct contact (solid lines) and incubated with LPS (1 µg/mL) for 0, 8, 24 and 48 h. Cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were measured in culture media following these incubation times by capture ELISA. All experimental points were performed in triplicate.

    Techniques Used: Isolation, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

    Cell contact-mediated enhancement of IL-6 and MCP-1 secretion requires TNFα signaling. (A and B) Differentiated adipocytes or murine splenocytes (black bars, isolated from wild type mice; gray bars, isolated from TNFα -/- mice) were cultured alone (individual culture, columns 1 and 2) or in co-culture with no contact (columns 3 and 4) or direct contact (columns 5 and 6) as in Figure 1 . Cells were incubated in the absence (−) or presence (+) of LPS (1 µg/mL) for 24 h as indicated. (C and D) Wild type (WT) or TNFα -/- (TNFα KO) splenocytes were incubated with adipocytes with direct contact in the presence of LPS (1 µg/mL) and co-cultures were supplemented with 0, 300 pg/mL or 10 ng/mL purified murine TNFα as indicated. Secreted IL-6 (A and C) and MCP-1 (B and D) were quantified by capture ELISA. Experimental points were measured in triplicate (A and B) or duplicate (C and D) for calculation of means and standard deviations. For (A and B) comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact from WT and TNFα KO were calculated using ANOVA and followed by the post-hoc Bonferroni test. For C and D all experimental points were compared statistically by ANOVA followed by the Bonferroni post-hoc test, and differed significantly (p
    Figure Legend Snippet: Cell contact-mediated enhancement of IL-6 and MCP-1 secretion requires TNFα signaling. (A and B) Differentiated adipocytes or murine splenocytes (black bars, isolated from wild type mice; gray bars, isolated from TNFα -/- mice) were cultured alone (individual culture, columns 1 and 2) or in co-culture with no contact (columns 3 and 4) or direct contact (columns 5 and 6) as in Figure 1 . Cells were incubated in the absence (−) or presence (+) of LPS (1 µg/mL) for 24 h as indicated. (C and D) Wild type (WT) or TNFα -/- (TNFα KO) splenocytes were incubated with adipocytes with direct contact in the presence of LPS (1 µg/mL) and co-cultures were supplemented with 0, 300 pg/mL or 10 ng/mL purified murine TNFα as indicated. Secreted IL-6 (A and C) and MCP-1 (B and D) were quantified by capture ELISA. Experimental points were measured in triplicate (A and B) or duplicate (C and D) for calculation of means and standard deviations. For (A and B) comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact from WT and TNFα KO were calculated using ANOVA and followed by the post-hoc Bonferroni test. For C and D all experimental points were compared statistically by ANOVA followed by the Bonferroni post-hoc test, and differed significantly (p

    Techniques Used: Isolation, Mouse Assay, Cell Culture, Co-Culture Assay, Incubation, Purification, Enzyme-linked Immunosorbent Assay

    Splenocytes and adipocytes differentially express pro-inflammatory markers. Differentiated 3T3-L1 adipocytes were co-cultured in direct contact with GFP-expressing murine splenocytes and activated by incubation with LPS (1 µg/mL) for 24 h. Cells were sorted for GFP-positive (splenocyte) and negative (adipocyte) cells by FACS. (A) Representative FACS is shown, with GFP-negative cells gated in R1 and GFP-positive cells gated in R2. (B) Quantitative real-time PCR (qRT-PCR) was used to measure adiponectin and F4/80 expression, specific markers for adipocytes and splenocytes, respectively, to confirm efficiency of cell sorting. (C) TNFα, IL-6 and MCP-1 mRNA expression levels were quantified by qRT-PCR in splenocytes and adipocytes following cell sorting to distinguish individual cytokine expression patterns. All qRT-PCR values were normalized to values obtained for 36B4, a ribosomal 60S subunit protein. Experimental points were measured in duplicate (B and C) for calculation of means and standard deviations. In (C), statistical significance of differing secretion levels between splenocytes and adipocytes was determined by an unpaired two-tailed Student's t-test. A significant effect was accepted when p
    Figure Legend Snippet: Splenocytes and adipocytes differentially express pro-inflammatory markers. Differentiated 3T3-L1 adipocytes were co-cultured in direct contact with GFP-expressing murine splenocytes and activated by incubation with LPS (1 µg/mL) for 24 h. Cells were sorted for GFP-positive (splenocyte) and negative (adipocyte) cells by FACS. (A) Representative FACS is shown, with GFP-negative cells gated in R1 and GFP-positive cells gated in R2. (B) Quantitative real-time PCR (qRT-PCR) was used to measure adiponectin and F4/80 expression, specific markers for adipocytes and splenocytes, respectively, to confirm efficiency of cell sorting. (C) TNFα, IL-6 and MCP-1 mRNA expression levels were quantified by qRT-PCR in splenocytes and adipocytes following cell sorting to distinguish individual cytokine expression patterns. All qRT-PCR values were normalized to values obtained for 36B4, a ribosomal 60S subunit protein. Experimental points were measured in duplicate (B and C) for calculation of means and standard deviations. In (C), statistical significance of differing secretion levels between splenocytes and adipocytes was determined by an unpaired two-tailed Student's t-test. A significant effect was accepted when p

    Techniques Used: Cell Culture, Expressing, Incubation, FACS, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

    Direct contact between splenocytes and adipocytes alters secreted levels of inflammatory cytokines. Differentiated 3T3-L1 adipocytes (columns 1 and 2) or isolated murine splenocytes (columns 3 and 4) were cultured alone or together with either direct contact (columns 7 and 8) or no contact (cells separated by a 0.4 µm transwell filter system; columns 5 and 6). Cells were additionally incubated in the absence (-) (columns 1, 3, 5 and 7) or presence (+) (columns 2, 4, 6 and 8) of LPS (1 µg/mL) for 24 h. Secreted cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were quantified by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. Comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact were calculated using ANOVA and followed by the post-hoc Bonferroni test. A significant effect was accepted when p
    Figure Legend Snippet: Direct contact between splenocytes and adipocytes alters secreted levels of inflammatory cytokines. Differentiated 3T3-L1 adipocytes (columns 1 and 2) or isolated murine splenocytes (columns 3 and 4) were cultured alone or together with either direct contact (columns 7 and 8) or no contact (cells separated by a 0.4 µm transwell filter system; columns 5 and 6). Cells were additionally incubated in the absence (-) (columns 1, 3, 5 and 7) or presence (+) (columns 2, 4, 6 and 8) of LPS (1 µg/mL) for 24 h. Secreted cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were quantified by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. Comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact were calculated using ANOVA and followed by the post-hoc Bonferroni test. A significant effect was accepted when p

    Techniques Used: Isolation, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

    NF-κB intracellular signaling pathway participates in the paracrine and cell contact-mediated enhancement of IL-6 and MCP-1 secretion. (A) Isolated splenocytes were incubated with LPS (1 µg/mL) in the absence (-) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of TNFα were measured by capture ELISA. (B and C) Differentiated 3T3-L1 adipocytes were co-cultured with isolated splenocytes with either no contact or direct contact as indicated. Cells were stimulated with LPS (1 µg/mL) in the absence (−) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of IL-6 (B) or MCP-1 (C) were measured by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. In (A) an unpaired two-tailed Student's t test was used to compare Bay11-7082 treated and untreated LPS-stimulated splenocytes. Comparisons between co-cultures (B and C) were calculated using ANOVA followed by the post-hoc Bonferroni test.
    Figure Legend Snippet: NF-κB intracellular signaling pathway participates in the paracrine and cell contact-mediated enhancement of IL-6 and MCP-1 secretion. (A) Isolated splenocytes were incubated with LPS (1 µg/mL) in the absence (-) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of TNFα were measured by capture ELISA. (B and C) Differentiated 3T3-L1 adipocytes were co-cultured with isolated splenocytes with either no contact or direct contact as indicated. Cells were stimulated with LPS (1 µg/mL) in the absence (−) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of IL-6 (B) or MCP-1 (C) were measured by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. In (A) an unpaired two-tailed Student's t test was used to compare Bay11-7082 treated and untreated LPS-stimulated splenocytes. Comparisons between co-cultures (B and C) were calculated using ANOVA followed by the post-hoc Bonferroni test.

