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TaKaRa β galactosidase
Dominant-negative Rab11 inhibits the effect of CIN85 on TGFβ signaling. (A) HEK293T cells were transfected with HA-TβRI, Flag-CIN85, and the dominant-negative myc-Rab11(S25N) mutant, as indicated. 48 h after transfection, cell surface proteins were biotinylated and isolated by neutravidin-agarose pull-down. The amount of biotinylated HA-TβRI was analyzed by immunoblotting with HA antibodies. Cells transfected with HA-TβRI, but not treated with biotin, were used as a control for specificity of neutravidin-agarose pull-down. The levels of HA-TβRI, Flag-CIN85, and myc-Rab11(S25N) expression were determined by immunoblotting of total cell lysates. (B) HEK293T cells that had been transfected with CAGA 12 -Luc, Flag-CIN85, and myc-Rab11(S25N), as indicated, were either treated with TGFβ (5 ng/ml) for 20 h (shaded bars) or not (open bars), and the luciferase activity in the cell lysates was measured. Transfection efficiency was normalized to <t>β-galactosidase</t> activity. The data are presented as the mean (in percentage of control) of six independent experiments. Error bars are SD. *, P
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1) Product Images from "CIN85 modulates TGFβ signaling by promoting the presentation of TGFβ receptors on the cell surface"

Article Title: CIN85 modulates TGFβ signaling by promoting the presentation of TGFβ receptors on the cell surface

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201411025

Dominant-negative Rab11 inhibits the effect of CIN85 on TGFβ signaling. (A) HEK293T cells were transfected with HA-TβRI, Flag-CIN85, and the dominant-negative myc-Rab11(S25N) mutant, as indicated. 48 h after transfection, cell surface proteins were biotinylated and isolated by neutravidin-agarose pull-down. The amount of biotinylated HA-TβRI was analyzed by immunoblotting with HA antibodies. Cells transfected with HA-TβRI, but not treated with biotin, were used as a control for specificity of neutravidin-agarose pull-down. The levels of HA-TβRI, Flag-CIN85, and myc-Rab11(S25N) expression were determined by immunoblotting of total cell lysates. (B) HEK293T cells that had been transfected with CAGA 12 -Luc, Flag-CIN85, and myc-Rab11(S25N), as indicated, were either treated with TGFβ (5 ng/ml) for 20 h (shaded bars) or not (open bars), and the luciferase activity in the cell lysates was measured. Transfection efficiency was normalized to β-galactosidase activity. The data are presented as the mean (in percentage of control) of six independent experiments. Error bars are SD. *, P
Figure Legend Snippet: Dominant-negative Rab11 inhibits the effect of CIN85 on TGFβ signaling. (A) HEK293T cells were transfected with HA-TβRI, Flag-CIN85, and the dominant-negative myc-Rab11(S25N) mutant, as indicated. 48 h after transfection, cell surface proteins were biotinylated and isolated by neutravidin-agarose pull-down. The amount of biotinylated HA-TβRI was analyzed by immunoblotting with HA antibodies. Cells transfected with HA-TβRI, but not treated with biotin, were used as a control for specificity of neutravidin-agarose pull-down. The levels of HA-TβRI, Flag-CIN85, and myc-Rab11(S25N) expression were determined by immunoblotting of total cell lysates. (B) HEK293T cells that had been transfected with CAGA 12 -Luc, Flag-CIN85, and myc-Rab11(S25N), as indicated, were either treated with TGFβ (5 ng/ml) for 20 h (shaded bars) or not (open bars), and the luciferase activity in the cell lysates was measured. Transfection efficiency was normalized to β-galactosidase activity. The data are presented as the mean (in percentage of control) of six independent experiments. Error bars are SD. *, P

Techniques Used: Dominant Negative Mutation, Transfection, Mutagenesis, Isolation, Expressing, Luciferase, Activity Assay

CIN85 enhances TGFβ transcriptional activity. (A) HEK293T cells, transfected with CAGA 12 -Luc (left) or xFAST/ARE-Luc (right) and different amounts of Flag-CIN85 plasmid, were not treated (open bars) or treated with 5 ng/ml TGFβ (shaded bars) for 20 h, and luciferase activity was measured. The results are presented as the mean (in percentage of control) of six independent experiments. Error bars represent SD. (B) qRT-PCR analysis for expression for PAI-1 was performed on mRNA extracted from PC-3U cells transiently transfected with empty pCDNA3 vector or Flag-CIN85 and treated with 5 ng/ml TGFβ for the indicated time periods. The results were normalized on the basis of GAPDH mRNA expression. The data are plotted as the mean fold induction of TGFβ-stimulated mRNA levels relative to unstimulated levels (0 h set to 1) with SD determined from triplicate measurements. Results of one representative experiment out of three performed are shown. (C) HEK293T cells transfected with CAGA 12 -Luc reporter and control plasmid (open bars) or Flag-CIN85 plasmid (shaded bars). The cells were pretreated for 1 h with DMSO or TβRI kinase inhibitor SB505124 (10 µM) and then incubated with 5 ng/ml TGFβ for 20 h as indicated. Luciferase activity was measured and transfection efficiency was normalized to β-galactosidase activity. The results are presented as the mean (in percentage of control) of three independent experiments. Error bars represent SD. *, P
Figure Legend Snippet: CIN85 enhances TGFβ transcriptional activity. (A) HEK293T cells, transfected with CAGA 12 -Luc (left) or xFAST/ARE-Luc (right) and different amounts of Flag-CIN85 plasmid, were not treated (open bars) or treated with 5 ng/ml TGFβ (shaded bars) for 20 h, and luciferase activity was measured. The results are presented as the mean (in percentage of control) of six independent experiments. Error bars represent SD. (B) qRT-PCR analysis for expression for PAI-1 was performed on mRNA extracted from PC-3U cells transiently transfected with empty pCDNA3 vector or Flag-CIN85 and treated with 5 ng/ml TGFβ for the indicated time periods. The results were normalized on the basis of GAPDH mRNA expression. The data are plotted as the mean fold induction of TGFβ-stimulated mRNA levels relative to unstimulated levels (0 h set to 1) with SD determined from triplicate measurements. Results of one representative experiment out of three performed are shown. (C) HEK293T cells transfected with CAGA 12 -Luc reporter and control plasmid (open bars) or Flag-CIN85 plasmid (shaded bars). The cells were pretreated for 1 h with DMSO or TβRI kinase inhibitor SB505124 (10 µM) and then incubated with 5 ng/ml TGFβ for 20 h as indicated. Luciferase activity was measured and transfection efficiency was normalized to β-galactosidase activity. The results are presented as the mean (in percentage of control) of three independent experiments. Error bars represent SD. *, P

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

CIN85 interacts with TGFβ receptors. (A) HEK293T cells were transfected with Flag-CIN85 in the absence or presence of HA-TβRI and incubated with 5 ng/ml TGFβ for 15 min, as indicated. The cell lysates were subjected to immunoprecipitation with HA antibodies followed by blotting with Flag antibodies. The levels of HA-TβRI and Flag-CIN85 expression were determined by immunoblotting of total cell lysates. (B) PC-3U cells were treated with 5 ng/ml TGFβ for 15 min and endogenous TβRI (left) or CIN85 (right) were immunoprecipitated with 1 µg of goat anti-TβRI or rabbit anti-CIN85 antibodies, respectively. Immunoprecipitates were analyzed by immunoblotting for the presence of CIN85 or TβRI, as indicated. Total cell lysate was also subjected to immunoblotting for CIN85 and TβRI, and for phosphorylated Smad2 to determine activation of TGFβ signaling (bottom). (C) HEK293T cells were transfected with Flag-CIN85 in the absence or presence of His-TβRII and incubated with 5 ng/ml TGFβ for 15 min, as indicated. TβRII was immunoprecipitated from the cell lysates with His antibodies. Coimmunoprecipitated CIN85 and immunoprecipitated TβRII were detected by immunoblotting with Flag and His antibodies, respectively. The levels of His-TβRII and Flag-CIN85 expression were determined by immunoblotting of total cell lysates. (D) Schematic illustration of the CIN85 molecule. The full-length molecule (FL) and the parts of CIN85 included into the deletion mutants containing three SH3 domains (3SH3) or proline-rich and coiled-coil domains (PcC) are indicated. (E) TβRI interacts with the N-terminal part of CIN85. HA-TβRI was transfected into HEK293T cells together with full-length Flag-CIN85 or with deletion mutants including three SH3 domains (3SH3) or proline-rich and coiled coil domains (PcC). The cells were incubated with 5 ng/ml TGFβ for 15 min and TβRI was immunoprecipitated from the cell lysates with HA antibodies. The coimmunoprecipitated CIN85 or CIN85 deletion mutants were detected by immunoblotting with Flag antibodies. The expression of HA-TβRI and Flag-CIN85 molecules was determined by immunoblotting of total cell lysates. (F) TβRII interacts with the N-terminal part of CIN85. HA-TβRII was transfected into HEK293T cells together with full-length Flag-CIN85 or with its deletion mutants. The cells were treated as in E and TβRII was immunoprecipitated from the cell lysates with HA antibodies. The coimmunoprecipitated CIN85 or CIN85 deletion mutants were detected by immunoblotting with Flag antibodies. (G) The transcriptional activity of TGFβ is enhanced by full-length CIN85 but not by its N- or C-terminal fragments. HEK293T cells, transfected with CAGA 12 -Luc and full-length CIN85 or its N- or C-terminal parts, were treated (shaded bars) or not (open bars) with TGFβ (5 ng/ml) for 20 h. Luciferase activity was measured and transfection efficiency was normalized to β-galactosidase activity. The results are presented as the mean (in percentage of control) of three independent experiments. Error bars are SD. *, P
Figure Legend Snippet: CIN85 interacts with TGFβ receptors. (A) HEK293T cells were transfected with Flag-CIN85 in the absence or presence of HA-TβRI and incubated with 5 ng/ml TGFβ for 15 min, as indicated. The cell lysates were subjected to immunoprecipitation with HA antibodies followed by blotting with Flag antibodies. The levels of HA-TβRI and Flag-CIN85 expression were determined by immunoblotting of total cell lysates. (B) PC-3U cells were treated with 5 ng/ml TGFβ for 15 min and endogenous TβRI (left) or CIN85 (right) were immunoprecipitated with 1 µg of goat anti-TβRI or rabbit anti-CIN85 antibodies, respectively. Immunoprecipitates were analyzed by immunoblotting for the presence of CIN85 or TβRI, as indicated. Total cell lysate was also subjected to immunoblotting for CIN85 and TβRI, and for phosphorylated Smad2 to determine activation of TGFβ signaling (bottom). (C) HEK293T cells were transfected with Flag-CIN85 in the absence or presence of His-TβRII and incubated with 5 ng/ml TGFβ for 15 min, as indicated. TβRII was immunoprecipitated from the cell lysates with His antibodies. Coimmunoprecipitated CIN85 and immunoprecipitated TβRII were detected by immunoblotting with Flag and His antibodies, respectively. The levels of His-TβRII and Flag-CIN85 expression were determined by immunoblotting of total cell lysates. (D) Schematic illustration of the CIN85 molecule. The full-length molecule (FL) and the parts of CIN85 included into the deletion mutants containing three SH3 domains (3SH3) or proline-rich and coiled-coil domains (PcC) are indicated. (E) TβRI interacts with the N-terminal part of CIN85. HA-TβRI was transfected into HEK293T cells together with full-length Flag-CIN85 or with deletion mutants including three SH3 domains (3SH3) or proline-rich and coiled coil domains (PcC). The cells were incubated with 5 ng/ml TGFβ for 15 min and TβRI was immunoprecipitated from the cell lysates with HA antibodies. The coimmunoprecipitated CIN85 or CIN85 deletion mutants were detected by immunoblotting with Flag antibodies. The expression of HA-TβRI and Flag-CIN85 molecules was determined by immunoblotting of total cell lysates. (F) TβRII interacts with the N-terminal part of CIN85. HA-TβRII was transfected into HEK293T cells together with full-length Flag-CIN85 or with its deletion mutants. The cells were treated as in E and TβRII was immunoprecipitated from the cell lysates with HA antibodies. The coimmunoprecipitated CIN85 or CIN85 deletion mutants were detected by immunoblotting with Flag antibodies. (G) The transcriptional activity of TGFβ is enhanced by full-length CIN85 but not by its N- or C-terminal fragments. HEK293T cells, transfected with CAGA 12 -Luc and full-length CIN85 or its N- or C-terminal parts, were treated (shaded bars) or not (open bars) with TGFβ (5 ng/ml) for 20 h. Luciferase activity was measured and transfection efficiency was normalized to β-galactosidase activity. The results are presented as the mean (in percentage of control) of three independent experiments. Error bars are SD. *, P

Techniques Used: Transfection, Incubation, Immunoprecipitation, Expressing, Activation Assay, Periodic Counter-current Chromatography, Activity Assay, Luciferase

2) Product Images from "The proapoptotic function of Drosophila Hid is conserved in mammalian cells"

Article Title: The proapoptotic function of Drosophila Hid is conserved in mammalian cells

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

doi:

Hid localizes to the mitochondria. HeLa cells were transfected with pcDNA3 Hid. Twenty-four hours posttransfection, the cells were stained with 100 nM MitoTracker for 30 minutes, fixed, and immunostained with a mAb to Hid (CL1C3) and a FITC-conjugated secondary antibody. A – C shows a single HeLa cell visualized with indirect fluorescence microscopy filtered to allow FITC ( A ), MitoTracker ( B ), or both ( C ) to be seen. ( D ) Enlarged detail of the area indicated by the white box in C .
Figure Legend Snippet: Hid localizes to the mitochondria. HeLa cells were transfected with pcDNA3 Hid. Twenty-four hours posttransfection, the cells were stained with 100 nM MitoTracker for 30 minutes, fixed, and immunostained with a mAb to Hid (CL1C3) and a FITC-conjugated secondary antibody. A – C shows a single HeLa cell visualized with indirect fluorescence microscopy filtered to allow FITC ( A ), MitoTracker ( B ), or both ( C ) to be seen. ( D ) Enlarged detail of the area indicated by the white box in C .

Techniques Used: Transfection, Staining, Fluorescence, Microscopy

Hid induces apoptotic morphology in HeLa cells. HeLa cells were transiently transfected with 0.5 μg of pcDNA3 empty vector ( A ) or 0.5 μg of pcDNA3 Hid ( B ) along with 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed, stained with X-Gal, and observed by using light microscopy. ( C – E ) HeLa cells were transfected with 0.5 μg of pcDNA3 Hid along with 0.5 μg of pEGFP-CMV. At 24 hours, they were fixed, and the cells were stained with Hoechst dye no. 33358 (5 μg/ml) for 30 minutes, washed, and observed by using fluorescence microscopy on a FITC filter set ( C ) and a blue filterset ( D ). ( E ) Artificially merged images. ( F ) Quantification of apoptosis: The indicated cell types were transfected with 0.5 μg of pcDNA3 Hid or pcDNA3 along with a 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed and stained with X-Gal. Two hundred blue cells were counted in each of two duplicate transfections in three independent experiments. The fraction of blue stained cells with apoptotic morphology as a percentage of all blue-stained cells was calculated. The percentage is expressed as the mean ± SEM.
Figure Legend Snippet: Hid induces apoptotic morphology in HeLa cells. HeLa cells were transiently transfected with 0.5 μg of pcDNA3 empty vector ( A ) or 0.5 μg of pcDNA3 Hid ( B ) along with 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed, stained with X-Gal, and observed by using light microscopy. ( C – E ) HeLa cells were transfected with 0.5 μg of pcDNA3 Hid along with 0.5 μg of pEGFP-CMV. At 24 hours, they were fixed, and the cells were stained with Hoechst dye no. 33358 (5 μg/ml) for 30 minutes, washed, and observed by using fluorescence microscopy on a FITC filter set ( C ) and a blue filterset ( D ). ( E ) Artificially merged images. ( F ) Quantification of apoptosis: The indicated cell types were transfected with 0.5 μg of pcDNA3 Hid or pcDNA3 along with a 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed and stained with X-Gal. Two hundred blue cells were counted in each of two duplicate transfections in three independent experiments. The fraction of blue stained cells with apoptotic morphology as a percentage of all blue-stained cells was calculated. The percentage is expressed as the mean ± SEM.

Techniques Used: Transfection, Plasmid Preparation, Staining, Light Microscopy, Fluorescence, Microscopy

3) Product Images from "Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription"

Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

Journal: eLife

doi: 10.7554/eLife.12785

ORC1 binds SUV39H1 to control Cyclin E gene transcription. ORC1, but not ORC3 or ORC4 can repress gene transcription. ( A ) The U2OS cells were transfected with a Gal4-driven luciferase reporter as shown in the schematic with increasing amounts of Gal4DBD-ORC1 or Gal4DBD-SUV39H1 together with pCMV-LacZ plasmids. Relative luciferase activity was determined and normalized to lacZ activity. Experiments were carried out in triplicate. The whole cell extract was immunoblotted with anti-Gal4 antibody for expression of Gal4DBD fusion plasmids. α-Tubulin served as a loading control. Statistical analysis was performed using the Student’s t test. **p
Figure Legend Snippet: ORC1 binds SUV39H1 to control Cyclin E gene transcription. ORC1, but not ORC3 or ORC4 can repress gene transcription. ( A ) The U2OS cells were transfected with a Gal4-driven luciferase reporter as shown in the schematic with increasing amounts of Gal4DBD-ORC1 or Gal4DBD-SUV39H1 together with pCMV-LacZ plasmids. Relative luciferase activity was determined and normalized to lacZ activity. Experiments were carried out in triplicate. The whole cell extract was immunoblotted with anti-Gal4 antibody for expression of Gal4DBD fusion plasmids. α-Tubulin served as a loading control. Statistical analysis was performed using the Student’s t test. **p

Techniques Used: Transfection, Luciferase, Activity Assay, Expressing

4) Product Images from "A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy"

Article Title: A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy

Journal: Brain

doi: 10.1093/brain/awx058

In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P
Figure Legend Snippet: In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P

Techniques Used: In Vitro, Mutagenesis, Reporter Assay, Luciferase, Dominant Negative Mutation, Transfection, Expressing

5) Product Images from "p66? and p66? of the Mi-2/NuRD complex mediate MBD2 and histone interaction"

Article Title: p66? and p66? of the Mi-2/NuRD complex mediate MBD2 and histone interaction

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkj437

p66α- or p66β-mediated repression acts downstream of MBD2. Mouse fibroblasts ( 30 ) expressing wild-type MBD2 (MBD2+/+) ( A ) or MBD2 knock-out fibroblasts (MBD2−/−) ( B ) were transfected with vectors expressing the Gal-DNA binding domain, Gal-p66α or Gal-p66β together with a 4xUAStk luciferase reporter. Significant changes relative to Gal are indicated by asterisk.
Figure Legend Snippet: p66α- or p66β-mediated repression acts downstream of MBD2. Mouse fibroblasts ( 30 ) expressing wild-type MBD2 (MBD2+/+) ( A ) or MBD2 knock-out fibroblasts (MBD2−/−) ( B ) were transfected with vectors expressing the Gal-DNA binding domain, Gal-p66α or Gal-p66β together with a 4xUAStk luciferase reporter. Significant changes relative to Gal are indicated by asterisk.

Techniques Used: Expressing, Knock-Out, Transfection, Binding Assay, Luciferase

Knock-down of endogenous p66α or p66β decreases MBD2-mediated repression. ( A ) Endogenous expression of p66α and p66β is reduced 96 h after RNAi treatment. HeLa cells were transfected with either pSilencer constructs targeting p66α (pSil-αX), p66β (pSil-βX), a non-targeting control sequence (pSil-neg) or left untransfected (n.trans). Nuclear extracts were prepared and subjected to western blotting using the p66 antibody. ( B ) MBD2-mediated repression is reduced by knock-down of endogenous p66α or p66β. HeLa cells were transfected with the indicated pSilencer constructs together with expression vectors for the Gal-DNA binding domain or Gal-MBD2b and the 4xUAStk luciferase reporter. Cell extracts were analyzed for reporter gene activity. Fold repression was determined relative to the Gal-DNA binding domain. Error bars represent variations within duplicate transfections, significant changes relative to pSil-neg are indicated by asterisk.
Figure Legend Snippet: Knock-down of endogenous p66α or p66β decreases MBD2-mediated repression. ( A ) Endogenous expression of p66α and p66β is reduced 96 h after RNAi treatment. HeLa cells were transfected with either pSilencer constructs targeting p66α (pSil-αX), p66β (pSil-βX), a non-targeting control sequence (pSil-neg) or left untransfected (n.trans). Nuclear extracts were prepared and subjected to western blotting using the p66 antibody. ( B ) MBD2-mediated repression is reduced by knock-down of endogenous p66α or p66β. HeLa cells were transfected with the indicated pSilencer constructs together with expression vectors for the Gal-DNA binding domain or Gal-MBD2b and the 4xUAStk luciferase reporter. Cell extracts were analyzed for reporter gene activity. Fold repression was determined relative to the Gal-DNA binding domain. Error bars represent variations within duplicate transfections, significant changes relative to pSil-neg are indicated by asterisk.

Techniques Used: Expressing, Transfection, Construct, Sequencing, Western Blot, Binding Assay, Luciferase, Activity Assay

K149 of p66α is required for the MBD2 interaction as well as for the MBD2-mediated repression. ( A ) HEK293 cells were harvested 48 h after transfection with various combinations of DNA constructs, as indicated above the figure. Nuclear protein extracts were prepared (input) and purified with glutathione–Sepharose beads. The bound protein together with the input fractions were analyzed by western blotting using the anti-Gal antibody. ( B ) K149R mutant of p66α decreases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with vectors expressing the Gal-DNA binding domain, or Gal-MBD2b and increasing amount of pSG5-p66α or pSG5-p66αK149R. Fold repression was determined relative to the Gal-DNA binding domain, significant changes relative to Gal-MBD2b (asterisk) and relative to comparable amounts of p66α (open triangle) are indicated.
Figure Legend Snippet: K149 of p66α is required for the MBD2 interaction as well as for the MBD2-mediated repression. ( A ) HEK293 cells were harvested 48 h after transfection with various combinations of DNA constructs, as indicated above the figure. Nuclear protein extracts were prepared (input) and purified with glutathione–Sepharose beads. The bound protein together with the input fractions were analyzed by western blotting using the anti-Gal antibody. ( B ) K149R mutant of p66α decreases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with vectors expressing the Gal-DNA binding domain, or Gal-MBD2b and increasing amount of pSG5-p66α or pSG5-p66αK149R. Fold repression was determined relative to the Gal-DNA binding domain, significant changes relative to Gal-MBD2b (asterisk) and relative to comparable amounts of p66α (open triangle) are indicated.

