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ATCC vegf promoter reporter plasmid

FIGURE 1. Effects of BRCA1 and HIF-1 on hypoxia-induced <t>VEGF</t> promoter activity. A, effect of exogenous BRCA1 and HIF-1 on VEGF reporter activity. Exponentially pro- liferating cells (MCF-7) were transfected with various combinations of pVEGF-kpnI-Luc (125 ng), HIF-1 (125 ng), and either of two doses of a BRCA1 expression vector (125 ng or 250 ng), incubated for 24 h and then exposed to hypoxia (0.1%, O2) for 6 h, harvested, and assayed for luciferase activity as described under “Experimental Procedures.” Trans- fection of HIF-1 alone into MCF-7 cells induced statistically significant pVEGF-kpnI-Luc activity under hypoxic condition (p 0.005 for comparisons of cells transfected with or without the HIF-1 expression vector in 0.1% O2) and co-transfections with both the BRCA1 and HIF-1 expression vectors further enhanced this expression (p 0.005 for comparisons between cells transfected with and without the BRCA1 expression vector, in the presence of exogenous HIF-1). B, role of the HRE in BRCA1-enhanced, HIF-1- induced VEGF promoter activity during hypoxia. To determine if the ability of BRCA1 to affectVEGFpromoteractivityduringhypoxiamightrequireinteractionbetweentheHRE and HIF-1, cells were transfected with either p11wt-Luc, p11mt-Luc, or empty vector (125 ng) plus HIF-1 and BRCA1 expression vectors (125 ng of each) as indicated in the figure, incubated for 24 h, and then exposed to hypoxia (0.1%, O2) for 6 h before meas- uring luciferase activities. As in A, BRCA1 significantly enhanced HIF-1-induced Luc activity under hypoxic conditions when the VEGF promoter contained a wild-type HRE (p 0.005 for comparisons of cells transfected with and without the BRCA1 expression vector). In contrast, neither HIF-1 alone nor HIF-1 plus BRCA1 induced statistically significant increased amounts of reporter activity in cells transfected with the HRE mutant reporter plasmid (p11mt-Luc).

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1) Product Images from "BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression"

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

Journal: Journal of Biological Chemistry

doi: 10.1074/jbc.m513033200

FIGURE 1. Effects of BRCA1 and HIF-1 on hypoxia-induced VEGF promoter activity. A, effect of exogenous BRCA1 and HIF-1 on VEGF reporter activity. Exponentially pro- liferating cells (MCF-7) were transfected with various combinations of pVEGF-kpnI-Luc (125 ng), HIF-1 (125 ng), and either of two doses of a BRCA1 expression vector (125 ng or 250 ng), incubated for 24 h and then exposed to hypoxia (0.1%, O2) for 6 h, harvested, and assayed for luciferase activity as described under “Experimental Procedures.” Trans- fection of HIF-1 alone into MCF-7 cells induced statistically significant pVEGF-kpnI-Luc activity under hypoxic condition (p 0.005 for comparisons of cells transfected with or without the HIF-1 expression vector in 0.1% O2) and co-transfections with both the BRCA1 and HIF-1 expression vectors further enhanced this expression (p 0.005 for comparisons between cells transfected with and without the BRCA1 expression vector, in the presence of exogenous HIF-1). B, role of the HRE in BRCA1-enhanced, HIF-1- induced VEGF promoter activity during hypoxia. To determine if the ability of BRCA1 to affectVEGFpromoteractivityduringhypoxiamightrequireinteractionbetweentheHRE and HIF-1, cells were transfected with either p11wt-Luc, p11mt-Luc, or empty vector (125 ng) plus HIF-1 and BRCA1 expression vectors (125 ng of each) as indicated in the figure, incubated for 24 h, and then exposed to hypoxia (0.1%, O2) for 6 h before meas- uring luciferase activities. As in A, BRCA1 significantly enhanced HIF-1-induced Luc activity under hypoxic conditions when the VEGF promoter contained a wild-type HRE (p 0.005 for comparisons of cells transfected with and without the BRCA1 expression vector). In contrast, neither HIF-1 alone nor HIF-1 plus BRCA1 induced statistically significant increased amounts of reporter activity in cells transfected with the HRE mutant reporter plasmid (p11mt-Luc).

