etoposide vp 16  (Millipore)


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  • 99
    Name:
    Etoposide
    Description:
    Etoposide is synthesised from podophyllotoxins of plants
    Catalog Number:
    e1383
    Price:
    None
    Applications:
    Etoposide has been used:. to prepare drug stock solution in dimethyl sulfoxide (DMSO) and also to profile and compare the sensitivity of DT40 mutant cells. to incubate cells for cell viability assay . to treat neuro-2A cells to induce programmed cell death
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    Structured Review

    Millipore etoposide vp 16
    Etoposide
    Etoposide is synthesised from podophyllotoxins of plants
    https://www.bioz.com/result/etoposide vp 16/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    etoposide vp 16 - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2"

    Article Title: MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2

    Journal: Oncotarget

    doi:

    Silencing of SNAI2 phenocopies the effects of miR-203 re-expression on sensitization of U251AR cells to anticancer drugs and reversion of EMT (A) a. U251AR/shNC cells; b. U251AR/shSNAI2 cells. Light microscopy, 100× (a, b); Fluorescent microscopy, 100× (a, b). shSNAI2 and negative vector (shNC) were transfected into U251AR cells. At 48 h after transfection, fluorescent microscopy showed emission green fluorescence. (B) qRT-PCR validate the downregulation of SNAI2 after shRNA knockdown in U251AR cells. (C) Immunofluorescence analysis of the endogenous SNAI2 protein (red, left panels) in U251AR cells transfected with shSNAI2 or negative vector. Nuclei are stained in blue with DAPI. Scale bar, 20 μm. (D) The sensitivities of U251AR and U251AR/shSNAI2 to different concentrations of TMZ, imatinib and VP-16. (E) Morphology of U251AR cells transfected with negative vector or shSNAI2 vector. Scale bar, 100 μm. (F) SNAI2 knockdown reduces the invasion capacity of U251AR cells. Scale bar, 200 μm. (G) U251AR cell monolayer was transfected as indicated and scratched, then the migration of the cells towards the wound was visualised. Images were taken at various time points and Image J was used to determine the migration distance. (H) Western blotting show that silencing of SNAI2 can modulate the expression of EMT markers. VP-16, etoposide; TMZ, temozolomide. Data are presented as mean±s.d. of three independent experiments. * P
    Figure Legend Snippet: Silencing of SNAI2 phenocopies the effects of miR-203 re-expression on sensitization of U251AR cells to anticancer drugs and reversion of EMT (A) a. U251AR/shNC cells; b. U251AR/shSNAI2 cells. Light microscopy, 100× (a, b); Fluorescent microscopy, 100× (a, b). shSNAI2 and negative vector (shNC) were transfected into U251AR cells. At 48 h after transfection, fluorescent microscopy showed emission green fluorescence. (B) qRT-PCR validate the downregulation of SNAI2 after shRNA knockdown in U251AR cells. (C) Immunofluorescence analysis of the endogenous SNAI2 protein (red, left panels) in U251AR cells transfected with shSNAI2 or negative vector. Nuclei are stained in blue with DAPI. Scale bar, 20 μm. (D) The sensitivities of U251AR and U251AR/shSNAI2 to different concentrations of TMZ, imatinib and VP-16. (E) Morphology of U251AR cells transfected with negative vector or shSNAI2 vector. Scale bar, 100 μm. (F) SNAI2 knockdown reduces the invasion capacity of U251AR cells. Scale bar, 200 μm. (G) U251AR cell monolayer was transfected as indicated and scratched, then the migration of the cells towards the wound was visualised. Images were taken at various time points and Image J was used to determine the migration distance. (H) Western blotting show that silencing of SNAI2 can modulate the expression of EMT markers. VP-16, etoposide; TMZ, temozolomide. Data are presented as mean±s.d. of three independent experiments. * P

    Techniques Used: Expressing, Light Microscopy, Microscopy, Plasmid Preparation, Transfection, Fluorescence, Quantitative RT-PCR, shRNA, Immunofluorescence, Staining, Migration, Western Blot

    Re-expression of miR-203 in U251AR and U87AR cells sensitizes cells to anticancer drugs and reverses EMT while knockdown of miR-203 promotes resistance to anticancer drugs in U251 and U87 cells (A) qRT-PCR data validation of the downregulation of miR-203 in imatinib-resistant GBM cells compared with their parental cells, normalized to U6RNA, which was obtained from miRNA microarrays. (B, C) The sensitivities of U251AR and U87AR cells to imatinib, VP-16 and TMZ after transfected with miR-203 or miRNAs control. (D, E) Transfection with anti-miR-203 promotes resistance to imatinib, VP-16 and TMZ in U251 and U87 cells. (F) Morphology of U251AR and U87AR cells transfected with miRNA control or miR-203. Scale bar, 100 μm. (G) Western blotting show that re-expression of miR-203 modulates the expression of EMT markers. (H, I) U251AR and U87AR cells were transfected with miR-203 or anti-miR-203, and then collected for transwell invasion assay or wound healing assay. Shown were pictures of representative fields for each experiment. Scale bar, 200 μm. Data were expressed as mean±s.d. from three independent experiments. VP-16, etoposide; TMZ, temozolomide. * P
    Figure Legend Snippet: Re-expression of miR-203 in U251AR and U87AR cells sensitizes cells to anticancer drugs and reverses EMT while knockdown of miR-203 promotes resistance to anticancer drugs in U251 and U87 cells (A) qRT-PCR data validation of the downregulation of miR-203 in imatinib-resistant GBM cells compared with their parental cells, normalized to U6RNA, which was obtained from miRNA microarrays. (B, C) The sensitivities of U251AR and U87AR cells to imatinib, VP-16 and TMZ after transfected with miR-203 or miRNAs control. (D, E) Transfection with anti-miR-203 promotes resistance to imatinib, VP-16 and TMZ in U251 and U87 cells. (F) Morphology of U251AR and U87AR cells transfected with miRNA control or miR-203. Scale bar, 100 μm. (G) Western blotting show that re-expression of miR-203 modulates the expression of EMT markers. (H, I) U251AR and U87AR cells were transfected with miR-203 or anti-miR-203, and then collected for transwell invasion assay or wound healing assay. Shown were pictures of representative fields for each experiment. Scale bar, 200 μm. Data were expressed as mean±s.d. from three independent experiments. VP-16, etoposide; TMZ, temozolomide. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Transwell Invasion Assay, Wound Healing Assay

