non targeting sirna  (Santa Cruz Biotechnology)

 
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    Name:
    HPV16 E6 siRNA
    Description:
    Target species human papillomavirus Gene Silencers generally consist of pools of three to five target specific 19 25 nucleotide sequences in length For independent verification of HPV16 E6 gene silencing results individual duplex components or plasmids are also available upon request
    Catalog Number:
    SC-156008
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    Gene Editing siRNA shRNA Gene Silencers Non Mammalian siRNA shRNA Plasmid and Lentiviral Particle Gene Silencers HPV siRNA shRNA Plasmid and Lentiviral Particle Gene Silencers HPV16 E6 siRNA and shRNA Plasmids hpv
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    Structured Review

    Santa Cruz Biotechnology non targeting sirna
    Depletion of Epac1 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. <t>HPAEC</t> were treated with Epac1 specific <t>siRNA</t> or non-targeting siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Epac1 significantly attenuated adenosine-induced endothelial barrier enhancement. n=3; mean +/− SEM; * p
    Target species human papillomavirus Gene Silencers generally consist of pools of three to five target specific 19 25 nucleotide sequences in length For independent verification of HPV16 E6 gene silencing results individual duplex components or plasmids are also available upon request
    https://www.bioz.com/result/non targeting sirna/product/Santa Cruz Biotechnology
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    non targeting sirna - by Bioz Stars, 2021-09
    93/100 stars

    Images

    1) Product Images from "Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: molecular mechanisms and barrier-protective role"

    Article Title: Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: molecular mechanisms and barrier-protective role

    Journal: Journal of cellular physiology

    doi: 10.1002/jcp.26281

    Depletion of Epac1 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Epac1 specific siRNA or non-targeting siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Epac1 significantly attenuated adenosine-induced endothelial barrier enhancement. n=3; mean +/− SEM; * p
    Figure Legend Snippet: Depletion of Epac1 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Epac1 specific siRNA or non-targeting siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Epac1 significantly attenuated adenosine-induced endothelial barrier enhancement. n=3; mean +/− SEM; * p

    Techniques Used: Activation Assay

    Depletion of Vav2 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Vav2, specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in the TER measurement assay. Depletion of Vav2 significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM;*** p
    Figure Legend Snippet: Depletion of Vav2 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Vav2, specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in the TER measurement assay. Depletion of Vav2 significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM;*** p

    Techniques Used: Activation Assay

    Depletion of Rap1b modestly affects adenosine-induced increase in TER while substantially decreases Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Rap1b specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1b modestly affected maximal adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine in Rac1 activation in Rap1b depleted cells. HPAEC were transfected with Rap1b specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **p
    Figure Legend Snippet: Depletion of Rap1b modestly affects adenosine-induced increase in TER while substantially decreases Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Rap1b specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1b modestly affected maximal adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine in Rac1 activation in Rap1b depleted cells. HPAEC were transfected with Rap1b specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **p

    Techniques Used: Activation Assay, Transfection, Activity Assay

    Tiam1 depletion has no effect on adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Tiam1 specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Tiam1 did not affect adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine on Rac1 activation in Tiam1 depleted cells. HPAEC were transfected with Tiam1 specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **P
    Figure Legend Snippet: Tiam1 depletion has no effect on adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Tiam1 specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Tiam1 did not affect adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine on Rac1 activation in Tiam1 depleted cells. HPAEC were transfected with Tiam1 specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **P

    Techniques Used: Activation Assay, Transfection, Activity Assay

    Depletion of Rap1a attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Rap1a specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1a significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; **p
    Figure Legend Snippet: Depletion of Rap1a attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Rap1a specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1a significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; **p

    Techniques Used: Activation Assay

    2) Product Images from "Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells"

    Article Title: Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056666

    Cbl is required for berberine inhibition of proliferation by berberine in IMCE cells. Cells were transfected with Cbl siRNA or non-targeting siRNA. (A) at the 30-hour of posttransfection, cells were treated with berberine at 50 µM for 18 hours under non-permissive condition in the presence or absence of EGF (30 ng/ml) treatment for 5 minutes. Cellular lysates were collected for Western blot analysis to detect Cbl expression and indicated signaling pathways. (B) at the 24-hour of posttransfection, cells were treated with berberine at 50 µM for 24 hours under non-permissive condition in the presence or absence of EGF (30 ng/ml) treatment for 24 hours to detect cell proliferation, as described in Figure 2A . Density plots with BrdU-FITC vs PI and percentages of cell s in G1, S, and G2 phase are shown. Data in this Figure are representative of three separate experiments.
    Figure Legend Snippet: Cbl is required for berberine inhibition of proliferation by berberine in IMCE cells. Cells were transfected with Cbl siRNA or non-targeting siRNA. (A) at the 30-hour of posttransfection, cells were treated with berberine at 50 µM for 18 hours under non-permissive condition in the presence or absence of EGF (30 ng/ml) treatment for 5 minutes. Cellular lysates were collected for Western blot analysis to detect Cbl expression and indicated signaling pathways. (B) at the 24-hour of posttransfection, cells were treated with berberine at 50 µM for 24 hours under non-permissive condition in the presence or absence of EGF (30 ng/ml) treatment for 24 hours to detect cell proliferation, as described in Figure 2A . Density plots with BrdU-FITC vs PI and percentages of cell s in G1, S, and G2 phase are shown. Data in this Figure are representative of three separate experiments.

