short hairpin rna shrna plasmid Search Results


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  • 99
    Millipore plasmids short hairpin rnas shrna
    Silencing of C/EBPα prevents Elk-1 and PGN induction of HO-1. A : mouse macrophages (RAW 264.7) were stably transfected with short hairpin <t>RNA</t> <t>(shRNA)</t> plasmids for C/EBPα (2 separate transfections) or vector control. Protein was extracted,
    Plasmids Short Hairpin Rnas Shrna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Genechem cpeb3 short hairpin rna shrna plasmid
    The antitumor effect of lidocaine was reversed by <t>CPEB3</t> knockdown. (a) HepG2 tumor cells were transfected with E. coli <t>TOP10-EGFP-shRNA-CPEB3.</t> Images of the same field were taken to show both the bright-field image (cell morphology) and the GFP fluorescence (100x magnification). (b) qPCR was used to quantify CPEB3 mRNA expression in the control group (normal tumor cells), negative control (scramble shRNA), and shRNA-CPEB3 group. (c, d) HepG2 cell survival rate in shRNA-CPEB3 group after lidocaine treatment compared with control group and negative control group. Compared with control group, ∗ indicates P
    Cpeb3 Short Hairpin Rna Shrna Plasmid, supplied by Genechem, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    OriGene rassf1a short hairpin rna shrna plasmids
    The antitumor effect of lidocaine was reversed by <t>CPEB3</t> knockdown. (a) HepG2 tumor cells were transfected with E. coli <t>TOP10-EGFP-shRNA-CPEB3.</t> Images of the same field were taken to show both the bright-field image (cell morphology) and the GFP fluorescence (100x magnification). (b) qPCR was used to quantify CPEB3 mRNA expression in the control group (normal tumor cells), negative control (scramble shRNA), and shRNA-CPEB3 group. (c, d) HepG2 cell survival rate in shRNA-CPEB3 group after lidocaine treatment compared with control group and negative control group. Compared with control group, ∗ indicates P
    Rassf1a Short Hairpin Rna Shrna Plasmids, supplied by OriGene, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher control short hairpin rna shrna plasmids
    The antitumor effect of lidocaine was reversed by <t>CPEB3</t> knockdown. (a) HepG2 tumor cells were transfected with E. coli <t>TOP10-EGFP-shRNA-CPEB3.</t> Images of the same field were taken to show both the bright-field image (cell morphology) and the GFP fluorescence (100x magnification). (b) qPCR was used to quantify CPEB3 mRNA expression in the control group (normal tumor cells), negative control (scramble shRNA), and shRNA-CPEB3 group. (c, d) HepG2 cell survival rate in shRNA-CPEB3 group after lidocaine treatment compared with control group and negative control group. Compared with control group, ∗ indicates P
    Control Short Hairpin Rna Shrna Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    SABiosciences control short hairpin rna shrna plasmids
    The antitumor effect of lidocaine was reversed by <t>CPEB3</t> knockdown. (a) HepG2 tumor cells were transfected with E. coli <t>TOP10-EGFP-shRNA-CPEB3.</t> Images of the same field were taken to show both the bright-field image (cell morphology) and the GFP fluorescence (100x magnification). (b) qPCR was used to quantify CPEB3 mRNA expression in the control group (normal tumor cells), negative control (scramble shRNA), and shRNA-CPEB3 group. (c, d) HepG2 cell survival rate in shRNA-CPEB3 group after lidocaine treatment compared with control group and negative control group. Compared with control group, ∗ indicates P
    Control Short Hairpin Rna Shrna Plasmids, supplied by SABiosciences, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology ddb2 short hairpin rna shrna plasmids
    CRL4 <t>DDB2</t> ubiquitylates HBO1 in response to UV damage. (A) Depletion of CUL4A/B in HEK293 cells suppressed ubiquitylation of HBO1. CUL1, CUL2, and CUL4A/B were targeted by <t>siRNA</t> transfection for 48 h. Cell lysates were subjected to Western blotting with the indicated antibodies. (B) Depletion of CUL4A/B protected HBO1 phosphorylated at Ser50 and Ser53 from degradation after UV irradiation. CUL4A/B or negative-control siRNA was transfected into HEK293 cells. After 48 h of transfection, cells were exposed to 40 J/m 2 UV light. Cells were incubated for 6 h, and Western blotting was performed with the indicated antibodies. β-Actin was used for normalization of HBO1 expression. The graph indicates the average HBO1 expression levels for three independent experiments. Error bars indicate standard errors (**, P
    Ddb2 Short Hairpin Rna Shrna Plasmids, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology dysf short hairpin rna shrna plasmid
    CRL4 <t>DDB2</t> ubiquitylates HBO1 in response to UV damage. (A) Depletion of CUL4A/B in HEK293 cells suppressed ubiquitylation of HBO1. CUL1, CUL2, and CUL4A/B were targeted by <t>siRNA</t> transfection for 48 h. Cell lysates were subjected to Western blotting with the indicated antibodies. (B) Depletion of CUL4A/B protected HBO1 phosphorylated at Ser50 and Ser53 from degradation after UV irradiation. CUL4A/B or negative-control siRNA was transfected into HEK293 cells. After 48 h of transfection, cells were exposed to 40 J/m 2 UV light. Cells were incubated for 6 h, and Western blotting was performed with the indicated antibodies. β-Actin was used for normalization of HBO1 expression. The graph indicates the average HBO1 expression levels for three independent experiments. Error bars indicate standard errors (**, P
    Dysf Short Hairpin Rna Shrna Plasmid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology bcl 2 short hairpin rna shrna plasmid
    Regulation of expression of Bax and <t>Bcl‐2</t> proteins in SK‐N‐DZ and IMR‐32 cells by KLF4. Treatments (24 h): control (CTL) cells, 100 nM Bcl‐2 <t>shRNA,</t> 100 nM KLF4 vector, and 100 nM
    Bcl 2 Short Hairpin Rna Shrna Plasmid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology padi2 short hairpin rna shrna plasmid
    Regulation of expression of Bax and <t>Bcl‐2</t> proteins in SK‐N‐DZ and IMR‐32 cells by KLF4. Treatments (24 h): control (CTL) cells, 100 nM Bcl‐2 <t>shRNA,</t> 100 nM KLF4 vector, and 100 nM
    Padi2 Short Hairpin Rna Shrna Plasmid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Silencing of C/EBPα prevents Elk-1 and PGN induction of HO-1. A : mouse macrophages (RAW 264.7) were stably transfected with short hairpin RNA (shRNA) plasmids for C/EBPα (2 separate transfections) or vector control. Protein was extracted,