    SNAI2 contributes to chemoresistance and EMT in GBM cells (A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. * P
    Figure Legend Snippet: SNAI2 contributes to chemoresistance and EMT in GBM cells (A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. * P

    Techniques Used: Over Expression, Transfection, Expressing, Western Blot, Construct, Invasion Assay, Quantitative RT-PCR

    2) Product Images from "Inhibition of tumor angiogenesis by oral etoposide"

    Article Title: Inhibition of tumor angiogenesis by oral etoposide

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2010.127

    VEGF-induced vascular permeability is inhibited in mice treated with etoposide. (A and B) Evan's blue dye leakage in dorsal skin and ears after injection with VEGF or saline in control mice (n=10 mice/group) and etoposide (VP16)-treated mice (n=10 mice/group).
    Figure Legend Snippet: VEGF-induced vascular permeability is inhibited in mice treated with etoposide. (A and B) Evan's blue dye leakage in dorsal skin and ears after injection with VEGF or saline in control mice (n=10 mice/group) and etoposide (VP16)-treated mice (n=10 mice/group).

    Techniques Used: Permeability, Mouse Assay, Injection

    Etoposide has anti-angiogenic and anti-tumor cell effects in vitro . (A) The proliferation percentage of BCE cells was determined by comparing cells exposed to an angiogenic stimulus (FGF2) alone with those exposed to FGF2 and etoposide, relative to unstimulated
    Figure Legend Snippet: Etoposide has anti-angiogenic and anti-tumor cell effects in vitro . (A) The proliferation percentage of BCE cells was determined by comparing cells exposed to an angiogenic stimulus (FGF2) alone with those exposed to FGF2 and etoposide, relative to unstimulated

    Techniques Used: In Vitro

    Systemic therapy with etoposide (VP16) inhibits primary tumor growth and metastasis by inhibiting angiogenesis. After the tumors reached 100–150 mm 3 in size, etoposide treatment (40 or 80 mg/kg/day) was initiated (Day 0). On the last day of treatment,
    Figure Legend Snippet: Systemic therapy with etoposide (VP16) inhibits primary tumor growth and metastasis by inhibiting angiogenesis. After the tumors reached 100–150 mm 3 in size, etoposide treatment (40 or 80 mg/kg/day) was initiated (Day 0). On the last day of treatment,

    Techniques Used:

    Etoposide (VP16) exhibits synergistic anti-tumor activity with oral anti-tumor and anti-angiogenic inhibitors. After the LLC tumors reached 100–150 mm 3 in size, low-dose etoposide treatment (10 mg/kg/day) together with celecoxib (30 mg/kg/day)
    Figure Legend Snippet: Etoposide (VP16) exhibits synergistic anti-tumor activity with oral anti-tumor and anti-angiogenic inhibitors. After the LLC tumors reached 100–150 mm 3 in size, low-dose etoposide treatment (10 mg/kg/day) together with celecoxib (30 mg/kg/day)

    Techniques Used: Activity Assay

    Etoposide inhibits endothelial cell tube formation (HUVEC morphogenesis on Matrigel) and FGF2- and VEGF-induced corneal neovascularization. (A) Representative photomicrograph of control tubes documenting the formation of a network of tube-like structures
    Figure Legend Snippet: Etoposide inhibits endothelial cell tube formation (HUVEC morphogenesis on Matrigel) and FGF2- and VEGF-induced corneal neovascularization. (A) Representative photomicrograph of control tubes documenting the formation of a network of tube-like structures

    Techniques Used:

    Related Articles

    Staining:

    Article Title: The DRY Box and C-Terminal Domain of the Human Cytomegalovirus US27 Gene Product Play a Role in Promoting Cell Growth and Survival
    Article Snippet: .. Apoptosis Assays Cells were seeded into 6-well dishes and treated with 10 µM etoposide (Sigma-Aldrich) for 48 hours, then harvested and stained with Annexin V and propidium iodide using the TACS Annexin V-FITC Staining Kit (Trevigen, Gaithersburg, MD) before analysis by flow cytometry. .. In addition, cells were seeded into white 96 well plates at a density of 5×103 cells per well in the presence of 10 µM etoposide, and cell viability determined at indicated time points via the addition of CellTiter-Glo reagent as above.