    Techniques Used: Inhibition, Transfection, Western Blot, Expressing

    3) Product Images from "NF-κB and Androgen Receptor Variant 7 Induce Expression of SRD5A Isoforms and Confer 5ARI Resistance"

    Article Title: NF-κB and Androgen Receptor Variant 7 Induce Expression of SRD5A Isoforms and Confer 5ARI Resistance

    Journal: The Prostate

    doi: 10.1002/pros.23195

    NF-κB inhibition affects SRD5A2 expression. ( A ) Inhibition of NF-κB activation by BMS-345541 in NHPrE1-EE and ( C ) BHPrS1-EE cell lines resulted in a significant decrease in SRD5A2 expression. ( B ) Silencing of NF-κB using an siRNA
    Figure Legend Snippet: NF-κB inhibition affects SRD5A2 expression. ( A ) Inhibition of NF-κB activation by BMS-345541 in NHPrE1-EE and ( C ) BHPrS1-EE cell lines resulted in a significant decrease in SRD5A2 expression. ( B ) Silencing of NF-κB using an siRNA

    Techniques Used: Inhibition, Expressing, Activation Assay

    Knock-down of SRD5A2 decreased viability in cells with activated NF-κB. NHPrE1-EV (empty vector control— A ) -EE (constitutively active NF-κB— B ), and -AR-V7 (expressing AR-V7— C ) cell lines were transfected with siRNA
    Figure Legend Snippet: Knock-down of SRD5A2 decreased viability in cells with activated NF-κB. NHPrE1-EV (empty vector control— A ) -EE (constitutively active NF-κB— B ), and -AR-V7 (expressing AR-V7— C ) cell lines were transfected with siRNA

    Techniques Used: Plasmid Preparation, Expressing, Transfection

    4) Product Images from "mtDNA as a Mediator for Expression of Hypoxia-Inducible Factor 1α and ROS in Hypoxic Neuroblastoma Cells"

    Article Title: mtDNA as a Mediator for Expression of Hypoxia-Inducible Factor 1α and ROS in Hypoxic Neuroblastoma Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18061220

    The effect of HIF-1α siRNA. Cells were transiently transfected with siRNA targeted for HIF-1α (HIF-1α siRNA) or non-specific siRNA (Mock) for 24 h, and then incubated in normoxia or 1% O 2 for 4 h. The expression of HIF-1α, DRP1 and β-actin proteins was revealed by Western blot analysis, and the quantification analyses are shown below. β-Actin was used as a loading control. The results are shown as mean ± SD of at least three experiments. # Represented p
    Figure Legend Snippet: The effect of HIF-1α siRNA. Cells were transiently transfected with siRNA targeted for HIF-1α (HIF-1α siRNA) or non-specific siRNA (Mock) for 24 h, and then incubated in normoxia or 1% O 2 for 4 h. The expression of HIF-1α, DRP1 and β-actin proteins was revealed by Western blot analysis, and the quantification analyses are shown below. β-Actin was used as a loading control. The results are shown as mean ± SD of at least three experiments. # Represented p

    Techniques Used: Transfection, Incubation, Expressing, Western Blot

    The effect of DRP1 siRNA. Cells were transiently transfected with siRNA targeted for DRP1 (DRP1 siRNA) or non-specific siRNA (Mock) for 24 h, and then incubated in normoxia or 1% O 2 for 4 h. The expression of HIF-1α, DRP1 and β-actin proteins were revealed by Western blot analysis, and the quantification analyses are shown below. β-Actin was used as a loading control. The results are shown as mean ± SD of at least three experiments. * Represented p
    Figure Legend Snippet: The effect of DRP1 siRNA. Cells were transiently transfected with siRNA targeted for DRP1 (DRP1 siRNA) or non-specific siRNA (Mock) for 24 h, and then incubated in normoxia or 1% O 2 for 4 h. The expression of HIF-1α, DRP1 and β-actin proteins were revealed by Western blot analysis, and the quantification analyses are shown below. β-Actin was used as a loading control. The results are shown as mean ± SD of at least three experiments. * Represented p