    Techniques Used: Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

    25) Product Images from "Evaluation of Specificity of BP3385 for Bordetella pertussis Detection ▿"

    Article Title: Evaluation of Specificity of BP3385 for Bordetella pertussis Detection ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00945-10

    Southern blot with EcoRV-digested Bordetella genomic DNA hybridized to a BP3385 probe. Lanes: 1, B. pertussis Tohama; 2, B. bronchiseptica RB50; 3, B. bronchiseptica PV6; 4, B. bronchiseptica OSU-083; 5, B. hinzii ATCC 51783; and 6, B. hinzii 4135. Relative positions of DNA size markers are indicated to the left.
    Figure Legend Snippet: Southern blot with EcoRV-digested Bordetella genomic DNA hybridized to a BP3385 probe. Lanes: 1, B. pertussis Tohama; 2, B. bronchiseptica RB50; 3, B. bronchiseptica PV6; 4, B. bronchiseptica OSU-083; 5, B. hinzii ATCC 51783; and 6, B. hinzii 4135. Relative positions of DNA size markers are indicated to the left.

    Techniques Used: Southern Blot

    26) Product Images from "Molecular cloning and characterization of a novel tomato xylosyltransferase specific for gentisic acid"

    Article Title: Molecular cloning and characterization of a novel tomato xylosyltransferase specific for gentisic acid

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erq234

    Xylosyltransferase activity of recombinant GAGT. Left panel: GAGT cDNA was expressed in Pichia pastoris cells under the control of a methanol-inducible promoter (see the Materials and methods). Extracts from the uninduced (lane 1) and methanol-induced cells (lane 2) were incubated with UDP[ 14 C]-xylose and GA. Lane 3 corresponds to UDP[ 14 C]-xylose. Right panel: samples of UDP-xylose (lane 1), standard GA-5- O -β- D -xyloside, previously obtained in our laboratory (lane 2; Fayos et al. , 2006 ) and the GA xyloside produced using the crude tomato leaf extracts (lane 3) were separated by TLC under the same conditions, and were chemically revealed as described in the text.
    Figure Legend Snippet: Xylosyltransferase activity of recombinant GAGT. Left panel: GAGT cDNA was expressed in Pichia pastoris cells under the control of a methanol-inducible promoter (see the Materials and methods). Extracts from the uninduced (lane 1) and methanol-induced cells (lane 2) were incubated with UDP[ 14 C]-xylose and GA. Lane 3 corresponds to UDP[ 14 C]-xylose. Right panel: samples of UDP-xylose (lane 1), standard GA-5- O -β- D -xyloside, previously obtained in our laboratory (lane 2; Fayos et al. , 2006 ) and the GA xyloside produced using the crude tomato leaf extracts (lane 3) were separated by TLC under the same conditions, and were chemically revealed as described in the text.

    Techniques Used: Activity Assay, Recombinant, Incubation, Produced, Thin Layer Chromatography

    27) Product Images from "Phenotypic and functional evaluation of CD3+CD4−CD8− T cells in human CD8 immunodeficiency"

    Article Title: Phenotypic and functional evaluation of CD3+CD4−CD8− T cells in human CD8 immunodeficiency

    Journal: Haematologica

    doi: 10.3324/haematol.2011.041301

    Analysis of TREC and TCR V repertoire in patients’ DN T cells. (A) Patients CD4 + and DN T cells and control CD4 + and CD8 + T cells were magnetically sorted, and TRECs levels were analyzed by qPCR. (B) TCR V families were analyzed by flow cytometry in gated CD3 + CD4 + T cells from controls and patients. (C) TCR V families were analyzed by flow cytometry in gated CD3 + CD8 + T cells from controls or CD3 + CD4 − CD8 − DN T cells from patients.
    Figure Legend Snippet: Analysis of TREC and TCR V repertoire in patients’ DN T cells. (A) Patients CD4 + and DN T cells and control CD4 + and CD8 + T cells were magnetically sorted, and TRECs levels were analyzed by qPCR. (B) TCR V families were analyzed by flow cytometry in gated CD3 + CD4 + T cells from controls and patients. (C) TCR V families were analyzed by flow cytometry in gated CD3 + CD8 + T cells from controls or CD3 + CD4 − CD8 − DN T cells from patients.

    Techniques Used: Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    28) Product Images from "Modulators of Hepatic Lipoprotein Metabolism Identified in a Search for Small-Molecule Inducers of Tribbles Pseudokinase 1 Expression"

    Article Title: Modulators of Hepatic Lipoprotein Metabolism Identified in a Search for Small-Molecule Inducers of Tribbles Pseudokinase 1 Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120295

    Effects of BRD0418 on expression of selected marker genes in HepG2 ΔTRIB1 :: Neo R cell line carrying chromosomal deletion/disruption of TRIB1 locus. (A) Schematic representation of CRISPR-Cas9 induced inactivation of TRIB1 gene in HepG2 cells. The indicated 22 bp target sequence in exon 2 of TRIB1 gene was used to design Cas9 guide RNA. The replacement of the target sequence in the chromosome with the Neo R gene was induced by co-transfecting cells with the CRISPR-Cas9 plasmid together with the homologous recombination repair template consisting of Neo R gene cassette and two ∼800 bp fragments from the TRIB1 locus that flank the target sequence. Location of PCR primers used for identifying the Geneticin resistant clone that carries the designed chromosomal rearrangement is indicated by arrows. (B) Changes in the steady state transcript levels in HepG2 ΔTRIB1 :: Neo R cells comparing to parental HepG2 cells. The relative expression level of indicated lipid metabolic genes normalized to expression level of GAPDH1 was measured by qRT-PCR 48 hours after plating cells in 384-well plate. The mean values for six replicates ± S.D. ( error bars ) are shown for the representative of three independent experiments. (C) Changes in transcript levels in HepG2 and in HepG2 ΔTRIB1 :: Neo R cells in response to 24 h treatment with 10 μM BRD0418. The values are normalized to DMSO controls for each strain and represent mean fold change for six replicates ± S.D. ( error bars ).
    Figure Legend Snippet: Effects of BRD0418 on expression of selected marker genes in HepG2 ΔTRIB1 :: Neo R cell line carrying chromosomal deletion/disruption of TRIB1 locus. (A) Schematic representation of CRISPR-Cas9 induced inactivation of TRIB1 gene in HepG2 cells. The indicated 22 bp target sequence in exon 2 of TRIB1 gene was used to design Cas9 guide RNA. The replacement of the target sequence in the chromosome with the Neo R gene was induced by co-transfecting cells with the CRISPR-Cas9 plasmid together with the homologous recombination repair template consisting of Neo R gene cassette and two ∼800 bp fragments from the TRIB1 locus that flank the target sequence. Location of PCR primers used for identifying the Geneticin resistant clone that carries the designed chromosomal rearrangement is indicated by arrows. (B) Changes in the steady state transcript levels in HepG2 ΔTRIB1 :: Neo R cells comparing to parental HepG2 cells. The relative expression level of indicated lipid metabolic genes normalized to expression level of GAPDH1 was measured by qRT-PCR 48 hours after plating cells in 384-well plate. The mean values for six replicates ± S.D. ( error bars ) are shown for the representative of three independent experiments. (C) Changes in transcript levels in HepG2 and in HepG2 ΔTRIB1 :: Neo R cells in response to 24 h treatment with 10 μM BRD0418. The values are normalized to DMSO controls for each strain and represent mean fold change for six replicates ± S.D. ( error bars ).