    Flow Cytometry:

    Article Title: The DRY Box and C-Terminal Domain of the Human Cytomegalovirus US27 Gene Product Play a Role in Promoting Cell Growth and Survival
    Article Snippet: .. Apoptosis Assays Cells were seeded into 6-well dishes and treated with 10 µM etoposide (Sigma-Aldrich) for 48 hours, then harvested and stained with Annexin V and propidium iodide using the TACS Annexin V-FITC Staining Kit (Trevigen, Gaithersburg, MD) before analysis by flow cytometry. .. In addition, cells were seeded into white 96 well plates at a density of 5×103 cells per well in the presence of 10 µM etoposide, and cell viability determined at indicated time points via the addition of CellTiter-Glo reagent as above.

    Selection:

    Article Title: Low Dose Ionizing Radiation Strongly Stimulates Insertional Mutagenesis in a γH2AX Dependent Manner
    Article Snippet: .. For etoposide treatment, electroporated cells were seeded into 10 cm dishes containing a range of etoposide (Sigma E1383, stock solution 10 mM in DMSO) concentrations in 10 ml media; after seeding 5 µl and 50 µl aliquots were taken and plated in triplicate into 6-well plates containing 2 ml media with the same etoposide concentration; media was replaced next day and selection started in 10 cm dishes. ..

    Cytometry:

    Article Title: The DRY Box and C-Terminal Domain of the Human Cytomegalovirus US27 Gene Product Play a Role in Promoting Cell Growth and Survival
    Article Snippet: .. Apoptosis Assays Cells were seeded into 6-well dishes and treated with 10 µM etoposide (Sigma-Aldrich) for 48 hours, then harvested and stained with Annexin V and propidium iodide using the TACS Annexin V-FITC Staining Kit (Trevigen, Gaithersburg, MD) before analysis by flow cytometry. .. In addition, cells were seeded into white 96 well plates at a density of 5×103 cells per well in the presence of 10 µM etoposide, and cell viability determined at indicated time points via the addition of CellTiter-Glo reagent as above.

    Concentration Assay:

    Article Title: Low Dose Ionizing Radiation Strongly Stimulates Insertional Mutagenesis in a γH2AX Dependent Manner
    Article Snippet: .. For etoposide treatment, electroporated cells were seeded into 10 cm dishes containing a range of etoposide (Sigma E1383, stock solution 10 mM in DMSO) concentrations in 10 ml media; after seeding 5 µl and 50 µl aliquots were taken and plated in triplicate into 6-well plates containing 2 ml media with the same etoposide concentration; media was replaced next day and selection started in 10 cm dishes. ..

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    Millipore etoposide
    Role of Egr-1 in <t>etoposide-induced</t> BRCA1 expression. (A) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total RNA was isolated and Egr-1 mRNA expression was assessed by Northern blotting with 32 P-labeled Egr-1 cDNA. The same blot was re-probed with 32 P-labeled GAPDH cDNA as an internal control. (B) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total cell lysates were prepared and subjected to Western blot analysis with rabbit anti-Egr-1 antibody. The same blot was reprobed with anti-GAPDH antibody as an internal control. (C) HeLa cells were transiently co-transfected with 0.5 μg pBRCA1-Luc(–1066/+135) and an shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ), along with 50 ng of the pRL-null vector plasmid. After 24 h, the cells were left untreated or treated with 100 μM etoposide for 8 h, and the luciferase activity was measured. Egr-1 is indicated by an arrow. The knockdown of Egr-1 expression was verified by Western blot analysis ( upper panel ). Luciferase activity is shown as the mean ± SD of three independent experiments performed in triplicate (bottom graph). **P < 0.01. (D) HeLa cells were transiently transfected with 0.5 μg shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ). After 24 h, the cells were left untreated or treated with 100 μM etoposide for 3 h. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against Egr-1 and BRCA1. Egr-1 is indicated by an arrow. The same blot was reprobed with anti-GAPDH antibody as an internal control. The relative band intensities were measured by quantitative scanning densitometer ( bottom graph ).
    Etoposide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/etoposide/product/Millipore
    Average 99 stars, based on 483 article reviews
    Price from $9.99 to $1999.99
    etoposide - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

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    Role of Egr-1 in etoposide-induced BRCA1 expression. (A) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total RNA was isolated and Egr-1 mRNA expression was assessed by Northern blotting with 32 P-labeled Egr-1 cDNA. The same blot was re-probed with 32 P-labeled GAPDH cDNA as an internal control. (B) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total cell lysates were prepared and subjected to Western blot analysis with rabbit anti-Egr-1 antibody. The same blot was reprobed with anti-GAPDH antibody as an internal control. (C) HeLa cells were transiently co-transfected with 0.5 μg pBRCA1-Luc(–1066/+135) and an shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ), along with 50 ng of the pRL-null vector plasmid. After 24 h, the cells were left untreated or treated with 100 μM etoposide for 8 h, and the luciferase activity was measured. Egr-1 is indicated by an arrow. The knockdown of Egr-1 expression was verified by Western blot analysis ( upper panel ). Luciferase activity is shown as the mean ± SD of three independent experiments performed in triplicate (bottom graph). **P < 0.01. (D) HeLa cells were transiently transfected with 0.5 μg shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ). After 24 h, the cells were left untreated or treated with 100 μM etoposide for 3 h. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against Egr-1 and BRCA1. Egr-1 is indicated by an arrow. The same blot was reprobed with anti-GAPDH antibody as an internal control. The relative band intensities were measured by quantitative scanning densitometer ( bottom graph ).