    Techniques Used: Transfection, Incubation, Expressing, Western Blot

    5) Product Images from "Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis"

    Article Title: Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-40553-2

    Knocking down CTMP in TNFα-treated differentiated C2C12 myotubes using siRNA rescued the decrease in Akt phosphorylation. Incubating differentiated TNFα-treated C2C12 myotubes with siRNA against CTMP for 73 hours significantly decreased CTMP expression and increased Akt phosphorylation, indicating a direct relationship between CTMP expression and Akt phosphorylation in this muscle atrophy model. Blot images are cropped from different membranes used for data collection and analysis (See Supplemental Figures). * P
    Figure Legend Snippet: Knocking down CTMP in TNFα-treated differentiated C2C12 myotubes using siRNA rescued the decrease in Akt phosphorylation. Incubating differentiated TNFα-treated C2C12 myotubes with siRNA against CTMP for 73 hours significantly decreased CTMP expression and increased Akt phosphorylation, indicating a direct relationship between CTMP expression and Akt phosphorylation in this muscle atrophy model. Blot images are cropped from different membranes used for data collection and analysis (See Supplemental Figures). * P

    Techniques Used: Expressing

    6) Product Images from "Heme oxygenase-1 promotes tumor progression and metastasis of colorectal carcinoma cells by inhibiting antitumor immunity"

    Article Title: Heme oxygenase-1 promotes tumor progression and metastasis of colorectal carcinoma cells by inhibiting antitumor immunity

    Journal: Oncotarget

    doi:

    Effect of HO-1 on TPA-induced ICAM-1 expression A. HT-29 cells were treated with TPA (10 ng/ml) and then harvested for ICAM-1 mRNA ( left panel ) and ICAM-1 protein ( right panel ), which was measured using real time PCR and western blot analysis, respectively. The values represent mRNA levels normalized to that of GAPDH mRNA. B. The effect of HO-1 on TPA-induced ICAM-1 mRNA expression in HT-29 cells. The cells were pretreated with 0–20 μM hemin for 1 h followed by stimulation with 10 ng/ml TPA for an additional 10 h after which the cells were harvested for RNA preparation. RNA samples were analyzed by real time PCR to determine the levels of ICAM-1 mRNA. The values represent mRNA levels normalized to that of GAPDH mRNA. C. The effect of HO-1 on the p65 intranuclear translocation ( upper panel ) and on the NF-κB DNA binding activity ( lower panel ) in TPA-stimulated HT-29 cells. HT-29 cells were pretreated with or without 20 μM hemin for 1 h followed by incubation with 10 ng/ml TPA for the indicated intervals. The cells were harvested and the nuclear levels of NF-κBp65 and NF-κB DNA binding activity were determined by western blot and DNA binding assay, respectively. D. The effect of HO-1 on TPA-induced ICAM-1 protein expression in HT-29 cells. The cells were pretreated with 0–20 μM hemin for 1 h and were subsequently stimulated with 10 ng/ml TPA for an additional 24 h. The cells were harvested for ICAM-1 protein expression by western blot analysis. E. The effects of HO-1 on TPA-induced ICAM-1 mRNA and protein expression in Caco-2 cells. The cells were pretreated with 0–20 μM hemin for 1 h and were subsequently stimulated with 10 ng/ml TPA. The cells were harvested for ICAM-1 mRNA ( left panel ) and protein ( right panel ) expression by RT-PCR and western blot analysis, respectively. F. The effect of HO-1 silencing on the hemin-mediated reduction of ICAM-1 in TPA-induced HT-29 cells. HT-29 cells were transfected with siRNA against HO-1 (HO-1 si) or control siRNA (Ctrl si). After pretreatment with 20 μM hemin, and then stimulated with 10 ng/ml TPA for an additional 24 h. The expression levels of ICAM-1 and HO-1 were analyzed by western blot analysis. G. The effect of ZnPP on HO-1 activity ( left panel ) and TPA-induced ICAM-1 protein expression ( right panel ) in HT-29 cells. The cells were pretreated with 0–20 μM ZnPP for 1 h, followed by stimulation with 10 ng/ml TPA for an additional 24 h. The cells were harvested for measurement of HO activity and western blot analysis. The results were repeated in at least three independent experiments. The data are expressed as mean ± SD. ** P
    Figure Legend Snippet: Effect of HO-1 on TPA-induced ICAM-1 expression A. HT-29 cells were treated with TPA (10 ng/ml) and then harvested for ICAM-1 mRNA ( left panel ) and ICAM-1 protein ( right panel ), which was measured using real time PCR and western blot analysis, respectively. The values represent mRNA levels normalized to that of GAPDH mRNA. B. The effect of HO-1 on TPA-induced ICAM-1 mRNA expression in HT-29 cells. The cells were pretreated with 0–20 μM hemin for 1 h followed by stimulation with 10 ng/ml TPA for an additional 10 h after which the cells were harvested for RNA preparation. RNA samples were analyzed by real time PCR to determine the levels of ICAM-1 mRNA. The values represent mRNA levels normalized to that of GAPDH mRNA. C. The effect of HO-1 on the p65 intranuclear translocation ( upper panel ) and on the NF-κB DNA binding activity ( lower panel ) in TPA-stimulated HT-29 cells. HT-29 cells were pretreated with or without 20 μM hemin for 1 h followed by incubation with 10 ng/ml TPA for the indicated intervals. The cells were harvested and the nuclear levels of NF-κBp65 and NF-κB DNA binding activity were determined by western blot and DNA binding assay, respectively. D. The effect of HO-1 on TPA-induced ICAM-1 protein expression in HT-29 cells. The cells were pretreated with 0–20 μM hemin for 1 h and were subsequently stimulated with 10 ng/ml TPA for an additional 24 h. The cells were harvested for ICAM-1 protein expression by western blot analysis. E. The effects of HO-1 on TPA-induced ICAM-1 mRNA and protein expression in Caco-2 cells. The cells were pretreated with 0–20 μM hemin for 1 h and were subsequently stimulated with 10 ng/ml TPA. The cells were harvested for ICAM-1 mRNA ( left panel ) and protein ( right panel ) expression by RT-PCR and western blot analysis, respectively. F. The effect of HO-1 silencing on the hemin-mediated reduction of ICAM-1 in TPA-induced HT-29 cells. HT-29 cells were transfected with siRNA against HO-1 (HO-1 si) or control siRNA (Ctrl si). After pretreatment with 20 μM hemin, and then stimulated with 10 ng/ml TPA for an additional 24 h. The expression levels of ICAM-1 and HO-1 were analyzed by western blot analysis. G. The effect of ZnPP on HO-1 activity ( left panel ) and TPA-induced ICAM-1 protein expression ( right panel ) in HT-29 cells. The cells were pretreated with 0–20 μM ZnPP for 1 h, followed by stimulation with 10 ng/ml TPA for an additional 24 h. The cells were harvested for measurement of HO activity and western blot analysis. The results were repeated in at least three independent experiments. The data are expressed as mean ± SD. ** P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Translocation Assay, Binding Assay, Activity Assay, Incubation, DNA Binding Assay, Reverse Transcription Polymerase Chain Reaction, Transfection