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Regulation of heme oxygenase-1 gene by peptidoglycan involves the interaction of Elk-1 and C/EBP? to increase expression

    doi: 10.1152/ajplung.00382.2009

    Figure Lengend Snippet: Silencing of C/EBPα prevents Elk-1 and PGN induction of HO-1. A : mouse macrophages (RAW 264.7) were stably transfected with short hairpin RNA (shRNA) plasmids for C/EBPα (2 separate transfections) or vector control. Protein was extracted,

    Article Snippet: Short hairpin RNA (shRNA) plasmids for C/EBPα (Clone .1-898s1c1; Sigma-Aldrich) and vector control (pLKO.1-puro) were transfected into RAW 264.7 cells, and stable clones were selected using puromycin (5 μg/ml) resistance.

    Techniques: Stable Transfection, Transfection, shRNA, Plasmid Preparation

    Inhibition of mitochondrial translation phenocopies synthetic lethality resulting from POLRMT inhibition A. HCT116 cells were infected with lentiviral MYC shRNA and/or TFB1M shRNA or Luciferase (Luc) shRNA. Five days post-infection cells were harvested and stained with Annexin V and 7-AAD. A representative flow cytometry analysis (left) and quantification of percent Annexin V positive cells (right) is shown. Error bars represent SEM, n = 5. * p

    Journal: Oncotarget

    Article Title: Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

    doi: 10.18632/oncotarget.11718

    Figure Lengend Snippet: Inhibition of mitochondrial translation phenocopies synthetic lethality resulting from POLRMT inhibition A. HCT116 cells were infected with lentiviral MYC shRNA and/or TFB1M shRNA or Luciferase (Luc) shRNA. Five days post-infection cells were harvested and stained with Annexin V and 7-AAD. A representative flow cytometry analysis (left) and quantification of percent Annexin V positive cells (right) is shown. Error bars represent SEM, n = 5. * p

    Article Snippet: Lentiviral short hairpin RNA (shRNA) plasmids corresponding to MYC, POLRMT, TFB1M, SHMT2 and control luciferase shRNAs were obtained from TRC (Sigma).

    Techniques: Inhibition, Infection, shRNA, Luciferase, Staining, Flow Cytometry, Cytometry

    Mitochondrial gene expression protects MYC-overexpressing cells from toxic levels of ROS A. U2OS MYC/ER cells were infected with lentiviral POLRMT shRNA or Luciferase (Luc) shRNA and treated with NAC. MYC activity was induced via 4-OHT treatment (MYC On). Three days post-treatment cells were harvested and whole cell lysates were analyzed by Western blot for the indicated proteins (left). Relative mRNA expression was measured by qRT-PCR (right). Error bars represent SD, n = 3. B. U2OS MYC/ER cells were treated with tigecycline (Tig) and/or NAC. MYC activity was induced via 4-OHT treatment. Three days post-treatment cells were harvested and whole cell lysates were analyzed by Western blot for the indicated proteins. C. U2OS MYC/ER cells were treated with chloramphenicol (CAM). MYC activity was induced via 4-OHT treatment. Whole cell lysates were analyzed by Western blot using an antibody cocktail for subunits of electron transport chain (ETC) complexes, as indicated (I-IV). D. U2OS MYC/ER cells were infected with lentiviral POLRMT shRNA or Luciferase (Luc) shRNA and treated with MnTBAP. MYC activity was induced via 4-OHT treatment. Three days post-treatment cells were harvested. Whole cell lysates were analyzed by Western blot for the indicated proteins. Casp-3, caspase-3; FL, full length; CL, cleaved; UT, untreated.