Figure Legend Snippet: FIGURE 1. Effects of BRCA1 and HIF-1 on hypoxia-induced VEGF promoter activity. A, effect of exogenous BRCA1 and HIF-1 on VEGF reporter activity. Exponentially pro- liferating cells (MCF-7) were transfected with various combinations of pVEGF-kpnI-Luc (125 ng), HIF-1 (125 ng), and either of two doses of a BRCA1 expression vector (125 ng or 250 ng), incubated for 24 h and then exposed to hypoxia (0.1%, O2) for 6 h, harvested, and assayed for luciferase activity as described under “Experimental Procedures.” Trans- fection of HIF-1 alone into MCF-7 cells induced statistically significant pVEGF-kpnI-Luc activity under hypoxic condition (p 0.005 for comparisons of cells transfected with or without the HIF-1 expression vector in 0.1% O2) and co-transfections with both the BRCA1 and HIF-1 expression vectors further enhanced this expression (p 0.005 for comparisons between cells transfected with and without the BRCA1 expression vector, in the presence of exogenous HIF-1). B, role of the HRE in BRCA1-enhanced, HIF-1- induced VEGF promoter activity during hypoxia. To determine if the ability of BRCA1 to affectVEGFpromoteractivityduringhypoxiamightrequireinteractionbetweentheHRE and HIF-1, cells were transfected with either p11wt-Luc, p11mt-Luc, or empty vector (125 ng) plus HIF-1 and BRCA1 expression vectors (125 ng of each) as indicated in the figure, incubated for 24 h, and then exposed to hypoxia (0.1%, O2) for 6 h before meas- uring luciferase activities. As in A, BRCA1 significantly enhanced HIF-1-induced Luc activity under hypoxic conditions when the VEGF promoter contained a wild-type HRE (p 0.005 for comparisons of cells transfected with and without the BRCA1 expression vector). In contrast, neither HIF-1 alone nor HIF-1 plus BRCA1 induced statistically significant increased amounts of reporter activity in cells transfected with the HRE mutant reporter plasmid (p11mt-Luc).

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

FIGURE 2. Effects of BRCA1 on the endogenous levels of hypoxia-induced VEGF121 and VEGF165 mRNA. A, agarose gel analysis. Exponentially proliferating HEK293T cells were transiently transfected with empty vector (pCDNA3) or the BRCA1 expression vec- tor,incubatedfor24handthenexposedtohypoxia(0.1%,O2)for6hwhentotalRNAwas extracted used for semiquantitative RT-PCR. The gel image presented is representative of three independent experiments. B, quantitative analysis of agarose gel densitometry data. Each PCR band on photographs of the three agarose gels was quantitated using densitometry, and means S.E. of these values were calculated. BRCA1 overexpression significantly enhanced hypoxia-induced VEGF121 and VEGF165 mRNA expression (p 0.005 for comparisons of cells transfected with or without BRCA1 in 0.1% O2). C, confir- mation of PCR results. Real time PCR were performed in quadruplicate in three inde- pendent experiments to confirm the findings shown in A and B. -Actin was used as the loading control for semiquantitative RT-PCR and to normalize the quantitative real time PCR results. The results were normalized to the control (reporter only) in 21% O2 and are presented as bar graphs showing the means S.E. The effect of overexpressing BRCA1 on hypoxia-induced VEGF expression is statistically significant for both VEGF mRNAs (p 0.005).