    7) Product Images from "Sodium hydrosulfide alleviates dexamethasone-induced cell senescence and dysfunction through targeting the miR-22/sirt1 pathway in osteoblastic MC3T3-E1 cells"

    Article Title: Sodium hydrosulfide alleviates dexamethasone-induced cell senescence and dysfunction through targeting the miR-22/sirt1 pathway in osteoblastic MC3T3-E1 cells

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2021.9669

    Impact of sirt1 siRNA on the protective role of NaHS against Dex-induced damage and senescence in osteoblastic MC3T3-E1 cells. Osteoblasts were transfected with control siRNA or sirt1 siRNA for 24 h, then subjected to treatment with vehicle; Dex and vehicle; or NaHS for 48 h. (A) Cell viability and (B) ALP activity as well as changes in the (C) mRNA and protein expression levels of sirt1 were assessed. Data are shown as the mean ± SEM (n=4). ** P
    Figure Legend Snippet: Impact of sirt1 siRNA on the protective role of NaHS against Dex-induced damage and senescence in osteoblastic MC3T3-E1 cells. Osteoblasts were transfected with control siRNA or sirt1 siRNA for 24 h, then subjected to treatment with vehicle; Dex and vehicle; or NaHS for 48 h. (A) Cell viability and (B) ALP activity as well as changes in the (C) mRNA and protein expression levels of sirt1 were assessed. Data are shown as the mean ± SEM (n=4). ** P

    Techniques Used: Transfection, Activity Assay, Expressing

    8) Product Images from "Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab"

    Article Title: Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab

    Journal: Cell Cycle

    doi: 10.4161/cc.10.14.16418

    β-catenin is required for SP enrichment by IGF-1. (A) side population analysis of DLD1 cells (left panel) and CA-Akt DLD1 cells (right panel) with β-catenin knockdown by siRNA, (B) side population analysis of DLD1 cells treated with lithium
    Figure Legend Snippet: β-catenin is required for SP enrichment by IGF-1. (A) side population analysis of DLD1 cells (left panel) and CA-Akt DLD1 cells (right panel) with β-catenin knockdown by siRNA, (B) side population analysis of DLD1 cells treated with lithium

    Techniques Used:

    9) Product Images from "Up-regulation of BMP-2 antagonizes TGF-β1/ROCK-enhanced cardiac fibrotic signalling through activation of Smurf1/Smad6 complex"

    Article Title: Up-regulation of BMP-2 antagonizes TGF-β1/ROCK-enhanced cardiac fibrotic signalling through activation of Smurf1/Smad6 complex

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2012.01538.x

    BMP-2 inhibited TGF-β1-mediated PKC-δ and Smad3 signalling cascade through Smurf1/Smad6 interaction and activating Smad6. (A) Effect of rhBMP-2 (50 ng/ml) on time-dependent Smad6 and Smad7 expression in cultured cardiomyocytes. (B) Effect of TGF-β1 (10 ng/ml) on phosphorylation levels of PKC-δ and Smad3 in cells after 24 hr transfecting with or without Smad6 expression plasmid or pEZ-M29 plasmid, respectively. (C) Effect of TGF-β1 (10 ng/ml) on expression of TGF-β RI receptor and phosphorylated PKC-δ and Smad3 in the presence or absence of Smad6 expression plasmid in BMP-2 knock-down cardiomyocytes. (D) The effects of rhBMP-2 (50 ng/ml) supply or BMP-2 knock-down on TGF-β1 (10 ng/ml)-induced activation of PKC-δ and Smad3 in cells after 24 hr transfecting with or without Smad6 siRNA. (E) Effect of TGF-β1 (10 ng/ml) on expression of TGF-β RI receptor and phosphorylated PKC-δ and Smad3 in BMP-2 knock-down cardiomyocytes in the absence or presence of Smurf1 siRNA. Each bottom blot indicated equal loading of proteins normalized by β-actin. (F) Expression of Smad6 was examined in immunoprecipitated (IP) complex with anti-Smurf-1 antibody, and Smurf-1 in total cell lysates.
    Figure Legend Snippet: BMP-2 inhibited TGF-β1-mediated PKC-δ and Smad3 signalling cascade through Smurf1/Smad6 interaction and activating Smad6. (A) Effect of rhBMP-2 (50 ng/ml) on time-dependent Smad6 and Smad7 expression in cultured cardiomyocytes. (B) Effect of TGF-β1 (10 ng/ml) on phosphorylation levels of PKC-δ and Smad3 in cells after 24 hr transfecting with or without Smad6 expression plasmid or pEZ-M29 plasmid, respectively. (C) Effect of TGF-β1 (10 ng/ml) on expression of TGF-β RI receptor and phosphorylated PKC-δ and Smad3 in the presence or absence of Smad6 expression plasmid in BMP-2 knock-down cardiomyocytes. (D) The effects of rhBMP-2 (50 ng/ml) supply or BMP-2 knock-down on TGF-β1 (10 ng/ml)-induced activation of PKC-δ and Smad3 in cells after 24 hr transfecting with or without Smad6 siRNA. (E) Effect of TGF-β1 (10 ng/ml) on expression of TGF-β RI receptor and phosphorylated PKC-δ and Smad3 in BMP-2 knock-down cardiomyocytes in the absence or presence of Smurf1 siRNA. Each bottom blot indicated equal loading of proteins normalized by β-actin. (F) Expression of Smad6 was examined in immunoprecipitated (IP) complex with anti-Smurf-1 antibody, and Smurf-1 in total cell lysates.

    Techniques Used: Expressing, Cell Culture, Plasmid Preparation, Activation Assay, Immunoprecipitation

    Knocking down BMP-2 enhanced TGF-β1-mediated phosphorylation levels of PKC-δ and Smad3. (A) Quantitative PCR analyses of BMP-2 level in cardiomyocytes treated with special lentivirus-mediated anti-BMP-2 siRNA (LV-BMP2-siRNA) or LV-scramble siRNA, respectively for 24 hr. (B) TGF-β1 (0∼10 ng/ml)-induced phosphorylation levels of PKC-δ at Tyr 155 and Smad3 at Ser 423/425 were determined by Western blotting in cultured cardiomyocytes pre-treated with LV-BMP2-siRNA or LV-scramble-siRNA for 24 hr. (C) Western blotting analyses of the protein level of ROCK induced by TGF-β1 (0∼10 ng/ml) for 24 hr. (D) Quantitative PCR analyses of the mRNA level of BMP-2. Each bottom blot indicated equal loading of proteins normalized by total PKC, Smad3 or β-actin. Data were representative as means ± S.E.M. of n = 4 independent experiments.
    Figure Legend Snippet: Knocking down BMP-2 enhanced TGF-β1-mediated phosphorylation levels of PKC-δ and Smad3. (A) Quantitative PCR analyses of BMP-2 level in cardiomyocytes treated with special lentivirus-mediated anti-BMP-2 siRNA (LV-BMP2-siRNA) or LV-scramble siRNA, respectively for 24 hr. (B) TGF-β1 (0∼10 ng/ml)-induced phosphorylation levels of PKC-δ at Tyr 155 and Smad3 at Ser 423/425 were determined by Western blotting in cultured cardiomyocytes pre-treated with LV-BMP2-siRNA or LV-scramble-siRNA for 24 hr. (C) Western blotting analyses of the protein level of ROCK induced by TGF-β1 (0∼10 ng/ml) for 24 hr. (D) Quantitative PCR analyses of the mRNA level of BMP-2. Each bottom blot indicated equal loading of proteins normalized by total PKC, Smad3 or β-actin. Data were representative as means ± S.E.M. of n = 4 independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Cell Culture