    Journal: BMB Reports

    Article Title: Egr-1 regulates the transcription of the BRCA1 gene by etoposide

    doi: 10.5483/BMBRep.2013.46.2.202

    Figure Lengend Snippet: Role of Egr-1 in etoposide-induced BRCA1 expression. (A) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total RNA was isolated and Egr-1 mRNA expression was assessed by Northern blotting with 32 P-labeled Egr-1 cDNA. The same blot was re-probed with 32 P-labeled GAPDH cDNA as an internal control. (B) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total cell lysates were prepared and subjected to Western blot analysis with rabbit anti-Egr-1 antibody. The same blot was reprobed with anti-GAPDH antibody as an internal control. (C) HeLa cells were transiently co-transfected with 0.5 μg pBRCA1-Luc(–1066/+135) and an shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ), along with 50 ng of the pRL-null vector plasmid. After 24 h, the cells were left untreated or treated with 100 μM etoposide for 8 h, and the luciferase activity was measured. Egr-1 is indicated by an arrow. The knockdown of Egr-1 expression was verified by Western blot analysis ( upper panel ). Luciferase activity is shown as the mean ± SD of three independent experiments performed in triplicate (bottom graph). **P < 0.01. (D) HeLa cells were transiently transfected with 0.5 μg shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ). After 24 h, the cells were left untreated or treated with 100 μM etoposide for 3 h. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against Egr-1 and BRCA1. Egr-1 is indicated by an arrow. The same blot was reprobed with anti-GAPDH antibody as an internal control. The relative band intensities were measured by quantitative scanning densitometer ( bottom graph ).

    Article Snippet: Etoposide was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Expressing, Isolation, Northern Blot, Labeling, Western Blot, Transfection, shRNA, Plasmid Preparation, Luciferase, Activity Assay

    Egr-1 transactivates the BRCA1 promoter through direct binding to the EBS. (A) HeLa cells were co-transfected with 0.5 μg wild-type (WT) p BRCA1 -Luc(–1066/+135) and different concentrations of the empty vector (pcDNA3.1zeo) or Egr-1 expression plasmid (pcDNA3.1zeo/Egr1), as indicated. After 48 h, the cells were collected and analyzed for luciferase activity. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. (B and C) Purified recombinant Egr-1 protein (B) and nuclear extracts from HeLa cells treated with 100 μM etoposide for 1 h (C) were incubated with 32 P-labeled oligodeoxynucleotide probes that contain EBS. For competition, unlabeled oligodeoxynucleotides (Competitor) were added at 10-fold or 100-fold excess. Arrowheads indicate DNA-Egr-1 complexes.

    Journal: BMB Reports

    Article Title: Egr-1 regulates the transcription of the BRCA1 gene by etoposide

    doi: 10.5483/BMBRep.2013.46.2.202

    Figure Lengend Snippet: Egr-1 transactivates the BRCA1 promoter through direct binding to the EBS. (A) HeLa cells were co-transfected with 0.5 μg wild-type (WT) p BRCA1 -Luc(–1066/+135) and different concentrations of the empty vector (pcDNA3.1zeo) or Egr-1 expression plasmid (pcDNA3.1zeo/Egr1), as indicated. After 48 h, the cells were collected and analyzed for luciferase activity. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. (B and C) Purified recombinant Egr-1 protein (B) and nuclear extracts from HeLa cells treated with 100 μM etoposide for 1 h (C) were incubated with 32 P-labeled oligodeoxynucleotide probes that contain EBS. For competition, unlabeled oligodeoxynucleotides (Competitor) were added at 10-fold or 100-fold excess. Arrowheads indicate DNA-Egr-1 complexes.

    Article Snippet: Etoposide was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Binding Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Purification, Recombinant, Incubation, Labeling

    Effect of etoposide on the induction of BRCA1 expression. (A) HeLa cells were treated with 100 μM etoposide for different time periods. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against BRCA1, p53 and p21. The ∼220-kDa full length and ∼90-kDa fragment of BRCA1 are indicated by an arrow and an arrowhead, respectively. The same blot was reprobed with anti-GAPDH antibody as an internal control. The blots shown are representative of the results obtained from three independent experiments. (B) Total RNA was isolated and the levels of BRCA1 mRNA were measured by QRT-PCR. Relative levels are normalized to the level of gapdh mRNA. The data shown represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, compared with the untreated control cells. (C) HeLa cells grown in 12-well plates were transfected with 0.5 μg of the BRCA1 promoter reporter plasmid, p BRCA1 -Luc(–1066/+135), along with 50 ng of the pRL-null vector. After 24 h, the cells were either untreated or treated with 50 μM or 100 μM etoposide for 8 h. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. *P < 0.05; **P < 0.01, compared with the untreated control cells.