    Journal: Oncotarget

    Article Title: Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

    doi: 10.18632/oncotarget.11718

    Figure Lengend Snippet: Mitochondrial gene expression protects MYC-overexpressing cells from toxic levels of ROS A. U2OS MYC/ER cells were infected with lentiviral POLRMT shRNA or Luciferase (Luc) shRNA and treated with NAC. MYC activity was induced via 4-OHT treatment (MYC On). Three days post-treatment cells were harvested and whole cell lysates were analyzed by Western blot for the indicated proteins (left). Relative mRNA expression was measured by qRT-PCR (right). Error bars represent SD, n = 3. B. U2OS MYC/ER cells were treated with tigecycline (Tig) and/or NAC. MYC activity was induced via 4-OHT treatment. Three days post-treatment cells were harvested and whole cell lysates were analyzed by Western blot for the indicated proteins. C. U2OS MYC/ER cells were treated with chloramphenicol (CAM). MYC activity was induced via 4-OHT treatment. Whole cell lysates were analyzed by Western blot using an antibody cocktail for subunits of electron transport chain (ETC) complexes, as indicated (I-IV). D. U2OS MYC/ER cells were infected with lentiviral POLRMT shRNA or Luciferase (Luc) shRNA and treated with MnTBAP. MYC activity was induced via 4-OHT treatment. Three days post-treatment cells were harvested. Whole cell lysates were analyzed by Western blot for the indicated proteins. Casp-3, caspase-3; FL, full length; CL, cleaved; UT, untreated.

    Article Snippet: Lentiviral short hairpin RNA (shRNA) plasmids corresponding to MYC, POLRMT, TFB1M, SHMT2 and control luciferase shRNAs were obtained from TRC (Sigma).

    Techniques: Expressing, Infection, shRNA, Luciferase, Activity Assay, Western Blot, Quantitative RT-PCR, Chick Chorioallantoic Membrane Assay

    Synthetic Lethal Dependency of Deregulated MYC on POLRMT A. U2OS MYC/ER cells were infected with lentiviral POLRMT shRNA or Luciferase (Luc) shRNA as a control. Five days post-infection cells were seeded at equal densities and MYC activity was induced via treatment with 4-OHT (MYC On). Cells were counted on day three and day five post MYC-activation. shPOLRMT cell counts are shown with an adjusted scale (right). Error bars represent SD, n = 3. *** p

    Journal: Oncotarget

    Article Title: Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

    doi: 10.18632/oncotarget.11718

    Figure Lengend Snippet: Synthetic Lethal Dependency of Deregulated MYC on POLRMT A. U2OS MYC/ER cells were infected with lentiviral POLRMT shRNA or Luciferase (Luc) shRNA as a control. Five days post-infection cells were seeded at equal densities and MYC activity was induced via treatment with 4-OHT (MYC On). Cells were counted on day three and day five post MYC-activation. shPOLRMT cell counts are shown with an adjusted scale (right). Error bars represent SD, n = 3. *** p

    Article Snippet: Lentiviral short hairpin RNA (shRNA) plasmids corresponding to MYC, POLRMT, TFB1M, SHMT2 and control luciferase shRNAs were obtained from TRC (Sigma).

    Techniques: Infection, shRNA, Luciferase, Activity Assay, Activation Assay

    POLRMT is a direct transcriptional target of the MYC oncoprotein A. 2091 MYC/ER cells were treated with 4-hydroxytamoxifen (4-OHT) to induce MYC activity. Cells were harvested at the indicated time points and analyzed by quantitative real-time PCR (qRT-PCR) to show the kinetics of gene induction. Error bars represent SD, n = 3. B. H1299 cells were infected with lentiviral MYC shRNA or Luciferase (Luc) shRNA as a control. Cells were harvested and whole cells lysates were analyzed by Western blot for the indicated proteins. C. Raji cells were treated with JQ1 to deplete MYC levels. Cells were harvested and whole cell lysates were analyzed by Western blot (left) for the indicated proteins and qRT-PCR (right) for POLRMT expression. Error bars represent SD, n = 3. D. Mammary tumors were induced in six week old female bitransgenic MMTV-rtTA;TetO-MYC mice via administration of 2 mg/mL doxycycline in drinking water. After mammary tumor formation, doxycycline was withdrawn from the water of 4 mice (MYC OFF) while the other mice were maintained on doxycycline (MYC ON). 96 hours after doxycycline withdrawal, tumors were harvested and mRNA analyzed by qRT-PCR. Error bars represent SD, n = 4. *** p