Techniques Used: Transfection, Construct, Purification, Western Blot, Mutagenesis, Luciferase, Expressing, Binding Assay

Expression of p66α or p66β increases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with plasmids coding for the Gal-DNA binding domain, Gal-MBD2b and no (mock) or increasing amounts of p66α ( A ) or p66β ( B ). Cell extracts were analyzed for reporter gene activity. Fold repression was determined relative to the Gal-DNA binding domain. Error bars represent variations within duplicate transfections, significant changes relative to mock are indicated by asterisk. Luciferase activity was measured 48 h after transfection.
Figure Legend Snippet: Expression of p66α or p66β increases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with plasmids coding for the Gal-DNA binding domain, Gal-MBD2b and no (mock) or increasing amounts of p66α ( A ) or p66β ( B ). Cell extracts were analyzed for reporter gene activity. Fold repression was determined relative to the Gal-DNA binding domain. Error bars represent variations within duplicate transfections, significant changes relative to mock are indicated by asterisk. Luciferase activity was measured 48 h after transfection.

Techniques Used: Expressing, Luciferase, Binding Assay, Activity Assay, Transfection

6) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

Journal: Scientific Reports

doi: 10.1038/srep16435

Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.
Figure Legend Snippet: Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.

Techniques Used: Reporter Assay, Activity Assay, Sequencing, Plasmid Preparation, Construct, Derivative Assay, Negative Control, Expressing, Transfection, Standard Deviation

7) Product Images from "Defect in the p53-Mdm2 Autoregulatory Loop Resulting from Inactivation of TAFII250 in Cell Cycle Mutant tsBN462 Cells"

Article Title: Defect in the p53-Mdm2 Autoregulatory Loop Resulting from Inactivation of TAFII250 in Cell Cycle Mutant tsBN462 Cells

Journal: Molecular and Cellular Biology

doi:

Effect of Mdm2, TAF II 250, and inhibiting Mdm2-p53 interactions on the cell cycle. tsBN462 cells were transfected in 9-cm dishes with vectors that express CD20 (5 μg, all samples) and, in addition, Hdm2 (10 μg), Hdm2 G58A (10 μg), Hdm2 V75A (10 μg), and CTS1 (10 μg) (A) and TAF II 250 (10 μg), pBC (10 μg, control for IP3.2), IP3.2 (pBC-IP3.2 10 μg), and TAF II 250 plus IP3.2 (10 μg of each) (B); after 24 h at 39°C the cells were harvested and analyzed by flow cytometry. The results presented are the average of two values, each of which was derived from two independently transfected plates. One representative experiment of three is shown. The percentage of transfected cells in each phase of the cell cycle was calculated and subtracted from the control transfected with empty vector. The control (zero change) is presented as small bars for illustration only.
Figure Legend Snippet: Effect of Mdm2, TAF II 250, and inhibiting Mdm2-p53 interactions on the cell cycle. tsBN462 cells were transfected in 9-cm dishes with vectors that express CD20 (5 μg, all samples) and, in addition, Hdm2 (10 μg), Hdm2 G58A (10 μg), Hdm2 V75A (10 μg), and CTS1 (10 μg) (A) and TAF II 250 (10 μg), pBC (10 μg, control for IP3.2), IP3.2 (pBC-IP3.2 10 μg), and TAF II 250 plus IP3.2 (10 μg of each) (B); after 24 h at 39°C the cells were harvested and analyzed by flow cytometry. The results presented are the average of two values, each of which was derived from two independently transfected plates. One representative experiment of three is shown. The percentage of transfected cells in each phase of the cell cycle was calculated and subtracted from the control transfected with empty vector. The control (zero change) is presented as small bars for illustration only.

Techniques Used: Transfection, Flow Cytometry, Cytometry, Derivative Assay, Plasmid Preparation

8) Product Images from "The proapoptotic function of Drosophila Hid is conserved in mammalian cells"

Article Title: The proapoptotic function of Drosophila Hid is conserved in mammalian cells

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

doi:

Hid localizes to the mitochondria. HeLa cells were transfected with pcDNA3 Hid. Twenty-four hours posttransfection, the cells were stained with 100 nM MitoTracker for 30 minutes, fixed, and immunostained with a mAb to Hid (CL1C3) and a FITC-conjugated secondary antibody. A – C shows a single HeLa cell visualized with indirect fluorescence microscopy filtered to allow FITC ( A ), MitoTracker ( B ), or both ( C ) to be seen. ( D ) Enlarged detail of the area indicated by the white box in C .
Figure Legend Snippet: Hid localizes to the mitochondria. HeLa cells were transfected with pcDNA3 Hid. Twenty-four hours posttransfection, the cells were stained with 100 nM MitoTracker for 30 minutes, fixed, and immunostained with a mAb to Hid (CL1C3) and a FITC-conjugated secondary antibody. A – C shows a single HeLa cell visualized with indirect fluorescence microscopy filtered to allow FITC ( A ), MitoTracker ( B ), or both ( C ) to be seen. ( D ) Enlarged detail of the area indicated by the white box in C .

Techniques Used: Transfection, Staining, Fluorescence, Microscopy

Hid induces apoptotic morphology in HeLa cells. HeLa cells were transiently transfected with 0.5 μg of pcDNA3 empty vector ( A ) or 0.5 μg of pcDNA3 Hid ( B ) along with 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed, stained with X-Gal, and observed by using light microscopy. ( C – E ) HeLa cells were transfected with 0.5 μg of pcDNA3 Hid along with 0.5 μg of pEGFP-CMV. At 24 hours, they were fixed, and the cells were stained with Hoechst dye no. 33358 (5 μg/ml) for 30 minutes, washed, and observed by using fluorescence microscopy on a FITC filter set ( C ) and a blue filterset ( D ). ( E ) Artificially merged images. ( F ) Quantification of apoptosis: The indicated cell types were transfected with 0.5 μg of pcDNA3 Hid or pcDNA3 along with a 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed and stained with X-Gal. Two hundred blue cells were counted in each of two duplicate transfections in three independent experiments. The fraction of blue stained cells with apoptotic morphology as a percentage of all blue-stained cells was calculated. The percentage is expressed as the mean ± SEM.
Figure Legend Snippet: Hid induces apoptotic morphology in HeLa cells. HeLa cells were transiently transfected with 0.5 μg of pcDNA3 empty vector ( A ) or 0.5 μg of pcDNA3 Hid ( B ) along with 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed, stained with X-Gal, and observed by using light microscopy. ( C – E ) HeLa cells were transfected with 0.5 μg of pcDNA3 Hid along with 0.5 μg of pEGFP-CMV. At 24 hours, they were fixed, and the cells were stained with Hoechst dye no. 33358 (5 μg/ml) for 30 minutes, washed, and observed by using fluorescence microscopy on a FITC filter set ( C ) and a blue filterset ( D ). ( E ) Artificially merged images. ( F ) Quantification of apoptosis: The indicated cell types were transfected with 0.5 μg of pcDNA3 Hid or pcDNA3 along with a 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed and stained with X-Gal. Two hundred blue cells were counted in each of two duplicate transfections in three independent experiments. The fraction of blue stained cells with apoptotic morphology as a percentage of all blue-stained cells was calculated. The percentage is expressed as the mean ± SEM.

Techniques Used: Transfection, Plasmid Preparation, Staining, Light Microscopy, Fluorescence, Microscopy

9) Product Images from "Transient Disruption of Intercellular Junctions Enables Baculovirus Entry into Nondividing Hepatocytes"

Article Title: Transient Disruption of Intercellular Junctions Enables Baculovirus Entry into Nondividing Hepatocytes

Journal: Journal of Virology

doi: 10.1128/JVI.75.20.9857-9871.2001

Increased dose-dependent gene transfer into primary rat hepatocytes under calcium-depleted conditions. Twenty-one days postseeding, primary rat hepatocytes were pretreated in either control medium (light shaded bars), calcium-free DMEM (dark shaded bars), or calcium-free DMEM supplemented with either 25 (open bars) or 100 (solid bars) μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with the indicated MOI of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA. At 24 h p.i., cultures were stained for β-Gal activity. The percent β-Gal-positive cells was determined for the total culture (A), the peripheral cells (B), and the internal cells (C). (D) Fold increase in internal β-Gal-positive cells following extracellular calcium depletion over the level in control medium. Each bar represents the mean percent β-Gal-positive primary rat hepatocytes at the indicated PFU of CMV- lacZ baculovirus/cell, with the standard deviation.
Figure Legend Snippet: Increased dose-dependent gene transfer into primary rat hepatocytes under calcium-depleted conditions. Twenty-one days postseeding, primary rat hepatocytes were pretreated in either control medium (light shaded bars), calcium-free DMEM (dark shaded bars), or calcium-free DMEM supplemented with either 25 (open bars) or 100 (solid bars) μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with the indicated MOI of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA. At 24 h p.i., cultures were stained for β-Gal activity. The percent β-Gal-positive cells was determined for the total culture (A), the peripheral cells (B), and the internal cells (C). (D) Fold increase in internal β-Gal-positive cells following extracellular calcium depletion over the level in control medium. Each bar represents the mean percent β-Gal-positive primary rat hepatocytes at the indicated PFU of CMV- lacZ baculovirus/cell, with the standard deviation.

Techniques Used: Infection, Staining, Activity Assay, Standard Deviation

Determination of the proper pretreatment time prior to infection affording the maximal baculovirus-mediated gene delivery efficiency. At 20 days postseeding, primary rat hepatocytes were pretreated for the indicated times in either control medium (shaded bars), calcium-free DMEM (open bars), or calcium-free DMEM supplemented with 25 μM EGTA (solid bars). Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with 25 μM EGTA. At 24 h p.i., cultures were stained for β-Gal activity. The percent β-Gal-positive cells was determined for the total culture (A), the peripheral cells (B), and the internal cells (C). Each bar represents the mean percent β-Gal-positive primary rat hepatocytes, with the standard deviation, at the indicated pretreatment time prior to CMV- lacZ baculovirus infection.
Figure Legend Snippet: Determination of the proper pretreatment time prior to infection affording the maximal baculovirus-mediated gene delivery efficiency. At 20 days postseeding, primary rat hepatocytes were pretreated for the indicated times in either control medium (shaded bars), calcium-free DMEM (open bars), or calcium-free DMEM supplemented with 25 μM EGTA (solid bars). Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with 25 μM EGTA. At 24 h p.i., cultures were stained for β-Gal activity. The percent β-Gal-positive cells was determined for the total culture (A), the peripheral cells (B), and the internal cells (C). Each bar represents the mean percent β-Gal-positive primary rat hepatocytes, with the standard deviation, at the indicated pretreatment time prior to CMV- lacZ baculovirus infection.

Techniques Used: Infection, Staining, Activity Assay, Standard Deviation

Detection of β-Gal activity at various times after infection of long-term cultures of primary hepatocytes following calcium depletion. Twenty days postseeding, primary rat hepatocytes were pretreated in either control medium (light shaded bars), calcium-free DMEM (dark shaded bars), or calcium-free DMEM supplemented with either 25 (open bars) or 100 (solid bars) μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA. At the indicated times p.i., cultures were stained for β-Gal activity. The percent β-Gal-positive cells was determined for the total culture (A), the peripheral cells (B), and the internal cells (C). (D) Fold increase in internal β-Gal-positive cells following extracellular calcium depletion relative to β-Gal-positive internal cells in the control medium. Each bar represents the mean percent β-Gal-positive primary rat hepatocytes at the indicated time p.i., with the standard deviation.
Figure Legend Snippet: Detection of β-Gal activity at various times after infection of long-term cultures of primary hepatocytes following calcium depletion. Twenty days postseeding, primary rat hepatocytes were pretreated in either control medium (light shaded bars), calcium-free DMEM (dark shaded bars), or calcium-free DMEM supplemented with either 25 (open bars) or 100 (solid bars) μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA. At the indicated times p.i., cultures were stained for β-Gal activity. The percent β-Gal-positive cells was determined for the total culture (A), the peripheral cells (B), and the internal cells (C). (D) Fold increase in internal β-Gal-positive cells following extracellular calcium depletion relative to β-Gal-positive internal cells in the control medium. Each bar represents the mean percent β-Gal-positive primary rat hepatocytes at the indicated time p.i., with the standard deviation.

Techniques Used: Activity Assay, Infection, Staining, Standard Deviation

Effect of calcium depletion on baculovirus-mediated gene delivery to internal hepatocytes. At day 20 postseeding, primary rat hepatocytes were pretreated in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium (A), calcium-free DMEM (B), or calcium-free DMEM supplemented with either 25 (C) or 100 (D) μM EGTA. At 24 h p.i., cultures were stained for β-Gal activity.
Figure Legend Snippet: Effect of calcium depletion on baculovirus-mediated gene delivery to internal hepatocytes. At day 20 postseeding, primary rat hepatocytes were pretreated in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium (A), calcium-free DMEM (B), or calcium-free DMEM supplemented with either 25 (C) or 100 (D) μM EGTA. At 24 h p.i., cultures were stained for β-Gal activity.

Techniques Used: Infection, Staining, Activity Assay

Baculovirus-mediated gene transfer into primary rat hepatocytes in short-term culture. (A) Dose dependence of baculovirus-mediated gene transfer into short-term culture. Primary rat hepatocytes were perfused and seeded on collagen-coated dishes. Cultures were infected with CMV- lacZ baculovirus at the indicated multiplicities 24 h postseeding. At 24 h p.i., primary hepatocyte cultures were stained for β-Gal activity as described in Materials and Methods, and the percent β-Gal-positive cells was determined. The mean percent β-Gal-positive cells, with the standard deviation, is shown as a function of the PFU of CMV- lacZ baculovirus/cell used to infect primary rat hepatocyte cultures. (B) Effect of time in culture on susceptibility of primary rat hepatocytes to baculovirus-mediated gene transfer. Primary rat hepatocytes were perfused, seeded on collagen-coated dishes, and infected with 300 PFU of CMV- lacZ baculovirus/cell on the indicated days postseeding. At 24 h p.i., cultures were stained for β-Gal activity and the percent positive cells was determined. Each symbol represents the mean percentage of primary rat hepatocytes at the indicated day postseeding that were β-Gal positive, with the standard deviation. (C) Detection of β-Gal activity at various times after infection of hepatocytes in short-term culture. Primary rat hepatocytes were seeded on collagen-coated dishes and infected with 200 PFU of CMV- lacZ baculovirus/cell 24 h postseeding. Cultures were stained for β-Gal activity at the indicated number of days p.i., and the percent β-Gal-positive cells was determined. Each symbol represents the mean percentage of primary rat hepatocytes that stained β-Gal positive at the indicated day p.i., with the standard deviation.
Figure Legend Snippet: Baculovirus-mediated gene transfer into primary rat hepatocytes in short-term culture. (A) Dose dependence of baculovirus-mediated gene transfer into short-term culture. Primary rat hepatocytes were perfused and seeded on collagen-coated dishes. Cultures were infected with CMV- lacZ baculovirus at the indicated multiplicities 24 h postseeding. At 24 h p.i., primary hepatocyte cultures were stained for β-Gal activity as described in Materials and Methods, and the percent β-Gal-positive cells was determined. The mean percent β-Gal-positive cells, with the standard deviation, is shown as a function of the PFU of CMV- lacZ baculovirus/cell used to infect primary rat hepatocyte cultures. (B) Effect of time in culture on susceptibility of primary rat hepatocytes to baculovirus-mediated gene transfer. Primary rat hepatocytes were perfused, seeded on collagen-coated dishes, and infected with 300 PFU of CMV- lacZ baculovirus/cell on the indicated days postseeding. At 24 h p.i., cultures were stained for β-Gal activity and the percent positive cells was determined. Each symbol represents the mean percentage of primary rat hepatocytes at the indicated day postseeding that were β-Gal positive, with the standard deviation. (C) Detection of β-Gal activity at various times after infection of hepatocytes in short-term culture. Primary rat hepatocytes were seeded on collagen-coated dishes and infected with 200 PFU of CMV- lacZ baculovirus/cell 24 h postseeding. Cultures were stained for β-Gal activity at the indicated number of days p.i., and the percent β-Gal-positive cells was determined. Each symbol represents the mean percentage of primary rat hepatocytes that stained β-Gal positive at the indicated day p.i., with the standard deviation.

Techniques Used: Infection, Staining, Activity Assay, Standard Deviation

Dose-dependent baculovirus-mediated gene transfer into primary rat hepatocytes in long-term DMSO culture. (A) Fifty-five days postseeding, primary rat hepatocytes in long-term DMSO culture were infected with 3, 6, 12, 25, 50, or 100 PFU of CMV- lacZ baculovirus/cell. At 24 h p.i., cultures were stained for β-Gal activity and the percent positive peripheral (edge) cells was determined (□). In a separate experiment, primary rat hepatocytes in long-term DMSO culture for 56 days were infected with 50, 100, 200, 400, or 800 PFU of CMV- lacZ baculovirus/cell (●). At 24 h p.i., cultures were stained for β-Gal activity. Results show the percentage of peripheral hepatocytes positive for β-Gal activity, with the standard deviation, at the indicated PFU of CMV- lacZ baculovirus/cell. (B) Primary rat hepatocytes in long-term DMSO culture for 56 days were mock infected and stained for β-Gal activity 24 h later. (C) Primary hepatocytes in long-term DMSO culture for 56 days were infected with 800 PFU of CMV- lacZ baculovirus/cell and stained for β-Gal activity at 24 h p.i.
Figure Legend Snippet: Dose-dependent baculovirus-mediated gene transfer into primary rat hepatocytes in long-term DMSO culture. (A) Fifty-five days postseeding, primary rat hepatocytes in long-term DMSO culture were infected with 3, 6, 12, 25, 50, or 100 PFU of CMV- lacZ baculovirus/cell. At 24 h p.i., cultures were stained for β-Gal activity and the percent positive peripheral (edge) cells was determined (□). In a separate experiment, primary rat hepatocytes in long-term DMSO culture for 56 days were infected with 50, 100, 200, 400, or 800 PFU of CMV- lacZ baculovirus/cell (●). At 24 h p.i., cultures were stained for β-Gal activity. Results show the percentage of peripheral hepatocytes positive for β-Gal activity, with the standard deviation, at the indicated PFU of CMV- lacZ baculovirus/cell. (B) Primary rat hepatocytes in long-term DMSO culture for 56 days were mock infected and stained for β-Gal activity 24 h later. (C) Primary hepatocytes in long-term DMSO culture for 56 days were infected with 800 PFU of CMV- lacZ baculovirus/cell and stained for β-Gal activity at 24 h p.i.

Techniques Used: Infection, Staining, Activity Assay, Standard Deviation

Detection of β-Gal activity at various times after infection of long-term cultures of primary hepatocytes and susceptibility of peripheral cells to reinfection with CMV- lacZ baculovirus. (A) Detection of β-Gal activity at various times after infection of a long-term DMSO culture. Thirty days postseeding, primary rat hepatocytes in DMSO culture were infected with 400 PFU of CMV- lacZ baculovirus/cell. At the indicated time p.i., cultures were stained for β-Gal activity and the percent β-Gal-positive peripheral (edge) cells was determined. The results show the percentage of peripheral cells positive for β-Gal activity at the indicated time p.i., with the standard deviation. Where no error bar is shown, the error falls within the size of the symbol. (B) Susceptibility of peripheral cells to reinfection. At day 30 postseeding, hepatocytes were either mock infected or infected with 400 PFU of CMV- lacZ baculovirus/cell. Cultures were divided into three groups. One group of cultures infected on day 30 was mock infected at day 49 (+, −), one group of cultures infected at day 30 was reinfected at day 49 (+, +), and the cultures mock infected at day 30 were infected at day 49 (−, +). All cultures were fixed and stained for β-Gal activity at 50 days postseeding. Each bar represents the mean percentage of peripheral (edge) hepatocytes positive for β-Gal activity, with the standard deviation, following infection at the indicated day postseeding.
Figure Legend Snippet: Detection of β-Gal activity at various times after infection of long-term cultures of primary hepatocytes and susceptibility of peripheral cells to reinfection with CMV- lacZ baculovirus. (A) Detection of β-Gal activity at various times after infection of a long-term DMSO culture. Thirty days postseeding, primary rat hepatocytes in DMSO culture were infected with 400 PFU of CMV- lacZ baculovirus/cell. At the indicated time p.i., cultures were stained for β-Gal activity and the percent β-Gal-positive peripheral (edge) cells was determined. The results show the percentage of peripheral cells positive for β-Gal activity at the indicated time p.i., with the standard deviation. Where no error bar is shown, the error falls within the size of the symbol. (B) Susceptibility of peripheral cells to reinfection. At day 30 postseeding, hepatocytes were either mock infected or infected with 400 PFU of CMV- lacZ baculovirus/cell. Cultures were divided into three groups. One group of cultures infected on day 30 was mock infected at day 49 (+, −), one group of cultures infected at day 30 was reinfected at day 49 (+, +), and the cultures mock infected at day 30 were infected at day 49 (−, +). All cultures were fixed and stained for β-Gal activity at 50 days postseeding. Each bar represents the mean percentage of peripheral (edge) hepatocytes positive for β-Gal activity, with the standard deviation, following infection at the indicated day postseeding.

Techniques Used: Activity Assay, Infection, Staining, Standard Deviation

Motility of primary hepatocytes in long-term DMSO culture. Primary rat hepatocytes in long-term DMSO culture for 30 days were infected with 400 PFU of CMV- lacZ baculovirus/cell. Cultures were stained for β-Gal activity after 1 (A), 5 (B), 10 (C), 15 (D), and 30 (E) days p.i. (F) Susceptibility to reinfection of peripheral (edge) cells in long-term primary rat hepatocyte cultures maintained in DMSO. A culture infected on day 30 postseeding was reinfected with an additional 400 PFU of CMV- lacZ baculovirus/cell on day 49 and was stained for β-Gal activity at 24 h p.i.
Figure Legend Snippet: Motility of primary hepatocytes in long-term DMSO culture. Primary rat hepatocytes in long-term DMSO culture for 30 days were infected with 400 PFU of CMV- lacZ baculovirus/cell. Cultures were stained for β-Gal activity after 1 (A), 5 (B), 10 (C), 15 (D), and 30 (E) days p.i. (F) Susceptibility to reinfection of peripheral (edge) cells in long-term primary rat hepatocyte cultures maintained in DMSO. A culture infected on day 30 postseeding was reinfected with an additional 400 PFU of CMV- lacZ baculovirus/cell on day 49 and was stained for β-Gal activity at 24 h p.i.