    Journal: BMB Reports

    Article Title: Egr-1 regulates the transcription of the BRCA1 gene by etoposide

    doi: 10.5483/BMBRep.2013.46.2.202

    Figure Lengend Snippet: Effect of etoposide on the induction of BRCA1 expression. (A) HeLa cells were treated with 100 μM etoposide for different time periods. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against BRCA1, p53 and p21. The ∼220-kDa full length and ∼90-kDa fragment of BRCA1 are indicated by an arrow and an arrowhead, respectively. The same blot was reprobed with anti-GAPDH antibody as an internal control. The blots shown are representative of the results obtained from three independent experiments. (B) Total RNA was isolated and the levels of BRCA1 mRNA were measured by QRT-PCR. Relative levels are normalized to the level of gapdh mRNA. The data shown represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, compared with the untreated control cells. (C) HeLa cells grown in 12-well plates were transfected with 0.5 μg of the BRCA1 promoter reporter plasmid, p BRCA1 -Luc(–1066/+135), along with 50 ng of the pRL-null vector. After 24 h, the cells were either untreated or treated with 50 μM or 100 μM etoposide for 8 h. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. *P < 0.05; **P < 0.01, compared with the untreated control cells.

    Article Snippet: Etoposide was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Expressing, Western Blot, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay

    The M 3 -muscarinic-receptor-mediated anti-apoptotic response and transcriptional activation are both attenuated by actinomycin D treatment ( A ) CHO-M 3 cells seeded on to 6-well plates were serum-starved for 2 h before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for the indicated times. [ 3 H]UTP incorporation was determined by scintillation counting. Data shown is the agonist-induced incorporation of [ 3 H]UTP subtracted from that observed in the absence of agonist, and represent the means±S.E.M. ( n =3). ( B ) CHO-M 3 cells seeded on 6-well plates were serum-starved for 2 h and then incubated with or without actinomycin D (100 ng/ml) for 30 min before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for 30 min. [ 3 H]UTP incorporation was determined by scintillation counting. Results are means±S.E.M. ( n =3). ( C ) CHO-M 3 cells seeded on to 10 cm 2 plates were pre-incubated with or without actinomycin D (100 ng/ml) for 30 min. Cells were then treated with carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h and cell lysates processed for caspase activity. Caspase activity of 100% is given as the increase in caspase activity in the presence of etoposide. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: The M 3 -muscarinic-receptor-mediated anti-apoptotic response and transcriptional activation are both attenuated by actinomycin D treatment ( A ) CHO-M 3 cells seeded on to 6-well plates were serum-starved for 2 h before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for the indicated times. [ 3 H]UTP incorporation was determined by scintillation counting. Data shown is the agonist-induced incorporation of [ 3 H]UTP subtracted from that observed in the absence of agonist, and represent the means±S.E.M. ( n =3). ( B ) CHO-M 3 cells seeded on 6-well plates were serum-starved for 2 h and then incubated with or without actinomycin D (100 ng/ml) for 30 min before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for 30 min. [ 3 H]UTP incorporation was determined by scintillation counting. Results are means±S.E.M. ( n =3). ( C ) CHO-M 3 cells seeded on to 10 cm 2 plates were pre-incubated with or without actinomycin D (100 ng/ml) for 30 min. Cells were then treated with carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h and cell lysates processed for caspase activity. Caspase activity of 100% is given as the increase in caspase activity in the presence of etoposide. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Activation Assay, Incubation, Activity Assay

    Decreased mRNA levels of Bcl-2 following etoposide treatment could not be inhibited by muscarinic receptor stimulation in CHO-M 3 cells CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with vehicle or etoposide (250 μM) for 16 h in the absence or presence of carbachol (1 mM). Total RNA was then extracted and reverse transcribed. RT-PCR using primers specific for Bcl-2 and the internal control β-actin was performed using SYBR® Green mastermix. Levels of the Bcl-2 transcript were then normalized to β-actin and expressed as a percentage of the control vehicle-treated cells. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: Decreased mRNA levels of Bcl-2 following etoposide treatment could not be inhibited by muscarinic receptor stimulation in CHO-M 3 cells CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with vehicle or etoposide (250 μM) for 16 h in the absence or presence of carbachol (1 mM). Total RNA was then extracted and reverse transcribed. RT-PCR using primers specific for Bcl-2 and the internal control β-actin was performed using SYBR® Green mastermix. Levels of the Bcl-2 transcript were then normalized to β-actin and expressed as a percentage of the control vehicle-treated cells. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay

    The decrease in the expression levels of the anti-apoptotic Bcl-2 protein following incubation of cells with etoposide can be inhibited by activation of the M 3 -muscarinic receptor CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with or without carbachol (CCh; 1mM) either for 2 min before a 16-h treatment with etoposide (250 μM) or incubated with etoposide and carbachol for 16 h. In the case of cells treated with carbachol for 2 min, the carbachol stimulation was stopped by the addition of the muscarinic receptor antagonist atropine (0.5 μM) followed by three washes in α-MEM before being incubated with etoposide for 16 h. Cell lysates were then prepared and separated by SDS/PAGE (12% gels; 100 μg of protein/well). The gel was then probed for Bcl-2 immunoreactivity. The nitrocellulose was then stripped and re-probed with ERK antisera in order to check for equal lane loading (right-hand panels). Gels are representative of three separate experiments.