    Journal: Oncotarget

    Article Title: Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

    doi: 10.18632/oncotarget.11718

    Figure Lengend Snippet: POLRMT is a direct transcriptional target of the MYC oncoprotein A. 2091 MYC/ER cells were treated with 4-hydroxytamoxifen (4-OHT) to induce MYC activity. Cells were harvested at the indicated time points and analyzed by quantitative real-time PCR (qRT-PCR) to show the kinetics of gene induction. Error bars represent SD, n = 3. B. H1299 cells were infected with lentiviral MYC shRNA or Luciferase (Luc) shRNA as a control. Cells were harvested and whole cells lysates were analyzed by Western blot for the indicated proteins. C. Raji cells were treated with JQ1 to deplete MYC levels. Cells were harvested and whole cell lysates were analyzed by Western blot (left) for the indicated proteins and qRT-PCR (right) for POLRMT expression. Error bars represent SD, n = 3. D. Mammary tumors were induced in six week old female bitransgenic MMTV-rtTA;TetO-MYC mice via administration of 2 mg/mL doxycycline in drinking water. After mammary tumor formation, doxycycline was withdrawn from the water of 4 mice (MYC OFF) while the other mice were maintained on doxycycline (MYC ON). 96 hours after doxycycline withdrawal, tumors were harvested and mRNA analyzed by qRT-PCR. Error bars represent SD, n = 4. *** p

    Article Snippet: Lentiviral short hairpin RNA (shRNA) plasmids corresponding to MYC, POLRMT, TFB1M, SHMT2 and control luciferase shRNAs were obtained from TRC (Sigma).

    Techniques: Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection, shRNA, Luciferase, Western Blot, Expressing, Mouse Assay

    Generation and validation of ERβ KO, ERβ1 and ERβ5 cells. U251 ERβ KO (A) and U87 ERβ KO (B) cells were generated using the CRISPR/Cas9 system, and ERβ knockout was confirmed by genomic PCR, and validated by western blotting. C. ERβ was knocked down in GBM-040815 cells using lentiviral transduction of ERβ shRNA. ERβ knockdown was validated by RT-qPCR and western blotting. D. U251-WT or U251-ERβ KO cells were injected orthotopically and the number of survival days of the mice were recorded and analyzed using Kaplan-Meier graph. E. U87 cells and U251 cells stably expressing ERβ1 and ERβ5 isoforms were generated by lentivirus transduction in the ERβ KO background. Primary (GBM-040815) cells were transduced with ERβ1 and ERβ5 lentiviral plasmids. The expression of ERβ1 and ERβ5 was validated by western blotting. The cell viability of U251 (F) and GBM-040815 cells (G) expressing ERβ1 and ERβ5 were measured by MTT and MTS assay respectively. H, I. U251 WT, ERβ KO, ERβ1 and ERβ5 cells were seeded in 6-well plates (500 cells/well) and after 14 days the colonies were stained with 0.5% crystal violet and the percentage of colony area was determined. J. NIH3T3 cells were transfected with empty vector or ERβ5 vector, and focus formation assays were performed as described in methods. K. NIH3T3 cells were transfected with indicated plasmids and subjected to soft agar colony formation assay. Data are represented as mean ± SE. * p

    Journal: Cancer research

    Article Title: Differential effects of estrogen receptor beta isoforms on glioblastoma progression

    doi: 10.1158/0008-5472.CAN-17-3470

    Figure Lengend Snippet: Generation and validation of ERβ KO, ERβ1 and ERβ5 cells. U251 ERβ KO (A) and U87 ERβ KO (B) cells were generated using the CRISPR/Cas9 system, and ERβ knockout was confirmed by genomic PCR, and validated by western blotting. C. ERβ was knocked down in GBM-040815 cells using lentiviral transduction of ERβ shRNA. ERβ knockdown was validated by RT-qPCR and western blotting. D. U251-WT or U251-ERβ KO cells were injected orthotopically and the number of survival days of the mice were recorded and analyzed using Kaplan-Meier graph. E. U87 cells and U251 cells stably expressing ERβ1 and ERβ5 isoforms were generated by lentivirus transduction in the ERβ KO background. Primary (GBM-040815) cells were transduced with ERβ1 and ERβ5 lentiviral plasmids. The expression of ERβ1 and ERβ5 was validated by western blotting. The cell viability of U251 (F) and GBM-040815 cells (G) expressing ERβ1 and ERβ5 were measured by MTT and MTS assay respectively. H, I. U251 WT, ERβ KO, ERβ1 and ERβ5 cells were seeded in 6-well plates (500 cells/well) and after 14 days the colonies were stained with 0.5% crystal violet and the percentage of colony area was determined. J. NIH3T3 cells were transfected with empty vector or ERβ5 vector, and focus formation assays were performed as described in methods. K. NIH3T3 cells were transfected with indicated plasmids and subjected to soft agar colony formation assay. Data are represented as mean ± SE. * p

    Article Snippet: ERβ-specific short hairpin RNA (shRNA) lentiviral plasmids, β-actin and all secondary antibodies were purchased from Sigma Chemical Co (St. Louis, MO).