Techniques Used: Infection, Staining, Activity Assay

Immunoperoxidase staining for Ac M NPV in primary rat hepatocytes. Twenty-one days postseeding, primary rat hepatocytes were pretreated in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA. At 3 h p.i., cultures were fixed and incubated with an antibody to Ac M NPV (A, C, D, E, and F) or a control antibody (B) as described in Materials and Methods. (A) Primary rat hepatocytes at 21 days postseeding that were pretreated and mock infected in control medium. (B) Cultures pretreated and infected with the CMV- lacZ baculovirus in control medium and incubated with a rabbit IgG control antibody. (C) Cultures pretreated and infected in control medium. (D) Cultures pretreated and infected in calcium-free DMEM. (E and F) Cultures pretreated and infected under calcium depletion conditions of 25 and 100 μM, respectively.
Figure Legend Snippet: Immunoperoxidase staining for Ac M NPV in primary rat hepatocytes. Twenty-one days postseeding, primary rat hepatocytes were pretreated in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25 or 100 μM EGTA. At 3 h p.i., cultures were fixed and incubated with an antibody to Ac M NPV (A, C, D, E, and F) or a control antibody (B) as described in Materials and Methods. (A) Primary rat hepatocytes at 21 days postseeding that were pretreated and mock infected in control medium. (B) Cultures pretreated and infected with the CMV- lacZ baculovirus in control medium and incubated with a rabbit IgG control antibody. (C) Cultures pretreated and infected in control medium. (D) Cultures pretreated and infected in calcium-free DMEM. (E and F) Cultures pretreated and infected under calcium depletion conditions of 25 and 100 μM, respectively.

Techniques Used: Immunoperoxidase Staining, Infection, Incubation

Effect of calcium depletion on baculovirus-mediated gene delivery of CMV- lacZ to primary rat hepatocyte cultures. At the indicated days postseeding, primary rat hepatocytes were pretreated in either control medium (DMEM–F12; designated Control), calcium-free DMEM (Ca-free), or calcium-free DMEM supplemented with either 25 (25 μM), 100 (100 μM), 250 (250 μM), or 500 (500 μM) μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25, 100, 250, or 500 μM EGTA. At 24 h p.i., cultures were stained for β-Gal activity. Percent β-Gal-positive cells was determined for the total culture (A), the peripheral cells (B), and the internal cells (C). (D) Fold increase in internal β-Gal-positive cells following extracellular calcium depletion relative to the control medium. Each bar represents the mean percent β-Gal-positive primary rat hepatocytes, with the standard deviation, at the indicated medium condition. (E) Percent cytopathic cells present in the primary rat hepatocyte cultures following calcium depletion prior to and during baculovirus infection. Each bar represents the mean percent cytopathic cells at the indicated medium condition, with the standard deviation.
Figure Legend Snippet: Effect of calcium depletion on baculovirus-mediated gene delivery of CMV- lacZ to primary rat hepatocyte cultures. At the indicated days postseeding, primary rat hepatocytes were pretreated in either control medium (DMEM–F12; designated Control), calcium-free DMEM (Ca-free), or calcium-free DMEM supplemented with either 25 (25 μM), 100 (100 μM), 250 (250 μM), or 500 (500 μM) μM EGTA for 1 h. Subsequently, cultures were infected for 1 h with 400 PFU of CMV- lacZ baculovirus/cell diluted in either control medium, calcium-free DMEM, or calcium-free DMEM supplemented with either 25, 100, 250, or 500 μM EGTA. At 24 h p.i., cultures were stained for β-Gal activity. Percent β-Gal-positive cells was determined for the total culture (A), the peripheral cells (B), and the internal cells (C). (D) Fold increase in internal β-Gal-positive cells following extracellular calcium depletion relative to the control medium. Each bar represents the mean percent β-Gal-positive primary rat hepatocytes, with the standard deviation, at the indicated medium condition. (E) Percent cytopathic cells present in the primary rat hepatocyte cultures following calcium depletion prior to and during baculovirus infection. Each bar represents the mean percent cytopathic cells at the indicated medium condition, with the standard deviation.

Techniques Used: Infection, Staining, Activity Assay, Standard Deviation

10) Product Images from "A Zebrafish Cell Culture Assay for the Identification of MicroRNA Targets"

Article Title: A Zebrafish Cell Culture Assay for the Identification of MicroRNA Targets

Journal: Zebrafish

doi: 10.1089/zeb.2010.0674

HEK293 cells fail to support the interaction of zebrafish miR-430b with known mRNA targets. (A) Time course of miR-430b-mediated inhibition of luciferase expression using the reporter construct pGL3-3xPT, which carries three tandom copies of the perfect target sequence. pGL3-3xPT and miR-430b were cotransfected into the HEK293 cells and luciferase expression was measured at the times indicated post-transfection. (B) miR-430b-mediated inhibition of luciferase expression using reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, and pGl3-3′nanos that carry 3′UTRs containing known miR-430b target sequences. Luciferase expression was measured at 48 h post-transfection (hpt). pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively, and the transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar on the graph represents the average of four separate transfections.
Figure Legend Snippet: HEK293 cells fail to support the interaction of zebrafish miR-430b with known mRNA targets. (A) Time course of miR-430b-mediated inhibition of luciferase expression using the reporter construct pGL3-3xPT, which carries three tandom copies of the perfect target sequence. pGL3-3xPT and miR-430b were cotransfected into the HEK293 cells and luciferase expression was measured at the times indicated post-transfection. (B) miR-430b-mediated inhibition of luciferase expression using reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, and pGl3-3′nanos that carry 3′UTRs containing known miR-430b target sequences. Luciferase expression was measured at 48 h post-transfection (hpt). pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively, and the transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar on the graph represents the average of four separate transfections.

Techniques Used: Inhibition, Luciferase, Expressing, Construct, Sequencing, Transfection

ZSSJ cells support the interaction of zebrafish miR-430b and mRNA target sequences. ZSSJ cells were transfected with miR-430b or control miRNA along with one of three luciferase reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, or pGl3-3′nanos, that carry known 3′UTR target sequences for miR-430b. Luciferase expression was measured 48 hpt. pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively. The transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar represents the average of three separate transfections. *A significant difference ( p
Figure Legend Snippet: ZSSJ cells support the interaction of zebrafish miR-430b and mRNA target sequences. ZSSJ cells were transfected with miR-430b or control miRNA along with one of three luciferase reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, or pGl3-3′nanos, that carry known 3′UTR target sequences for miR-430b. Luciferase expression was measured 48 hpt. pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively. The transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar represents the average of three separate transfections. *A significant difference ( p

Techniques Used: Transfection, Luciferase, Construct, Expressing

miRNA transfection efficiency of ZF and ZSSJ cells. ZF and ZSSJ cell cultures were transfected with siRNA coupled with a red fluorophore in a 12-well plate. Cell fluorescence was observed under a Nikon fluorescence microscope equipped with a digital camera. The pictures were taken 48 hpt.
Figure Legend Snippet: miRNA transfection efficiency of ZF and ZSSJ cells. ZF and ZSSJ cell cultures were transfected with siRNA coupled with a red fluorophore in a 12-well plate. Cell fluorescence was observed under a Nikon fluorescence microscope equipped with a digital camera. The pictures were taken 48 hpt.

Techniques Used: Transfection, Fluorescence, Microscopy

11) Product Images from "A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissue-specific amplification of gene expression"

Article Title: A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissue-specific amplification of gene expression

Journal: Nucleic Acids Research

doi:

Construction and functional testing of single vectors containing both modified Cre recombinase and LOX-dependent conditional reporter cassettes. ( A ) pPr-CREM/CMV-STOP-luc vector is a fusion of pPr-CREM and CMV-STOP-luc cassettes. pPr-CREM/Ins/CMV-STOP-luc contains pPr-CREM/Ins separated from CMV-STOP-luc by two chicken globin insulators. ( B ) Evaluation of inducible and amplified gene expression using single vector constructs. PC-3 cells were transfected with pCMV-mAR in the presence or absence of 4 nM mibolerone and additional plasmids (0.1 pmol) as indicated. Assays were performed 48 h post-transfection.
Figure Legend Snippet: Construction and functional testing of single vectors containing both modified Cre recombinase and LOX-dependent conditional reporter cassettes. ( A ) pPr-CREM/CMV-STOP-luc vector is a fusion of pPr-CREM and CMV-STOP-luc cassettes. pPr-CREM/Ins/CMV-STOP-luc contains pPr-CREM/Ins separated from CMV-STOP-luc by two chicken globin insulators. ( B ) Evaluation of inducible and amplified gene expression using single vector constructs. PC-3 cells were transfected with pCMV-mAR in the presence or absence of 4 nM mibolerone and additional plasmids (0.1 pmol) as indicated. Assays were performed 48 h post-transfection.

Techniques Used: Functional Assay, Modification, Plasmid Preparation, Amplification, Expressing, Construct, Transfection

Amplification of gene expression using a weak, tissue-specific promoter to activate transcription from a strong promoter. ( A ) Luciferase reporter gene vectors. SV, SV40 early promoter; CMVe-βAc, CMV enhancer, β-actin promoter. ( B ) PC-3 cells were transfected with the reporter constructs pPr-luc, pCMV-STOP-luc or pCMV-STOP-luc with pPr-cre. Luciferase expression increased 15-fold using this amplification strategy. Assays performed 48 h post-transfection.
Figure Legend Snippet: Amplification of gene expression using a weak, tissue-specific promoter to activate transcription from a strong promoter. ( A ) Luciferase reporter gene vectors. SV, SV40 early promoter; CMVe-βAc, CMV enhancer, β-actin promoter. ( B ) PC-3 cells were transfected with the reporter constructs pPr-luc, pCMV-STOP-luc or pCMV-STOP-luc with pPr-cre. Luciferase expression increased 15-fold using this amplification strategy. Assays performed 48 h post-transfection.

Techniques Used: Amplification, Expressing, Luciferase, Transfection, Construct

12) Product Images from "Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein"

Article Title: Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein

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

doi:

High-powered section of skeletal muscle 2 months after injection with AAV-lacZ. Tibialis anterior muscle was processed for in situ detection of β-gal and photographed with diffraction-interference contrast optics at ×400.
Figure Legend Snippet: High-powered section of skeletal muscle 2 months after injection with AAV-lacZ. Tibialis anterior muscle was processed for in situ detection of β-gal and photographed with diffraction-interference contrast optics at ×400.

Techniques Used: Injection, In Situ

( A ) pAAV-lacZ. ITR, inverted terminal repeat; CMV, CMV promoter; lacZ, bacterial β-gal; An, simian virus 40 (SV40) early polyadenylylation signal. ( B ) pAAV-Epo. Epo, human Epo coding sequence; An, SV40 polyadenylylation signal; noncoding, 2.2-kb noncoding fragment from the lacZ gene. See text for details.
Figure Legend Snippet: ( A ) pAAV-lacZ. ITR, inverted terminal repeat; CMV, CMV promoter; lacZ, bacterial β-gal; An, simian virus 40 (SV40) early polyadenylylation signal. ( B ) pAAV-Epo. Epo, human Epo coding sequence; An, SV40 polyadenylylation signal; noncoding, 2.2-kb noncoding fragment from the lacZ gene. See text for details.

Techniques Used: Sequencing

13) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

Journal: Scientific Reports

doi: 10.1038/srep16435

Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.
Figure Legend Snippet: Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.

Techniques Used: Reporter Assay, Activity Assay, Sequencing, Plasmid Preparation, Construct, Derivative Assay, Negative Control, Expressing, Transfection, Standard Deviation

14) Product Images from "A Zebrafish Cell Culture Assay for the Identification of MicroRNA Targets"

Article Title: A Zebrafish Cell Culture Assay for the Identification of MicroRNA Targets

Journal: Zebrafish

doi: 10.1089/zeb.2010.0674

HEK293 cells fail to support the interaction of zebrafish miR-430b with known mRNA targets. (A) Time course of miR-430b-mediated inhibition of luciferase expression using the reporter construct pGL3-3xPT, which carries three tandom copies of the perfect target sequence. pGL3-3xPT and miR-430b were cotransfected into the HEK293 cells and luciferase expression was measured at the times indicated post-transfection. (B) miR-430b-mediated inhibition of luciferase expression using reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, and pGl3-3′nanos that carry 3′UTRs containing known miR-430b target sequences. Luciferase expression was measured at 48 h post-transfection (hpt). pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively, and the transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar on the graph represents the average of four separate transfections.
Figure Legend Snippet: HEK293 cells fail to support the interaction of zebrafish miR-430b with known mRNA targets. (A) Time course of miR-430b-mediated inhibition of luciferase expression using the reporter construct pGL3-3xPT, which carries three tandom copies of the perfect target sequence. pGL3-3xPT and miR-430b were cotransfected into the HEK293 cells and luciferase expression was measured at the times indicated post-transfection. (B) miR-430b-mediated inhibition of luciferase expression using reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, and pGl3-3′nanos that carry 3′UTRs containing known miR-430b target sequences. Luciferase expression was measured at 48 h post-transfection (hpt). pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively, and the transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar on the graph represents the average of four separate transfections.

Techniques Used: Inhibition, Luciferase, Expressing, Construct, Sequencing, Transfection

ZSSJ cells support the interaction of zebrafish miR-430b and mRNA target sequences. ZSSJ cells were transfected with miR-430b or control miRNA along with one of three luciferase reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, or pGl3-3′nanos, that carry known 3′UTR target sequences for miR-430b. Luciferase expression was measured 48 hpt. pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively. The transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar represents the average of three separate transfections. *A significant difference ( p
Figure Legend Snippet: ZSSJ cells support the interaction of zebrafish miR-430b and mRNA target sequences. ZSSJ cells were transfected with miR-430b or control miRNA along with one of three luciferase reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, or pGl3-3′nanos, that carry known 3′UTR target sequences for miR-430b. Luciferase expression was measured 48 hpt. pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively. The transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar represents the average of three separate transfections. *A significant difference ( p

Techniques Used: Transfection, Luciferase, Construct, Expressing

15) Product Images from "Electroporation Enhances Immunogenicity of a DNA Vaccine Expressing Woodchuck Hepatitis Virus Surface Antigen in Woodchucks ▿"

Article Title: Electroporation Enhances Immunogenicity of a DNA Vaccine Expressing Woodchuck Hepatitis Virus Surface Antigen in Woodchucks ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02437-10

Expression of reporter genes following non-EP or EP injection of pDNA into the tibialis cranialis muscle of woodchucks. (A) LacZ expression. Three woodchucks received a single dose of vector pCMV-beta (0.5 mg pDNA in 0.5 ml PBS) into the right tibialis
Figure Legend Snippet: Expression of reporter genes following non-EP or EP injection of pDNA into the tibialis cranialis muscle of woodchucks. (A) LacZ expression. Three woodchucks received a single dose of vector pCMV-beta (0.5 mg pDNA in 0.5 ml PBS) into the right tibialis

Techniques Used: Expressing, Injection, Plasmid Preparation

16) Product Images from "A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy"

Article Title: A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy

Journal: Brain

doi: 10.1093/brain/awx058

In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P
Figure Legend Snippet: In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P

Techniques Used: In Vitro, Mutagenesis, Reporter Assay, Luciferase, Dominant Negative Mutation, Transfection, Expressing

His257Arg (H257R) TrpRS inhibits neurite outgrowth and leads to neurite degeneration. Neuro-2a (N2a) ( A ) or SH-SY5Y ( B ) cells were transfected with expression vector containing wild-type (WT) or H257R TrpRS or empty vector (vector control), grown under differentiation conditions, and immunostained against Myc (exogenous TrpRS staining) and neurofilament heavy polypeptide (NFH; neurite staining) at 72 h post-transfection. The representative fluorescence-immunostaining and phase-contrast images were shown, along with statistical results of per cent of cells bearing neurites and average primary neurite length. Scale bar = 50 μm. At least 100 cells from three independent experiments were measured for each preparation and data were expressed as the mean ± standard error of the mean (SEM). * P -value
Figure Legend Snippet: His257Arg (H257R) TrpRS inhibits neurite outgrowth and leads to neurite degeneration. Neuro-2a (N2a) ( A ) or SH-SY5Y ( B ) cells were transfected with expression vector containing wild-type (WT) or H257R TrpRS or empty vector (vector control), grown under differentiation conditions, and immunostained against Myc (exogenous TrpRS staining) and neurofilament heavy polypeptide (NFH; neurite staining) at 72 h post-transfection. The representative fluorescence-immunostaining and phase-contrast images were shown, along with statistical results of per cent of cells bearing neurites and average primary neurite length. Scale bar = 50 μm. At least 100 cells from three independent experiments were measured for each preparation and data were expressed as the mean ± standard error of the mean (SEM). * P -value

Techniques Used: Transfection, Expressing, Plasmid Preparation, Staining, Fluorescence, Immunostaining

17) Product Images from "Treatment and Prevention of Lung Cancer Using a Virus-Infected Reprogrammed Somatic Cell-Derived Tumor Cell Vaccination (VIReST) Regime"

Article Title: Treatment and Prevention of Lung Cancer Using a Virus-Infected Reprogrammed Somatic Cell-Derived Tumor Cell Vaccination (VIReST) Regime

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.01996

AdV and VV can infect and replicate in transformed iPSCs and mitomycin-C treatment inhibits ongoing replication and tumor cell proliferation. (A) Cytotoxicity of Ad5 or VV on iPSC-derived KP-LC cells and KPL 160302S lung tumor cells. Cell death was determined by MTS assay 144 h post-infection. Mean EC50 values ± SEM are shown. (B) Production of infectious Ad5 or VV virions in KP-LC cells. Cells were infected with virus and were untreated or treated with mitomycin C. Mean viral replication ± SEM was determined at 24 h intervals for 96 h by TCID50 assay. JH293 cells for Ad5 or CV1 cells for VVL15-RFP. (C) Cell proliferation of KP-LC cells after infection and mitomycin-C treatment was determined using MTS assay at 24 and 72 h post-mitomycin C treatment. Mean OD 490 nm values ± SEM are shown. n = 3/group. (D) Cell proliferation of KP-LC cells after infection and mitomycin-C treatment was determined by cell counting at 48 and 96 h post-mitomycin C treatment. n = 3/group. (E) Plate colony formation of KP-LC cells after infection and mitomycin-C treatment. (F) Viral protein expression was determined in KP-LC or KPL 160302S cells at 24 and 72 h post-infection +/– mitomycin C treatment of cells. Anti-E1A was used to confirm AdV protein expression. Anti-VV coat protein was used to confirm VV protein expression. GAPDH was used as a loading control. ** p
Figure Legend Snippet: AdV and VV can infect and replicate in transformed iPSCs and mitomycin-C treatment inhibits ongoing replication and tumor cell proliferation. (A) Cytotoxicity of Ad5 or VV on iPSC-derived KP-LC cells and KPL 160302S lung tumor cells. Cell death was determined by MTS assay 144 h post-infection. Mean EC50 values ± SEM are shown. (B) Production of infectious Ad5 or VV virions in KP-LC cells. Cells were infected with virus and were untreated or treated with mitomycin C. Mean viral replication ± SEM was determined at 24 h intervals for 96 h by TCID50 assay. JH293 cells for Ad5 or CV1 cells for VVL15-RFP. (C) Cell proliferation of KP-LC cells after infection and mitomycin-C treatment was determined using MTS assay at 24 and 72 h post-mitomycin C treatment. Mean OD 490 nm values ± SEM are shown. n = 3/group. (D) Cell proliferation of KP-LC cells after infection and mitomycin-C treatment was determined by cell counting at 48 and 96 h post-mitomycin C treatment. n = 3/group. (E) Plate colony formation of KP-LC cells after infection and mitomycin-C treatment. (F) Viral protein expression was determined in KP-LC or KPL 160302S cells at 24 and 72 h post-infection +/– mitomycin C treatment of cells. Anti-E1A was used to confirm AdV protein expression. Anti-VV coat protein was used to confirm VV protein expression. GAPDH was used as a loading control. ** p

Techniques Used: Transformation Assay, Derivative Assay, MTS Assay, Infection, TCID50 Assay, Cell Counting, Expressing

18) Product Images from "Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals"

Article Title: Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals

Journal: Retrovirology

doi: 10.1186/1742-4690-6-107

Fusion assay between ASCT2 -transduced and syncytin-Ory1 -transduced co-cultured cells demonstrates that ASCT2 is the syncytin-Ory1 receptor . Left panel: Cell-cell fusion was assayed upon independent transfections of a set of A23 cells with an empty vector (none) or an expression vector for either the syncytin-Ory1, syncytin-1 or syncytin-2 protein together with an nls- LacZ gene-expression vector, and another set of A23 cells with an expression vector for the syncytin-1 receptor ASCT2, the syncytin-2 receptor MFSD2 [ 13 ] or an empty vector (none). One day after transfection, cells were resuspended and pairs of transfected cells from each set were cocultured for 1-2 days, fixed and X-Gal stained. Right panel: Syncytia can be easily detected (arrows) for the syncytin-Ory1/ASCT2, syncytin-1/ASCT2 and syncytin-2/MFSD2 pairs, with only mononucleated cells visible in the other cases. Abbreviations: syn-Ory1, syncytin-Ory1; syn1, syncytin-1; syn2, syncytin-2.
Figure Legend Snippet: Fusion assay between ASCT2 -transduced and syncytin-Ory1 -transduced co-cultured cells demonstrates that ASCT2 is the syncytin-Ory1 receptor . Left panel: Cell-cell fusion was assayed upon independent transfections of a set of A23 cells with an empty vector (none) or an expression vector for either the syncytin-Ory1, syncytin-1 or syncytin-2 protein together with an nls- LacZ gene-expression vector, and another set of A23 cells with an expression vector for the syncytin-1 receptor ASCT2, the syncytin-2 receptor MFSD2 [ 13 ] or an empty vector (none). One day after transfection, cells were resuspended and pairs of transfected cells from each set were cocultured for 1-2 days, fixed and X-Gal stained. Right panel: Syncytia can be easily detected (arrows) for the syncytin-Ory1/ASCT2, syncytin-1/ASCT2 and syncytin-2/MFSD2 pairs, with only mononucleated cells visible in the other cases. Abbreviations: syn-Ory1, syncytin-Ory1; syn1, syncytin-1; syn2, syncytin-2.