Figure Legend Snippet: FIGURE 2. Effects of BRCA1 on the endogenous levels of hypoxia-induced VEGF121 and VEGF165 mRNA. A, agarose gel analysis. Exponentially proliferating HEK293T cells were transiently transfected with empty vector (pCDNA3) or the BRCA1 expression vec- tor,incubatedfor24handthenexposedtohypoxia(0.1%,O2)for6hwhentotalRNAwas extracted used for semiquantitative RT-PCR. The gel image presented is representative of three independent experiments. B, quantitative analysis of agarose gel densitometry data. Each PCR band on photographs of the three agarose gels was quantitated using densitometry, and means S.E. of these values were calculated. BRCA1 overexpression significantly enhanced hypoxia-induced VEGF121 and VEGF165 mRNA expression (p 0.005 for comparisons of cells transfected with or without BRCA1 in 0.1% O2). C, confir- mation of PCR results. Real time PCR were performed in quadruplicate in three inde- pendent experiments to confirm the findings shown in A and B. -Actin was used as the loading control for semiquantitative RT-PCR and to normalize the quantitative real time PCR results. The results were normalized to the control (reporter only) in 21% O2 and are presented as bar graphs showing the means S.E. The effect of overexpressing BRCA1 on hypoxia-induced VEGF expression is statistically significant for both VEGF mRNAs (p 0.005).

Techniques Used: Agarose Gel Electrophoresis, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Real-time Polymerase Chain Reaction, Control

FIGURE 3. The effect of hypoxia on ChIP assays for genomic VEGF promoter DNA. A, agarose gel analysis of PCR products. Exponentially proliferating cells exposed to hypoxia for the times indicated were harvested for ChIP assays as described under “Experimental Procedures.” (Protein-DNA cross-linked samples were immunoprecipi- tated with the indicated antibodies or control IgG, processed, and used as templates for PCR reactions to measure relative amounts of the HRE element-containing region of the genomicVEGFpromoterDNAthathadbeenimmunoprecipitated.)B,densitometertrac- ings of the results in A. The amount of each PCR product band was quantitated by densitometry for three independent experiments, and means S.E. were calculated. Statistically significant increases (p 0.005) of VEGF promoter DNA were observed for chromatinimmunoprecipitatedwiththeBRCA1,ARNT,andHIF-1antibodiesinhypoxic versus normoxic conditions. C, negative controls: as in A except that the primers were for genomic VEGF promoter sequences lacking known HRE sites.

Figure Legend Snippet: FIGURE 3. The effect of hypoxia on ChIP assays for genomic VEGF promoter DNA. A, agarose gel analysis of PCR products. Exponentially proliferating cells exposed to hypoxia for the times indicated were harvested for ChIP assays as described under “Experimental Procedures.” (Protein-DNA cross-linked samples were immunoprecipi- tated with the indicated antibodies or control IgG, processed, and used as templates for PCR reactions to measure relative amounts of the HRE element-containing region of the genomicVEGFpromoterDNAthathadbeenimmunoprecipitated.)B,densitometertrac- ings of the results in A. The amount of each PCR product band was quantitated by densitometry for three independent experiments, and means S.E. were calculated. Statistically significant increases (p 0.005) of VEGF promoter DNA were observed for chromatinimmunoprecipitatedwiththeBRCA1,ARNT,andHIF-1antibodiesinhypoxic versus normoxic conditions. C, negative controls: as in A except that the primers were for genomic VEGF promoter sequences lacking known HRE sites.

Techniques Used: Agarose Gel Electrophoresis, Control

FIGURE 4. Effect of BRCA1 mutants on hypoxia- stimulated VEGF promoter activity. A, genetic maps. Diagrams of wtBRCA1 and mutant BRCA1 (5382insC, C5365G, 5677insA, and T300G) expres- sion vector constructs. B, effect of BRCA mutants on reporter activity. MCF-7 cells were co-trans- fected with pVEGF-kpnI-Luc, HIF-1, and either wtBRCA1 or one the mutant BRCA1s as indicated. 24 h later, cells were treated with or without 0.1% hypoxic gas for 6 h. pCMV--gal and pCDNA3 vec- tors were included as controls for data normaliza- tion. The data are presented as bar graphs of the means S.E. of quadruplicate wells of three inde- pendent experiments. C, Western blot of the rela- tive wild-type and mutant BRCA1 protein levels in the cells analyzed in B.