    Techniques: Generated, CRISPR, Knock-Out, Polymerase Chain Reaction, Western Blot, Transduction, shRNA, Quantitative RT-PCR, Injection, Mouse Assay, Stable Transfection, Expressing, MTT Assay, MTS Assay, Staining, Transfection, Plasmid Preparation, Soft Agar Assay

    Alteration of mitochondria by adenylate kinase 4 (AK4) knockdown. a Representative electron microscope images of cells in normoxic condition. Left panel: HeLa cells transfected with control short hairpin (sh)RNA. Right panel: HeLa cells expressing adenylate kinase 4 (AK4) shRNA. The arrows indicate mitochondria. Right: The graphs show the relative mitochondrial counts and cross-sectional area, as indicated. Mitochondrial numbers were counted in ten random pictures taken at a magnification of 10,000. Mitochondrial size was determined using BZ-II analyzer (Keyence) software. We examined 70 mitochondria in control shRNA cells and 100 mitochondria for in AK4 shRNA cells. b Relative mitochondrial (mt)DNA copy number under the conditions indicated. mtDNA was measured by real-time PCR using the Human mtDNA Monitoring Primer Set (Takara Japan). Briefly, we measured mtDNA ( ND1 and ND5 ), then normalized to the nuclear DNA level ( SLCO2B1 and SERPINA1 ). The mtDNA content index was the ratio of mtDNA/nuclear DNA, calculated by dividing the ND1 and ND5 signals by the SLCO2B1 and SERPINA1 signals. P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Modulation of anti-cancer drug sensitivity through the regulation of mitochondrial activity by adenylate kinase 4

    doi: 10.1186/s13046-016-0322-2

    Figure Lengend Snippet: Alteration of mitochondria by adenylate kinase 4 (AK4) knockdown. a Representative electron microscope images of cells in normoxic condition. Left panel: HeLa cells transfected with control short hairpin (sh)RNA. Right panel: HeLa cells expressing adenylate kinase 4 (AK4) shRNA. The arrows indicate mitochondria. Right: The graphs show the relative mitochondrial counts and cross-sectional area, as indicated. Mitochondrial numbers were counted in ten random pictures taken at a magnification of 10,000. Mitochondrial size was determined using BZ-II analyzer (Keyence) software. We examined 70 mitochondria in control shRNA cells and 100 mitochondria for in AK4 shRNA cells. b Relative mitochondrial (mt)DNA copy number under the conditions indicated. mtDNA was measured by real-time PCR using the Human mtDNA Monitoring Primer Set (Takara Japan). Briefly, we measured mtDNA ( ND1 and ND5 ), then normalized to the nuclear DNA level ( SLCO2B1 and SERPINA1 ). The mtDNA content index was the ratio of mtDNA/nuclear DNA, calculated by dividing the ND1 and ND5 signals by the SLCO2B1 and SERPINA1 signals. P

    Article Snippet: Expression and suppression of AK4 HeLa cells were transiently transfected with small interfering RNA (siRNA) or with and AK4 short hairpin RNA (shRNA) plasmid (TRCN37554), a control shRNA plasmid, a green fluorescent protein (GFP)-tagged AK4 expression vector (RG220572), and a control expression vector, purchased from Sigma-Aldrich, Tokyo, Japan or from Origene, Rockville, USA, respectively.

    Techniques: Microscopy, Transfection, Expressing, shRNA, Software, Real-time Polymerase Chain Reaction

    Oxygen consumption rate (OCR) and metabolite analysis in HeLa cells expressing adenylate kinase 4 (AK4) or control short hairpin (sh)RNA. a Relative ATP level per cell. 10,000 cells were seeded in 96 wells, and measured 3 days after siRNA treatment. ATP level was normalized by cell number deduced from cyquant assay. * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Modulation of anti-cancer drug sensitivity through the regulation of mitochondrial activity by adenylate kinase 4

    doi: 10.1186/s13046-016-0322-2

    Figure Lengend Snippet: Oxygen consumption rate (OCR) and metabolite analysis in HeLa cells expressing adenylate kinase 4 (AK4) or control short hairpin (sh)RNA. a Relative ATP level per cell. 10,000 cells were seeded in 96 wells, and measured 3 days after siRNA treatment. ATP level was normalized by cell number deduced from cyquant assay. * p

    Article Snippet: Expression and suppression of AK4 HeLa cells were transiently transfected with small interfering RNA (siRNA) or with and AK4 short hairpin RNA (shRNA) plasmid (TRCN37554), a control shRNA plasmid, a green fluorescent protein (GFP)-tagged AK4 expression vector (RG220572), and a control expression vector, purchased from Sigma-Aldrich, Tokyo, Japan or from Origene, Rockville, USA, respectively.