Techniques Used: Single Vesicle Fusion Assay, Cell Culture, Transfection, Plasmid Preparation, Expressing, Staining

Putative entry date of syncytin-Ory1 during lagomorph evolution . Schematized phylogenetic tree with the evolutionary timeline of four lagomorph genus (adapted from [ 16 ]) and the rodent and primate outgroups depicted, with average divergence times indicated for the nodes. The presence of syncytin-Ory1 sequences in each genus, detected either by PCR experiments (a) or database screening (b), is indicated on the right.
Figure Legend Snippet: Putative entry date of syncytin-Ory1 during lagomorph evolution . Schematized phylogenetic tree with the evolutionary timeline of four lagomorph genus (adapted from [ 16 ]) and the rodent and primate outgroups depicted, with average divergence times indicated for the nodes. The presence of syncytin-Ory1 sequences in each genus, detected either by PCR experiments (a) or database screening (b), is indicated on the right.

Techniques Used: Polymerase Chain Reaction

Fusogenic activity of syncytin-Ory1 . (A) Assay for cell-cell fusion mediated by syncytin-Ory1. The indicated cell lines were transfected with an expression vector for syncytin-Ory1 or an empty vector (none) together with a LacZ expression vector. Cells were cultured for 1-2 days after transfection, fixed and X-gal-stained. Syncytia (arrows) were detected in syncytin-Ory1 -transfected SH-SY5Y cells, with only mononucleated cells visible in the other cases. (B) Assay for cell infection mediated by syncytin-Ory1-pseudotyped virus particles. Pseudotypes were produced by cotransfection of human 293T cells with expression vectors for the SIV core, the syncytin-Ory1 protein (or an empty vector) and a LacZ -containing retroviral transcript. Supernatants were used to infect the indicated target cells, which were X-gal stained 3 days after infection. Abbreviation: Syn-Ory1, syncytin-Ory1.
Figure Legend Snippet: Fusogenic activity of syncytin-Ory1 . (A) Assay for cell-cell fusion mediated by syncytin-Ory1. The indicated cell lines were transfected with an expression vector for syncytin-Ory1 or an empty vector (none) together with a LacZ expression vector. Cells were cultured for 1-2 days after transfection, fixed and X-gal-stained. Syncytia (arrows) were detected in syncytin-Ory1 -transfected SH-SY5Y cells, with only mononucleated cells visible in the other cases. (B) Assay for cell infection mediated by syncytin-Ory1-pseudotyped virus particles. Pseudotypes were produced by cotransfection of human 293T cells with expression vectors for the SIV core, the syncytin-Ory1 protein (or an empty vector) and a LacZ -containing retroviral transcript. Supernatants were used to infect the indicated target cells, which were X-gal stained 3 days after infection. Abbreviation: Syn-Ory1, syncytin-Ory1.

Techniques Used: Activity Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Staining, Infection, Produced, Cotransfection

Structure and in situ hybridization for syncytin-Ory1 expression of day 12 rabbit placenta: (A) Schematic representation of a rabbit placenta (right) and haematoxylin and eosin staining of a day 12 placenta section (left) with the 3 main layers of the placenta indicated . (B) Higher magnification of the areas framed in A. Abbreviations: frbc: fetal red blood cell, fv: fetal blood vessel, ml: maternal blood lacuna, mrbc: maternal red blood cell. (C) In situ hybridization on sections of a day 12 rabbit placenta (serial sections of the HES in B) with digoxigenin-labeled syncytin-Ory1 sense (lower panel, negative control) and antisense (upper panel) riboprobes, revealed with an alkaline phosphatase-conjugated anti-digoxigenin antibody. Brackets and arrows highlight the positive labeling of trophoblast cells surrounding the invading fetal vessels in the junctional zone.
Figure Legend Snippet: Structure and in situ hybridization for syncytin-Ory1 expression of day 12 rabbit placenta: (A) Schematic representation of a rabbit placenta (right) and haematoxylin and eosin staining of a day 12 placenta section (left) with the 3 main layers of the placenta indicated . (B) Higher magnification of the areas framed in A. Abbreviations: frbc: fetal red blood cell, fv: fetal blood vessel, ml: maternal blood lacuna, mrbc: maternal red blood cell. (C) In situ hybridization on sections of a day 12 rabbit placenta (serial sections of the HES in B) with digoxigenin-labeled syncytin-Ory1 sense (lower panel, negative control) and antisense (upper panel) riboprobes, revealed with an alkaline phosphatase-conjugated anti-digoxigenin antibody. Brackets and arrows highlight the positive labeling of trophoblast cells surrounding the invading fetal vessels in the junctional zone.

Techniques Used: In Situ Hybridization, Expressing, Staining, Labeling, Negative Control

19) Product Images from "Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms"

Article Title: Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1001-13.2013

The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p
Figure Legend Snippet: The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p

Techniques Used: Luciferase, Activity Assay, Transfection, Mutagenesis, Construct

Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p
Figure Legend Snippet: Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p

Techniques Used: Activity Assay, Plasmid Preparation, Luciferase, Expressing, Transfection, Construct, Mutagenesis

slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p
Figure Legend Snippet: slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p

Techniques Used: Activity Assay, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Construct

20) Product Images from "Regulated expression and temporal induction of the tail-anchored sarcolemmal-membrane-associated protein is critical for myoblast fusion"

Article Title: Regulated expression and temporal induction of the tail-anchored sarcolemmal-membrane-associated protein is critical for myoblast fusion

Journal: Biochemical Journal

doi: 10.1042/BJ20031723

LZ motifs mediate SLMAP homodimer formation ( A ) SLMAP3 sequences encoding amino acids 1–450 (SLMAPN) or 450–771 (SLMAPC) were fused in-frame with either the GAL4 DNA-BD or the VP16 AD. ( B ) C2C12 myoblasts were cotransfected with the CAT reporter plasmid pCMV-LacZ, the DNA-BD recombinant plasmid or the AD recombinant plasmid. Lane 1, DNA-BD+AD; lane 2, SLMAPN-(DNA-BD)+AD; lane 3, DNA-BD+SLMAPC-(AD); lane 4, SLMAPC-(DNA-BD)+AD; lane 5, SLMAPN-(DNA-BD)+SLMAPN-(AD); lane 6, SLMAPN-(DNA-BD)+SLMAPC-(AD); lane 7, SLMAPC-(DNA-BD)+SLMAPN-(AD); lane 8, SLMAPC-(DNA-BD)+SLMAPC-(AD); lane 9, SLMAPCΔLZ-(DNA-BD)+SLMAPCΔLZ-(AD); lane 10, SLMAPCΔLZ-(DNA-BD)+SLMAPC-(AD); lane 11, SLMAPC-(DNA-BD)+SLMAPCΔLZ (AD); lane 12, ninein-LZ-(DNA-BD)+SLMAPC-(AD); lane 13, ninein-LZ-(AD)+SLMAPC-(DNA-BO) and lane 14, positive control. The level of CAT activity measured for each sample was normalized based on transfection efficiency as described in the Experimental section. SLMAP homodimer formation was mediated by C-terminal sequences (lane 8) encompassing the LZ motifs. Deletion of the LZs (lanes 9–11) failed to activate CAT reporter expression above basal levels. Error bars represent S.D. ( C ) Cells stably expressing either 6Myc-SLMAP1-ΔLZ or the control vector (6Myc-pcDNA3) were cultured in differentiation medium for 6 days, fixed with 4% PFA and stained with haematoxylin and eosin. Fusion indices were calculated for three independent clones. Error bars represent S.D.
Figure Legend Snippet: LZ motifs mediate SLMAP homodimer formation ( A ) SLMAP3 sequences encoding amino acids 1–450 (SLMAPN) or 450–771 (SLMAPC) were fused in-frame with either the GAL4 DNA-BD or the VP16 AD. ( B ) C2C12 myoblasts were cotransfected with the CAT reporter plasmid pCMV-LacZ, the DNA-BD recombinant plasmid or the AD recombinant plasmid. Lane 1, DNA-BD+AD; lane 2, SLMAPN-(DNA-BD)+AD; lane 3, DNA-BD+SLMAPC-(AD); lane 4, SLMAPC-(DNA-BD)+AD; lane 5, SLMAPN-(DNA-BD)+SLMAPN-(AD); lane 6, SLMAPN-(DNA-BD)+SLMAPC-(AD); lane 7, SLMAPC-(DNA-BD)+SLMAPN-(AD); lane 8, SLMAPC-(DNA-BD)+SLMAPC-(AD); lane 9, SLMAPCΔLZ-(DNA-BD)+SLMAPCΔLZ-(AD); lane 10, SLMAPCΔLZ-(DNA-BD)+SLMAPC-(AD); lane 11, SLMAPC-(DNA-BD)+SLMAPCΔLZ (AD); lane 12, ninein-LZ-(DNA-BD)+SLMAPC-(AD); lane 13, ninein-LZ-(AD)+SLMAPC-(DNA-BO) and lane 14, positive control. The level of CAT activity measured for each sample was normalized based on transfection efficiency as described in the Experimental section. SLMAP homodimer formation was mediated by C-terminal sequences (lane 8) encompassing the LZ motifs. Deletion of the LZs (lanes 9–11) failed to activate CAT reporter expression above basal levels. Error bars represent S.D. ( C ) Cells stably expressing either 6Myc-SLMAP1-ΔLZ or the control vector (6Myc-pcDNA3) were cultured in differentiation medium for 6 days, fixed with 4% PFA and stained with haematoxylin and eosin. Fusion indices were calculated for three independent clones. Error bars represent S.D.

Techniques Used: Plasmid Preparation, Recombinant, Positive Control, Activity Assay, Transfection, Expressing, Stable Transfection, Cell Culture, Staining, Clone Assay

21) Product Images from "Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma"

Article Title: Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085976

Effect of immunization protocol on T cells cytokines secretion of Der f mice (open bar, n = 6), pCMV-βgal (grey bar, n = 7) and pVAX-Der f1 (black bar, n = 7) when using 10 µg of DNA. One day after the last airway allergen challenge, mice were sacrificed and lung cells were cultured. The concentration of IL-4, IL-5, IL-13, IFN-γ, IL-10 and IL-17 were measured by flow cytometry. Results are expressed as the mean and standard deviation for each group. *p
Figure Legend Snippet: Effect of immunization protocol on T cells cytokines secretion of Der f mice (open bar, n = 6), pCMV-βgal (grey bar, n = 7) and pVAX-Der f1 (black bar, n = 7) when using 10 µg of DNA. One day after the last airway allergen challenge, mice were sacrificed and lung cells were cultured. The concentration of IL-4, IL-5, IL-13, IFN-γ, IL-10 and IL-17 were measured by flow cytometry. Results are expressed as the mean and standard deviation for each group. *p

Techniques Used: Mouse Assay, Cell Culture, Concentration Assay, Flow Cytometry, Cytometry, Standard Deviation

Immunization protocols against Der f1 in asthmatic mice. pVAX-Der f1 or pCMV-βgal plasmid were injected i.m at days −28 and −7. Mice were then epicutaneously sensitized and intranasally challenged with total extract of HDM. Analyses were performed on day 35.
Figure Legend Snippet: Immunization protocols against Der f1 in asthmatic mice. pVAX-Der f1 or pCMV-βgal plasmid were injected i.m at days −28 and −7. Mice were then epicutaneously sensitized and intranasally challenged with total extract of HDM. Analyses were performed on day 35.

Techniques Used: Mouse Assay, Plasmid Preparation, Injection

Effect of immunization protocol on the immune response of asthmatic mice. A. Splenocytes were stimulated overnight with a pool of Der f1 immunodominant peptides. The number of IFN-γ SFCs was determined. Der f (n = 2), pCMV-βgal (n = 5), pVAX-Der f1 (n = 6). B. Lung cells were stimulated overnight with a pool of Der f1 immunodominant peptides. n = 3 mice per group. The number of IFNγ spot forming colonies (SFCs) was determined. C. Humoral response was measured in sera one day after the last challenge in Der f (n = 6), pCMV-βgal (n = 7) and pVAX-Der f1 (n = 7) mice and additionally in control mice for IgE. Results are expressed as IgE, IgG1, IgG2a antibody titer. The mean number and standard deviation are shown for each group. *p
Figure Legend Snippet: Effect of immunization protocol on the immune response of asthmatic mice. A. Splenocytes were stimulated overnight with a pool of Der f1 immunodominant peptides. The number of IFN-γ SFCs was determined. Der f (n = 2), pCMV-βgal (n = 5), pVAX-Der f1 (n = 6). B. Lung cells were stimulated overnight with a pool of Der f1 immunodominant peptides. n = 3 mice per group. The number of IFNγ spot forming colonies (SFCs) was determined. C. Humoral response was measured in sera one day after the last challenge in Der f (n = 6), pCMV-βgal (n = 7) and pVAX-Der f1 (n = 7) mice and additionally in control mice for IgE. Results are expressed as IgE, IgG1, IgG2a antibody titer. The mean number and standard deviation are shown for each group. *p

Techniques Used: Mouse Assay, Standard Deviation

Effect of prophylactic immunization protocol with 10 µg of Der f1 DNA on respiratory function. Airway resistance and Compliance was measured at day 35 using Flexivent with instillation of 5 to 20/ml methacholine in non asthmatic non vaccinated mice (n = 6, ) Der f (n = 7, O ), pCMV-βgal mice (n = 9, ) and pVAX-Der f1 (n = 9,▾) mice. Results are expressed in increased fold, as a mean for each group ± standard deviation. *p
Figure Legend Snippet: Effect of prophylactic immunization protocol with 10 µg of Der f1 DNA on respiratory function. Airway resistance and Compliance was measured at day 35 using Flexivent with instillation of 5 to 20/ml methacholine in non asthmatic non vaccinated mice (n = 6, ) Der f (n = 7, O ), pCMV-βgal mice (n = 9, ) and pVAX-Der f1 (n = 9,▾) mice. Results are expressed in increased fold, as a mean for each group ± standard deviation. *p

Techniques Used: Mouse Assay, Standard Deviation

Effect of immunization protocol on BAL inflammation of non asthmatic non vaccinated mice (light grey bar, n = 8), Der f mice (open bar, n = 9), pCMV-βgal (grey bar, n = 7) and pVAX-Der f1 mice (black bar, n = 7) when using 10 µg of DNA. The total number of cells was determined by cell count on Kova slides. The cellular composition was established by flow cytometry. Results are expressed as absolute number of cells, as the mean and standard deviation for each group. **p
Figure Legend Snippet: Effect of immunization protocol on BAL inflammation of non asthmatic non vaccinated mice (light grey bar, n = 8), Der f mice (open bar, n = 9), pCMV-βgal (grey bar, n = 7) and pVAX-Der f1 mice (black bar, n = 7) when using 10 µg of DNA. The total number of cells was determined by cell count on Kova slides. The cellular composition was established by flow cytometry. Results are expressed as absolute number of cells, as the mean and standard deviation for each group. **p

Techniques Used: Mouse Assay, Cell Counting, Flow Cytometry, Cytometry, Standard Deviation

22) Product Images from "Targeting Artificial Transcription Factors to the Utrophin A Promoter"

Article Title: Targeting Artificial Transcription Factors to the Utrophin A Promoter

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M804518200

Analysis of activation of mouse utrophin A promoter in vitro . A , a schematic illustration of ZFP/VP16 transcription factor construct. The expression cassette was driven by the CMV promoter and contained a nuclear localization signal ( NLS ) and was tagged with the FLAG epitope. B , expression levels of various ZFPs after transfection. Western blotting with an anti-FLAG antibody was used to monitor the levels of the FLAG-tagged ZFP after transfection into the different cell lines. The examples shown are the results obtained in COS-1 cells. Sample loading was verified by an anti-β-actin antibody. C , ability of various ZFP transcription factors to activate a luciferase reporter linked to 2.98 kb of utrophin A upstream sequences. Results are expressed relative to the control NLS plasmid and are normalized to transfection efficiency as described under “Experimental Procedures.” Error bar : mean ± S.E., n = 3.
Figure Legend Snippet: Analysis of activation of mouse utrophin A promoter in vitro . A , a schematic illustration of ZFP/VP16 transcription factor construct. The expression cassette was driven by the CMV promoter and contained a nuclear localization signal ( NLS ) and was tagged with the FLAG epitope. B , expression levels of various ZFPs after transfection. Western blotting with an anti-FLAG antibody was used to monitor the levels of the FLAG-tagged ZFP after transfection into the different cell lines. The examples shown are the results obtained in COS-1 cells. Sample loading was verified by an anti-β-actin antibody. C , ability of various ZFP transcription factors to activate a luciferase reporter linked to 2.98 kb of utrophin A upstream sequences. Results are expressed relative to the control NLS plasmid and are normalized to transfection efficiency as described under “Experimental Procedures.” Error bar : mean ± S.E., n = 3.

Techniques Used: Activation Assay, In Vitro, Construct, Expressing, FLAG-tag, Transfection, Western Blot, Luciferase, Plasmid Preparation

23) Product Images from "The transcription factor Net regulates the angiogenic switch"

Article Title: The transcription factor Net regulates the angiogenic switch

Journal: Genes & Development

doi: 10.1101/gad.272503

Net regulation of VEGF expression in cell culture. ( A ) Endogenous Net is phosphorylated by GPCR, Ras, and FGF-2 through MAP kinase cascades. NIH3T3 cells were transfected with expression vectors for KSHV-GPCR or Ras-V12, or induced with FGF-2. Cell extracts were analyzed by Western blotting for P-Net, activated P-ERK, and TBP (control for loading). ( B ) Down-regulation of Net inhibits VEGF expression induced by GPCR and Ras-V12. NIH3T3 cells were transfected with GPCR, Ras-V12, antisense net, and corresponding control vectors, as indicated. Transfected cells were kept in 0.5% FCS medium for 48 h after the wash. VEGF in this conditioned media was measured by ELISA. Results are averaged from four independent transfection experiments (corrected for cell numbers). ( C ) P-Net regulates VEGF promoter activity and stimulates endogenous VEGF expression. NIH3T3 cells were transfected with the mouse VEGF promoter luciferase reporter (-1217/+370), the pCMV LacZ internal control, and expression vectors for Net and Ras-V12. VEGF promoter activity relative to the transfections with empty vectors was determined (graph). Cell extracts were analyzed by Western blotting for the expression of P-Net, endogenous VEGF, and TBP (loading control; lower panels). ( D ) Coexpression of P-Net and VEGF in transfected cells. NIH3T3 cells were transfected with expression vectors for P-Net and Ras-V12. The cells were stained with antibodies against VEGF and P-Net, corresponding fluorescent secondary antibodies, and DAPI (nuclei), and examined by confocal microscopy. Transfected cells fluoresced brightly (white arrowheads), with VEGF (red) in the cytoplasm and P-Net (green) in the nucleus. Nontransfected cells gave clearly lower fluorescence signals (yellow arrows).
Figure Legend Snippet: Net regulation of VEGF expression in cell culture. ( A ) Endogenous Net is phosphorylated by GPCR, Ras, and FGF-2 through MAP kinase cascades. NIH3T3 cells were transfected with expression vectors for KSHV-GPCR or Ras-V12, or induced with FGF-2. Cell extracts were analyzed by Western blotting for P-Net, activated P-ERK, and TBP (control for loading). ( B ) Down-regulation of Net inhibits VEGF expression induced by GPCR and Ras-V12. NIH3T3 cells were transfected with GPCR, Ras-V12, antisense net, and corresponding control vectors, as indicated. Transfected cells were kept in 0.5% FCS medium for 48 h after the wash. VEGF in this conditioned media was measured by ELISA. Results are averaged from four independent transfection experiments (corrected for cell numbers). ( C ) P-Net regulates VEGF promoter activity and stimulates endogenous VEGF expression. NIH3T3 cells were transfected with the mouse VEGF promoter luciferase reporter (-1217/+370), the pCMV LacZ internal control, and expression vectors for Net and Ras-V12. VEGF promoter activity relative to the transfections with empty vectors was determined (graph). Cell extracts were analyzed by Western blotting for the expression of P-Net, endogenous VEGF, and TBP (loading control; lower panels). ( D ) Coexpression of P-Net and VEGF in transfected cells. NIH3T3 cells were transfected with expression vectors for P-Net and Ras-V12. The cells were stained with antibodies against VEGF and P-Net, corresponding fluorescent secondary antibodies, and DAPI (nuclei), and examined by confocal microscopy. Transfected cells fluoresced brightly (white arrowheads), with VEGF (red) in the cytoplasm and P-Net (green) in the nucleus. Nontransfected cells gave clearly lower fluorescence signals (yellow arrows).

Techniques Used: Expressing, Cell Culture, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Luciferase, Staining, Confocal Microscopy, Fluorescence

24) Product Images from "PIM-1 kinase interacts with the DNA binding domain of the vitamin D receptor: a further kinase implicated in 1,25-(OH)2D3 signaling"

Article Title: PIM-1 kinase interacts with the DNA binding domain of the vitamin D receptor: a further kinase implicated in 1,25-(OH)2D3 signaling

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-13-18

Illustration of the used VDR/Pim-1 variants. ( A ) In addition to full-length VDR the DNA Binding Domain (DBD: AA 16–125) and the Ligand Binding Domain (LBD: AA 111–427) of the VDR were subjected for Y2H analysis as bait molecules. They were tested with ( B ) full-length PIM-1 isoform 2 as well as the truncated version of PIM-1 (AA 135–313).
Figure Legend Snippet: Illustration of the used VDR/Pim-1 variants. ( A ) In addition to full-length VDR the DNA Binding Domain (DBD: AA 16–125) and the Ligand Binding Domain (LBD: AA 111–427) of the VDR were subjected for Y2H analysis as bait molecules. They were tested with ( B ) full-length PIM-1 isoform 2 as well as the truncated version of PIM-1 (AA 135–313).

Techniques Used: Binding Assay, Ligand Binding Assay

25) Product Images from "Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein"

Article Title: Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein

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

doi:

( A ) pAAV-lacZ. ITR, inverted terminal repeat; CMV, CMV promoter; lacZ, bacterial β-gal; An, simian virus 40 (SV40) early polyadenylylation signal. ( B ) pAAV-Epo. Epo, human Epo coding sequence; An, SV40 polyadenylylation signal; noncoding, 2.2-kb noncoding fragment from the lacZ gene. See text for details.
Figure Legend Snippet: ( A ) pAAV-lacZ. ITR, inverted terminal repeat; CMV, CMV promoter; lacZ, bacterial β-gal; An, simian virus 40 (SV40) early polyadenylylation signal. ( B ) pAAV-Epo. Epo, human Epo coding sequence; An, SV40 polyadenylylation signal; noncoding, 2.2-kb noncoding fragment from the lacZ gene. See text for details.