Figure Legend Snippet: FIGURE 4. Effect of BRCA1 mutants on hypoxia- stimulated VEGF promoter activity. A, genetic maps. Diagrams of wtBRCA1 and mutant BRCA1 (5382insC, C5365G, 5677insA, and T300G) expres- sion vector constructs. B, effect of BRCA mutants on reporter activity. MCF-7 cells were co-trans- fected with pVEGF-kpnI-Luc, HIF-1, and either wtBRCA1 or one the mutant BRCA1s as indicated. 24 h later, cells were treated with or without 0.1% hypoxic gas for 6 h. pCMV--gal and pCDNA3 vec- tors were included as controls for data normaliza- tion. The data are presented as bar graphs of the means S.E. of quadruplicate wells of three inde- pendent experiments. C, Western blot of the rela- tive wild-type and mutant BRCA1 protein levels in the cells analyzed in B.

Techniques Used: Activity Assay, Mutagenesis, Plasmid Preparation, Construct, Western Blot

FIGURE 6. The effect of endogenous BRCA1 levels on hypoxia-induced VEGF pro- moter activity and VEGF secretion. A, effect of BRCA1 siRNAs on VEGF promoter activ- ity. HEK293T cells were transiently transfected with control-siRNA, BRCA1-siRNA-1 or BRCA1-siRNA-3 for 48 h when the remaining siRNA was removed. The cells were then immediately co-transfected with the pVEGF-kpnI-Luc reporter construct and the same siRNAs,(freshlyprepared),incubatedfor24h,andthenexposedtohypoxiafor6hbefore luciferase activity was measured. The activity levels, relative to the control luciferase activity (control-siRNA in normoxia), are presented as bar graphs of the means S.E. of four wells from each of three independent experiments. The effects of both BRCA1 siR- NAs on hypoxia-induced VEGF-Luc reporter activity was statistically significant (p 0.005). B, effect of BRCA1 siRNAs on hypoxia-induced secretion of endogenous VEGF. VEGF secretion into the medium was measured with a VEGF ELISA kit (see “Experimental Procedures”). Cells (T47D) transiently transfected with control-siRNA, BRCA1-siRNA-1, or BRCA1-siRNA-3 for 48 h were exposed to hypoxia or normoxia for an additional 16 h (without removing the siRNAs) when the amount of VEGF secreted into the medium was measured. The values presented as bar graphs give the means S.E. of quadruplicate wells from three independent experiments. The effects of both BRCA1 siRNAs on the secretion of endogenous VEGF protein are statistically significant under hypoxic condi- tions (p 0.005) but not under normoxic conditions.

Figure Legend Snippet: FIGURE 6. The effect of endogenous BRCA1 levels on hypoxia-induced VEGF pro- moter activity and VEGF secretion. A, effect of BRCA1 siRNAs on VEGF promoter activ- ity. HEK293T cells were transiently transfected with control-siRNA, BRCA1-siRNA-1 or BRCA1-siRNA-3 for 48 h when the remaining siRNA was removed. The cells were then immediately co-transfected with the pVEGF-kpnI-Luc reporter construct and the same siRNAs,(freshlyprepared),incubatedfor24h,andthenexposedtohypoxiafor6hbefore luciferase activity was measured. The activity levels, relative to the control luciferase activity (control-siRNA in normoxia), are presented as bar graphs of the means S.E. of four wells from each of three independent experiments. The effects of both BRCA1 siR- NAs on hypoxia-induced VEGF-Luc reporter activity was statistically significant (p 0.005). B, effect of BRCA1 siRNAs on hypoxia-induced secretion of endogenous VEGF. VEGF secretion into the medium was measured with a VEGF ELISA kit (see “Experimental Procedures”). Cells (T47D) transiently transfected with control-siRNA, BRCA1-siRNA-1, or BRCA1-siRNA-3 for 48 h were exposed to hypoxia or normoxia for an additional 16 h (without removing the siRNAs) when the amount of VEGF secreted into the medium was measured. The values presented as bar graphs give the means S.E. of quadruplicate wells from three independent experiments. The effects of both BRCA1 siRNAs on the secretion of endogenous VEGF protein are statistically significant under hypoxic condi- tions (p 0.005) but not under normoxic conditions.