    Techniques: Expressing, CyQUANT Assay

    ATF4 expression promotes RET ubiquitination. (A and B) HEK 293T cells were transfected with the indicated plasmids, and western blot (WB) analysis was performed with the indicated antibodies. (C) Western blot analysis of TT cells infected with the lentivirus harboring ATF4 with the indicated antibodies. (D) RNA was isolated from cells treated as in (C) and subjected to real-time PCR using ATF4 or RET Taqman primers probes. (E) MTC cells expressing lentiviral-ATF4 were treated with MG132 (4 h), and denatured extracts were immunoprecipitated with an anti-RET antibody followed by western blot analysis with antiubiquitin antibody. (F) ATF4-shRNA (clones of 74 and 76) and nontargeting shRNA (Con) are treated with tunicamycin (2 μg/mL) for 24 h, and western blot analysis was performed with the indicated antibodies. (G) ATF4 expression inhibits RET signaling. MTC cells expressing lentiviral-ATF4 were immunoblotted with the indicated antibodies. IP, immunoprecipitation.

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: ATF4 Targets RET for Degradation and Is a Candidate Tumor Suppressor Gene in Medullary Thyroid Cancer

    doi: 10.1210/jc.2016-2878

    Figure Lengend Snippet: ATF4 expression promotes RET ubiquitination. (A and B) HEK 293T cells were transfected with the indicated plasmids, and western blot (WB) analysis was performed with the indicated antibodies. (C) Western blot analysis of TT cells infected with the lentivirus harboring ATF4 with the indicated antibodies. (D) RNA was isolated from cells treated as in (C) and subjected to real-time PCR using ATF4 or RET Taqman primers probes. (E) MTC cells expressing lentiviral-ATF4 were treated with MG132 (4 h), and denatured extracts were immunoprecipitated with an anti-RET antibody followed by western blot analysis with antiubiquitin antibody. (F) ATF4-shRNA (clones of 74 and 76) and nontargeting shRNA (Con) are treated with tunicamycin (2 μg/mL) for 24 h, and western blot analysis was performed with the indicated antibodies. (G) ATF4 expression inhibits RET signaling. MTC cells expressing lentiviral-ATF4 were immunoblotted with the indicated antibodies. IP, immunoprecipitation.

    Article Snippet: Lentiviral ATF4 short hairpin RNA (shRNA) plasmids were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Expressing, Transfection, Western Blot, Infection, Isolation, Real-time Polymerase Chain Reaction, Immunoprecipitation, shRNA

    Proliferation of primary MEFs in the absence of Cip/Kip inhibitors. (a) The proliferation of wild-type (Wt), p21 −/− p27 −/− (DKO), and Cdk2 −/− p21 −/− p27 −/− (TKO) primary MEFs in 10% fetal bovine serum (FBS) was assessed upon infection with lentiviral particles expressing an shRNA against p57 Kip2 (sh p57) or a scramble control (sh Ctrl). Data are shown as means ± SDs ( n = 3). (b) Same as for panel a, but MEFs were maintained under limiting serum conditions (2% fetal bovine serum). arb., arbitrary.

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Characterization of the Role of the Cip/Kip Family of Proteins as Cyclin-Dependent Kinase Inhibitors and Assembly Factors

    doi: 10.1128/MCB.01163-13

    Figure Lengend Snippet: Proliferation of primary MEFs in the absence of Cip/Kip inhibitors. (a) The proliferation of wild-type (Wt), p21 −/− p27 −/− (DKO), and Cdk2 −/− p21 −/− p27 −/− (TKO) primary MEFs in 10% fetal bovine serum (FBS) was assessed upon infection with lentiviral particles expressing an shRNA against p57 Kip2 (sh p57) or a scramble control (sh Ctrl). Data are shown as means ± SDs ( n = 3). (b) Same as for panel a, but MEFs were maintained under limiting serum conditions (2% fetal bovine serum). arb., arbitrary.

    Article Snippet: Knockdown of p57 Kip2 was mediated with lentiviral Mission short hairpin RNA (shRNA) plasmids (Sigma no. SHGLY- ) according to the manufacturer's instructions.