    Techniques Used: Expressing, Marker, CRISPR, Sequencing, Plasmid Preparation, Homologous Recombination, Polymerase Chain Reaction, Quantitative RT-PCR

    29) Product Images from "A dehydrin gene isolated from feral olive enhances drought tolerance in Arabidopsis transgenic plants"

    Article Title: A dehydrin gene isolated from feral olive enhances drought tolerance in Arabidopsis transgenic plants

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.00392

    (A) Relative expression levels of OesDHN , estimated by qRT-PCR, in 35S::OesDHN:GFP lines. The levels of OesDHN were normalized to TIP41 and PP2 housekeeping genes. Data shown are averages of three biological replicates, with error bars representing SD. (B,C) subcellular localization of the Oes DHN protein fused in N and C-terminal GFP under the control of the CaMV35S promoter in stable T2 35S:: OesDHN :GFP line A 4-1, 35S::GFP: OesDHN line C11-5 lines of Arabidopsis with one T-DNA locus. (B) Root maturation zone; (C) Guard cells of stomata, located on the abaxial side of leaves. cm, cell membrane; n, nucleus. dhn1, dhn2, dhn3 indicate three pairs of gene specific primers located in different points of the OesDNH ORF.
    Figure Legend Snippet: (A) Relative expression levels of OesDHN , estimated by qRT-PCR, in 35S::OesDHN:GFP lines. The levels of OesDHN were normalized to TIP41 and PP2 housekeeping genes. Data shown are averages of three biological replicates, with error bars representing SD. (B,C) subcellular localization of the Oes DHN protein fused in N and C-terminal GFP under the control of the CaMV35S promoter in stable T2 35S:: OesDHN :GFP line A 4-1, 35S::GFP: OesDHN line C11-5 lines of Arabidopsis with one T-DNA locus. (B) Root maturation zone; (C) Guard cells of stomata, located on the abaxial side of leaves. cm, cell membrane; n, nucleus. dhn1, dhn2, dhn3 indicate three pairs of gene specific primers located in different points of the OesDNH ORF.

    Techniques Used: Expressing, Quantitative RT-PCR

    30) Product Images from "A novel autoantibody against fibronectin leucine-rich transmembrane protein 2 expressed on the endothelial cell surface identified by retroviral vector system in systemic lupus erythematosus"

    Article Title: A novel autoantibody against fibronectin leucine-rich transmembrane protein 2 expressed on the endothelial cell surface identified by retroviral vector system in systemic lupus erythematosus

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar3897

    Identification of FLRT2 as a novel autoantigen of AECAs . (A) Unsorted, C9, and C18 were stained with isotype control or anti-FLRT2 antibody, followed by secondary antibody, and analyzed with flow cytometry (left). Western blotting of proteins from unsorted, C9, and C18 was performed, and they were stained with anti-FLRT2 antibody followed by secondary antibody (right). Arrows indicate the bands of FLRT2. Both of the two bands are FLRT2 because some of FLRT2 proteins were glycosylated. (B) Expression vector, empty-IRES-GFP, or FLRT2-IRES-GFP was transfected into HEK293T cells, and these cells were stained with 0.5 mg/ml of control IgG or E10-19 IgG, followed by secondary antibody, and analyzed with flow cytometry. Binding activities of IgG to cell-surface FLRT2 were analyzed in histograms (right) by gating for the GFP-positive transfected population (left). (C) HUVECs were stained with isotype control or anti-FLRT2 antibody followed by secondary antibody, and analyzed with flow cytometry (left). Western blotting of proteins from HUVECs was performed, and they were stained with anti-FLRT2 antibody followed by secondary antibody (right). The arrows indicate the bands of FLRT2. (D) HAECs, HRGECs, and HMVEC-Ls were stained with isotype control or anti-FLRT2 antibody followed by secondary antibody, and analyzed with flow cytometry. (E) HUVECs were stained with isotype control, anti-FLRT1 antibody, or anti-FLRT3 antibody followed by secondary antibody, and analyzed with flow cytometry. (F) Expression vector, FLRT1-IRES-GFP (left), or FLRT3-IRES-GFP (right) was transfected into HEK293T cells, and these cells were stained with 0.5 mg/ml of control IgG or E10-19 IgG, followed by secondary antibody, and analyzed with flow cytometry.
    Figure Legend Snippet: Identification of FLRT2 as a novel autoantigen of AECAs . (A) Unsorted, C9, and C18 were stained with isotype control or anti-FLRT2 antibody, followed by secondary antibody, and analyzed with flow cytometry (left). Western blotting of proteins from unsorted, C9, and C18 was performed, and they were stained with anti-FLRT2 antibody followed by secondary antibody (right). Arrows indicate the bands of FLRT2. Both of the two bands are FLRT2 because some of FLRT2 proteins were glycosylated. (B) Expression vector, empty-IRES-GFP, or FLRT2-IRES-GFP was transfected into HEK293T cells, and these cells were stained with 0.5 mg/ml of control IgG or E10-19 IgG, followed by secondary antibody, and analyzed with flow cytometry. Binding activities of IgG to cell-surface FLRT2 were analyzed in histograms (right) by gating for the GFP-positive transfected population (left). (C) HUVECs were stained with isotype control or anti-FLRT2 antibody followed by secondary antibody, and analyzed with flow cytometry (left). Western blotting of proteins from HUVECs was performed, and they were stained with anti-FLRT2 antibody followed by secondary antibody (right). The arrows indicate the bands of FLRT2. (D) HAECs, HRGECs, and HMVEC-Ls were stained with isotype control or anti-FLRT2 antibody followed by secondary antibody, and analyzed with flow cytometry. (E) HUVECs were stained with isotype control, anti-FLRT1 antibody, or anti-FLRT3 antibody followed by secondary antibody, and analyzed with flow cytometry. (F) Expression vector, FLRT1-IRES-GFP (left), or FLRT3-IRES-GFP (right) was transfected into HEK293T cells, and these cells were stained with 0.5 mg/ml of control IgG or E10-19 IgG, followed by secondary antibody, and analyzed with flow cytometry.

    Techniques Used: Staining, Flow Cytometry, Cytometry, Western Blot, Expressing, Plasmid Preparation, Transfection, Binding Assay

    31) Product Images from "Molecular analysis of sister chromatid recombination in mammalian cells"

    Article Title: Molecular analysis of sister chromatid recombination in mammalian cells

    Journal:

    doi: 10.1016/j.dnarep.2004.08.010

    Southern blot analysis of I-SceI-induced BsdR+ colonies. (A–D) Southern blotting was performed on U2OS reporter Clones #18 (A, C) and #24 (B, D) using Pst I (A, B) or Hin dIII (C, D) digested genomic DNA. The probe was the 700 bp GFP cDNA. See restriction
    Figure Legend Snippet: Southern blot analysis of I-SceI-induced BsdR+ colonies. (A–D) Southern blotting was performed on U2OS reporter Clones #18 (A, C) and #24 (B, D) using Pst I (A, B) or Hin dIII (C, D) digested genomic DNA. The probe was the 700 bp GFP cDNA. See restriction

    Techniques Used: Southern Blot, Clone Assay

    PCR analysis of the reporter locus in BsdR+ colonies. (A) Scheme for PCR analysis, showing parental reporter ( top ) and “ GFP triplication” outcome ( bottom ). Duplication of the BsdR cassette in the rearranged reporter allows a 2.6 kb fragment
    Figure Legend Snippet: PCR analysis of the reporter locus in BsdR+ colonies. (A) Scheme for PCR analysis, showing parental reporter ( top ) and “ GFP triplication” outcome ( bottom ). Duplication of the BsdR cassette in the rearranged reporter allows a 2.6 kb fragment

    Techniques Used: Polymerase Chain Reaction

    32) Product Images from "The Alphaherpesvirus US3/ORF66 Protein Kinases Direct Phosphorylation of the Nuclear Matrix Protein Matrin 3 ▿"

    Article Title: The Alphaherpesvirus US3/ORF66 Protein Kinases Direct Phosphorylation of the Nuclear Matrix Protein Matrin 3 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01611-10