    10) Product Images from "Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy"

    Article Title: Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2015.08.003

    ) (A) Quantitative PCR of FACS-purified HB9 + MNs derived from wild-type and SMA cultures at day 31. SMA MNs from 1-38G and 1-51N show higher expression of ER stress markers. Only 1-38G MNs show increased expression of markers characteristic of chronic ER stress: PERK , CHOP and CASP3 . Gene expression is normalized to GAPDH . (B) Co-staining of MN marker ISL1 (green) and ER stress marker ATF6 (red) in wild-type and SMA MN cultures. Scale bar indicates 25 μm. Nuclear ATF6 intensities of ISL1 + and ISL1 − cells are also measured. (C) Co-staining of motor neuron marker ISL1 (green) and ER stress marker ATF4 (red) in wild-type and SMA MN cultures. Scale bar indicates 100 μm. The graph depicts percentage of ATF4 + cells in whole cultures, as well as percentage of ISL1 + MNs co-expressing ATF4. (D–E) Quantification of ISL1 + MNs co-expressing ATF4 and cCASP3 respectively at days 23, 28 and 31, showing increasing ER stress over time, co-incident with increased apoptosis in SMA MNs. (F) Immunostaining analysis indicating that majority of MNs undergoing ER stress (ISL1 + ATF4 + MNs) also co-express ChAT. (G–H) qPCR and western blot analyses respectively show that knockdown of SMN in wild-type BJ-riPS MNs increased UPR target gene expression in a dose-dependent manner. (I) Co-staining of ISL1 (green) and ATF6 (red) in wild-type MNs transfected with non-targeting siRNA (si-NT) and SMN siRNA (si-SMN) at the indicated dose. The scale bar indicates 50 μm. Increase in nuclear ATF6 intensity upon SMN knockdown is graphically represented. (J) Co-staining of ISL1 (green) and ATF4 (red) in SMN knockdown BJ-riPS cultures. Scale bar indicates 100 μm. The graph shows increase in percentage of total cells and ISL1 + MNs co-expressing ATF4 in SMN knockdown conditions.
    Figure Legend Snippet: ) (A) Quantitative PCR of FACS-purified HB9 + MNs derived from wild-type and SMA cultures at day 31. SMA MNs from 1-38G and 1-51N show higher expression of ER stress markers. Only 1-38G MNs show increased expression of markers characteristic of chronic ER stress: PERK , CHOP and CASP3 . Gene expression is normalized to GAPDH . (B) Co-staining of MN marker ISL1 (green) and ER stress marker ATF6 (red) in wild-type and SMA MN cultures. Scale bar indicates 25 μm. Nuclear ATF6 intensities of ISL1 + and ISL1 − cells are also measured. (C) Co-staining of motor neuron marker ISL1 (green) and ER stress marker ATF4 (red) in wild-type and SMA MN cultures. Scale bar indicates 100 μm. The graph depicts percentage of ATF4 + cells in whole cultures, as well as percentage of ISL1 + MNs co-expressing ATF4. (D–E) Quantification of ISL1 + MNs co-expressing ATF4 and cCASP3 respectively at days 23, 28 and 31, showing increasing ER stress over time, co-incident with increased apoptosis in SMA MNs. (F) Immunostaining analysis indicating that majority of MNs undergoing ER stress (ISL1 + ATF4 + MNs) also co-express ChAT. (G–H) qPCR and western blot analyses respectively show that knockdown of SMN in wild-type BJ-riPS MNs increased UPR target gene expression in a dose-dependent manner. (I) Co-staining of ISL1 (green) and ATF6 (red) in wild-type MNs transfected with non-targeting siRNA (si-NT) and SMN siRNA (si-SMN) at the indicated dose. The scale bar indicates 50 μm. Increase in nuclear ATF6 intensity upon SMN knockdown is graphically represented. (J) Co-staining of ISL1 (green) and ATF4 (red) in SMN knockdown BJ-riPS cultures. Scale bar indicates 100 μm. The graph shows increase in percentage of total cells and ISL1 + MNs co-expressing ATF4 in SMN knockdown conditions.