Techniques Used: Activity Assay, Transfection, Control, Construct, Luciferase, Enzyme-linked Immunosorbent Assay

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Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 1. Effects of BRCA1 and HIF-1 on hypoxia-induced VEGF promoter activity. A, effect of exogenous BRCA1 and HIF-1 on VEGF reporter activity. Exponentially pro- liferating cells (MCF-7) were transfected with various combinations of pVEGF-kpnI-Luc (125 ng), HIF-1 (125 ng), and either of two doses of a BRCA1 expression vector (125 ng or 250 ng), incubated for 24 h and then exposed to hypoxia (0.1%, O2) for 6 h, harvested, and assayed for luciferase activity as described under “Experimental Procedures.” Trans- fection of HIF-1 alone into MCF-7 cells induced statistically significant pVEGF-kpnI-Luc activity under hypoxic condition (p 0.005 for comparisons of cells transfected with or without the HIF-1 expression vector in 0.1% O2) and co-transfections with both the BRCA1 and HIF-1 expression vectors further enhanced this expression (p 0.005 for comparisons between cells transfected with and without the BRCA1 expression vector, in the presence of exogenous HIF-1). B, role of the HRE in BRCA1-enhanced, HIF-1- induced VEGF promoter activity during hypoxia. To determine if the ability of BRCA1 to affectVEGFpromoteractivityduringhypoxiamightrequireinteractionbetweentheHRE and HIF-1, cells were transfected with either p11wt-Luc, p11mt-Luc, or empty vector (125 ng) plus HIF-1 and BRCA1 expression vectors (125 ng of each) as indicated in the figure, incubated for 24 h, and then exposed to hypoxia (0.1%, O2) for 6 h before meas- uring luciferase activities. As in A, BRCA1 significantly enhanced HIF-1-induced Luc activity under hypoxic conditions when the VEGF promoter contained a wild-type HRE (p 0.005 for comparisons of cells transfected with and without the BRCA1 expression vector). In contrast, neither HIF-1 alone nor HIF-1 plus BRCA1 induced statistically significant increased amounts of reporter activity in cells transfected with the HRE mutant reporter plasmid (p11mt-Luc).

Article Snippet: Transient Transfection of DNA and Luciferase Assays—The HIF-1 expression vector (pCEP4-HIF-1 ), the VEGF promoter-reporter plasmid (pVEGF-kpnI-Luc containing the 2.6-kbVEGFpromoter in pGL2), p11wt (the VEGF promoter containing the wild-type HRE in the pGL2 vector), and p11mt (the VEGF promoter containing a mutated HRE in the pGL2 vector) were purchased from ATCC.

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

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 2. Effects of BRCA1 on the endogenous levels of hypoxia-induced VEGF121 and VEGF165 mRNA. A, agarose gel analysis. Exponentially proliferating HEK293T cells were transiently transfected with empty vector (pCDNA3) or the BRCA1 expression vec- tor,incubatedfor24handthenexposedtohypoxia(0.1%,O2)for6hwhentotalRNAwas extracted used for semiquantitative RT-PCR. The gel image presented is representative of three independent experiments. B, quantitative analysis of agarose gel densitometry data. Each PCR band on photographs of the three agarose gels was quantitated using densitometry, and means S.E. of these values were calculated. BRCA1 overexpression significantly enhanced hypoxia-induced VEGF121 and VEGF165 mRNA expression (p 0.005 for comparisons of cells transfected with or without BRCA1 in 0.1% O2). C, confir- mation of PCR results. Real time PCR were performed in quadruplicate in three inde- pendent experiments to confirm the findings shown in A and B. -Actin was used as the loading control for semiquantitative RT-PCR and to normalize the quantitative real time PCR results. The results were normalized to the control (reporter only) in 21% O2 and are presented as bar graphs showing the means S.E. The effect of overexpressing BRCA1 on hypoxia-induced VEGF expression is statistically significant for both VEGF mRNAs (p 0.005).