    Anti-PKAps profiles for cells infected with alphaherpesviruses (VZV, HSV-1, or PRV) with or without expression of their US3 kinases. MeWo cells were either mock infected, infected at an MOI of 0.1 with VZV.GFP-66 or VZV.GFP-66kd (expressing kinase-inactivated
    Figure Legend Snippet: Anti-PKAps profiles for cells infected with alphaherpesviruses (VZV, HSV-1, or PRV) with or without expression of their US3 kinases. MeWo cells were either mock infected, infected at an MOI of 0.1 with VZV.GFP-66 or VZV.GFP-66kd (expressing kinase-inactivated

    Techniques Used: Infection, Expressing

    33) Product Images from "Change of the Expression of Human Telomerase Reverse Transcriptase mRNA and Human Telomerase RNA after Cisplatin and 5-Fluorouracil Exposure in Head and Neck Squamous Cell Carcinoma Cell Lines"

    Article Title: Change of the Expression of Human Telomerase Reverse Transcriptase mRNA and Human Telomerase RNA after Cisplatin and 5-Fluorouracil Exposure in Head and Neck Squamous Cell Carcinoma Cell Lines

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2007.22.S.S73

    Detection of hTERT mRNA in PNUH-12 ( A ) and SNU-899 ( B ) samples by real-time RT-PCR analysis using the LightCycler. Concomitant detection of PBGD mRNA served as a reference for relative quantification. The normalized hTERT mRNA levels decreased after cisplatin and 5-fluorouracil (5-FU) exposure in both cell lines.
    Figure Legend Snippet: Detection of hTERT mRNA in PNUH-12 ( A ) and SNU-899 ( B ) samples by real-time RT-PCR analysis using the LightCycler. Concomitant detection of PBGD mRNA served as a reference for relative quantification. The normalized hTERT mRNA levels decreased after cisplatin and 5-fluorouracil (5-FU) exposure in both cell lines.

    Techniques Used: Quantitative RT-PCR

    34) Product Images from "Cyclin D regulation of a sexually dimorphic asymmetric cell division"

    Article Title: Cyclin D regulation of a sexually dimorphic asymmetric cell division

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2005.09.004

    q626 , a missense mutation in cyd-1 that delays SGP division
    Figure Legend Snippet: q626 , a missense mutation in cyd-1 that delays SGP division

    Techniques Used: Mutagenesis

    35) Product Images from "Generation of transgenic chickens expressing the human erythropoietin (hEPO) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites"

    Article Title: Generation of transgenic chickens expressing the human erythropoietin (hEPO) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194721

    (A) Structure of the FIV-Ov19-hEPO provirus. CMV/ΔLTR, a hybrid CMV promoter fused to the FIV long terminal repeat devoid of U3 region; Gag, cis-acting sequence helping efficient packaging of virus transcripts; RRE, coding sequence for the rev response element involved in packaging of the virus particles; cPPT, central polypurine track involved in integration of proviruses into the host cell genome; Ov19p, ovalbumin promoter, hEPO, human erythropoietin gene cDNA; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element sequence; ΔLTR, long terminal repeat with deletion of the U3 region to prevent replication-competent virus production after integration into the host cell genome. The approximate position of the probe for Southern blotting is shown just above the hEPO gene. Selected restriction enzyme sites in the provirus sequence are also indicated. In this construct, expression of the hEPO gene is driven by the ovalbumin promoter. Drawing is not to scale. (B C) PCR analysis of G o (B) and G 1 (C) transgenics. lane P, plasmid pFIV-Ov19-hEPO; lane N, non-transgenic control chicken, lane Ov19-1, a founder transgenic rooster of G 0 generation; lanes marked with Ov19-1-1 ~ Ov19-1-4, four transgenic G 1 generation chickens sired by a transgenic G 0 rooster. (D E) Southern blot analysis of G 1 transgenic chickens. Genomic DNA of chickens was double-digested with Spe I and Sbf I (D) or single-digested with Spe I (E), then hybridized with the hEPO gene probe. Lane M, molecular size markers; the remaining lanes are same as indicated in (B C).
    Figure Legend Snippet: (A) Structure of the FIV-Ov19-hEPO provirus. CMV/ΔLTR, a hybrid CMV promoter fused to the FIV long terminal repeat devoid of U3 region; Gag, cis-acting sequence helping efficient packaging of virus transcripts; RRE, coding sequence for the rev response element involved in packaging of the virus particles; cPPT, central polypurine track involved in integration of proviruses into the host cell genome; Ov19p, ovalbumin promoter, hEPO, human erythropoietin gene cDNA; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element sequence; ΔLTR, long terminal repeat with deletion of the U3 region to prevent replication-competent virus production after integration into the host cell genome. The approximate position of the probe for Southern blotting is shown just above the hEPO gene. Selected restriction enzyme sites in the provirus sequence are also indicated. In this construct, expression of the hEPO gene is driven by the ovalbumin promoter. Drawing is not to scale. (B C) PCR analysis of G o (B) and G 1 (C) transgenics. lane P, plasmid pFIV-Ov19-hEPO; lane N, non-transgenic control chicken, lane Ov19-1, a founder transgenic rooster of G 0 generation; lanes marked with Ov19-1-1 ~ Ov19-1-4, four transgenic G 1 generation chickens sired by a transgenic G 0 rooster. (D E) Southern blot analysis of G 1 transgenic chickens. Genomic DNA of chickens was double-digested with Spe I and Sbf I (D) or single-digested with Spe I (E), then hybridized with the hEPO gene probe. Lane M, molecular size markers; the remaining lanes are same as indicated in (B C).

    Techniques Used: Sequencing, Southern Blot, Construct, Expressing, Polymerase Chain Reaction, Plasmid Preparation, Transgenic Assay

    36) Product Images from "Shugoshin forms a specialized chromatin domain at subtelomeres that regulates transcription and replication timing"

    Article Title: Shugoshin forms a specialized chromatin domain at subtelomeres that regulates transcription and replication timing

    Journal: Nature Communications

    doi: 10.1038/ncomms10393

    Sgo2 regulates replication timing at the subtelomeres by limiting Sld3 loading. ( a ) Replication kinetics of the early and late origins in wt and sgo2 Δ cells. The locations of the origins on chromosome 2 are shown schematically (top). ars2004 is an early origin and AT2088 is a late origin. tel-21.2 is located at 21.2 kb from tel2R. The non-origin region ( non-ori ) is located at 30 kb from ars2004 . Temperature-sensitive cdc25-22 mutant cells expressing thymidine kinase and the nucleotide transporter were arrested at the G 2 /M boundary at the restrictive temperature (36 °C) and then released from arrest by a temperature shift to 25 °C in the presence of BrdU (200 μM). At the indicated time points, the amount of HL DNA and LL DNA, which were separated by caesium chloride (CsCl) density gradient centrifugation, was determined by qPCR (middle and bottom). ( b ) Replication of the subtelomeric late origins is affected in sgo2 Δ cells. The strains used in a were released from G 2 /M arrest and their DNA was labelled with BrdU for 90 min in the presence of hydroxyurea (HU; 10 mM). The BrdU-labelled DNA was purified by immunoprecipitation and quantified by quantitative PCR. Each value was normalized to that of ars2004 . Error bars indicate the s.d. ( n =3). Asterisks indicate a significant change as compared with the wt strain ( P
    Figure Legend Snippet: Sgo2 regulates replication timing at the subtelomeres by limiting Sld3 loading. ( a ) Replication kinetics of the early and late origins in wt and sgo2 Δ cells. The locations of the origins on chromosome 2 are shown schematically (top). ars2004 is an early origin and AT2088 is a late origin. tel-21.2 is located at 21.2 kb from tel2R. The non-origin region ( non-ori ) is located at 30 kb from ars2004 . Temperature-sensitive cdc25-22 mutant cells expressing thymidine kinase and the nucleotide transporter were arrested at the G 2 /M boundary at the restrictive temperature (36 °C) and then released from arrest by a temperature shift to 25 °C in the presence of BrdU (200 μM). At the indicated time points, the amount of HL DNA and LL DNA, which were separated by caesium chloride (CsCl) density gradient centrifugation, was determined by qPCR (middle and bottom). ( b ) Replication of the subtelomeric late origins is affected in sgo2 Δ cells. The strains used in a were released from G 2 /M arrest and their DNA was labelled with BrdU for 90 min in the presence of hydroxyurea (HU; 10 mM). The BrdU-labelled DNA was purified by immunoprecipitation and quantified by quantitative PCR. Each value was normalized to that of ars2004 . Error bars indicate the s.d. ( n =3). Asterisks indicate a significant change as compared with the wt strain ( P