    Techniques Used: Real-time Polymerase Chain Reaction, FACS, Purification, Derivative Assay, Expressing, Staining, Marker, Immunostaining, Western Blot, Transfection

    11) Product Images from "Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signaling"

    Article Title: Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signaling

    Journal: Nature communications

    doi: 10.1038/ncomms5421

    JZL184 alters Slit2 expression and localization in oligodendrocytes. ( a ) Oligodendrocytes ( solid arrowheads ), but not astrocytes, ( open arrowheads ) contained Slit2. ( b ) GFAP and CNPase were used to validate the purity of isolated astrocytes and oligodendrocytes, respectively. ( c ) Oligodendrocytes, but not astrocytes, contained mature Slit2. An additional antibody raised against a phylogenetically conserved epitope of Drosophila Slit confirmed this finding ( Slit ). ( d-f ) Slit2-like immunoreactivity localized to oligodendrocyte somas ( s ) and processes. Membranous staining pattern is shown in both e and f ( arrowheads ). Asterisks (*) indicate location of the nucleus. ( g,h ) Oligodendrocyte differentiation and increased Slit2 immunoreactivity, particularly in end-feet ( arrowheads ), upon JZL184 exposure. ( i ) Slit2 protein content in oligodendrocytes after JZL184 treatment. ( j ) JZL184-induced Slit2 accumulation in oligodendrocyte end-feet as measured by quantitative morphometry. ( k-k ) Effect of AM630, a CB 2 R antagonist, on JZL184-induced Slit2 accumulation in oligodendrocyte end-feet ( open and closed arrowheads ). ( o,p ) The effect of siRNA-mediated MGL inhibition on Slit2 expression in end-feet ( arrowheads ). ( q ) CNPase + oligodendroglial end-feet in the interbundle space (ibs) and in apposition to enlarged fascicles of Robo1 + corticofugal axons (cfa) in JZL184-treated mouse embryos. ( r ) Three-dimensional reconstruction of Slit2 in oligodendrocyte end-feet ( arrowheads ) adjacent to corticofugal axons. ( s ) Schematic of JZL184-induced remodeling of axonal pathfinding upon premature oligodendrocyte differentiation. Data were expressed as means ± s.e.m., n = 8 - 32 cells/group for each condition in duplicate experiments; * p
    Figure Legend Snippet: JZL184 alters Slit2 expression and localization in oligodendrocytes. ( a ) Oligodendrocytes ( solid arrowheads ), but not astrocytes, ( open arrowheads ) contained Slit2. ( b ) GFAP and CNPase were used to validate the purity of isolated astrocytes and oligodendrocytes, respectively. ( c ) Oligodendrocytes, but not astrocytes, contained mature Slit2. An additional antibody raised against a phylogenetically conserved epitope of Drosophila Slit confirmed this finding ( Slit ). ( d-f ) Slit2-like immunoreactivity localized to oligodendrocyte somas ( s ) and processes. Membranous staining pattern is shown in both e and f ( arrowheads ). Asterisks (*) indicate location of the nucleus. ( g,h ) Oligodendrocyte differentiation and increased Slit2 immunoreactivity, particularly in end-feet ( arrowheads ), upon JZL184 exposure. ( i ) Slit2 protein content in oligodendrocytes after JZL184 treatment. ( j ) JZL184-induced Slit2 accumulation in oligodendrocyte end-feet as measured by quantitative morphometry. ( k-k ) Effect of AM630, a CB 2 R antagonist, on JZL184-induced Slit2 accumulation in oligodendrocyte end-feet ( open and closed arrowheads ). ( o,p ) The effect of siRNA-mediated MGL inhibition on Slit2 expression in end-feet ( arrowheads ). ( q ) CNPase + oligodendroglial end-feet in the interbundle space (ibs) and in apposition to enlarged fascicles of Robo1 + corticofugal axons (cfa) in JZL184-treated mouse embryos. ( r ) Three-dimensional reconstruction of Slit2 in oligodendrocyte end-feet ( arrowheads ) adjacent to corticofugal axons. ( s ) Schematic of JZL184-induced remodeling of axonal pathfinding upon premature oligodendrocyte differentiation. Data were expressed as means ± s.e.m., n = 8 - 32 cells/group for each condition in duplicate experiments; * p