Techniques: Agarose Gel Electrophoresis, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Real-time Polymerase Chain Reaction, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 3. The effect of hypoxia on ChIP assays for genomic VEGF promoter DNA. A, agarose gel analysis of PCR products. Exponentially proliferating cells exposed to hypoxia for the times indicated were harvested for ChIP assays as described under “Experimental Procedures.” (Protein-DNA cross-linked samples were immunoprecipi- tated with the indicated antibodies or control IgG, processed, and used as templates for PCR reactions to measure relative amounts of the HRE element-containing region of the genomicVEGFpromoterDNAthathadbeenimmunoprecipitated.)B,densitometertrac- ings of the results in A. The amount of each PCR product band was quantitated by densitometry for three independent experiments, and means S.E. were calculated. Statistically significant increases (p 0.005) of VEGF promoter DNA were observed for chromatinimmunoprecipitatedwiththeBRCA1,ARNT,andHIF-1antibodiesinhypoxic versus normoxic conditions. C, negative controls: as in A except that the primers were for genomic VEGF promoter sequences lacking known HRE sites.

Techniques: Agarose Gel Electrophoresis, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 4. Effect of BRCA1 mutants on hypoxia- stimulated VEGF promoter activity. A, genetic maps. Diagrams of wtBRCA1 and mutant BRCA1 (5382insC, C5365G, 5677insA, and T300G) expres- sion vector constructs. B, effect of BRCA mutants on reporter activity. MCF-7 cells were co-trans- fected with pVEGF-kpnI-Luc, HIF-1, and either wtBRCA1 or one the mutant BRCA1s as indicated. 24 h later, cells were treated with or without 0.1% hypoxic gas for 6 h. pCMV--gal and pCDNA3 vec- tors were included as controls for data normaliza- tion. The data are presented as bar graphs of the means S.E. of quadruplicate wells of three inde- pendent experiments. C, Western blot of the rela- tive wild-type and mutant BRCA1 protein levels in the cells analyzed in B.

Techniques: Activity Assay, Mutagenesis, Plasmid Preparation, Construct, Western Blot

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 6. The effect of endogenous BRCA1 levels on hypoxia-induced VEGF pro- moter activity and VEGF secretion. A, effect of BRCA1 siRNAs on VEGF promoter activ- ity. HEK293T cells were transiently transfected with control-siRNA, BRCA1-siRNA-1 or BRCA1-siRNA-3 for 48 h when the remaining siRNA was removed. The cells were then immediately co-transfected with the pVEGF-kpnI-Luc reporter construct and the same siRNAs,(freshlyprepared),incubatedfor24h,andthenexposedtohypoxiafor6hbefore luciferase activity was measured. The activity levels, relative to the control luciferase activity (control-siRNA in normoxia), are presented as bar graphs of the means S.E. of four wells from each of three independent experiments. The effects of both BRCA1 siR- NAs on hypoxia-induced VEGF-Luc reporter activity was statistically significant (p 0.005). B, effect of BRCA1 siRNAs on hypoxia-induced secretion of endogenous VEGF. VEGF secretion into the medium was measured with a VEGF ELISA kit (see “Experimental Procedures”). Cells (T47D) transiently transfected with control-siRNA, BRCA1-siRNA-1, or BRCA1-siRNA-3 for 48 h were exposed to hypoxia or normoxia for an additional 16 h (without removing the siRNAs) when the amount of VEGF secreted into the medium was measured. The values presented as bar graphs give the means S.E. of quadruplicate wells from three independent experiments. The effects of both BRCA1 siRNAs on the secretion of endogenous VEGF protein are statistically significant under hypoxic condi- tions (p 0.005) but not under normoxic conditions.