    Techniques Used: Mutagenesis, Expressing, Gradient Centrifugation, Real-time Polymerase Chain Reaction, Purification, Immunoprecipitation

    37) Product Images from "Analysis of 2-LTR circle junctions of Viral DNA in infected cells"

    Article Title: Analysis of 2-LTR circle junctions of Viral DNA in infected cells

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-59745-170-3_6

    Quantitation of 2-LTR circle Junction DNA by Real-time PCR. Jurkat cells were infected with WT R3B virus and infected cells DNA was isolated at 2h, 24h and 48h post-infection. Virus infected cell DNA was analyzed for amount of 2-LTR circle junction DNA
    Figure Legend Snippet: Quantitation of 2-LTR circle Junction DNA by Real-time PCR. Jurkat cells were infected with WT R3B virus and infected cells DNA was isolated at 2h, 24h and 48h post-infection. Virus infected cell DNA was analyzed for amount of 2-LTR circle junction DNA

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Infection, Isolation

    38) Product Images from "BRAF and TERT promoter mutations in the aggressiveness of papillary thyroid carcinoma: a study of 653 patients"

    Article Title: BRAF and TERT promoter mutations in the aggressiveness of papillary thyroid carcinoma: a study of 653 patients

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7811

    Distribution of BRAF V600E and TERT promoter C228T and C250T mutations and their relationship in papillary thyroid cancer A total of 653 cases of papillary thyroid cancer were tested for the indicated mutations. Shown are the case numbers and the corresponding mutation status. There was no overlap between the two TERT promoter mutations but there was a considerable overlap between BRAF V600E and the TERT promoter mutations.
    Figure Legend Snippet: Distribution of BRAF V600E and TERT promoter C228T and C250T mutations and their relationship in papillary thyroid cancer A total of 653 cases of papillary thyroid cancer were tested for the indicated mutations. Shown are the case numbers and the corresponding mutation status. There was no overlap between the two TERT promoter mutations but there was a considerable overlap between BRAF V600E and the TERT promoter mutations.

    Techniques Used: Mutagenesis

    39) Product Images from "Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells"

    Article Title: Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms14059751

    Sp1 acted on the motif of VEGF promoter. ( A ) Effects of resistin on Sp1 activation in HO-8910 cells. Cells were incubated with 100 ng/mL resistin for 2 h and whole-cell lysates underwent Western blotting to detect the phosphorylation of Sp1 at either Thr-453 or Thr-739; ( B ) Scheme of the series of plasmid constructs containing dissected fragments of VEGF promoter; ( C ) Relative luciferase activity in transient transfection assays using the series of plasmid constructs (schematically shown in B ) and pSp1-luciferase reporter. Relative luciferase activity was normalized with respect to the activity of PGL3-basic in untreated cells. Data are mean ± SEM from four independent triplicated experiments; ( D ) Chromatin immunoprecipitation of VEGF promoter complexes. HO-8910 cells underwent immunoprecipitation with anti-Sp1 antibody or control IgG. Immunoprecipitated chromatin fragments were amplified by PCR with specific primers targeting the predicted Sp1 binding motifs within the VEGF promoter; ( E ) Effects of various competitors in EMSA of resistin-stimulated HO-8910 cells. Competitors, including Sp1, AP-2 and Egr-1 consensus sequences, were added to the reaction mixture at a 100-fold molar excess to labeled probe, the VEGF promoter fragment containing predicted Sp1 binding motifs. Lane 2 contained no competitor. Nuclear extracts were prepared from HO-8910 cells and underwent EMSA. Cells were pre-incubated with 100 ng/mL resistin for 24 h.
    Figure Legend Snippet: Sp1 acted on the motif of VEGF promoter. ( A ) Effects of resistin on Sp1 activation in HO-8910 cells. Cells were incubated with 100 ng/mL resistin for 2 h and whole-cell lysates underwent Western blotting to detect the phosphorylation of Sp1 at either Thr-453 or Thr-739; ( B ) Scheme of the series of plasmid constructs containing dissected fragments of VEGF promoter; ( C ) Relative luciferase activity in transient transfection assays using the series of plasmid constructs (schematically shown in B ) and pSp1-luciferase reporter. Relative luciferase activity was normalized with respect to the activity of PGL3-basic in untreated cells. Data are mean ± SEM from four independent triplicated experiments; ( D ) Chromatin immunoprecipitation of VEGF promoter complexes. HO-8910 cells underwent immunoprecipitation with anti-Sp1 antibody or control IgG. Immunoprecipitated chromatin fragments were amplified by PCR with specific primers targeting the predicted Sp1 binding motifs within the VEGF promoter; ( E ) Effects of various competitors in EMSA of resistin-stimulated HO-8910 cells. Competitors, including Sp1, AP-2 and Egr-1 consensus sequences, were added to the reaction mixture at a 100-fold molar excess to labeled probe, the VEGF promoter fragment containing predicted Sp1 binding motifs. Lane 2 contained no competitor. Nuclear extracts were prepared from HO-8910 cells and underwent EMSA. Cells were pre-incubated with 100 ng/mL resistin for 24 h.

    Techniques Used: Activation Assay, Incubation, Western Blot, Plasmid Preparation, Construct, Luciferase, Activity Assay, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Binding Assay, Labeling

    Activated PI3K/Akt upon resistin interacted with Sp1 and activated it through phosphorylation. ( A ) Blockage of resistin-induced Sp1 activation by LY294002 and wortmannin in HO-8910 cells. Cells were pretreated with (+) or without (−) LY294002 (20 μM) and wortmannin (2 μM) for 1 h, then incubated in the presence (+) or in the absence (−) of resistin 100 ng/mL for 2 h. The top panel is a representative blot obtained from three independent experiments. The lower panel represents the summary results of Sp1 phosphorylation at either Thr-453 or Thr-739. Relative phosphorylated Sp1 protein levels were normalized to that of untreated cells; ( B ) Attenuation of resistin-induced Sp1 binding activity by LY294002 and wortmannin in HO-8910 cells. Cells were transfected with pSp1-luciferase reporter together with control plasmid. After being pretreated with (+) or without (−) LY294002 (20 μM) and wortmannin (2 μM) for 1 h, cells were incubated in the presence (+) or in the absence (−) of resistin 100 ng/mL for 24 h. Data are mean ± SEM from four independent triplicated experiments. The basal binding activity of each test plasmid is indicated as luciferase activity normalized by each internal control activity (PRL-TK). Relative luciferase activity was normalized with respect to the activity in untreated cells. ** p
    Figure Legend Snippet: Activated PI3K/Akt upon resistin interacted with Sp1 and activated it through phosphorylation. ( A ) Blockage of resistin-induced Sp1 activation by LY294002 and wortmannin in HO-8910 cells. Cells were pretreated with (+) or without (−) LY294002 (20 μM) and wortmannin (2 μM) for 1 h, then incubated in the presence (+) or in the absence (−) of resistin 100 ng/mL for 2 h. The top panel is a representative blot obtained from three independent experiments. The lower panel represents the summary results of Sp1 phosphorylation at either Thr-453 or Thr-739. Relative phosphorylated Sp1 protein levels were normalized to that of untreated cells; ( B ) Attenuation of resistin-induced Sp1 binding activity by LY294002 and wortmannin in HO-8910 cells. Cells were transfected with pSp1-luciferase reporter together with control plasmid. After being pretreated with (+) or without (−) LY294002 (20 μM) and wortmannin (2 μM) for 1 h, cells were incubated in the presence (+) or in the absence (−) of resistin 100 ng/mL for 24 h. Data are mean ± SEM from four independent triplicated experiments. The basal binding activity of each test plasmid is indicated as luciferase activity normalized by each internal control activity (PRL-TK). Relative luciferase activity was normalized with respect to the activity in untreated cells. ** p