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: The decrease in the expression levels of the anti-apoptotic Bcl-2 protein following incubation of cells with etoposide can be inhibited by activation of the M 3 -muscarinic receptor CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with or without carbachol (CCh; 1mM) either for 2 min before a 16-h treatment with etoposide (250 μM) or incubated with etoposide and carbachol for 16 h. In the case of cells treated with carbachol for 2 min, the carbachol stimulation was stopped by the addition of the muscarinic receptor antagonist atropine (0.5 μM) followed by three washes in α-MEM before being incubated with etoposide for 16 h. Cell lysates were then prepared and separated by SDS/PAGE (12% gels; 100 μg of protein/well). The gel was then probed for Bcl-2 immunoreactivity. The nitrocellulose was then stripped and re-probed with ERK antisera in order to check for equal lane loading (right-hand panels). Gels are representative of three separate experiments.

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Expressing, Incubation, Activation Assay, SDS Page

    The M 3 -muscarinic receptor anti-apoptotic response is independent of extracellular calcium CHO-M 3 cells were seeded on 10 cm 2 plates. Cells were incubated in Krebs buffer (+calcium) or in Krebs buffer lacking calcium and supplemented with 3 mM EDTA (−calcium) for 3 min. CHO-M 3 cells were treated with carbachol (CCh; 1 mM) for 5 min and cells were washed three times with α-MEM. CHO-M 3 cells were then incubated for 16 h with etoposide (250 μM) and cell lysates processed for caspase activity. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: The M 3 -muscarinic receptor anti-apoptotic response is independent of extracellular calcium CHO-M 3 cells were seeded on 10 cm 2 plates. Cells were incubated in Krebs buffer (+calcium) or in Krebs buffer lacking calcium and supplemented with 3 mM EDTA (−calcium) for 3 min. CHO-M 3 cells were treated with carbachol (CCh; 1 mM) for 5 min and cells were washed three times with α-MEM. CHO-M 3 cells were then incubated for 16 h with etoposide (250 μM) and cell lysates processed for caspase activity. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Incubation, Activity Assay

    Both M 3 -muscarinic and vasopressin V 1 A receptors increase inositol phosphate production, but only the M 3 -muscarinic receptor mediates an anti-apoptotic response ( A ) CHO-M 3 and CHO-V 1 A cells plated on 24-well plates were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) for 25 min. The reactions were terminated with 1 M trichloroacetic acid and inositol phosphate production was determined as described in the Materials and methods section. ( B ) CHO-M 3 and CHO-V 1 A cells were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) and etoposide (250 μM). Cells were incubated for 16 h at 37 °C. Floating and attached cells were harvested and pooled, and cell lysates were processed for caspase activity as described in the Materials and methods section. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: Both M 3 -muscarinic and vasopressin V 1 A receptors increase inositol phosphate production, but only the M 3 -muscarinic receptor mediates an anti-apoptotic response ( A ) CHO-M 3 and CHO-V 1 A cells plated on 24-well plates were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) for 25 min. The reactions were terminated with 1 M trichloroacetic acid and inositol phosphate production was determined as described in the Materials and methods section. ( B ) CHO-M 3 and CHO-V 1 A cells were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) and etoposide (250 μM). Cells were incubated for 16 h at 37 °C. Floating and attached cells were harvested and pooled, and cell lysates were processed for caspase activity as described in the Materials and methods section. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Incubation, Activity Assay

    Time-course of etoposide-induced decrease in Bcl-2 levels and caspase activation CHO-M 3 cells seeded on 10 cm 2 plates were incubated with etoposide (250 μM) for the times indicated. Cell lysates were prepared and run on a SDS/PAGE (12% gels) and Western blotted for Bcl-2. Data shown is representative of three separate experiments (upper panel). In parallel experiments, a time course for caspase activity was determined following etoposide (Et; 250 μM) treatment for the indicated times. Results for caspase activation are means±S.E.M. ( n =3) (lower panel).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: Time-course of etoposide-induced decrease in Bcl-2 levels and caspase activation CHO-M 3 cells seeded on 10 cm 2 plates were incubated with etoposide (250 μM) for the times indicated. Cell lysates were prepared and run on a SDS/PAGE (12% gels) and Western blotted for Bcl-2. Data shown is representative of three separate experiments (upper panel). In parallel experiments, a time course for caspase activity was determined following etoposide (Et; 250 μM) treatment for the indicated times. Results for caspase activation are means±S.E.M. ( n =3) (lower panel).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Activation Assay, Incubation, SDS Page, Western Blot, Activity Assay

    Attenuation of etoposide-mediated caspase activation by M 3 -muscarinic receptors in CHO-M 3 and SHSY-5Y cells ( A ) CHO-M 2 and CHO-M 3 cells seeded on 10 cm 2 plates were treated with vehicle (Control), carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h, and cell lysates were processed for caspase activity. ( B ) SHSY-5Y neuroblastoma cells seeded on 10 cm 2 plates were treated with vehicle, carbachol (CCh; 1 mM), etoposide (25 μM) and/or atropine (0.5 μM) for 16 h, and cell lysates were processed for caspase assays. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: Attenuation of etoposide-mediated caspase activation by M 3 -muscarinic receptors in CHO-M 3 and SHSY-5Y cells ( A ) CHO-M 2 and CHO-M 3 cells seeded on 10 cm 2 plates were treated with vehicle (Control), carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h, and cell lysates were processed for caspase activity. ( B ) SHSY-5Y neuroblastoma cells seeded on 10 cm 2 plates were treated with vehicle, carbachol (CCh; 1 mM), etoposide (25 μM) and/or atropine (0.5 μM) for 16 h, and cell lysates were processed for caspase assays. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Activation Assay, Activity Assay