Techniques: Activity Assay, Transfection, Control, Construct, Luciferase, Enzyme-linked Immunosorbent Assay

FIGURE 5 Araliadiol enhances hair growth-promoting signals and anagen phase markers in human hair follicle cells. (A) HHFSCs were treated with different concentrations of araliadiol (0–1.2 μg/mL) for 48 h. Protein levels of hair follicle stem cell-speciﬁc markers (K15 and K19), anagen phase markers (CD34 and p63α), and hair growth-promoting proteins (VEGF and ANGPTL4) were analyzed by Western blotting, with β-Actin serving as a loading control. (B) HDPCs were stimulated with indicated concentrations of araliadiol (0–2.5 μg/mL) for 48 h. Protein levels of hair growth-promoting proteins (FGF2, FGF7, FGF10, VEGF, IGF-1, and NOG) were analyzed by Western blotting, with β-Actin serving as a loading control. The protein levels in all blots were quantiﬁed using ImageJ software version 1.53t. K15, cytokeratin 15; K19, cytokeratin 19; CD34, cluster of differentiation 34; p63α, tumor protein 63 alpha; VEGF, vascular endothelial growth factor; ANGPTL4, angiopoietin like 4; FGF2, ﬁbroblast growth factor 2; FGF7, ﬁbroblast growth factor 7; FGF10, ﬁbroblast growth factor 10; IGF-1, insulin-like growth factor 1; NOG, noggin.

Journal: Frontiers in pharmacology

Article Title: In vitro hair growth-promoting effects of araliadiol via the p38/PPAR-γ signaling pathway in human hair follicle stem cells and dermal papilla cells.

doi: 10.3389/fphar.2024.1482898

Figure Lengend Snippet: FIGURE 5 Araliadiol enhances hair growth-promoting signals and anagen phase markers in human hair follicle cells. (A) HHFSCs were treated with different concentrations of araliadiol (0–1.2 μg/mL) for 48 h. Protein levels of hair follicle stem cell-speciﬁc markers (K15 and K19), anagen phase markers (CD34 and p63α), and hair growth-promoting proteins (VEGF and ANGPTL4) were analyzed by Western blotting, with β-Actin serving as a loading control. (B) HDPCs were stimulated with indicated concentrations of araliadiol (0–2.5 μg/mL) for 48 h. Protein levels of hair growth-promoting proteins (FGF2, FGF7, FGF10, VEGF, IGF-1, and NOG) were analyzed by Western blotting, with β-Actin serving as a loading control. The protein levels in all blots were quantiﬁed using ImageJ software version 1.53t. K15, cytokeratin 15; K19, cytokeratin 19; CD34, cluster of differentiation 34; p63α, tumor protein 63 alpha; VEGF, vascular endothelial growth factor; ANGPTL4, angiopoietin like 4; FGF2, ﬁbroblast growth factor 2; FGF7, ﬁbroblast growth factor 7; FGF10, ﬁbroblast growth factor 10; IGF-1, insulin-like growth factor 1; NOG, noggin.

Article Snippet: The pSV-βgalactosidase plasmid (#E1081) was sourced from Promega, whereas the Gli response element-driven luciferase reporter plasmid (#113712), TCF/LEF response element-driven luciferase reporter plasmid (#12456), and VEGF promoter-driven luciferase reporter plasmid (#66128) were acquired from Addgene (MA, United States).