Techniques Used: Sequencing

26) Product Images from "A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissue-specific amplification of gene expression"

Article Title: A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissue-specific amplification of gene expression

Journal: Nucleic Acids Research

doi:

Gene-switch strategy using cre/LOX induced translational frameshift. ( A ) Gene 1 flanked by LOX sites is inserted in frame within the open reading frame of gene 2 resulting in appropriate translation of Gene 1, but not Gene 2. In the presence of Cre recombinase Gene 1 is excised and the correct reading frame of Gene 2 is restored which is now expressed. ( B ) EGFP/β-gal gene-switch under the transcriptional control of the SV40 early promoter. Translational start site and first 18 amino acids of E.coli gtp are upstream of the floxed EGFP followed by E.coli gtp amino acids 19–47 fused in-frame to β-gal. The insertion of a cytosine at the 3′ end of LOX I results in a disruption of the reading frame of the β-gal gene. The Cre-mediated excision of the floxed EGFP restores the correct reading frame for β-gal . ( C – F ) Transfection of CHO-K1 cells with pSV-EGFP/β-gal results in expression of only EGFP without cre whereas co-transfection with pCMV- cre results in loss of EGFP expression but gain of β-gal expression. (C and D) Fluorescent microscopy, GFP filter; (E and F) bright field (X 320). Assays performed 48 h post-transfection.
Figure Legend Snippet: Gene-switch strategy using cre/LOX induced translational frameshift. ( A ) Gene 1 flanked by LOX sites is inserted in frame within the open reading frame of gene 2 resulting in appropriate translation of Gene 1, but not Gene 2. In the presence of Cre recombinase Gene 1 is excised and the correct reading frame of Gene 2 is restored which is now expressed. ( B ) EGFP/β-gal gene-switch under the transcriptional control of the SV40 early promoter. Translational start site and first 18 amino acids of E.coli gtp are upstream of the floxed EGFP followed by E.coli gtp amino acids 19–47 fused in-frame to β-gal. The insertion of a cytosine at the 3′ end of LOX I results in a disruption of the reading frame of the β-gal gene. The Cre-mediated excision of the floxed EGFP restores the correct reading frame for β-gal . ( C – F ) Transfection of CHO-K1 cells with pSV-EGFP/β-gal results in expression of only EGFP without cre whereas co-transfection with pCMV- cre results in loss of EGFP expression but gain of β-gal expression. (C and D) Fluorescent microscopy, GFP filter; (E and F) bright field (X 320). Assays performed 48 h post-transfection.

Techniques Used: Transfection, Expressing, Cotransfection, Microscopy

27) Product Images from "Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ"

Article Title: Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ

Journal: Toxicology

doi: 10.1016/j.tox.2015.01.007

Isoflavone-induced enhancement of RORα- and RORγ-mediated Il17a promoter activation in Jurkat cells. Cells were co-transfected with the pCMV-β-Gal and pCMV10-3xFlag-RORα (A) or pCMV10-3xFlag-RORγ (B) and pGL4.14 reporter plasmid under the control of the Il17a promoter and treated with increasing concentrations of the isoflavones at 0.1, 1, and 10 μM. After 24 h, relative LUC activity was determined as described in Materials and Methods. The firefly luciferase activity was normalized against β-galactosidase activity. BA, GE, FN, and DZ represent each isoflavone, biochanin A, genistein, formononetin, and daidzein, respectively. Values represent the means ± SEM ( n = 3). Significant differences from the vehicle control (DMSO) plus RORα or RORγ are indicated by asterisks (* P
Figure Legend Snippet: Isoflavone-induced enhancement of RORα- and RORγ-mediated Il17a promoter activation in Jurkat cells. Cells were co-transfected with the pCMV-β-Gal and pCMV10-3xFlag-RORα (A) or pCMV10-3xFlag-RORγ (B) and pGL4.14 reporter plasmid under the control of the Il17a promoter and treated with increasing concentrations of the isoflavones at 0.1, 1, and 10 μM. After 24 h, relative LUC activity was determined as described in Materials and Methods. The firefly luciferase activity was normalized against β-galactosidase activity. BA, GE, FN, and DZ represent each isoflavone, biochanin A, genistein, formononetin, and daidzein, respectively. Values represent the means ± SEM ( n = 3). Significant differences from the vehicle control (DMSO) plus RORα or RORγ are indicated by asterisks (* P

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

Effects of isoflavones on interactions between ROR-LBD and the co-activator LXXLL peptide in mammalian two-hybrid assays. The analysis was performed by co-transfecting CHO-K1 cells with a pGL4.27-(UAS) 5 reporter plasmid, pCMV-β-Gal pM-EBIP96 peptide, and either VP16-RORα(LBD) (A) or VP16-RORγ(LBD) (B). Cells were treated in the presence of the vehicle (DMSO), or increasing concentrations of the four isoflavones as indicated. After 24 h, relative LUC activity was determined as described in Section 2. The firefly luciferase activity was normalized against β-galactosidase activity. BA, GE, FN, and DZ represent each isoflavone, biochanin A, genistein, formononetin, and daidzein, respectively. Values represent the means ± SEM ( n = 3) and are presented as the mean n -fold induction over the vehicle control. Significant differences from the vehicle control (DMSO) are indicated by asterisks (* P
Figure Legend Snippet: Effects of isoflavones on interactions between ROR-LBD and the co-activator LXXLL peptide in mammalian two-hybrid assays. The analysis was performed by co-transfecting CHO-K1 cells with a pGL4.27-(UAS) 5 reporter plasmid, pCMV-β-Gal pM-EBIP96 peptide, and either VP16-RORα(LBD) (A) or VP16-RORγ(LBD) (B). Cells were treated in the presence of the vehicle (DMSO), or increasing concentrations of the four isoflavones as indicated. After 24 h, relative LUC activity was determined as described in Section 2. The firefly luciferase activity was normalized against β-galactosidase activity. BA, GE, FN, and DZ represent each isoflavone, biochanin A, genistein, formononetin, and daidzein, respectively. Values represent the means ± SEM ( n = 3) and are presented as the mean n -fold induction over the vehicle control. Significant differences from the vehicle control (DMSO) are indicated by asterisks (* P

Techniques Used: Plasmid Preparation, Activity Assay, Luciferase

28) Product Images from "Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms"

Article Title: Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1001-13.2013

The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p
Figure Legend Snippet: The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p

Techniques Used: Luciferase, Activity Assay, Transfection, Mutagenesis, Construct

Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p
Figure Legend Snippet: Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p

Techniques Used: Activity Assay, Plasmid Preparation, Luciferase, Expressing, Transfection, Construct, Mutagenesis

slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p
Figure Legend Snippet: slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p

Techniques Used: Activity Assay, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Construct

29) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

Journal: Scientific Reports

doi: 10.1038/srep16435

Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.
Figure Legend Snippet: Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.

Techniques Used: Reporter Assay, Activity Assay, Sequencing, Plasmid Preparation, Construct, Derivative Assay, Negative Control, Expressing, Transfection, Standard Deviation

30) Product Images from "Glycosylation-dependent galectin-1/neuropilin-1 interactions promote liver fibrosis through activation of TGF-β- and PDGF-like signals in hepatic stellate cells"

Article Title: Glycosylation-dependent galectin-1/neuropilin-1 interactions promote liver fibrosis through activation of TGF-β- and PDGF-like signals in hepatic stellate cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-11212-1

Blocking core 2 branching O-glycosylation suppressed Gal-1-induced hepatic stellate cell (HSC) migration. ( A ) Schematic representation of core 1 and core 2 O-glycan biosynthesis. ( B ) An O-glycan inhibitor (benzyl-N-acetyl-α-galactosaminide; BαG) increases PNA binding to LX-2 cells. ( C ) BαG increases the binding of Gal-1 and LX-2 cells. ( D ) BαG suppresses Gal-1-induced LX-2 migration. ( E ) Knockdown of core 2 N-acetylglucosaminyltransferase 1 (GCNT1) suppresses GCNT1 expression. ( F ) Knockdown of GCNT1 inhibits Gal-1-induced HSC migration. LX-2 cells were treated with BαG or infected with a lentivirus carrying GCNT1 shRNA for 48 h. After treatment, cells were incubated with PNA and Gal-1-488 to detect their binding to LX-2 cells using flow cytometry. To measure the migratory ability, cells were treated with 500 nM Gal-1 for 16 h. Migrated cells were counted, and results are presented as the mean ± SEM of three independent experiments. * p
Figure Legend Snippet: Blocking core 2 branching O-glycosylation suppressed Gal-1-induced hepatic stellate cell (HSC) migration. ( A ) Schematic representation of core 1 and core 2 O-glycan biosynthesis. ( B ) An O-glycan inhibitor (benzyl-N-acetyl-α-galactosaminide; BαG) increases PNA binding to LX-2 cells. ( C ) BαG increases the binding of Gal-1 and LX-2 cells. ( D ) BαG suppresses Gal-1-induced LX-2 migration. ( E ) Knockdown of core 2 N-acetylglucosaminyltransferase 1 (GCNT1) suppresses GCNT1 expression. ( F ) Knockdown of GCNT1 inhibits Gal-1-induced HSC migration. LX-2 cells were treated with BαG or infected with a lentivirus carrying GCNT1 shRNA for 48 h. After treatment, cells were incubated with PNA and Gal-1-488 to detect their binding to LX-2 cells using flow cytometry. To measure the migratory ability, cells were treated with 500 nM Gal-1 for 16 h. Migrated cells were counted, and results are presented as the mean ± SEM of three independent experiments. * p

Techniques Used: Blocking Assay, Migration, Binding Assay, Expressing, Infection, shRNA, Incubation, Flow Cytometry, Cytometry

The glycome of activated hepatic stellate cells (HSCs) facilitated Gal-1 binding which induces HSC migration and activation. ( A ) Gal-1 induces the migration of LX-2 cells in a dose-dependent manner. The cell migratory ability was measured using a Boyden chamber assay. ( B ) Gal-1 induces HSC activation. LX-2 cells were starved for 24 h and then treated with different doses of the recombinant Gal-1 protein for 24 h. α-smooth muscle actin (α-SMA) expression was detected using an RT-qPCR. Relative expression levels were calculated by comparing the ΔCT values of Gal-1-treated cells to those of cells without treatment, and results are shown as folds of change. ( C ) Thiodigalactoside (TDG) inhibits Gal-1 binding with LX-2 cells. TDG (200 µM) was pre-incubated with Gal-1-488 (500 nM) for 30 min, and then LX-2 cells were incubated with the mixture for 30 min. The binding of LX-2 and Gal-1-488 was analyzed using flow cytometry. ( D ) TDG inhibits Gal-1-induced HSC migration. TDG (200 µM) was pre-incubated with Gal-1 (500 nM) for 30 min, and then LX-2 cells were treated with the mixture for 16 h. The cell migratory ability was measured using a Boyden chamber assay. ( E , F ) Knockdown of Gal-1 normalizes activated HSCs. LX-2 cells were infected with a lentivirus carrying Gal-1 and luciferase shRNAs (sh-B09, D09, and sh-Luc). Western blotting and an RT-qPCR were used to analyze Gal-1, α-SMA, fibroblast activation protein (FAP), and α-1 type I collagen (COL1A1) expression. Relative expression levels of individual genes were calculated by comparing the ΔCT values of sh-B09 cells to those of sh-Luc cells, and results are shown as folds of change. All of the experiments were performed in duplicate. Results are shown the mean ± SEM of three independent assays. * p
Figure Legend Snippet: The glycome of activated hepatic stellate cells (HSCs) facilitated Gal-1 binding which induces HSC migration and activation. ( A ) Gal-1 induces the migration of LX-2 cells in a dose-dependent manner. The cell migratory ability was measured using a Boyden chamber assay. ( B ) Gal-1 induces HSC activation. LX-2 cells were starved for 24 h and then treated with different doses of the recombinant Gal-1 protein for 24 h. α-smooth muscle actin (α-SMA) expression was detected using an RT-qPCR. Relative expression levels were calculated by comparing the ΔCT values of Gal-1-treated cells to those of cells without treatment, and results are shown as folds of change. ( C ) Thiodigalactoside (TDG) inhibits Gal-1 binding with LX-2 cells. TDG (200 µM) was pre-incubated with Gal-1-488 (500 nM) for 30 min, and then LX-2 cells were incubated with the mixture for 30 min. The binding of LX-2 and Gal-1-488 was analyzed using flow cytometry. ( D ) TDG inhibits Gal-1-induced HSC migration. TDG (200 µM) was pre-incubated with Gal-1 (500 nM) for 30 min, and then LX-2 cells were treated with the mixture for 16 h. The cell migratory ability was measured using a Boyden chamber assay. ( E , F ) Knockdown of Gal-1 normalizes activated HSCs. LX-2 cells were infected with a lentivirus carrying Gal-1 and luciferase shRNAs (sh-B09, D09, and sh-Luc). Western blotting and an RT-qPCR were used to analyze Gal-1, α-SMA, fibroblast activation protein (FAP), and α-1 type I collagen (COL1A1) expression. Relative expression levels of individual genes were calculated by comparing the ΔCT values of sh-B09 cells to those of sh-Luc cells, and results are shown as folds of change. All of the experiments were performed in duplicate. Results are shown the mean ± SEM of three independent assays. * p

Techniques Used: Binding Assay, Migration, Activation Assay, Boyden Chamber Assay, Recombinant, Expressing, Quantitative RT-PCR, Incubation, Flow Cytometry, Cytometry, Infection, Luciferase, Western Blot

Knockdown of Gal-1 attenuates PDGF- and TGF-β-induced LX-2 cell signaling, gene expression, and migration. ( A ) Silencing Gal-1 suppresses PDGF-induced signaling. LX-2 cells were infected with a luciferase (sh-Luc) and Gal-1 shRNA (sh-B09, D09) lentivirus and were treated with PDGF for 10 min. The cellular phosphorylation of extracellular sign-regulated kinase 1/2 (Erk1/2), and Akt was measured using Western blotting. ( B ) Knockdown of Gal-1 suppresses the TGF-β-induced Smad2/3 transactivation ability. LX-2-sh-GFP, sh-B09, sh-D09 cells were transfected with a SBE4-Luc plasmid (luciferase reporter containing four copies of the Smad-binding site). Cells were starved for 24 h followed by TGF-β (1 ng/ml) treatment for 24 h, and luciferase activities were measured using the Luciferase Assay system (Promega). ( C , D ) Knockdown of Gal-1 inhibits PDGF- and TGF-β-induced gene expression. α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and α-1 type I collagen (COL1A1) expressions in LX-2 cells were analyzed using RT-qPCR. Relative gene expression levels were calculated by comparing ∆CT values of each group to those of sh-Luc cells without treatment. Data are shown as folds of change. ( E ) Knockdown of Gal-1 expression inhibits PDGF- and TGF-β-induced HSC migration. Cell migration was measured using a Boyden chamber assay. Results are presented as the mean ± SEM of three independent experiments. * p
Figure Legend Snippet: Knockdown of Gal-1 attenuates PDGF- and TGF-β-induced LX-2 cell signaling, gene expression, and migration. ( A ) Silencing Gal-1 suppresses PDGF-induced signaling. LX-2 cells were infected with a luciferase (sh-Luc) and Gal-1 shRNA (sh-B09, D09) lentivirus and were treated with PDGF for 10 min. The cellular phosphorylation of extracellular sign-regulated kinase 1/2 (Erk1/2), and Akt was measured using Western blotting. ( B ) Knockdown of Gal-1 suppresses the TGF-β-induced Smad2/3 transactivation ability. LX-2-sh-GFP, sh-B09, sh-D09 cells were transfected with a SBE4-Luc plasmid (luciferase reporter containing four copies of the Smad-binding site). Cells were starved for 24 h followed by TGF-β (1 ng/ml) treatment for 24 h, and luciferase activities were measured using the Luciferase Assay system (Promega). ( C , D ) Knockdown of Gal-1 inhibits PDGF- and TGF-β-induced gene expression. α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and α-1 type I collagen (COL1A1) expressions in LX-2 cells were analyzed using RT-qPCR. Relative gene expression levels were calculated by comparing ∆CT values of each group to those of sh-Luc cells without treatment. Data are shown as folds of change. ( E ) Knockdown of Gal-1 expression inhibits PDGF- and TGF-β-induced HSC migration. Cell migration was measured using a Boyden chamber assay. Results are presented as the mean ± SEM of three independent experiments. * p

Techniques Used: Expressing, Migration, Infection, Luciferase, shRNA, Western Blot, Transfection, Plasmid Preparation, Binding Assay, Activation Assay, Quantitative RT-PCR, Boyden Chamber Assay

Blocking MGAT5-mediated N-glycosylation suppresses Gal-1-induced hepatic stellate cell (HSC) migration. ( A ) Schematic representation of N-glycan biosynthesis. ( B ) An N-glycan inhibitor (swainsonine, SW) inhibits L-PHA binding to LX-2 cells. ( C ) SW inhibits Gal-1 binding to LX-2 cells. ( D ) SW suppresses Gal-1-induced LX-2 cell migration. ( E ) Knockdown of MGAT5 inhibits L-PHA binding to LX-2 cells. ( F ) MGAT5 siRNAs suppress MGAT5 expression. ( G ) MGAT5 siRNAs inhibit Gal-1-induced LX-2 cell migration. LX-2 cells were treated with SW or transfected with MGAT5 siRNA for 48 h followed by incubation with L-PHA and Gal-1-488 to detect their binding to LX-2 cells using flow cytometry. The black line represents the binding of LX-2 cells with DyLight® 488 strepavidin alone and the brown line represents LX-2 cells without staining. To measure the migratory ability, cells were treated with 500 nM Gal-1 for 16 h. Migrated cells were counted, and results are presented as the mean ± SEM of three independent experiments. * p
Figure Legend Snippet: Blocking MGAT5-mediated N-glycosylation suppresses Gal-1-induced hepatic stellate cell (HSC) migration. ( A ) Schematic representation of N-glycan biosynthesis. ( B ) An N-glycan inhibitor (swainsonine, SW) inhibits L-PHA binding to LX-2 cells. ( C ) SW inhibits Gal-1 binding to LX-2 cells. ( D ) SW suppresses Gal-1-induced LX-2 cell migration. ( E ) Knockdown of MGAT5 inhibits L-PHA binding to LX-2 cells. ( F ) MGAT5 siRNAs suppress MGAT5 expression. ( G ) MGAT5 siRNAs inhibit Gal-1-induced LX-2 cell migration. LX-2 cells were treated with SW or transfected with MGAT5 siRNA for 48 h followed by incubation with L-PHA and Gal-1-488 to detect their binding to LX-2 cells using flow cytometry. The black line represents the binding of LX-2 cells with DyLight® 488 strepavidin alone and the brown line represents LX-2 cells without staining. To measure the migratory ability, cells were treated with 500 nM Gal-1 for 16 h. Migrated cells were counted, and results are presented as the mean ± SEM of three independent experiments. * p

Techniques Used: Blocking Assay, Migration, Binding Assay, Expressing, Transfection, Incubation, Flow Cytometry, Cytometry, Staining

Glycosylation-dependent Gal-1/NRP-1 interactions induce hepatic stellate cell (HSC) migration. ( A ) NRP-1 shRNAs (sh-N1 and N2) suppress NRP-1 expression. ( B ) Knockdown of NRP-1 suppresses Gal-1 binding to LX-2 cells. ( C ) Knockdown of NRP-1 suppresses Gal-1-induced HSC migration. LX-2 cells were infected with a lentivirus carrying luciferase (sh-Luc) and NRP-1 shRNA (sh-N1). Gal-1 binding to LX-2 sh-Luc and sh-N1 cells was determined by flow cytometry. To measure the migratory ability, LX-2 sh-Luc and sh-N1 cells were treated with 500 nM Gal-1 for 16 h, and migrated cells were counted. Results are shown as the mean ± SEM of three independent experiments. * p
Figure Legend Snippet: Glycosylation-dependent Gal-1/NRP-1 interactions induce hepatic stellate cell (HSC) migration. ( A ) NRP-1 shRNAs (sh-N1 and N2) suppress NRP-1 expression. ( B ) Knockdown of NRP-1 suppresses Gal-1 binding to LX-2 cells. ( C ) Knockdown of NRP-1 suppresses Gal-1-induced HSC migration. LX-2 cells were infected with a lentivirus carrying luciferase (sh-Luc) and NRP-1 shRNA (sh-N1). Gal-1 binding to LX-2 sh-Luc and sh-N1 cells was determined by flow cytometry. To measure the migratory ability, LX-2 sh-Luc and sh-N1 cells were treated with 500 nM Gal-1 for 16 h, and migrated cells were counted. Results are shown as the mean ± SEM of three independent experiments. * p

Techniques Used: Migration, Expressing, Binding Assay, Infection, Luciferase, shRNA, Flow Cytometry, Cytometry

The expression of galectin-1 and its binding lectins are associated with hepatic stellate cells (HSC) activation. ( A ) Gal-1 expression is upregulated in fibrotic livers. Mouse liver fibrosis was induced by an injection of thioacetamide and carbon chloride or by feeding a methionine- and choline-deficient (MCD) diet as described in “Materials and methods”. Liver tissues were homogenized for Western blotting. ( B ) Gal-1 is expressed in areas around the portal vein and areas of bridging fibrosis in fibrotic livers of mice. Tissue slides were stained with anti-Gal-1 and anti-α-SMA antibodies and Masson’s trichrome stain. The blue color indicates collagen. The brown color indicates a region positive for Gal-1 and α-smooth muscle actin (α-SMA). For immunofluorescence analysis, the slides were incubated with anti-Gal-1 and anti-α-SMA antibodies, and the signal was visualized by Alexa Fluor-488 and -594 secondary antibodies. ( C ) Gal-1 is overexpressed in cirrhotic liver tissues compared to normal liver tissues. Normal liver and cirrhotic tissues were obtained from US Biomax (LV805). P’t represents patient. Three representative samples of normal and cirrhotic livers are respectively shown in the left and right panel. Asterisks indicate parenchymal cells, and arrowheads indicate non-parenchymal cells. ( D ) Glycosylation signatures of LX-2 cells, an activated hepatic stellate cell line. Cell surface glycans of LX-2 cells were detected by different types of lectins as described in “Materials and methods”. ( E ) Quantitation of the relative mean fluorescence intensity (rMFI) of different lectins. The rMFI was calculated by comparing the mean fluorescence intensity of different lectins to that of DyLight® 488 streptavidin alone, and results are shown as folds of change.
Figure Legend Snippet: The expression of galectin-1 and its binding lectins are associated with hepatic stellate cells (HSC) activation. ( A ) Gal-1 expression is upregulated in fibrotic livers. Mouse liver fibrosis was induced by an injection of thioacetamide and carbon chloride or by feeding a methionine- and choline-deficient (MCD) diet as described in “Materials and methods”. Liver tissues were homogenized for Western blotting. ( B ) Gal-1 is expressed in areas around the portal vein and areas of bridging fibrosis in fibrotic livers of mice. Tissue slides were stained with anti-Gal-1 and anti-α-SMA antibodies and Masson’s trichrome stain. The blue color indicates collagen. The brown color indicates a region positive for Gal-1 and α-smooth muscle actin (α-SMA). For immunofluorescence analysis, the slides were incubated with anti-Gal-1 and anti-α-SMA antibodies, and the signal was visualized by Alexa Fluor-488 and -594 secondary antibodies. ( C ) Gal-1 is overexpressed in cirrhotic liver tissues compared to normal liver tissues. Normal liver and cirrhotic tissues were obtained from US Biomax (LV805). P’t represents patient. Three representative samples of normal and cirrhotic livers are respectively shown in the left and right panel. Asterisks indicate parenchymal cells, and arrowheads indicate non-parenchymal cells. ( D ) Glycosylation signatures of LX-2 cells, an activated hepatic stellate cell line. Cell surface glycans of LX-2 cells were detected by different types of lectins as described in “Materials and methods”. ( E ) Quantitation of the relative mean fluorescence intensity (rMFI) of different lectins. The rMFI was calculated by comparing the mean fluorescence intensity of different lectins to that of DyLight® 488 streptavidin alone, and results are shown as folds of change.