    Techniques: Infection, Expressing, shRNA

    Stability of Cdk-cyclin D complexes in the absence of Cip/Kip inhibitors. (a) Early-passage primary MEFs were infected with lentiviral particles expressing either an shRNA against p57 Kip2 (p57) or a scramble control (Ct). Protein extracts were prepared, and the levels of a panel of cell cycle regulators were analyzed by immunoblotting with antibodies elicited against the indicated proteins. Expression of β-actin served as a loading control. Results from two independent MEF cultures are shown for p21 −/− p27 −/− (DKO) and Cdk2 −/− p21 −/− p27 −/− (TKO) mice. Cyc, cyclin. (b) Wild-type, p21 −/− p27 −/− (DKO), and Cdk2 −/− p21 −/− p27 −/− (TKO) primary MEFs were infected with lentiviral particles expressing either an shRNA against p57 Kip2 (p57) or a scramble control (Ct). Exponentially growing cells were used to prepare whole-cell extracts, and the amount of Cdk4-cyclin D complexes was estimated by coprecipitation of cyclin D1 with anti-Cdk4 antibodies. The levels of Cdk4, used as a loading control, are shown. WB, Western blotting. (c) The amount of starting whole-cell extract from both p21 −/− p27 −/− (DKO) and Cdk2 −/− p21 −/− p27 −/− (TKO) MEFs was augmented to equal the cyclin D1 levels in the wild-type controls. The amount of Cdk4 (top) and Cdk6 (middle) that was coimmunoprecipitated with cyclin D1 antibodies was detected by Western blotting. Cyclin D1 levels (bottom) are shown to demonstrate equal estimation of the amount of starting material. Results from two independent MEF cultures are shown. (d) Western blotting of Cdk1 protein levels in whole-cell extracts from wild-type, p21 −/− p27 −/− (DKO), and Cdk2 −/− p21 −/− p27 −/− (TKO) MEFs. Expression of β-actin, which served as a loading control, is shown. Results from two independent MEF cultures are shown. (e) In vitro kinase activity associated with Cdk1 immunoprecipitates obtained from wild-type, p21 −/− p27 −/− (DKO), and Cdk2 −/− p21 −/− p27 −/− (TKO) MEFs. Histone H1 was used as the substrate (S). Results from two independent MEF cultures are shown. Lanes WCE, whole-cell extract at a 1:10 dilution before immunoprecipitation (IP); lanes M, mock immunoprecipitate.

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Characterization of the Role of the Cip/Kip Family of Proteins as Cyclin-Dependent Kinase Inhibitors and Assembly Factors

    doi: 10.1128/MCB.01163-13

    Figure Lengend Snippet: Stability of Cdk-cyclin D complexes in the absence of Cip/Kip inhibitors. (a) Early-passage primary MEFs were infected with lentiviral particles expressing either an shRNA against p57 Kip2 (p57) or a scramble control (Ct). Protein extracts were prepared, and the levels of a panel of cell cycle regulators were analyzed by immunoblotting with antibodies elicited against the indicated proteins. Expression of β-actin served as a loading control. Results from two independent MEF cultures are shown for p21 −/− p27 −/− (DKO) and Cdk2 −/− p21 −/− p27 −/− (TKO) mice. Cyc, cyclin. (b) Wild-type, p21 −/− p27 −/− (DKO), and Cdk2 −/− p21 −/− p27 −/− (TKO) primary MEFs were infected with lentiviral particles expressing either an shRNA against p57 Kip2 (p57) or a scramble control (Ct). Exponentially growing cells were used to prepare whole-cell extracts, and the amount of Cdk4-cyclin D complexes was estimated by coprecipitation of cyclin D1 with anti-Cdk4 antibodies. The levels of Cdk4, used as a loading control, are shown. WB, Western blotting. (c) The amount of starting whole-cell extract from both p21 −/− p27 −/− (DKO) and Cdk2 −/− p21 −/− p27 −/− (TKO) MEFs was augmented to equal the cyclin D1 levels in the wild-type controls. The amount of Cdk4 (top) and Cdk6 (middle) that was coimmunoprecipitated with cyclin D1 antibodies was detected by Western blotting. Cyclin D1 levels (bottom) are shown to demonstrate equal estimation of the amount of starting material. Results from two independent MEF cultures are shown. (d) Western blotting of Cdk1 protein levels in whole-cell extracts from wild-type, p21 −/− p27 −/− (DKO), and Cdk2 −/− p21 −/− p27 −/− (TKO) MEFs. Expression of β-actin, which served as a loading control, is shown. Results from two independent MEF cultures are shown. (e) In vitro kinase activity associated with Cdk1 immunoprecipitates obtained from wild-type, p21 −/− p27 −/− (DKO), and Cdk2 −/− p21 −/− p27 −/− (TKO) MEFs. Histone H1 was used as the substrate (S). Results from two independent MEF cultures are shown. Lanes WCE, whole-cell extract at a 1:10 dilution before immunoprecipitation (IP); lanes M, mock immunoprecipitate.

    Article Snippet: Knockdown of p57 Kip2 was mediated with lentiviral Mission short hairpin RNA (shRNA) plasmids (Sigma no. SHGLY- ) according to the manufacturer's instructions.