    Techniques Used: Activation Assay, Incubation, Binding Assay, Activity Assay, Transfection, Luciferase, Plasmid Preparation

    40) Product Images from "Diabetic Ephrin-B2-Stimulated Peripheral Blood Mononuclear Cells Enhance Poststroke Recovery in Mice"

    Article Title: Diabetic Ephrin-B2-Stimulated Peripheral Blood Mononuclear Cells Enhance Poststroke Recovery in Mice

    Journal: Stem Cells International

    doi: 10.1155/2018/2431567

    Cell tracking detection of human DNA in mice transplanted with human PB-MNC and PB-MNC+. DNA was extracted from mouse tissues and amplified using PCR to detect the human-specific P17H8 satellite sequence. Agarose gel analysis of PCR products demonstrated the presence of a 1171-bp human-specific fragment as early as one hour after cell injection mainly in the ipsilateral hemisphere and in the lungs of PB-MNC+ and unstimulated PB-MNC-treated mice. At D1 after injection, human DNA was readily detectable in the lungs, spleen, liver, and kidneys and in the contralateral hemisphere, whereas human DNA was still present in the ipsilateral hemisphere and had increased in the lungs. Signal disappeared from all organs, including brain, by D14 ( N = 5).
    Figure Legend Snippet: Cell tracking detection of human DNA in mice transplanted with human PB-MNC and PB-MNC+. DNA was extracted from mouse tissues and amplified using PCR to detect the human-specific P17H8 satellite sequence. Agarose gel analysis of PCR products demonstrated the presence of a 1171-bp human-specific fragment as early as one hour after cell injection mainly in the ipsilateral hemisphere and in the lungs of PB-MNC+ and unstimulated PB-MNC-treated mice. At D1 after injection, human DNA was readily detectable in the lungs, spleen, liver, and kidneys and in the contralateral hemisphere, whereas human DNA was still present in the ipsilateral hemisphere and had increased in the lungs. Signal disappeared from all organs, including brain, by D14 ( N = 5).

    Techniques Used: Cell Tracking Assay, Mouse Assay, Amplification, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Injection

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    Roche apoptosis assessment
    The effect of telomerase downregulation on <t>apoptosis</t> in breast cancer MCF7 and MDA-MB-231 cells MCF7 ( open bars ) and MDA-MB-231 ( grey bars ) were treated with 100 nM specific siRNA or with camptothecin (as positive control) and subjected to Annexin V and propidium iodide labeling followed by flow cytometry analysis. All experiments (three separate) were performed in triplicates, and error bars show the (±SEM) values. * p
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    Roche sinr4a1
    TXNDC5 is a novel NR4A1-regulated gene involved in ER stress-mediated apoptosis (A) Heat map of genes including ROS metabolism genes regulated by <t>siNR4A1</t> in Panc-1 cells (left panel). Each cell in the matrix represents the expression level of a gene feature. Red and green reflect relatively high and low expression levels of genes, respectively, as indicated in the scale bar (a log2-transformed scale). (A, right panel and B) Panc-1 cells were transfected with each indicated siRNA for 48 hr and mRNA levels were determined by real-time PCR, as described in the Materials and Methods. TATA-binding protein (TBP) was used as an internal control and mRNA levels are presented as means with s.d. of three experiments. * P
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    Roche hbv dna pcr fragment
    ( a ) Schematic representation of the intramolecular ligation technique to obtain the preCore and YMDD Pol motif close to each other. ( 1 ) The circle in a continuous line represents the complete <t>HBV</t> genome (3200 bp). The semicircles in discontinuous lines represent the first (first <t>PCR,</t> 2700 bp) and the nested (2194 bp for genotype A2 sequences and 2155 bp for genotype D sequences) PCR products. ( 2 ) The product obtained after intramolecular ligation and primers used to sequence the circular molecule ( Table 2 ) are indicated in the figure. ( b ) Agarose gel obtained after intramolecular digestion. Two markers (M-I and M-II) were loaded in the gel; band lengths are shown at the left side of the image. Two samples (S1 and S2) were loaded and four bands were detected; band lengths are indicated at the right side of the image. A band with an apparent size of 1700 bp was the circular molecule, with the preCore and YMDD Pol motif close to each other. Band B was the nested PCR product without ligation, and band D were dimers of nested PCR products without ligation. Band C corresponded to the ligated dimers. B , C and D bands were not used, because the main interest was to analyze regions from the same genome. ( c ) Sequence of the circular construct including the preCore region, YMDD polymerase motif (in box), and HindIII sequence. PreCore (1, 2, 14, 15, 28 and 29) and Core ( 1 ) codons analyzed in the text are also enclosed in boxes.
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    Roche thj 11t cells
    Immunocytochemical and Western blotting demonstration of resveratrol-induced alterations of RAR-β and PPAR-β/δ expression and their intracellular distribution patterns in <t>THJ-11T</t> cells. ( A ) Immunocytochemical staining (×40); ( B ) ImageJ-based quantification of nuclear labeling of RAR-β and PPAR-β/δ; ( C ) Levels of RAR-β and PPAR-β/δ determined by Western blotting. Control, 0.2% DMSO-treated cells; Resveratrol, 48 h 100 µM resveratrol treatment. Arrows indicate the portions in higher magnification in the insets (×80). Ratio, ratio between the levels of the target molecules and that of β-ACTIN; *, with statistical significance ( p
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    The effect of telomerase downregulation on apoptosis in breast cancer MCF7 and MDA-MB-231 cells MCF7 ( open bars ) and MDA-MB-231 ( grey bars ) were treated with 100 nM specific siRNA or with camptothecin (as positive control) and subjected to Annexin V and propidium iodide labeling followed by flow cytometry analysis. All experiments (three separate) were performed in triplicates, and error bars show the (±SEM) values. * p

    Journal: Molecular Biology Reports

    Article Title: Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner

    doi: 10.1007/s11033-013-2600-9

    Figure Lengend Snippet: The effect of telomerase downregulation on apoptosis in breast cancer MCF7 and MDA-MB-231 cells MCF7 ( open bars ) and MDA-MB-231 ( grey bars ) were treated with 100 nM specific siRNA or with camptothecin (as positive control) and subjected to Annexin V and propidium iodide labeling followed by flow cytometry analysis. All experiments (three separate) were performed in triplicates, and error bars show the (±SEM) values. * p

    Article Snippet: Apoptosis assessment: TUNEL assay In order to estimate the influence of telomerase subunits downregulation on apoptosis in breast cancer cells a DNA fragmentation assay (In Situ Cell Death Detection Kit, TUNEL; Roche Diagnostics, IN, USA) was performed as previously described [ ].

    Techniques: Multiple Displacement Amplification, Positive Control, Labeling, Flow Cytometry, Cytometry

    The effect of telomerase silencing on expression of genes contributing to apoptosis control Cells were treated with 100 nM of TERT targeting siRNA and after 72 h the expression profile of proapoptotic ( a ) and antiapoptotic ( b ) genes in MCF7 and MDA-MB-231 cancer cells was assessed using qPCR. The experiment was performed in duplicates. The graphs represent the increase or decrease of the expression of the indicated genes relative to untreated control cells, (baseline at “0″ level)

    Journal: Molecular Biology Reports

    Article Title: Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner

    doi: 10.1007/s11033-013-2600-9

    Figure Lengend Snippet: The effect of telomerase silencing on expression of genes contributing to apoptosis control Cells were treated with 100 nM of TERT targeting siRNA and after 72 h the expression profile of proapoptotic ( a ) and antiapoptotic ( b ) genes in MCF7 and MDA-MB-231 cancer cells was assessed using qPCR. The experiment was performed in duplicates. The graphs represent the increase or decrease of the expression of the indicated genes relative to untreated control cells, (baseline at “0″ level)

    Article Snippet: Apoptosis assessment: TUNEL assay In order to estimate the influence of telomerase subunits downregulation on apoptosis in breast cancer cells a DNA fragmentation assay (In Situ Cell Death Detection Kit, TUNEL; Roche Diagnostics, IN, USA) was performed as previously described [ ].