Techniques Used: Expressing, Binding Assay, Activation Assay, Injection, Western Blot, Mouse Assay, Staining, Immunofluorescence, Incubation, Quantitation Assay, Fluorescence

Gal-1 induces PDGF- and TGF-β-like signals through the NRP-1/PDGF receptor (PDGFR) and NRP-1/TGF-β receptor (TGF-βR) complex. ( A ) Gal-1 induced PDGF and TGF-β-like signaling in Gal-1 silencing cells (LX-2-shB09 cells). ( B ) Knockdown of NRP-1 suppressed Gal-1-induced signaling. LX-2 cells were serum-starved for 24 h followed by Gal-1 (500 nM) treatment for 10 min. The cell lysate was collected for Western blotting. ( C ) Sorafenib and SIS3 (a tyrosine kinase and a TGF-βR inhibitor) suppressed Gal-1-induced hepatic stellate cell (HSC) migration. For the migration assay, LX-2 cells were pretreated with sorafenib and SIS3 for 1 h. Then, cells were suspended and seeded into a transwell. After incubation for 24 h, migrated cells were counted, and results are presented as the mean ±SEM of three independent experiments. * p
Figure Legend Snippet: Gal-1 induces PDGF- and TGF-β-like signals through the NRP-1/PDGF receptor (PDGFR) and NRP-1/TGF-β receptor (TGF-βR) complex. ( A ) Gal-1 induced PDGF and TGF-β-like signaling in Gal-1 silencing cells (LX-2-shB09 cells). ( B ) Knockdown of NRP-1 suppressed Gal-1-induced signaling. LX-2 cells were serum-starved for 24 h followed by Gal-1 (500 nM) treatment for 10 min. The cell lysate was collected for Western blotting. ( C ) Sorafenib and SIS3 (a tyrosine kinase and a TGF-βR inhibitor) suppressed Gal-1-induced hepatic stellate cell (HSC) migration. For the migration assay, LX-2 cells were pretreated with sorafenib and SIS3 for 1 h. Then, cells were suspended and seeded into a transwell. After incubation for 24 h, migrated cells were counted, and results are presented as the mean ±SEM of three independent experiments. * p

Techniques Used: Western Blot, Migration, Incubation

31) Product Images from "Defect in the p53-Mdm2 Autoregulatory Loop Resulting from Inactivation of TAFII250 in Cell Cycle Mutant tsBN462 Cells"

Article Title: Defect in the p53-Mdm2 Autoregulatory Loop Resulting from Inactivation of TAFII250 in Cell Cycle Mutant tsBN462 Cells

Journal: Molecular and Cellular Biology

doi:

). tsBN462 cells were transfected with 5 μg (9-cm plates) of vectors expressing nothing (control), Hdm2, Hdm2 G58A, Hdm2 V75A, p53, p53 R175H, mBcl2, hBcl2, and TAF II 250, as indicated, and 1 μg of pSG5-lacZ (all plates). Parallel plates were transfected with 0.5 μg of pEGFP. After 16 h in the presence of the precipitate, the cells were washed, incubated for 24 h, trypsinized, and replated at half the density. An aliquot was removed to measure β-Gal activity (to correct for variations in transfection efficiency). The plates transfected with pEGFP were observed under a fluorescence microscope. The cells were incubated overnight at 32°C and then for 7 days at the nonpermissive temperature (39°C). The medium was changed every 2 days. To measure the number of surviving cells, they were trypsinized and the viable cells that excluded trypan blue were counted with a Bürker slide. The numbers of cells were adjusted for variations in transfection efficiency and expressed relative to the control empty vector. There were approximately 10 6 cells per 9-cm plate for TAF II 250 at the end of the experiment. Similar results were obtained in two independent experiments (±10%). (B) Following selection the cells were fixed, stained with specific antibodies (Hdm2, IF2; p53, DO1; TAF II 250, anti-HA, 12CA5; Bcl2, sc-509 [Santa Cruz]) followed by Cy3-labeled secondary antibodies and DAPI (nuclei). The cells were photographed under a fluorescence microscope. Control plates incubated with the specific antibodies and labeled secondary antibodies gave low levels of background fluorescence (not shown). The antibodies are specific for exogenous proteins.
Figure Legend Snippet: ). tsBN462 cells were transfected with 5 μg (9-cm plates) of vectors expressing nothing (control), Hdm2, Hdm2 G58A, Hdm2 V75A, p53, p53 R175H, mBcl2, hBcl2, and TAF II 250, as indicated, and 1 μg of pSG5-lacZ (all plates). Parallel plates were transfected with 0.5 μg of pEGFP. After 16 h in the presence of the precipitate, the cells were washed, incubated for 24 h, trypsinized, and replated at half the density. An aliquot was removed to measure β-Gal activity (to correct for variations in transfection efficiency). The plates transfected with pEGFP were observed under a fluorescence microscope. The cells were incubated overnight at 32°C and then for 7 days at the nonpermissive temperature (39°C). The medium was changed every 2 days. To measure the number of surviving cells, they were trypsinized and the viable cells that excluded trypan blue were counted with a Bürker slide. The numbers of cells were adjusted for variations in transfection efficiency and expressed relative to the control empty vector. There were approximately 10 6 cells per 9-cm plate for TAF II 250 at the end of the experiment. Similar results were obtained in two independent experiments (±10%). (B) Following selection the cells were fixed, stained with specific antibodies (Hdm2, IF2; p53, DO1; TAF II 250, anti-HA, 12CA5; Bcl2, sc-509 [Santa Cruz]) followed by Cy3-labeled secondary antibodies and DAPI (nuclei). The cells were photographed under a fluorescence microscope. Control plates incubated with the specific antibodies and labeled secondary antibodies gave low levels of background fluorescence (not shown). The antibodies are specific for exogenous proteins.

Techniques Used: Transfection, Expressing, Incubation, Activity Assay, Fluorescence, Microscopy, Plasmid Preparation, Selection, Staining, Labeling

Effect of TAF II 250, Hdm2, and p53 R175H expression on cell growth, cell cycle distribution, DNA synthesis, and long-term survival. Stable G418-resistant clones were established that express TAF II 250, Hdm2, Hdm2 G58A, Hdm2 V75A, or p53 R175H or contain the empty pCMV expression vector. (A and B) The cells were plated in six-well dishes (5 × 10 4 per well) and incubated at 32°C for 2 h before being incubated at either 39°C (A) or 32°C (B). At the indicated times, the cells were collected by trypsinization and viable cells (that exclude trypan blue) were counted in a Bürker cell. The clone numbers are indicated in parentheses. (C and D) Exponentially growing cells were incubated at 39°C (C) or 32°C (D) for 24 h and analyzed by flow cytometry. The scans for independent clones are shown in panel C (Control, clones 26.1, 28.2, and 28.3; TAF II 250, clones 6.1, 6.2, and 6.3; BHK21, two plates analyzed separately; Hdm2, clones 18.1, 18.2, and 18.3; Hdm2 V75A, clones 24.1 and 23.1; p53 R175H, clones 12.1, 12.2, and 11.1). One representative clone for each vector is shown in panel D. s-G1, sub-G 0 /G 1 (boxed and rotated); G1, G 0 /G 1 . (E) The clones were incubated for 12 h at 39°C, labeled with BrdU for 1 h, fixed, and incubated with anti-BrdU (Becton-Dickinson) and Texas red anti-mouse (Jackson) antibodies, and DAPI. Fluorescent cells in 10 different fields containing 10 to 50 cells were counted. The proportions of BrdU-positive cells were as follows: TAF II 250 (clones 6.1 and 6.3), 33% ± 5%; Hdm2 (clones 17.2 and 18.2), 35% ± 4%; Control (clones 27.2 and 28.3), 4% ± 2%. Representative photographs are shown. (F) Cells were fixed, stained with specific antibodies (Hdm2, IF2; p53R175H, DO1; TAF II 250, anti-HA, 12CA5) followed by Cy3-labeled secondary antibodies and DAPI (nuclei). The cells were photographed under a fluorescence microscope. Control clones incubated with the specific antibodies and labeled secondary antibodies gave low levels of background fluorescence (not shown), as expected for antibodies that are specific for human or viral proteins. One representative clone for each is shown. (G) Western blots of clones established with the selectable marker alone (lanes 1, 2, 5, and 6) or with expression vectors for Hdm2 (lanes 3 and 4) or p53R175H (lanes 7 and 8) and probed with antibodies against Hdm2 (Ab-1, Calbiochem OP46 [lanes 1 to 4]) or p53 (DO1 [lanes 5 to 8]) followed by enhanced chemiluminescence (ECL) (Amersham). (H) Exponentially growing cells were incubated at 39°C for 12, 24, and 60 h and analyzed by flow cytometry. One representative clone for each vector is shown.
Figure Legend Snippet: Effect of TAF II 250, Hdm2, and p53 R175H expression on cell growth, cell cycle distribution, DNA synthesis, and long-term survival. Stable G418-resistant clones were established that express TAF II 250, Hdm2, Hdm2 G58A, Hdm2 V75A, or p53 R175H or contain the empty pCMV expression vector. (A and B) The cells were plated in six-well dishes (5 × 10 4 per well) and incubated at 32°C for 2 h before being incubated at either 39°C (A) or 32°C (B). At the indicated times, the cells were collected by trypsinization and viable cells (that exclude trypan blue) were counted in a Bürker cell. The clone numbers are indicated in parentheses. (C and D) Exponentially growing cells were incubated at 39°C (C) or 32°C (D) for 24 h and analyzed by flow cytometry. The scans for independent clones are shown in panel C (Control, clones 26.1, 28.2, and 28.3; TAF II 250, clones 6.1, 6.2, and 6.3; BHK21, two plates analyzed separately; Hdm2, clones 18.1, 18.2, and 18.3; Hdm2 V75A, clones 24.1 and 23.1; p53 R175H, clones 12.1, 12.2, and 11.1). One representative clone for each vector is shown in panel D. s-G1, sub-G 0 /G 1 (boxed and rotated); G1, G 0 /G 1 . (E) The clones were incubated for 12 h at 39°C, labeled with BrdU for 1 h, fixed, and incubated with anti-BrdU (Becton-Dickinson) and Texas red anti-mouse (Jackson) antibodies, and DAPI. Fluorescent cells in 10 different fields containing 10 to 50 cells were counted. The proportions of BrdU-positive cells were as follows: TAF II 250 (clones 6.1 and 6.3), 33% ± 5%; Hdm2 (clones 17.2 and 18.2), 35% ± 4%; Control (clones 27.2 and 28.3), 4% ± 2%. Representative photographs are shown. (F) Cells were fixed, stained with specific antibodies (Hdm2, IF2; p53R175H, DO1; TAF II 250, anti-HA, 12CA5) followed by Cy3-labeled secondary antibodies and DAPI (nuclei). The cells were photographed under a fluorescence microscope. Control clones incubated with the specific antibodies and labeled secondary antibodies gave low levels of background fluorescence (not shown), as expected for antibodies that are specific for human or viral proteins. One representative clone for each is shown. (G) Western blots of clones established with the selectable marker alone (lanes 1, 2, 5, and 6) or with expression vectors for Hdm2 (lanes 3 and 4) or p53R175H (lanes 7 and 8) and probed with antibodies against Hdm2 (Ab-1, Calbiochem OP46 [lanes 1 to 4]) or p53 (DO1 [lanes 5 to 8]) followed by enhanced chemiluminescence (ECL) (Amersham). (H) Exponentially growing cells were incubated at 39°C for 12, 24, and 60 h and analyzed by flow cytometry. One representative clone for each vector is shown.

Techniques Used: Expressing, DNA Synthesis, Clone Assay, Plasmid Preparation, Incubation, Flow Cytometry, Cytometry, Labeling, Staining, Fluorescence, Microscopy, Western Blot, Marker

Effect of Mdm2, TAF II 250, and inhibiting Mdm2-p53 interactions on the cell cycle. tsBN462 cells were transfected in 9-cm dishes with vectors that express CD20 (5 μg, all samples) and, in addition, Hdm2 (10 μg), Hdm2 G58A (10 μg), Hdm2 V75A (10 μg), and CTS1 (10 μg) (A) and TAF II 250 (10 μg), pBC (10 μg, control for IP3.2), IP3.2 (pBC-IP3.2 10 μg), and TAF II 250 plus IP3.2 (10 μg of each) (B); after 24 h at 39°C the cells were harvested and analyzed by flow cytometry. The results presented are the average of two values, each of which was derived from two independently transfected plates. One representative experiment of three is shown. The percentage of transfected cells in each phase of the cell cycle was calculated and subtracted from the control transfected with empty vector. The control (zero change) is presented as small bars for illustration only.
Figure Legend Snippet: Effect of Mdm2, TAF II 250, and inhibiting Mdm2-p53 interactions on the cell cycle. tsBN462 cells were transfected in 9-cm dishes with vectors that express CD20 (5 μg, all samples) and, in addition, Hdm2 (10 μg), Hdm2 G58A (10 μg), Hdm2 V75A (10 μg), and CTS1 (10 μg) (A) and TAF II 250 (10 μg), pBC (10 μg, control for IP3.2), IP3.2 (pBC-IP3.2 10 μg), and TAF II 250 plus IP3.2 (10 μg of each) (B); after 24 h at 39°C the cells were harvested and analyzed by flow cytometry. The results presented are the average of two values, each of which was derived from two independently transfected plates. One representative experiment of three is shown. The percentage of transfected cells in each phase of the cell cycle was calculated and subtracted from the control transfected with empty vector. The control (zero change) is presented as small bars for illustration only.

Techniques Used: Transfection, Flow Cytometry, Cytometry, Derivative Assay, Plasmid Preparation

32) Product Images from "Increased Nuclear Thioredoxin-1 Potentiates Cadmium-Induced Cytotoxicity"

Article Title: Increased Nuclear Thioredoxin-1 Potentiates Cadmium-Induced Cytotoxicity

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfs271

Increased expression of redox active Trx1 in nuclei potentiated Cd-stimulated NF-κB activation. HeLa cells were cotransfected with plasmids containing NF-κB-luciferase, lacZ, and either empty vector (VC), NLS-wt Trx1, NLS-dn Trx1, NES-Trx1
Figure Legend Snippet: Increased expression of redox active Trx1 in nuclei potentiated Cd-stimulated NF-κB activation. HeLa cells were cotransfected with plasmids containing NF-κB-luciferase, lacZ, and either empty vector (VC), NLS-wt Trx1, NLS-dn Trx1, NES-Trx1

Techniques Used: Expressing, Activation Assay, Luciferase, Plasmid Preparation

Redox active Trx1 in nuclei is critical for Cd-induced cell death. Cells transfected with VC, NLS-wt Trx1, or NLS-dn Trx were exposed to Cd and analyzed for cell survival as described in . Nuclei-targeted expressions of both WT and DN Trx1 were
Figure Legend Snippet: Redox active Trx1 in nuclei is critical for Cd-induced cell death. Cells transfected with VC, NLS-wt Trx1, or NLS-dn Trx were exposed to Cd and analyzed for cell survival as described in . Nuclei-targeted expressions of both WT and DN Trx1 were

Techniques Used: Transfection

Low level of Cd-induced NF-κB activation in kidney was potentiated by NLS-Trx1 Tg mouse. Kidney tissues obtained from WT and NLS-Trx1 transgenic mice exposed to Cd (10mg/kg) or saline for 6h were used for subcellular fractionation and mRNA isolation.
Figure Legend Snippet: Low level of Cd-induced NF-κB activation in kidney was potentiated by NLS-Trx1 Tg mouse. Kidney tissues obtained from WT and NLS-Trx1 transgenic mice exposed to Cd (10mg/kg) or saline for 6h were used for subcellular fractionation and mRNA isolation.

Techniques Used: Activation Assay, Transgenic Assay, Mouse Assay, Fractionation, Isolation

33) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

Journal: Scientific Reports

doi: 10.1038/srep16435

Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.
Figure Legend Snippet: Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.

Techniques Used: Reporter Assay, Activity Assay, Sequencing, Plasmid Preparation, Construct, Derivative Assay, Negative Control, Expressing, Transfection, Standard Deviation

34) Product Images from "Increased Nuclear Thioredoxin-1 Potentiates Cadmium-Induced Cytotoxicity"

Article Title: Increased Nuclear Thioredoxin-1 Potentiates Cadmium-Induced Cytotoxicity

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfs271

Increased expression of redox active Trx1 in nuclei potentiated Cd-stimulated NF-κB activation. HeLa cells were cotransfected with plasmids containing NF-κB-luciferase, lacZ, and either empty vector (VC), NLS-wt Trx1, NLS-dn Trx1, NES-Trx1
Figure Legend Snippet: Increased expression of redox active Trx1 in nuclei potentiated Cd-stimulated NF-κB activation. HeLa cells were cotransfected with plasmids containing NF-κB-luciferase, lacZ, and either empty vector (VC), NLS-wt Trx1, NLS-dn Trx1, NES-Trx1

Techniques Used: Expressing, Activation Assay, Luciferase, Plasmid Preparation

Redox active Trx1 in nuclei is critical for Cd-induced cell death. Cells transfected with VC, NLS-wt Trx1, or NLS-dn Trx were exposed to Cd and analyzed for cell survival as described in . Nuclei-targeted expressions of both WT and DN Trx1 were
Figure Legend Snippet: Redox active Trx1 in nuclei is critical for Cd-induced cell death. Cells transfected with VC, NLS-wt Trx1, or NLS-dn Trx were exposed to Cd and analyzed for cell survival as described in . Nuclei-targeted expressions of both WT and DN Trx1 were

Techniques Used: Transfection

Low level of Cd-induced NF-κB activation in kidney was potentiated by NLS-Trx1 Tg mouse. Kidney tissues obtained from WT and NLS-Trx1 transgenic mice exposed to Cd (10mg/kg) or saline for 6h were used for subcellular fractionation and mRNA isolation.
Figure Legend Snippet: Low level of Cd-induced NF-κB activation in kidney was potentiated by NLS-Trx1 Tg mouse. Kidney tissues obtained from WT and NLS-Trx1 transgenic mice exposed to Cd (10mg/kg) or saline for 6h were used for subcellular fractionation and mRNA isolation.

Techniques Used: Activation Assay, Transgenic Assay, Mouse Assay, Fractionation, Isolation

35) Product Images from "Increased Nuclear Thioredoxin-1 Potentiates Cadmium-Induced Cytotoxicity"

Article Title: Increased Nuclear Thioredoxin-1 Potentiates Cadmium-Induced Cytotoxicity

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfs271

Increased expression of redox active Trx1 in nuclei potentiated Cd-stimulated NF-κB activation. HeLa cells were cotransfected with plasmids containing NF-κB-luciferase, lacZ, and either empty vector (VC), NLS-wt Trx1, NLS-dn Trx1, NES-Trx1
Figure Legend Snippet: Increased expression of redox active Trx1 in nuclei potentiated Cd-stimulated NF-κB activation. HeLa cells were cotransfected with plasmids containing NF-κB-luciferase, lacZ, and either empty vector (VC), NLS-wt Trx1, NLS-dn Trx1, NES-Trx1

Techniques Used: Expressing, Activation Assay, Luciferase, Plasmid Preparation

36) Product Images from "Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals"

Article Title: Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals

Journal: Retrovirology

doi: 10.1186/1742-4690-6-107

Fusion assay between ASCT2 -transduced and syncytin-Ory1 -transduced co-cultured cells demonstrates that ASCT2 is the syncytin-Ory1 receptor . Left panel: Cell-cell fusion was assayed upon independent transfections of a set of A23 cells with an empty vector (none) or an expression vector for either the syncytin-Ory1, syncytin-1 or syncytin-2 protein together with an nls- LacZ gene-expression vector, and another set of A23 cells with an expression vector for the syncytin-1 receptor ASCT2, the syncytin-2 receptor MFSD2 [ 13 ] or an empty vector (none). One day after transfection, cells were resuspended and pairs of transfected cells from each set were cocultured for 1-2 days, fixed and X-Gal stained. Right panel: Syncytia can be easily detected (arrows) for the syncytin-Ory1/ASCT2, syncytin-1/ASCT2 and syncytin-2/MFSD2 pairs, with only mononucleated cells visible in the other cases. Abbreviations: syn-Ory1, syncytin-Ory1; syn1, syncytin-1; syn2, syncytin-2.
Figure Legend Snippet: Fusion assay between ASCT2 -transduced and syncytin-Ory1 -transduced co-cultured cells demonstrates that ASCT2 is the syncytin-Ory1 receptor . Left panel: Cell-cell fusion was assayed upon independent transfections of a set of A23 cells with an empty vector (none) or an expression vector for either the syncytin-Ory1, syncytin-1 or syncytin-2 protein together with an nls- LacZ gene-expression vector, and another set of A23 cells with an expression vector for the syncytin-1 receptor ASCT2, the syncytin-2 receptor MFSD2 [ 13 ] or an empty vector (none). One day after transfection, cells were resuspended and pairs of transfected cells from each set were cocultured for 1-2 days, fixed and X-Gal stained. Right panel: Syncytia can be easily detected (arrows) for the syncytin-Ory1/ASCT2, syncytin-1/ASCT2 and syncytin-2/MFSD2 pairs, with only mononucleated cells visible in the other cases. Abbreviations: syn-Ory1, syncytin-Ory1; syn1, syncytin-1; syn2, syncytin-2.