Techniques: Western Blot, Control, Software

FIGURE 8 Araliadiol elevates hair growth-promoting effects in human hair follicle cells by upregulating the PPAR-γ signaling pathway. (A) Protein levels of PPAR-γ and hair growth-inductive proteins (K15, VEGF, and ANGPTL4) were assessed after treatment with araliadiol (1.2 μg/mL) with or without GW9662 (20 μM) in HHFSCs for 48 h. (B) Protein levels of PPAR-γ and hair growth-inductive proteins (FGF7, VEGF, and NOG) were assessed after treatment with araliadiol (2.5 μg/mL) with or without GW9662 (20 μM) in HDPCs for 48 h. Protein levels in all blots were quantiﬁed using ImageJ software version 1.53t.

Journal: Frontiers in pharmacology

Article Title: In vitro hair growth-promoting effects of araliadiol via the p38/PPAR-γ signaling pathway in human hair follicle stem cells and dermal papilla cells.

doi: 10.3389/fphar.2024.1482898

Figure Lengend Snippet: FIGURE 8 Araliadiol elevates hair growth-promoting effects in human hair follicle cells by upregulating the PPAR-γ signaling pathway. (A) Protein levels of PPAR-γ and hair growth-inductive proteins (K15, VEGF, and ANGPTL4) were assessed after treatment with araliadiol (1.2 μg/mL) with or without GW9662 (20 μM) in HHFSCs for 48 h. (B) Protein levels of PPAR-γ and hair growth-inductive proteins (FGF7, VEGF, and NOG) were assessed after treatment with araliadiol (2.5 μg/mL) with or without GW9662 (20 μM) in HDPCs for 48 h. Protein levels in all blots were quantiﬁed using ImageJ software version 1.53t.

Techniques: Software

FIGURE 10 Araliadiol enhances VEGF secretion via the p38/PPAR-γ signaling pathway in human hair follicle cells. (A, B) Human hair follicle cells were treated with araliadiol (0–2.5 μg/mL) for 48 h, either with or without SB202190 (20 μM) or GW9662 (20 μM). VEGF-A secretion levels were quantiﬁed using an ELISA on conditioned medium from the treated cells. Minoxidil (2 μg/mL) was included as a positive control. Results are presented as the mean ± SD of three independent experiments and analyzed using a one-way analysis of variance followed by Tukey’s test. ***p < 0.001 compared with the vehicle-treated group. ##p < 0.01; ###p < 0.001 compared with the araliadiol-treated group. ELISA, enzyme-linked immunosorbent assay.

Journal: Frontiers in pharmacology

Article Title: In vitro hair growth-promoting effects of araliadiol via the p38/PPAR-γ signaling pathway in human hair follicle stem cells and dermal papilla cells.

doi: 10.3389/fphar.2024.1482898

Figure Lengend Snippet: FIGURE 10 Araliadiol enhances VEGF secretion via the p38/PPAR-γ signaling pathway in human hair follicle cells. (A, B) Human hair follicle cells were treated with araliadiol (0–2.5 μg/mL) for 48 h, either with or without SB202190 (20 μM) or GW9662 (20 μM). VEGF-A secretion levels were quantiﬁed using an ELISA on conditioned medium from the treated cells. Minoxidil (2 μg/mL) was included as a positive control. Results are presented as the mean ± SD of three independent experiments and analyzed using a one-way analysis of variance followed by Tukey’s test. ***p < 0.001 compared with the vehicle-treated group. ##p < 0.01; ###p < 0.001 compared with the araliadiol-treated group. ELISA, enzyme-linked immunosorbent assay.

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control

Double-stranded RNA specific for the VEGF and HIVSF2-LTR promoters and genes encoding proteins that are regulators of VEGF transcription

Journal: RNA

Article Title: Intended transcriptional silencing with siRNA results in gene repression through sequence-specific off-targeting

doi: 10.1261/rna.1808510

Figure Lengend Snippet: Double-stranded RNA specific for the VEGF and HIVSF2-LTR promoters and genes encoding proteins that are regulators of VEGF transcription

Article Snippet: Reporter plasmid constructs containing either the human VEGF and HIV SF2 -LTR promoters, and a Tat-expression plasmid were based on the pGL3 and pGL4 series (Promega) expressing a firefly luciferase gene.

Techniques:

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