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

    TXNDC5 is a novel NR4A1-regulated gene involved in ER stress-mediated apoptosis (A) Heat map of genes including ROS metabolism genes regulated by siNR4A1 in Panc-1 cells (left panel). Each cell in the matrix represents the expression level of a gene feature. Red and green reflect relatively high and low expression levels of genes, respectively, as indicated in the scale bar (a log2-transformed scale). (A, right panel and B) Panc-1 cells were transfected with each indicated siRNA for 48 hr and mRNA levels were determined by real-time PCR, as described in the Materials and Methods. TATA-binding protein (TBP) was used as an internal control and mRNA levels are presented as means with s.d. of three experiments. * P

    Journal: Molecular cancer research : MCR

    Article Title: The Orphan Nuclear Receptor NR4A1 (Nur77) Regulates Oxidative and Endoplasmic Reticulum Stress in Pancreatic Cancer Cells

    doi: 10.1158/1541-7786.MCR-13-0567

    Figure Lengend Snippet: TXNDC5 is a novel NR4A1-regulated gene involved in ER stress-mediated apoptosis (A) Heat map of genes including ROS metabolism genes regulated by siNR4A1 in Panc-1 cells (left panel). Each cell in the matrix represents the expression level of a gene feature. Red and green reflect relatively high and low expression levels of genes, respectively, as indicated in the scale bar (a log2-transformed scale). (A, right panel and B) Panc-1 cells were transfected with each indicated siRNA for 48 hr and mRNA levels were determined by real-time PCR, as described in the Materials and Methods. TATA-binding protein (TBP) was used as an internal control and mRNA levels are presented as means with s.d. of three experiments. * P

    Article Snippet: After incubation with siNR4A1 or DIM-C-pPhOH, cells were subjected to DNA fragmentation assay according to the manufacturer’s instructions (Cell Death Detection ELISAPLUS, Roche).

    Techniques: Expressing, Transformation Assay, Transfection, Real-time Polymerase Chain Reaction, Binding Assay

    Knockdown of NR4A1 induces ER stress and apoptosis in multiple cancer cell lines (A) Panc-1 cells were transfected with either siScr or siNR4A1, and TEM analysis was performed at 60 hr after transfection. The structure of the ER (arrows) in cells transfected with siNR4A1 showed morphological disorders. (B, C, and D) Panc-1, L3.6pL, MCF-7/RKO cells, respectively, were transfected with either siScr or siNR4A1 for 72 hr, and whole cell lysates were analyzed by western blot analysis. β-Actin was used as a loading control.

    Journal: Molecular cancer research : MCR

    Article Title: The Orphan Nuclear Receptor NR4A1 (Nur77) Regulates Oxidative and Endoplasmic Reticulum Stress in Pancreatic Cancer Cells

    doi: 10.1158/1541-7786.MCR-13-0567

    Figure Lengend Snippet: Knockdown of NR4A1 induces ER stress and apoptosis in multiple cancer cell lines (A) Panc-1 cells were transfected with either siScr or siNR4A1, and TEM analysis was performed at 60 hr after transfection. The structure of the ER (arrows) in cells transfected with siNR4A1 showed morphological disorders. (B, C, and D) Panc-1, L3.6pL, MCF-7/RKO cells, respectively, were transfected with either siScr or siNR4A1 for 72 hr, and whole cell lysates were analyzed by western blot analysis. β-Actin was used as a loading control.

    Article Snippet: After incubation with siNR4A1 or DIM-C-pPhOH, cells were subjected to DNA fragmentation assay according to the manufacturer’s instructions (Cell Death Detection ELISAPLUS, Roche).

    Techniques: Transfection, Transmission Electron Microscopy, Western Blot

    Knockdown of NR4A1 induces ER stress-mediated apoptosis by increasing ROS production in pancreatic cancer cells (A) Panc-1 cells were transfected with each indicated siRNA for 72 hr, and whole cell lysates were analyzed by western blot analysis. (B-D) Cells were transfected with siScr or siNR4A1 for 6 hr. At 60 hr after transfection, ROS production was measured by the oxidation of non-fluorescent DCFH-DA to fluorescent DCF using a fluorescence plate reader (B). # P

    Journal: Molecular cancer research : MCR

    Article Title: The Orphan Nuclear Receptor NR4A1 (Nur77) Regulates Oxidative and Endoplasmic Reticulum Stress in Pancreatic Cancer Cells

    doi: 10.1158/1541-7786.MCR-13-0567

    Figure Lengend Snippet: Knockdown of NR4A1 induces ER stress-mediated apoptosis by increasing ROS production in pancreatic cancer cells (A) Panc-1 cells were transfected with each indicated siRNA for 72 hr, and whole cell lysates were analyzed by western blot analysis. (B-D) Cells were transfected with siScr or siNR4A1 for 6 hr. At 60 hr after transfection, ROS production was measured by the oxidation of non-fluorescent DCFH-DA to fluorescent DCF using a fluorescence plate reader (B). # P

    Article Snippet: After incubation with siNR4A1 or DIM-C-pPhOH, cells were subjected to DNA fragmentation assay according to the manufacturer’s instructions (Cell Death Detection ELISAPLUS, Roche).

    Techniques: Transfection, Western Blot, Fluorescence

    2D-PAGE gels showing differentially expressed proteins in Panc-1 cells transfected with siNR4A1 and functional classification of the proteins . Functional distribution (B) and canonical pathway (C) of the 38 identified proteins. Assignments were made based on information from the NCBI, the Swiss-Prot/TrEMBL Protein Knowledge Base, and the Ingenuity Pathways Knowledge Base. Enlarged images of protein spots for which image analysis software reported ≥2-fold differences in accumulation in siNR4A1-transfected cells are shown (D, left panel). Panc-1 cells were transfected with either siScr or siNR4A1 for 60 hr, and whole cell lysates were analyzed by western blot analysis (D, right panel).

    Journal: Molecular cancer research : MCR

    Article Title: The Orphan Nuclear Receptor NR4A1 (Nur77) Regulates Oxidative and Endoplasmic Reticulum Stress in Pancreatic Cancer Cells

    doi: 10.1158/1541-7786.MCR-13-0567

    Figure Lengend Snippet: 2D-PAGE gels showing differentially expressed proteins in Panc-1 cells transfected with siNR4A1 and functional classification of the proteins . Functional distribution (B) and canonical pathway (C) of the 38 identified proteins. Assignments were made based on information from the NCBI, the Swiss-Prot/TrEMBL Protein Knowledge Base, and the Ingenuity Pathways Knowledge Base. Enlarged images of protein spots for which image analysis software reported ≥2-fold differences in accumulation in siNR4A1-transfected cells are shown (D, left panel). Panc-1 cells were transfected with either siScr or siNR4A1 for 60 hr, and whole cell lysates were analyzed by western blot analysis (D, right panel).

    Article Snippet: After incubation with siNR4A1 or DIM-C-pPhOH, cells were subjected to DNA fragmentation assay according to the manufacturer’s instructions (Cell Death Detection ELISAPLUS, Roche).

    Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Functional Assay, Software, Western Blot

    ( a ) Schematic representation of the intramolecular ligation technique to obtain the preCore and YMDD Pol motif close to each other. ( 1 ) The circle in a continuous line represents the complete HBV genome (3200 bp). The semicircles in discontinuous lines represent the first (first PCR, 2700 bp) and the nested (2194 bp for genotype A2 sequences and 2155 bp for genotype D sequences) PCR products. ( 2 ) The product obtained after intramolecular ligation and primers used to sequence the circular molecule ( Table 2 ) are indicated in the figure. ( b ) Agarose gel obtained after intramolecular digestion. Two markers (M-I and M-II) were loaded in the gel; band lengths are shown at the left side of the image. Two samples (S1 and S2) were loaded and four bands were detected; band lengths are indicated at the right side of the image. A band with an apparent size of 1700 bp was the circular molecule, with the preCore and YMDD Pol motif close to each other. Band B was the nested PCR product without ligation, and band D were dimers of nested PCR products without ligation. Band C corresponded to the ligated dimers. B , C and D bands were not used, because the main interest was to analyze regions from the same genome. ( c ) Sequence of the circular construct including the preCore region, YMDD polymerase motif (in box), and HindIII sequence. PreCore (1, 2, 14, 15, 28 and 29) and Core ( 1 ) codons analyzed in the text are also enclosed in boxes.

    Journal: Nucleic Acids Research

    Article Title: Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome

    doi: 10.1093/nar/gkr451

    Figure Lengend Snippet: ( a ) Schematic representation of the intramolecular ligation technique to obtain the preCore and YMDD Pol motif close to each other. ( 1 ) The circle in a continuous line represents the complete HBV genome (3200 bp). The semicircles in discontinuous lines represent the first (first PCR, 2700 bp) and the nested (2194 bp for genotype A2 sequences and 2155 bp for genotype D sequences) PCR products. ( 2 ) The product obtained after intramolecular ligation and primers used to sequence the circular molecule ( Table 2 ) are indicated in the figure. ( b ) Agarose gel obtained after intramolecular digestion. Two markers (M-I and M-II) were loaded in the gel; band lengths are shown at the left side of the image. Two samples (S1 and S2) were loaded and four bands were detected; band lengths are indicated at the right side of the image. A band with an apparent size of 1700 bp was the circular molecule, with the preCore and YMDD Pol motif close to each other. Band B was the nested PCR product without ligation, and band D were dimers of nested PCR products without ligation. Band C corresponded to the ligated dimers. B , C and D bands were not used, because the main interest was to analyze regions from the same genome. ( c ) Sequence of the circular construct including the preCore region, YMDD polymerase motif (in box), and HindIII sequence. PreCore (1, 2, 14, 15, 28 and 29) and Core ( 1 ) codons analyzed in the text are also enclosed in boxes.

    Article Snippet: Optimal results were obtained with 0.5 ng/µ1 of the HBV DNA-PCR fragment, and 1 U of T4 DNA ligase (Roche Diagnostics GmbH).

    Techniques: Ligation, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Nested PCR, Construct

    Immunocytochemical and Western blotting demonstration of resveratrol-induced alterations of RAR-β and PPAR-β/δ expression and their intracellular distribution patterns in THJ-11T cells. ( A ) Immunocytochemical staining (×40); ( B ) ImageJ-based quantification of nuclear labeling of RAR-β and PPAR-β/δ; ( C ) Levels of RAR-β and PPAR-β/δ determined by Western blotting. Control, 0.2% DMSO-treated cells; Resveratrol, 48 h 100 µM resveratrol treatment. Arrows indicate the portions in higher magnification in the insets (×80). Ratio, ratio between the levels of the target molecules and that of β-ACTIN; *, with statistical significance ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Suppresses the Growth and Enhances Retinoic Acid Sensitivity of Anaplastic Thyroid Cancer Cells

    doi: 10.3390/ijms19041030

    Figure Lengend Snippet: Immunocytochemical and Western blotting demonstration of resveratrol-induced alterations of RAR-β and PPAR-β/δ expression and their intracellular distribution patterns in THJ-11T cells. ( A ) Immunocytochemical staining (×40); ( B ) ImageJ-based quantification of nuclear labeling of RAR-β and PPAR-β/δ; ( C ) Levels of RAR-β and PPAR-β/δ determined by Western blotting. Control, 0.2% DMSO-treated cells; Resveratrol, 48 h 100 µM resveratrol treatment. Arrows indicate the portions in higher magnification in the insets (×80). Ratio, ratio between the levels of the target molecules and that of β-ACTIN; *, with statistical significance ( p

    Article Snippet: The DNA fragmentation assay in THJ-11T cells on coverslips, treated with both RA and resveratrol, was performed by using a modification of the terminal deoxynucleotide transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNEL; Roche Inc., Basel, Switzerland).

    Techniques: Western Blot, Expressing, Staining, Labeling

    Resveratrol reverses the retinoic acid sensitivity of THJ-11T cells. ( A ) H/E staining (×40); ( B ) deoxynucleotidyl transferase-mediated dUTP-biotin nick and labeling assay (TUNEL) for apoptotic cell labeling (Green in color; ×40); ( C ) MTT cell proliferation assay; ( D ) viable cell counting; ( E ) immunocytochemical staining of thyroglobulin (Tg) and E-cadherin (×40). Control, cultured in 0.2% dimethylsulfoxide (DMSO)-containing medium; RA, retinoic acid treatment; Res, resveratrol treatment; Resveratrol plus RA, treated with a combination of 10 µM retinoic acid and 100 µM resveratrol for 48 h; Resveratrol-alone, 100 µM resveratrol; *, with statistical significance ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Suppresses the Growth and Enhances Retinoic Acid Sensitivity of Anaplastic Thyroid Cancer Cells

    doi: 10.3390/ijms19041030

    Figure Lengend Snippet: Resveratrol reverses the retinoic acid sensitivity of THJ-11T cells. ( A ) H/E staining (×40); ( B ) deoxynucleotidyl transferase-mediated dUTP-biotin nick and labeling assay (TUNEL) for apoptotic cell labeling (Green in color; ×40); ( C ) MTT cell proliferation assay; ( D ) viable cell counting; ( E ) immunocytochemical staining of thyroglobulin (Tg) and E-cadherin (×40). Control, cultured in 0.2% dimethylsulfoxide (DMSO)-containing medium; RA, retinoic acid treatment; Res, resveratrol treatment; Resveratrol plus RA, treated with a combination of 10 µM retinoic acid and 100 µM resveratrol for 48 h; Resveratrol-alone, 100 µM resveratrol; *, with statistical significance ( p

    Article Snippet: The DNA fragmentation assay in THJ-11T cells on coverslips, treated with both RA and resveratrol, was performed by using a modification of the terminal deoxynucleotide transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNEL; Roche Inc., Basel, Switzerland).

    Techniques: Staining, Labeling, TUNEL Assay, MTT Assay, Proliferation Assay, Cell Counting, Cell Culture

    Differential expression of CRABP2 and FARP5 in THJ-11T cells in the absence or presence of 100 µM resveratrol. ( A ) Double immunofluorescent labeling (×40); ( B ) ImageJ (Version 1.0, National Institutes of Health, Bethesda, MD, USA)-based quantification of nuclear labeling of CRABP2 and FABP5; ( C ) Western blotting; ( D ) RT-PCR. Control, 0.2% DMSO-treated cells; Resveratrol-alone, 100 µM resveratrol treatment; Ratio, ratio between the levels of the target molecules and that of β-ACTIN/ β-actin ; *, with statistical significance ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Suppresses the Growth and Enhances Retinoic Acid Sensitivity of Anaplastic Thyroid Cancer Cells

    doi: 10.3390/ijms19041030

    Figure Lengend Snippet: Differential expression of CRABP2 and FARP5 in THJ-11T cells in the absence or presence of 100 µM resveratrol. ( A ) Double immunofluorescent labeling (×40); ( B ) ImageJ (Version 1.0, National Institutes of Health, Bethesda, MD, USA)-based quantification of nuclear labeling of CRABP2 and FABP5; ( C ) Western blotting; ( D ) RT-PCR. Control, 0.2% DMSO-treated cells; Resveratrol-alone, 100 µM resveratrol treatment; Ratio, ratio between the levels of the target molecules and that of β-ACTIN/ β-actin ; *, with statistical significance ( p

    Article Snippet: The DNA fragmentation assay in THJ-11T cells on coverslips, treated with both RA and resveratrol, was performed by using a modification of the terminal deoxynucleotide transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNEL; Roche Inc., Basel, Switzerland).

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