Techniques Used: Single Vesicle Fusion Assay, Cell Culture, Transfection, Plasmid Preparation, Expressing, Staining

Fusogenic activity of syncytin-Ory1 . (A) Assay for cell-cell fusion mediated by syncytin-Ory1. The indicated cell lines were transfected with an expression vector for syncytin-Ory1 or an empty vector (none) together with a LacZ expression vector. Cells were cultured for 1-2 days after transfection, fixed and X-gal-stained. Syncytia (arrows) were detected in syncytin-Ory1 -transfected SH-SY5Y cells, with only mononucleated cells visible in the other cases. (B) Assay for cell infection mediated by syncytin-Ory1-pseudotyped virus particles. Pseudotypes were produced by cotransfection of human 293T cells with expression vectors for the SIV core, the syncytin-Ory1 protein (or an empty vector) and a LacZ -containing retroviral transcript. Supernatants were used to infect the indicated target cells, which were X-gal stained 3 days after infection. Abbreviation: Syn-Ory1, syncytin-Ory1.
Figure Legend Snippet: Fusogenic activity of syncytin-Ory1 . (A) Assay for cell-cell fusion mediated by syncytin-Ory1. The indicated cell lines were transfected with an expression vector for syncytin-Ory1 or an empty vector (none) together with a LacZ expression vector. Cells were cultured for 1-2 days after transfection, fixed and X-gal-stained. Syncytia (arrows) were detected in syncytin-Ory1 -transfected SH-SY5Y cells, with only mononucleated cells visible in the other cases. (B) Assay for cell infection mediated by syncytin-Ory1-pseudotyped virus particles. Pseudotypes were produced by cotransfection of human 293T cells with expression vectors for the SIV core, the syncytin-Ory1 protein (or an empty vector) and a LacZ -containing retroviral transcript. Supernatants were used to infect the indicated target cells, which were X-gal stained 3 days after infection. Abbreviation: Syn-Ory1, syncytin-Ory1.

Techniques Used: Activity Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Staining, Infection, Produced, Cotransfection

37) Product Images from "Accurately mapping the location of the binding site for the interaction between hepatitis B virus X protein and cytochrome c oxidase III"

Article Title: Accurately mapping the location of the binding site for the interaction between hepatitis B virus X protein and cytochrome c oxidase III

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2014.2018

Assays to detect β-galactosidase (β-gal) activity after mating. Panel 1, blue colonies on Whatman filter paper after mating between Y187-pACT2-COXIII and AH109-pAS2-1-X; panel 2, blue colonies on Whatman filter paper after mating between Y187-pACT2-COXIII and AH109-pAS2-1-X2; panel 3, white colonies on Whatman filter paper after mating between Y187-pACT2-COXIII and AH109-pAS2-1-X1.
Figure Legend Snippet: Assays to detect β-galactosidase (β-gal) activity after mating. Panel 1, blue colonies on Whatman filter paper after mating between Y187-pACT2-COXIII and AH109-pAS2-1-X; panel 2, blue colonies on Whatman filter paper after mating between Y187-pACT2-COXIII and AH109-pAS2-1-X2; panel 3, white colonies on Whatman filter paper after mating between Y187-pACT2-COXIII and AH109-pAS2-1-X1.

Techniques Used: Activity Assay

Hepatitis B viral virus X protein (HBx) X1 and X2 fragments amplified by PCR from AH109-pAS2-1-X1 and AH109-pAS2-1-X2. X1 and X2 fragments of the expected size can be seen after gel electrophoresis (0.6% agarose). Lane 1, 100-bp DNA ladder; lanes 2–4, HBx X1 (216 bp) amplified with primers P1 and P2; lane 5, negative controls; lanes 6 and 7, HBx X2 (352 bp) amplified with primers P1 and P3.
Figure Legend Snippet: Hepatitis B viral virus X protein (HBx) X1 and X2 fragments amplified by PCR from AH109-pAS2-1-X1 and AH109-pAS2-1-X2. X1 and X2 fragments of the expected size can be seen after gel electrophoresis (0.6% agarose). Lane 1, 100-bp DNA ladder; lanes 2–4, HBx X1 (216 bp) amplified with primers P1 and P2; lane 5, negative controls; lanes 6 and 7, HBx X2 (352 bp) amplified with primers P1 and P3.

Techniques Used: Amplification, Polymerase Chain Reaction, Nucleic Acid Electrophoresis

Hepatitis B viral virus X protein (HBx) fragments amplified by PCR from pAS2-1 recombinant plasmid and separated by gel electrophoresis (0.6% agarose). The different fragments have the expected molecular weights. Lane 1, 100-bp DNA ladder used as molecular weight marker; lane 2, full length (464 bp) HBx gene; lane 3, HBx X2 (352 bp) fragment; lane 4, negative controls; lanes 5–7, HBx X1 (216 bp) fragment.
Figure Legend Snippet: Hepatitis B viral virus X protein (HBx) fragments amplified by PCR from pAS2-1 recombinant plasmid and separated by gel electrophoresis (0.6% agarose). The different fragments have the expected molecular weights. Lane 1, 100-bp DNA ladder used as molecular weight marker; lane 2, full length (464 bp) HBx gene; lane 3, HBx X2 (352 bp) fragment; lane 4, negative controls; lanes 5–7, HBx X1 (216 bp) fragment.

Techniques Used: Amplification, Polymerase Chain Reaction, Recombinant, Plasmid Preparation, Nucleic Acid Electrophoresis, Molecular Weight, Marker

(A) DNA sequence of hepatitis B viral virus X protein (HBx) X1. Amplified HBx X1 fragments were ligated with linearized pAS2-1 and the recombinant plasmid designated pAS2-1-X1. Purified pAS2-1-X1 was sent to Life Technologies Corp. for sequencing. The X1 gene has 98% homology with the X gene from the GenBank database. The X1 gene runs from base 63 to base 278. (B) DNA sequence of HBx X2. Amplified HBx X2 fragments were ligated with linearized pAS2-1 and the recombinant plasmid designated pAS2-1-X2. The sequence shows that the X2 gene has 98% homology with X gene from the GenBank database. The X2 gene runs from base 61 to base 412.
Figure Legend Snippet: (A) DNA sequence of hepatitis B viral virus X protein (HBx) X1. Amplified HBx X1 fragments were ligated with linearized pAS2-1 and the recombinant plasmid designated pAS2-1-X1. Purified pAS2-1-X1 was sent to Life Technologies Corp. for sequencing. The X1 gene has 98% homology with the X gene from the GenBank database. The X1 gene runs from base 63 to base 278. (B) DNA sequence of HBx X2. Amplified HBx X2 fragments were ligated with linearized pAS2-1 and the recombinant plasmid designated pAS2-1-X2. The sequence shows that the X2 gene has 98% homology with X gene from the GenBank database. The X2 gene runs from base 61 to base 412.

Techniques Used: Sequencing, Amplification, Recombinant, Plasmid Preparation, Purification

38) Product Images from "Prothymosin ? overexpression contributes to the development of pulmonary emphysema"

Article Title: Prothymosin ? overexpression contributes to the development of pulmonary emphysema

Journal: Nature Communications

doi: 10.1038/ncomms2906

Overexpression of ProT increases NF-κB acetylation. ( a ) Detection of acetylated NF-κB p65 (Lys310). 293T cells that had been transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 (left) or transduced with lentiviruses expressing ProT shRNA-1 and -2 or GFP shRNA (right) were transfected with a p300 expression vector (pCMVb p300 HA delta33) or a control vector (pHM6-lacZ). After 24 h, total cell lysates were examined for the indicated proteins by immunoblotting. Equal levels of p300 and LacZ expression in the transfected cells were verified. ( b ) Reporter assay for NF-κB transactivation activity. 293T cells that had been transduced with lentiviruses expressing ProT-IRES-GFP or GFP alone (left), or ProT or GFP shRNA (right) were cotransfected with pNF-κB-Luc and pTK-Renilla reporter plasmids. Total cell lysates were harvested 48 h later, and their firefly and Renilla luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light activity. Values shown are the mean±s.e.m. ( n =4 for left and n =5 for right; Student’s t -test). ( c ) NF-κB and ProT binding assay. Cell lysates from 293T cells transduced with lentiviruses expressing GFP-tagged ProT or GFP alone were immuoprecipitated by anti-GFP monoclonal antibody followed by immunoblotting with anti-NF-κB p65 and anti-GFP antibody. Whole-cell lysates without immunoprecipitation (10% input) were estimated for the expression levels of NF-κB and ProT by immunoblotting. ( d ) Analysis of the effect of ProT on NF-κB and HDAC3 binding. 293T cells that had been transfected with an NF-κB p65 expression vector (pcDNA3-cFlag-RelA) or a control vector (pCMV-Tag2B) (left) or HDAC3 expression vector (pcDNA3.1-HDAC3-Flag) or a control vector (pCMV-Tag2B) (right) were transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1. After 24 h, total cell lysates were immuoprecipitated by Flag-M2 beads followed by immunoblotting with the indicated proteins. Whole-cell lysates without immunoprecipitation (10% input) were estimated by immunoblotting with indicated proteins. ( e ) p300 and NF-κB binding assay. 293T cells that had been transduced with lentiviruses expressing ProT shRNA-2 or luciferase shRNA as the control were transfected with the pCMVb p300 HA delta33 and pcDNA3-cFlag-RelA. Total cell lysates were immunoprecipitated by anti-HA antibody followed by immunoblotting with the indicated proteins. Results are representative of three independent experiments. ( f ) Proposed model for ProT-mediated NF-κB hyperacetylation via HDAC3/NF-κB dissociation and p300/NF-κB association.
Figure Legend Snippet: Overexpression of ProT increases NF-κB acetylation. ( a ) Detection of acetylated NF-κB p65 (Lys310). 293T cells that had been transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 (left) or transduced with lentiviruses expressing ProT shRNA-1 and -2 or GFP shRNA (right) were transfected with a p300 expression vector (pCMVb p300 HA delta33) or a control vector (pHM6-lacZ). After 24 h, total cell lysates were examined for the indicated proteins by immunoblotting. Equal levels of p300 and LacZ expression in the transfected cells were verified. ( b ) Reporter assay for NF-κB transactivation activity. 293T cells that had been transduced with lentiviruses expressing ProT-IRES-GFP or GFP alone (left), or ProT or GFP shRNA (right) were cotransfected with pNF-κB-Luc and pTK-Renilla reporter plasmids. Total cell lysates were harvested 48 h later, and their firefly and Renilla luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light activity. Values shown are the mean±s.e.m. ( n =4 for left and n =5 for right; Student’s t -test). ( c ) NF-κB and ProT binding assay. Cell lysates from 293T cells transduced with lentiviruses expressing GFP-tagged ProT or GFP alone were immuoprecipitated by anti-GFP monoclonal antibody followed by immunoblotting with anti-NF-κB p65 and anti-GFP antibody. Whole-cell lysates without immunoprecipitation (10% input) were estimated for the expression levels of NF-κB and ProT by immunoblotting. ( d ) Analysis of the effect of ProT on NF-κB and HDAC3 binding. 293T cells that had been transfected with an NF-κB p65 expression vector (pcDNA3-cFlag-RelA) or a control vector (pCMV-Tag2B) (left) or HDAC3 expression vector (pcDNA3.1-HDAC3-Flag) or a control vector (pCMV-Tag2B) (right) were transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1. After 24 h, total cell lysates were immuoprecipitated by Flag-M2 beads followed by immunoblotting with the indicated proteins. Whole-cell lysates without immunoprecipitation (10% input) were estimated by immunoblotting with indicated proteins. ( e ) p300 and NF-κB binding assay. 293T cells that had been transduced with lentiviruses expressing ProT shRNA-2 or luciferase shRNA as the control were transfected with the pCMVb p300 HA delta33 and pcDNA3-cFlag-RelA. Total cell lysates were immunoprecipitated by anti-HA antibody followed by immunoblotting with the indicated proteins. Results are representative of three independent experiments. ( f ) Proposed model for ProT-mediated NF-κB hyperacetylation via HDAC3/NF-κB dissociation and p300/NF-κB association.

Techniques Used: Over Expression, Transfection, Transduction, Expressing, shRNA, Plasmid Preparation, Reporter Assay, Activity Assay, Luciferase, Binding Assay, Immunoprecipitation

39) Product Images from "Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells"

Article Title: Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.22.7807-7816.2001

Northern analysis of cognate (EGFP) and the noncognate (β-galactosidase) mRNAs. Undifferentiated ES cells were grown on the feeder layer and transfected by three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 1, 2, and 3, respectively). As a control, ES cells were transfected with three plasmids, pCMV-lacZ (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 4, 5, and 6, respectively). Total RNA was isolated from transfected cells, and 25 μg of total RNA was loaded in each lane. The EGFP probe was a 0.7-kb fragment isolated from the pEGFP-C1 plasmid, and the lacZ probe was a 2.5-kb fragment from the pCMV-lacZ plasmid. The probes were labeled by [α- 32 P]dCTP. A cDNA probe corresponding to the mouse β-actin coding sequence was hybridized as a control.
Figure Legend Snippet: Northern analysis of cognate (EGFP) and the noncognate (β-galactosidase) mRNAs. Undifferentiated ES cells were grown on the feeder layer and transfected by three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 1, 2, and 3, respectively). As a control, ES cells were transfected with three plasmids, pCMV-lacZ (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 4, 5, and 6, respectively). Total RNA was isolated from transfected cells, and 25 μg of total RNA was loaded in each lane. The EGFP probe was a 0.7-kb fragment isolated from the pEGFP-C1 plasmid, and the lacZ probe was a 2.5-kb fragment from the pCMV-lacZ plasmid. The probes were labeled by [α- 32 P]dCTP. A cDNA probe corresponding to the mouse β-actin coding sequence was hybridized as a control.

Techniques Used: Northern Blot, Transfection, Isolation, Plasmid Preparation, Labeling, Sequencing

dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.
Figure Legend Snippet: dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.

Techniques Used: Produced, Sequencing, Inhibition, Expressing, In Situ, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation, In Vitro, Staining

Several mammalian cells do not show sequence-specific RNAi activity. Three mammalian cell lines, BsrT7/5 (A), STO (B), and CHO-K1 (C), were tested for RNAi activity by transient transfection of three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and increasing amounts of pGEMT-dsEGFP (0.25 to 2 μg). Throughout transfection, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfections of duplicate samples.
Figure Legend Snippet: Several mammalian cells do not show sequence-specific RNAi activity. Three mammalian cell lines, BsrT7/5 (A), STO (B), and CHO-K1 (C), were tested for RNAi activity by transient transfection of three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and increasing amounts of pGEMT-dsEGFP (0.25 to 2 μg). Throughout transfection, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfections of duplicate samples.

Techniques Used: Sequencing, Activity Assay, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation

Undifferentiated ES cells exhibit RNAi activity. (A) Sequence-specific and dose-dependent inhibition of EGFP by pGEMT-dsEGFP plasmid in ES cells grown on a feeder layer. ES cells were plated on STO feeder cells and transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfection experiments performed in duplicate. (B) Non-sequence-specific inhibition of EGFP by pGEMT-dsEGFP plasmid in differentiated ES cells cultured without the feeder layer. The same experiment was carried out in ES cells plated directly on a gelatin-coated plate with no feeder cells.
Figure Legend Snippet: Undifferentiated ES cells exhibit RNAi activity. (A) Sequence-specific and dose-dependent inhibition of EGFP by pGEMT-dsEGFP plasmid in ES cells grown on a feeder layer. ES cells were plated on STO feeder cells and transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfection experiments performed in duplicate. (B) Non-sequence-specific inhibition of EGFP by pGEMT-dsEGFP plasmid in differentiated ES cells cultured without the feeder layer. The same experiment was carried out in ES cells plated directly on a gelatin-coated plate with no feeder cells.

Techniques Used: Activity Assay, Sequencing, Inhibition, Plasmid Preparation, Transfection, Fluorescence, Standard Deviation, Cell Culture

Sequence-specific and dose-dependent inhibition of EGFP expression by in-vitro transcribed dsRNA in undifferentiated ES cells. (A) ES cells were plated on feeder cells and transfected with 1 μg of the pEGFP-C1 plasmid and increasing amounts, 0.25 to 1.0 μg, of the in vitro-transcribed dsRNA. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no dsRNA. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection performed in duplicate. (B) Fluorescence microscopy of undifferentiated ES cells transfected with 2.5 μg of pEGFP-C1 plasmid and an increasing amount, 0, 1, and 2 μg (lanes, 1, 2, and 3, respectively), of the in vitro-transcribed dsRNA-EGFP. Photographs were taken 72 h later, using a bright field (upper panel) and fluorescence (lower panel). (C) β-Galactosidase expression is not inhibited by in vitro-transcribed dsRNA-EGFP. ES cells were transfected with 2.5 μg of pCMV-lacZ and 0, 1, or 2 μg of in vitro-transcribed dsRNA-EGFP. Histochemical staining was carried out 72 h later.
Figure Legend Snippet: Sequence-specific and dose-dependent inhibition of EGFP expression by in-vitro transcribed dsRNA in undifferentiated ES cells. (A) ES cells were plated on feeder cells and transfected with 1 μg of the pEGFP-C1 plasmid and increasing amounts, 0.25 to 1.0 μg, of the in vitro-transcribed dsRNA. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no dsRNA. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection performed in duplicate. (B) Fluorescence microscopy of undifferentiated ES cells transfected with 2.5 μg of pEGFP-C1 plasmid and an increasing amount, 0, 1, and 2 μg (lanes, 1, 2, and 3, respectively), of the in vitro-transcribed dsRNA-EGFP. Photographs were taken 72 h later, using a bright field (upper panel) and fluorescence (lower panel). (C) β-Galactosidase expression is not inhibited by in vitro-transcribed dsRNA-EGFP. ES cells were transfected with 2.5 μg of pCMV-lacZ and 0, 1, or 2 μg of in vitro-transcribed dsRNA-EGFP. Histochemical staining was carried out 72 h later.

Techniques Used: Sequencing, Inhibition, Expressing, In Vitro, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation, Microscopy, Staining

40) Product Images from "Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells"

Article Title: Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.22.7807-7816.2001

Northern analysis of cognate (EGFP) and the noncognate (β-galactosidase) mRNAs. Undifferentiated ES cells were grown on the feeder layer and transfected by three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 1, 2, and 3, respectively). As a control, ES cells were transfected with three plasmids, pCMV-lacZ (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 4, 5, and 6, respectively). Total RNA was isolated from transfected cells, and 25 μg of total RNA was loaded in each lane. The EGFP probe was a 0.7-kb fragment isolated from the pEGFP-C1 plasmid, and the lacZ probe was a 2.5-kb fragment from the pCMV-lacZ plasmid. The probes were labeled by [α- 32 P]dCTP. A cDNA probe corresponding to the mouse β-actin coding sequence was hybridized as a control.
Figure Legend Snippet: Northern analysis of cognate (EGFP) and the noncognate (β-galactosidase) mRNAs. Undifferentiated ES cells were grown on the feeder layer and transfected by three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 1, 2, and 3, respectively). As a control, ES cells were transfected with three plasmids, pCMV-lacZ (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 4, 5, and 6, respectively). Total RNA was isolated from transfected cells, and 25 μg of total RNA was loaded in each lane. The EGFP probe was a 0.7-kb fragment isolated from the pEGFP-C1 plasmid, and the lacZ probe was a 2.5-kb fragment from the pCMV-lacZ plasmid. The probes were labeled by [α- 32 P]dCTP. A cDNA probe corresponding to the mouse β-actin coding sequence was hybridized as a control.

Techniques Used: Northern Blot, Transfection, Isolation, Plasmid Preparation, Labeling, Sequencing

dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.
Figure Legend Snippet: dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.

Techniques Used: Produced, Sequencing, Inhibition, Expressing, In Situ, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation, In Vitro, Staining

Several mammalian cells do not show sequence-specific RNAi activity. Three mammalian cell lines, BsrT7/5 (A), STO (B), and CHO-K1 (C), were tested for RNAi activity by transient transfection of three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and increasing amounts of pGEMT-dsEGFP (0.25 to 2 μg). Throughout transfection, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfections of duplicate samples.
Figure Legend Snippet: Several mammalian cells do not show sequence-specific RNAi activity. Three mammalian cell lines, BsrT7/5 (A), STO (B), and CHO-K1 (C), were tested for RNAi activity by transient transfection of three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and increasing amounts of pGEMT-dsEGFP (0.25 to 2 μg). Throughout transfection, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfections of duplicate samples.

Techniques Used: Sequencing, Activity Assay, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation

Undifferentiated ES cells exhibit RNAi activity. (A) Sequence-specific and dose-dependent inhibition of EGFP by pGEMT-dsEGFP plasmid in ES cells grown on a feeder layer. ES cells were plated on STO feeder cells and transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfection experiments performed in duplicate. (B) Non-sequence-specific inhibition of EGFP by pGEMT-dsEGFP plasmid in differentiated ES cells cultured without the feeder layer. The same experiment was carried out in ES cells plated directly on a gelatin-coated plate with no feeder cells.
Figure Legend Snippet: Undifferentiated ES cells exhibit RNAi activity. (A) Sequence-specific and dose-dependent inhibition of EGFP by pGEMT-dsEGFP plasmid in ES cells grown on a feeder layer. ES cells were plated on STO feeder cells and transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfection experiments performed in duplicate. (B) Non-sequence-specific inhibition of EGFP by pGEMT-dsEGFP plasmid in differentiated ES cells cultured without the feeder layer. The same experiment was carried out in ES cells plated directly on a gelatin-coated plate with no feeder cells.

Techniques Used: Activity Assay, Sequencing, Inhibition, Plasmid Preparation, Transfection, Fluorescence, Standard Deviation, Cell Culture

Sequence-specific and dose-dependent inhibition of EGFP expression by in-vitro transcribed dsRNA in undifferentiated ES cells. (A) ES cells were plated on feeder cells and transfected with 1 μg of the pEGFP-C1 plasmid and increasing amounts, 0.25 to 1.0 μg, of the in vitro-transcribed dsRNA. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no dsRNA. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection performed in duplicate. (B) Fluorescence microscopy of undifferentiated ES cells transfected with 2.5 μg of pEGFP-C1 plasmid and an increasing amount, 0, 1, and 2 μg (lanes, 1, 2, and 3, respectively), of the in vitro-transcribed dsRNA-EGFP. Photographs were taken 72 h later, using a bright field (upper panel) and fluorescence (lower panel). (C) β-Galactosidase expression is not inhibited by in vitro-transcribed dsRNA-EGFP. ES cells were transfected with 2.5 μg of pCMV-lacZ and 0, 1, or 2 μg of in vitro-transcribed dsRNA-EGFP. Histochemical staining was carried out 72 h later.
Figure Legend Snippet: Sequence-specific and dose-dependent inhibition of EGFP expression by in-vitro transcribed dsRNA in undifferentiated ES cells. (A) ES cells were plated on feeder cells and transfected with 1 μg of the pEGFP-C1 plasmid and increasing amounts, 0.25 to 1.0 μg, of the in vitro-transcribed dsRNA. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no dsRNA. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection performed in duplicate. (B) Fluorescence microscopy of undifferentiated ES cells transfected with 2.5 μg of pEGFP-C1 plasmid and an increasing amount, 0, 1, and 2 μg (lanes, 1, 2, and 3, respectively), of the in vitro-transcribed dsRNA-EGFP. Photographs were taken 72 h later, using a bright field (upper panel) and fluorescence (lower panel). (C) β-Galactosidase expression is not inhibited by in vitro-transcribed dsRNA-EGFP. ES cells were transfected with 2.5 μg of pCMV-lacZ and 0, 1, or 2 μg of in vitro-transcribed dsRNA-EGFP. Histochemical staining was carried out 72 h later.