    Techniques: Infection, Expressing, shRNA, Mouse Assay, Western Blot, In Vitro, Activity Assay, Immunoprecipitation

    The antitumor effect of lidocaine was reversed by CPEB3 knockdown. (a) HepG2 tumor cells were transfected with E. coli TOP10-EGFP-shRNA-CPEB3. Images of the same field were taken to show both the bright-field image (cell morphology) and the GFP fluorescence (100x magnification). (b) qPCR was used to quantify CPEB3 mRNA expression in the control group (normal tumor cells), negative control (scramble shRNA), and shRNA-CPEB3 group. (c, d) HepG2 cell survival rate in shRNA-CPEB3 group after lidocaine treatment compared with control group and negative control group. Compared with control group, ∗ indicates P

    Journal: BioMed Research International

    Article Title: Effects of Lidocaine-Mediated CPEB3 Upregulation in Human Hepatocellular Carcinoma Cell Proliferation In Vitro

    doi: 10.1155/2018/8403157

    Figure Lengend Snippet: The antitumor effect of lidocaine was reversed by CPEB3 knockdown. (a) HepG2 tumor cells were transfected with E. coli TOP10-EGFP-shRNA-CPEB3. Images of the same field were taken to show both the bright-field image (cell morphology) and the GFP fluorescence (100x magnification). (b) qPCR was used to quantify CPEB3 mRNA expression in the control group (normal tumor cells), negative control (scramble shRNA), and shRNA-CPEB3 group. (c, d) HepG2 cell survival rate in shRNA-CPEB3 group after lidocaine treatment compared with control group and negative control group. Compared with control group, ∗ indicates P

    Article Snippet: The CPEB3 short hairpin RNA (shRNA) plasmid was purchased from GeneChem Co., Ltd. (Shanghai, China).

    Techniques: Transfection, shRNA, Fluorescence, Real-time Polymerase Chain Reaction, Expressing, Negative Control

    CRL4 DDB2 ubiquitylates HBO1 in response to UV damage. (A) Depletion of CUL4A/B in HEK293 cells suppressed ubiquitylation of HBO1. CUL1, CUL2, and CUL4A/B were targeted by siRNA transfection for 48 h. Cell lysates were subjected to Western blotting with the indicated antibodies. (B) Depletion of CUL4A/B protected HBO1 phosphorylated at Ser50 and Ser53 from degradation after UV irradiation. CUL4A/B or negative-control siRNA was transfected into HEK293 cells. After 48 h of transfection, cells were exposed to 40 J/m 2 UV light. Cells were incubated for 6 h, and Western blotting was performed with the indicated antibodies. β-Actin was used for normalization of HBO1 expression. The graph indicates the average HBO1 expression levels for three independent experiments. Error bars indicate standard errors (**, P

    Journal: Molecular and Cellular Biology

    Article Title: UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation

    doi: 10.1128/MCB.00809-15

    Figure Lengend Snippet: CRL4 DDB2 ubiquitylates HBO1 in response to UV damage. (A) Depletion of CUL4A/B in HEK293 cells suppressed ubiquitylation of HBO1. CUL1, CUL2, and CUL4A/B were targeted by siRNA transfection for 48 h. Cell lysates were subjected to Western blotting with the indicated antibodies. (B) Depletion of CUL4A/B protected HBO1 phosphorylated at Ser50 and Ser53 from degradation after UV irradiation. CUL4A/B or negative-control siRNA was transfected into HEK293 cells. After 48 h of transfection, cells were exposed to 40 J/m 2 UV light. Cells were incubated for 6 h, and Western blotting was performed with the indicated antibodies. β-Actin was used for normalization of HBO1 expression. The graph indicates the average HBO1 expression levels for three independent experiments. Error bars indicate standard errors (**, P

    Article Snippet: HBO1 and DDB2 short hairpin RNA (shRNA) plasmids were purchased from Santa Cruz (sc-35530B-SH and sc-37799-SH).

    Techniques: Transfection, Western Blot, Irradiation, Negative Control, Incubation, Expressing

    Regulation of expression of Bax and Bcl‐2 proteins in SK‐N‐DZ and IMR‐32 cells by KLF4. Treatments (24 h): control (CTL) cells, 100 nM Bcl‐2 shRNA, 100 nM KLF4 vector, and 100 nM

    Journal: Molecular Oncology

    Article Title: KLF4 overexpression and apigenin treatment down regulated anti-apoptotic Bcl-2 proteins and matrix metalloproteinases to control growth of human malignant neuroblastoma SK-N-DZ and IMR-32 cells

    doi: 10.1016/j.molonc.2012.12.002

    Figure Lengend Snippet: Regulation of expression of Bax and Bcl‐2 proteins in SK‐N‐DZ and IMR‐32 cells by KLF4. Treatments (24 h): control (CTL) cells, 100 nM Bcl‐2 shRNA, 100 nM KLF4 vector, and 100 nM

    Article Snippet: With or without Bcl‐2 knockdown by transfection with Bcl‐2 short hairpin RNA (shRNA) plasmid (Santa Cruz Biotechnology, Santa Cruz, CA), we performed KLF4 plasmid transfection in SK‐N‐DZ and IMR‐32 cells.

    Techniques: Expressing, CTL Assay, shRNA, Plasmid Preparation