    SB203580 (SB) or SP600125 (SP) inhibit the formation of capillary-like structures and SB203580 cotreatment reduces migration and invasion of etoposide-treated cells. ( a ) Formation of capillary-like structures. Representative micrographs of the complete network of tubes formed by untreated (Ctr), treated (with etoposide, LY2940042, SB203580 or SP600125 alone) and cotreated cells (etoposide plus inhibitors). The negative control is obtained by cell exposure to 10 μ M sulforaphane. Original magnification × 10. The graph reports the number of branches of the tube network formed by cells under the treatment conditions as described above. Quantitative data are the means±S.D. of three independent experiments. °° P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: SB203580 (SB) or SP600125 (SP) inhibit the formation of capillary-like structures and SB203580 cotreatment reduces migration and invasion of etoposide-treated cells. ( a ) Formation of capillary-like structures. Representative micrographs of the complete network of tubes formed by untreated (Ctr), treated (with etoposide, LY2940042, SB203580 or SP600125 alone) and cotreated cells (etoposide plus inhibitors). The negative control is obtained by cell exposure to 10 μ M sulforaphane. Original magnification × 10. The graph reports the number of branches of the tube network formed by cells under the treatment conditions as described above. Quantitative data are the means±S.D. of three independent experiments. °° P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Migration, Negative Control

    SB203580 (SB) reduces COX-2, ICAM-1, CXCR4 levels and MMP9 activity in etoposide-treated cells. Immunoblot analyses of COX-2 ( a ), ICAM-1 ( b ) and CXCR4 ( c ). The histograms summarize quantitative data of means±S.D. of three independent experiments. °° P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: SB203580 (SB) reduces COX-2, ICAM-1, CXCR4 levels and MMP9 activity in etoposide-treated cells. Immunoblot analyses of COX-2 ( a ), ICAM-1 ( b ) and CXCR4 ( c ). The histograms summarize quantitative data of means±S.D. of three independent experiments. °° P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Activity Assay

    Etoposide activates p38MAPK, Akt and JNK. ( a ) Protein levels of PKC δ and α in cells treated with etoposide (1.25–100 μ M). Immunoblots shown are representative of three independent experiments. β -Actin is the internal loading control. ( b ) p38MAPK, JNK and Akt activation. Histograms summarize quantitative data of means±S.D. of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: Etoposide activates p38MAPK, Akt and JNK. ( a ) Protein levels of PKC δ and α in cells treated with etoposide (1.25–100 μ M). Immunoblots shown are representative of three independent experiments. β -Actin is the internal loading control. ( b ) p38MAPK, JNK and Akt activation. Histograms summarize quantitative data of means±S.D. of three independent experiments. * P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Western Blot, Activation Assay

    SB203580 cotreatment markedly reduces migration, invasion and MMP9 activity of etoposide-treated cells. ( a ) Formation of capillary-like structures. Representative micrographs of the complete network of tubes in untreated (Ctr) and treated cells. Original magnification × 10. ( b ) Immunoblot analysis of VEGF. The histogram summarizes quantitative data of means±S.D. of three independent experiments °° P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: SB203580 cotreatment markedly reduces migration, invasion and MMP9 activity of etoposide-treated cells. ( a ) Formation of capillary-like structures. Representative micrographs of the complete network of tubes in untreated (Ctr) and treated cells. Original magnification × 10. ( b ) Immunoblot analysis of VEGF. The histogram summarizes quantitative data of means±S.D. of three independent experiments °° P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Migration, Activity Assay

    Effects of SB203580 (SB) cotreatment on cell viability, clonogenicity and formation of NBSs. ( a ) Left panel, cell viability. Cells were pre-treated for 1 h with the different inhibitors (0.1 μ M chelerythrine chloride (Chele), 500 nM LY2940042 (LY), 50 μ M PD98059 (PD), 10 μ M SB203580 or 4 μ M SP600125 (SP)) and then exposed to 1.25 μ M etoposide for an additional 24 h. Histogram summarizes quantitative data of means±S.D. of five independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: Effects of SB203580 (SB) cotreatment on cell viability, clonogenicity and formation of NBSs. ( a ) Left panel, cell viability. Cells were pre-treated for 1 h with the different inhibitors (0.1 μ M chelerythrine chloride (Chele), 500 nM LY2940042 (LY), 50 μ M PD98059 (PD), 10 μ M SB203580 or 4 μ M SP600125 (SP)) and then exposed to 1.25 μ M etoposide for an additional 24 h. Histogram summarizes quantitative data of means±S.D. of five independent experiments. * P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques:

    Etoposide decreases cell viability and, at high drug concentrations, inhibits the tumorigenic potential of HTLA-230 NB cells and prevents NBS formation. ( a ) Cell viability was determined by MTT assays in cells exposed to increasing concentrations of etoposide (0.07–225 μ M) for 24 h. Histograms summarize quantitative data of means±S.D. of five independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: Etoposide decreases cell viability and, at high drug concentrations, inhibits the tumorigenic potential of HTLA-230 NB cells and prevents NBS formation. ( a ) Cell viability was determined by MTT assays in cells exposed to increasing concentrations of etoposide (0.07–225 μ M) for 24 h. Histograms summarize quantitative data of means±S.D. of five independent experiments. * P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: MTT Assay