Techniques Used: Sequencing, Inhibition, Expressing, In Vitro, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation, Microscopy, Staining

Related Articles

Transfection:

Article Title: Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors
Article Snippet: .. Following transfection, cells were cultured for 48 h, then harvested and assayed using the Luminescent β-galactosidase Detection kit II (Clontech), as previously described ( ). .. For ChIP, primary mouse embryo fibroblasts (MEFs) were transfected using Lipofectamine LTX and 8 μg of either pcDNA3.1-FLAG-FoxC2 or empty pCDNA3.1 vector in 10 cm dishes.

Luciferase:

Article Title: Polymorphism of the human ?1 immunoglobulin gene 3? enhancer hs1,2 and its relation to gene expression
Article Snippet: .. After 20 hr of culture in RPMI-1640 medium (Gibco, Cergy Pontoise, France) supplemented with 10% fetal calf serum (FCS), 2 m m l -glutamine, 100 µg/ml penicillin and 10 µg/ml streptomycin, cells were recovered, lysed and assayed for luciferase activity using the Luclite Plus Assay Kit (Packard Bio-Science B.V., Groningen, The Netherlands) and for β galactosidase activity with the luminescent β galactosidase detection kit (Clontech, Palo Alto, CA) to standardize the assay. ..

Article Title: Transforming growth factor ?-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells
Article Snippet: .. After 20 hr incubation in the absence or presence of 240 pM TGF-β1, luciferase activity was determined using the luciferase assay system (Promega) and β-galactosidase activity was measured using the Luminescent β-galactosidase detection kit (CLONTECH) with an Analytical Luminescent Laboratory luminometer. .. To address the physiological relevance of the Smad3 protein in cellular responses to TGF-β, we introduced the wild-type and various mutant Smad3 genes into a novel retroviral vector.

Article Title: Mesenchymal stem cells decrease lung inflammation during sepsis, acting through inhibition of the MAPK pathway
Article Snippet: .. Luciferase values were normalized using β-galactosidase, and activity measured with the Luminescent β-Galactosidase Detection Kit II (CLONTECH, Palo Alto, CA, USA). ..

Article Title: Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53
Article Snippet: .. Luciferase activity was measured using the Luciferase Assay System protocol (Promega, Madison, WI, USA) and a luminometer (Lumat LB 9507, Berthold). β-galactosidase activity was measured using a colorimetric enzyme assay using the Luminescent β-Galactosidase Detection Kit II according to the manufacturer’s instructions (Clontech Laboratories, Mountain View, CA). .. Luciferase expression levels were normalized to the levels of β-galactosidase expression.

Article Title: HIF2α signaling inhibits adherens junctional disruption in acute lung injury
Article Snippet: .. Anti–VE-cadherin (sc-9989, sc-6458, and sc-52751), anti–VE-PTP (SC-28905), anti-HIF2α (sc-13596), anti-PHD2 (sc-271835), anti-sFLT (sc-9029), and anti–β-actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology Inc.; mouse monoclonal anti–VE-PTP (610180) and anti-HIF1α (610959) antibodies were from BD; anti-HIF3α (ab10134) antibody was purchased from Abcam; anti-HIF2α (NB100-122) antibody was from Novus Biologicals; Lipofectamine 2000, ViraPower Lentiviral Expression System, and Alexa Fluor 488–, 594–, and 633–conjugated secondary antibodies and Alexa Fluor 555–albumin were obtained from Invitrogen; anti–VE-cadherin pY658, pY695, and pY731 antibodies, anti-phosphotyrosine antibody (4G10), and the ChIP assay kit were from EMD Millipore; the luciferase assay kit was purchased from Promega; and the Luminescent β-galactosidase Detection Kit was from Clontech. .. For in vivo experiments, we used EC-specific inducible Hif2a–/– mice generated by i . p . administration of tamoxifen (2 mg/day for 5 days) to Tie2-Cre Hif2afl/fl mice (129/B6 background), in which tamoxifen induced expression of a fusion protein of Cre recombinase with the modified estrogen receptor–binding domain ( CreERT2 ) under the control of the Tie2 promoter ( , ).

Enzymatic Assay:

Article Title: Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53
Article Snippet: .. Luciferase activity was measured using the Luciferase Assay System protocol (Promega, Madison, WI, USA) and a luminometer (Lumat LB 9507, Berthold). β-galactosidase activity was measured using a colorimetric enzyme assay using the Luminescent β-Galactosidase Detection Kit II according to the manufacturer’s instructions (Clontech Laboratories, Mountain View, CA). .. Luciferase expression levels were normalized to the levels of β-galactosidase expression.

Cell Culture:

Article Title: Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors
Article Snippet: .. Following transfection, cells were cultured for 48 h, then harvested and assayed using the Luminescent β-galactosidase Detection kit II (Clontech), as previously described ( ). .. For ChIP, primary mouse embryo fibroblasts (MEFs) were transfected using Lipofectamine LTX and 8 μg of either pcDNA3.1-FLAG-FoxC2 or empty pCDNA3.1 vector in 10 cm dishes.

Incubation:

Article Title: Transforming growth factor ?-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells
Article Snippet: .. After 20 hr incubation in the absence or presence of 240 pM TGF-β1, luciferase activity was determined using the luciferase assay system (Promega) and β-galactosidase activity was measured using the Luminescent β-galactosidase detection kit (CLONTECH) with an Analytical Luminescent Laboratory luminometer. .. To address the physiological relevance of the Smad3 protein in cellular responses to TGF-β, we introduced the wild-type and various mutant Smad3 genes into a novel retroviral vector.

Activity Assay:

Article Title: Polymorphism of the human ?1 immunoglobulin gene 3? enhancer hs1,2 and its relation to gene expression
Article Snippet: .. After 20 hr of culture in RPMI-1640 medium (Gibco, Cergy Pontoise, France) supplemented with 10% fetal calf serum (FCS), 2 m m l -glutamine, 100 µg/ml penicillin and 10 µg/ml streptomycin, cells were recovered, lysed and assayed for luciferase activity using the Luclite Plus Assay Kit (Packard Bio-Science B.V., Groningen, The Netherlands) and for β galactosidase activity with the luminescent β galactosidase detection kit (Clontech, Palo Alto, CA) to standardize the assay. ..

Article Title: Transforming growth factor ?-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells
Article Snippet: .. After 20 hr incubation in the absence or presence of 240 pM TGF-β1, luciferase activity was determined using the luciferase assay system (Promega) and β-galactosidase activity was measured using the Luminescent β-galactosidase detection kit (CLONTECH) with an Analytical Luminescent Laboratory luminometer. .. To address the physiological relevance of the Smad3 protein in cellular responses to TGF-β, we introduced the wild-type and various mutant Smad3 genes into a novel retroviral vector.

Article Title: Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites
Article Snippet: .. Cells were harvested 48 h post-transfection, and cellular extracts were prepared and assayed for β-galactosidase activity using the Luminescent β-galactosidase Detection Kit (Clontech) as previously described ( ). ..

Article Title: Mesenchymal stem cells decrease lung inflammation during sepsis, acting through inhibition of the MAPK pathway
Article Snippet: .. Luciferase values were normalized using β-galactosidase, and activity measured with the Luminescent β-Galactosidase Detection Kit II (CLONTECH, Palo Alto, CA, USA). ..

Article Title: Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53
Article Snippet: .. Luciferase activity was measured using the Luciferase Assay System protocol (Promega, Madison, WI, USA) and a luminometer (Lumat LB 9507, Berthold). β-galactosidase activity was measured using a colorimetric enzyme assay using the Luminescent β-Galactosidase Detection Kit II according to the manufacturer’s instructions (Clontech Laboratories, Mountain View, CA). .. Luciferase expression levels were normalized to the levels of β-galactosidase expression.

Chromatin Immunoprecipitation:

Article Title: HIF2α signaling inhibits adherens junctional disruption in acute lung injury
Article Snippet: .. Anti–VE-cadherin (sc-9989, sc-6458, and sc-52751), anti–VE-PTP (SC-28905), anti-HIF2α (sc-13596), anti-PHD2 (sc-271835), anti-sFLT (sc-9029), and anti–β-actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology Inc.; mouse monoclonal anti–VE-PTP (610180) and anti-HIF1α (610959) antibodies were from BD; anti-HIF3α (ab10134) antibody was purchased from Abcam; anti-HIF2α (NB100-122) antibody was from Novus Biologicals; Lipofectamine 2000, ViraPower Lentiviral Expression System, and Alexa Fluor 488–, 594–, and 633–conjugated secondary antibodies and Alexa Fluor 555–albumin were obtained from Invitrogen; anti–VE-cadherin pY658, pY695, and pY731 antibodies, anti-phosphotyrosine antibody (4G10), and the ChIP assay kit were from EMD Millipore; the luciferase assay kit was purchased from Promega; and the Luminescent β-galactosidase Detection Kit was from Clontech. .. For in vivo experiments, we used EC-specific inducible Hif2a–/– mice generated by i . p . administration of tamoxifen (2 mg/day for 5 days) to Tie2-Cre Hif2afl/fl mice (129/B6 background), in which tamoxifen induced expression of a fusion protein of Cre recombinase with the modified estrogen receptor–binding domain ( CreERT2 ) under the control of the Tie2 promoter ( , ).

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    TaKaRa pcmv lacz plasmid
    ORC1 binds SUV39H1 to control Cyclin E gene transcription. ORC1, but not ORC3 or ORC4 can repress gene transcription. ( A ) The U2OS cells were transfected with a Gal4-driven luciferase reporter as shown in the schematic with increasing amounts of Gal4DBD-ORC1 or Gal4DBD-SUV39H1 together with <t>pCMV-LacZ</t> plasmids. Relative luciferase activity was determined and normalized to lacZ activity. Experiments were carried out in triplicate. The whole cell extract was immunoblotted with anti-Gal4 antibody for expression of Gal4DBD fusion plasmids. α-Tubulin served as a loading control. Statistical analysis was performed using the Student’s t test. **p
    Pcmv Lacz Plasmid, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    89
    TaKaRa pcmv lacz
    HEK293 cells fail to support the interaction of zebrafish miR-430b with known mRNA targets. (A) Time course of miR-430b-mediated inhibition of luciferase expression using the reporter construct pGL3-3xPT, which carries three tandom copies of the perfect target sequence. pGL3-3xPT and miR-430b were cotransfected into the HEK293 cells and luciferase expression was measured at the times indicated post-transfection. (B) miR-430b-mediated inhibition of luciferase expression using reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, and pGl3-3′nanos that carry 3′UTRs containing known miR-430b target sequences. Luciferase expression was measured at 48 h post-transfection (hpt). pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively, and the transfection efficiency was normalized to β-galactosidase expression from <t>pCMV-lacZ.</t> Each bar on the graph represents the average of four separate transfections.
    Pcmv Lacz, supplied by TaKaRa, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ORC1 binds SUV39H1 to control Cyclin E gene transcription. ORC1, but not ORC3 or ORC4 can repress gene transcription. ( A ) The U2OS cells were transfected with a Gal4-driven luciferase reporter as shown in the schematic with increasing amounts of Gal4DBD-ORC1 or Gal4DBD-SUV39H1 together with pCMV-LacZ plasmids. Relative luciferase activity was determined and normalized to lacZ activity. Experiments were carried out in triplicate. The whole cell extract was immunoblotted with anti-Gal4 antibody for expression of Gal4DBD fusion plasmids. α-Tubulin served as a loading control. Statistical analysis was performed using the Student’s t test. **p

    Journal: eLife

    Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

    doi: 10.7554/eLife.12785

    Figure Lengend Snippet: ORC1 binds SUV39H1 to control Cyclin E gene transcription. ORC1, but not ORC3 or ORC4 can repress gene transcription. ( A ) The U2OS cells were transfected with a Gal4-driven luciferase reporter as shown in the schematic with increasing amounts of Gal4DBD-ORC1 or Gal4DBD-SUV39H1 together with pCMV-LacZ plasmids. Relative luciferase activity was determined and normalized to lacZ activity. Experiments were carried out in triplicate. The whole cell extract was immunoblotted with anti-Gal4 antibody for expression of Gal4DBD fusion plasmids. α-Tubulin served as a loading control. Statistical analysis was performed using the Student’s t test. **p

    Article Snippet: Plasmid construction and mutagenesis Plasmids expressing GFP-RB, 10–4 CCNE1 (Addgene: Cyclin E gene) promoter, E2F1, DP1, HDAC1 and pGL2-GAL4-UAS-Luc were purchased from Addgene. pCMV-LacZ plasmid was purchased from Clontech.

    Techniques: Transfection, Luciferase, Activity Assay, Expressing

    The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p

    Journal: The Journal of Neuroscience

    Article Title: Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

    doi: 10.1523/JNEUROSCI.1001-13.2013

    Figure Lengend Snippet: The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p

    Article Snippet: S2 cells were transfected with the luciferase reporter constructs (2 μg/well) along with a vector expressing β-galactosidase (β-gal; pCMV–LacZ vector from Clontech; 1 μg/well) in duplicate using Lipofectamine (Invitrogen).

    Techniques: Luciferase, Activity Assay, Transfection, Mutagenesis, Construct

    Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p

    Journal: The Journal of Neuroscience

    Article Title: Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

    doi: 10.1523/JNEUROSCI.1001-13.2013

    Figure Lengend Snippet: Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p

    Article Snippet: S2 cells were transfected with the luciferase reporter constructs (2 μg/well) along with a vector expressing β-galactosidase (β-gal; pCMV–LacZ vector from Clontech; 1 μg/well) in duplicate using Lipofectamine (Invitrogen).

    Techniques: Activity Assay, Plasmid Preparation, Luciferase, Expressing, Transfection, Construct, Mutagenesis

    slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p

    Journal: The Journal of Neuroscience

    Article Title: Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

    doi: 10.1523/JNEUROSCI.1001-13.2013

    Figure Lengend Snippet: slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p

    Article Snippet: S2 cells were transfected with the luciferase reporter constructs (2 μg/well) along with a vector expressing β-galactosidase (β-gal; pCMV–LacZ vector from Clontech; 1 μg/well) in duplicate using Lipofectamine (Invitrogen).

    Techniques: Activity Assay, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Construct

    HEK293 cells fail to support the interaction of zebrafish miR-430b with known mRNA targets. (A) Time course of miR-430b-mediated inhibition of luciferase expression using the reporter construct pGL3-3xPT, which carries three tandom copies of the perfect target sequence. pGL3-3xPT and miR-430b were cotransfected into the HEK293 cells and luciferase expression was measured at the times indicated post-transfection. (B) miR-430b-mediated inhibition of luciferase expression using reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, and pGl3-3′nanos that carry 3′UTRs containing known miR-430b target sequences. Luciferase expression was measured at 48 h post-transfection (hpt). pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively, and the transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar on the graph represents the average of four separate transfections.

    Journal: Zebrafish

    Article Title: A Zebrafish Cell Culture Assay for the Identification of MicroRNA Targets

    doi: 10.1089/zeb.2010.0674

    Figure Lengend Snippet: HEK293 cells fail to support the interaction of zebrafish miR-430b with known mRNA targets. (A) Time course of miR-430b-mediated inhibition of luciferase expression using the reporter construct pGL3-3xPT, which carries three tandom copies of the perfect target sequence. pGL3-3xPT and miR-430b were cotransfected into the HEK293 cells and luciferase expression was measured at the times indicated post-transfection. (B) miR-430b-mediated inhibition of luciferase expression using reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, and pGl3-3′nanos that carry 3′UTRs containing known miR-430b target sequences. Luciferase expression was measured at 48 h post-transfection (hpt). pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively, and the transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar on the graph represents the average of four separate transfections.

    Article Snippet: On the second day the medium was changed and transfection complex consisting of the Lipofectamine2000 reagent (2 μL), miRNA (50 nM), pCMV-lacZ (250 ng; Clontech), pGL3 plasmid (500 ng), and OPTI-MEMI (280 μL total volume; Gibco) was added to each well.

    Techniques: Inhibition, Luciferase, Expressing, Construct, Sequencing, Transfection

    ZSSJ cells support the interaction of zebrafish miR-430b and mRNA target sequences. ZSSJ cells were transfected with miR-430b or control miRNA along with one of three luciferase reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, or pGl3-3′nanos, that carry known 3′UTR target sequences for miR-430b. Luciferase expression was measured 48 hpt. pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively. The transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar represents the average of three separate transfections. *A significant difference ( p

    Journal: Zebrafish

    Article Title: A Zebrafish Cell Culture Assay for the Identification of MicroRNA Targets

    doi: 10.1089/zeb.2010.0674

    Figure Lengend Snippet: ZSSJ cells support the interaction of zebrafish miR-430b and mRNA target sequences. ZSSJ cells were transfected with miR-430b or control miRNA along with one of three luciferase reporter constructs, pGl3-3′zgc63829, pGl3-3′gstm, or pGl3-3′nanos, that carry known 3′UTR target sequences for miR-430b. Luciferase expression was measured 48 hpt. pGL3-3xPT and pGL-3 promoter were used as positive and negative controls, respectively. The transfection efficiency was normalized to β-galactosidase expression from pCMV-lacZ. Each bar represents the average of three separate transfections. *A significant difference ( p

    Article Snippet: On the second day the medium was changed and transfection complex consisting of the Lipofectamine2000 reagent (2 μL), miRNA (50 nM), pCMV-lacZ (250 ng; Clontech), pGL3 plasmid (500 ng), and OPTI-MEMI (280 μL total volume; Gibco) was added to each well.

    Techniques: Transfection, Luciferase, Construct, Expressing

    Net regulation of VEGF expression in cell culture. ( A ) Endogenous Net is phosphorylated by GPCR, Ras, and FGF-2 through MAP kinase cascades. NIH3T3 cells were transfected with expression vectors for KSHV-GPCR or Ras-V12, or induced with FGF-2. Cell extracts were analyzed by Western blotting for P-Net, activated P-ERK, and TBP (control for loading). ( B ) Down-regulation of Net inhibits VEGF expression induced by GPCR and Ras-V12. NIH3T3 cells were transfected with GPCR, Ras-V12, antisense net, and corresponding control vectors, as indicated. Transfected cells were kept in 0.5% FCS medium for 48 h after the wash. VEGF in this conditioned media was measured by ELISA. Results are averaged from four independent transfection experiments (corrected for cell numbers). ( C ) P-Net regulates VEGF promoter activity and stimulates endogenous VEGF expression. NIH3T3 cells were transfected with the mouse VEGF promoter luciferase reporter (-1217/+370), the pCMV LacZ internal control, and expression vectors for Net and Ras-V12. VEGF promoter activity relative to the transfections with empty vectors was determined (graph). Cell extracts were analyzed by Western blotting for the expression of P-Net, endogenous VEGF, and TBP (loading control; lower panels). ( D ) Coexpression of P-Net and VEGF in transfected cells. NIH3T3 cells were transfected with expression vectors for P-Net and Ras-V12. The cells were stained with antibodies against VEGF and P-Net, corresponding fluorescent secondary antibodies, and DAPI (nuclei), and examined by confocal microscopy. Transfected cells fluoresced brightly (white arrowheads), with VEGF (red) in the cytoplasm and P-Net (green) in the nucleus. Nontransfected cells gave clearly lower fluorescence signals (yellow arrows).

    Journal: Genes & Development

    Article Title: The transcription factor Net regulates the angiogenic switch

    doi: 10.1101/gad.272503

    Figure Lengend Snippet: Net regulation of VEGF expression in cell culture. ( A ) Endogenous Net is phosphorylated by GPCR, Ras, and FGF-2 through MAP kinase cascades. NIH3T3 cells were transfected with expression vectors for KSHV-GPCR or Ras-V12, or induced with FGF-2. Cell extracts were analyzed by Western blotting for P-Net, activated P-ERK, and TBP (control for loading). ( B ) Down-regulation of Net inhibits VEGF expression induced by GPCR and Ras-V12. NIH3T3 cells were transfected with GPCR, Ras-V12, antisense net, and corresponding control vectors, as indicated. Transfected cells were kept in 0.5% FCS medium for 48 h after the wash. VEGF in this conditioned media was measured by ELISA. Results are averaged from four independent transfection experiments (corrected for cell numbers). ( C ) P-Net regulates VEGF promoter activity and stimulates endogenous VEGF expression. NIH3T3 cells were transfected with the mouse VEGF promoter luciferase reporter (-1217/+370), the pCMV LacZ internal control, and expression vectors for Net and Ras-V12. VEGF promoter activity relative to the transfections with empty vectors was determined (graph). Cell extracts were analyzed by Western blotting for the expression of P-Net, endogenous VEGF, and TBP (loading control; lower panels). ( D ) Coexpression of P-Net and VEGF in transfected cells. NIH3T3 cells were transfected with expression vectors for P-Net and Ras-V12. The cells were stained with antibodies against VEGF and P-Net, corresponding fluorescent secondary antibodies, and DAPI (nuclei), and examined by confocal microscopy. Transfected cells fluoresced brightly (white arrowheads), with VEGF (red) in the cytoplasm and P-Net (green) in the nucleus. Nontransfected cells gave clearly lower fluorescence signals (yellow arrows).

    Article Snippet: We used pCEFL-KSHV-GPCR and control pCEFL ( ); p601D-antisense-net, pTL2-Net, pCMV LacZ ( ); pHa-Ras-V12 and control pΔRas ( ); pSG5-puromycin (IGBMC core facility); pEGFP-N1-antinet, Net cDNA in the 3′-5′ orientation between the Xho I and Bam HI sites of pEGFP-N1 (Clontech); TD-Net, [pTL2-Net (219-409) as described in Maira et al. ( )]; and mouse VEGF-Luc reporters (-1217/+370 and -449/+ 370; ).

    Techniques: Expressing, Cell Culture, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Luciferase, Staining, Confocal Microscopy, Fluorescence