    Effects of SB203580 (SB) cotreatment on viability, clonogenicity, CC133/Oct4 expression and p38MAPK activation in SK-N-SH and IMR-32 cells. ( a ) Cell viability. SK-N-SH (left panel) and IMR-32 (right panel) cells were exposed to increasing concentrations of etoposide (0.07–225 μ M) for 24 h. Histograms summarize quantitative data of means±S.D. of five independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: Effects of SB203580 (SB) cotreatment on viability, clonogenicity, CC133/Oct4 expression and p38MAPK activation in SK-N-SH and IMR-32 cells. ( a ) Cell viability. SK-N-SH (left panel) and IMR-32 (right panel) cells were exposed to increasing concentrations of etoposide (0.07–225 μ M) for 24 h. Histograms summarize quantitative data of means±S.D. of five independent experiments. * P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Expressing, Activation Assay

    ( A ) Western blot analysis of HO-1 protein expression after HO-1 siRNA addition to CoPP-treated macrophages. RAW 264.7 macrophages were transfected with two different HO-1 siRNA sequences (1 and 2) and nonspecific control siRNA before CoPP treatment. Lanes 1 and 2 represent HO-1 siRNA sequences 1 and 2, respectively. Note: HO-1 siRNA sequence 2 inhibited CoPP-induced HO-1 protein induction, whereas sequence 1 and nonspecific siRNA had no effect on HO-1 expression. ( B ) Western blot analysis of HO-1 and caspase-3 gene products in Ad-HO-1-transfected YPEN-1 cells. The expression of HO-1 and caspase-3 was probed with rabbit anti-mouse HO-1 ( a ) and caspase-3 ( b ) antibodies. Lane 1, YPEN-1 cells alone; lane 2, YPEN-1 cells plus etoposide (50 μ M ); lane 3, YPEN-1 cells transfected with Ad-HO-1 and HO-1 siRNA plus etoposide (50 μ M ); lane 4, YPEN-1 cells transfected with Ad-HO-1 and nonspecific siRNA plus etoposide (50 μ M ); lane 5, YPEN-1 cells transfected with Ad-HO-1; lane 6, YPEN-1 cells transfected with Ad-β-gal. Note the selectively inhibited expression of HO-1 in HO-1 siRNA-treated YPEN-1 cells (lane 3a), as compared with nonspecific siRNA and Ad-HO-1 (lanes 4a and 5a). In contrast, the expression of caspase-3 increased in cells treated with 50 μ M etoposide (lane 2b) or after HO-1 siRNA (lane 3b) or Ad-β-gal (lane 6b), as compared with nonspecific siRNA (lane 4b) or Ad-HO-1 (lane 5b). Anti-β-actin antibody was used to ensure equal protein amounts between the samples. Data shown are representative of three separate experiments.

    Journal: Human Gene Therapy

    Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

    doi: 10.1089/hum.2009.049

    Figure Lengend Snippet: ( A ) Western blot analysis of HO-1 protein expression after HO-1 siRNA addition to CoPP-treated macrophages. RAW 264.7 macrophages were transfected with two different HO-1 siRNA sequences (1 and 2) and nonspecific control siRNA before CoPP treatment. Lanes 1 and 2 represent HO-1 siRNA sequences 1 and 2, respectively. Note: HO-1 siRNA sequence 2 inhibited CoPP-induced HO-1 protein induction, whereas sequence 1 and nonspecific siRNA had no effect on HO-1 expression. ( B ) Western blot analysis of HO-1 and caspase-3 gene products in Ad-HO-1-transfected YPEN-1 cells. The expression of HO-1 and caspase-3 was probed with rabbit anti-mouse HO-1 ( a ) and caspase-3 ( b ) antibodies. Lane 1, YPEN-1 cells alone; lane 2, YPEN-1 cells plus etoposide (50 μ M ); lane 3, YPEN-1 cells transfected with Ad-HO-1 and HO-1 siRNA plus etoposide (50 μ M ); lane 4, YPEN-1 cells transfected with Ad-HO-1 and nonspecific siRNA plus etoposide (50 μ M ); lane 5, YPEN-1 cells transfected with Ad-HO-1; lane 6, YPEN-1 cells transfected with Ad-β-gal. Note the selectively inhibited expression of HO-1 in HO-1 siRNA-treated YPEN-1 cells (lane 3a), as compared with nonspecific siRNA and Ad-HO-1 (lanes 4a and 5a). In contrast, the expression of caspase-3 increased in cells treated with 50 μ M etoposide (lane 2b) or after HO-1 siRNA (lane 3b) or Ad-β-gal (lane 6b), as compared with nonspecific siRNA (lane 4b) or Ad-HO-1 (lane 5b). Anti-β-actin antibody was used to ensure equal protein amounts between the samples. Data shown are representative of three separate experiments.

    Article Snippet: After washing, cells were treated with 50 μ M etoposide (Calbiochem, San Diego, CA) in YPEN-1 cultures for 2 hr or cells were treated with cobalt protoporphyrin (CoPP, an HO-1 inducer, 10 μg/ml; Porphyrin Products, Logan, UT).

    Techniques: Western Blot, Expressing, Transfection, Sequencing