    Techniques Used: Expressing, Isolation, Staining, Inhibition

    CB 1 R stimulation regulates subcellular Robo1 positioning. ( a ) Robo1 and Robo2 immunoreactivity in the somata and neurites (including growth cone; arrowheads ) of CB 1 R + cortical neurons in vitro . ( b,c ) Cultured cortical neurons expressed both Robo1/Robo2 mRNA and protein. ( d ) Robo1 and Robo2 ( e ), fluorescence intensity in growth cones treated with JZL184 or O-2050, a CB 1 R antagonist. ( f ) Co-localization coefficient of Robo1 and CB 1 R in growth cones upon JZL184 exposure. ( g ) JZL184 induced Robo1 accumulation in growth cones ex vivo in isolated growth cone particles (GCPs). ( h ) Robo1 immunoreactivity adjacent to the growth cone in JZL184 treated neurons. ( i-k ) Robo1 immunoreactivity in growth cones upon siRNA-mediated MGL silencing in cultured primary neurons. ( l-n ) Effect of the genetic ablation of CB 1 Rs in cortical neurons on Robo1 receptor expression in growth cones after JZL184 or vehicle administration. ( o,p ) The effect of Erk1/2 (U0126) and JNK1 (SP600125) inhibition on Robo1 distribution. Data were expressed as means ± s.e.m., from triplicate experiments (sample size: 8-25 neurons/group in morphometric analysis), * p
    Figure Legend Snippet: CB 1 R stimulation regulates subcellular Robo1 positioning. ( a ) Robo1 and Robo2 immunoreactivity in the somata and neurites (including growth cone; arrowheads ) of CB 1 R + cortical neurons in vitro . ( b,c ) Cultured cortical neurons expressed both Robo1/Robo2 mRNA and protein. ( d ) Robo1 and Robo2 ( e ), fluorescence intensity in growth cones treated with JZL184 or O-2050, a CB 1 R antagonist. ( f ) Co-localization coefficient of Robo1 and CB 1 R in growth cones upon JZL184 exposure. ( g ) JZL184 induced Robo1 accumulation in growth cones ex vivo in isolated growth cone particles (GCPs). ( h ) Robo1 immunoreactivity adjacent to the growth cone in JZL184 treated neurons. ( i-k ) Robo1 immunoreactivity in growth cones upon siRNA-mediated MGL silencing in cultured primary neurons. ( l-n ) Effect of the genetic ablation of CB 1 Rs in cortical neurons on Robo1 receptor expression in growth cones after JZL184 or vehicle administration. ( o,p ) The effect of Erk1/2 (U0126) and JNK1 (SP600125) inhibition on Robo1 distribution. Data were expressed as means ± s.e.m., from triplicate experiments (sample size: 8-25 neurons/group in morphometric analysis), * p

    Techniques Used: In Vitro, Cell Culture, Fluorescence, Ex Vivo, Isolation, Expressing, Inhibition

    12) Product Images from "Down-Regulation of Deacetylase HDAC6 Inhibits the Melanoma Cell Line A375.S2 Growth through ROS-Dependent Mitochondrial Pathway"

    Article Title: Down-Regulation of Deacetylase HDAC6 Inhibits the Melanoma Cell Line A375.S2 Growth through ROS-Dependent Mitochondrial Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121247

    NAC blocked HDAC6 siRNA-induced apoptosis and mitochondrial dysfunction in A375.S2 cells. ( A ) ROS generation and ( B ) mitochondrial membrane potential (MMP) level were detected using DCFH2 and JC-1 staining respectively in H6-si-1 or-3-treated and NC-si-treated A375.S2 cells after pretreatment with or not with 5 mM NAC for 60 min. ( C ) The growth condition of H6-si-1 or-3 treated A375.S2 cells after pretreatment with or not with NAC ( ## P
    Figure Legend Snippet: NAC blocked HDAC6 siRNA-induced apoptosis and mitochondrial dysfunction in A375.S2 cells. ( A ) ROS generation and ( B ) mitochondrial membrane potential (MMP) level were detected using DCFH2 and JC-1 staining respectively in H6-si-1 or-3-treated and NC-si-treated A375.S2 cells after pretreatment with or not with 5 mM NAC for 60 min. ( C ) The growth condition of H6-si-1 or-3 treated A375.S2 cells after pretreatment with or not with NAC ( ## P

    Techniques Used: Staining

    13) Product Images from "Simulated ischemia/reperfusion-induced p65-Beclin 1-dependent autophagic cell death in human umbilical vein endothelial cells"

    Article Title: Simulated ischemia/reperfusion-induced p65-Beclin 1-dependent autophagic cell death in human umbilical vein endothelial cells

    Journal: Scientific Reports

    doi: 10.1038/srep37448

    Suppression of NF-κB p65 by PDTC or p65 siRNA diminished sI/R-induced development of autophagic vacuoles. ( A ) HUVECs subjected to normal media (control) or I/R for 4 h after either transfected with a scramble or p65 siRNA for 48 h or treated with 0.5 mM PDTC for 1 h, were labeled either with 50 μM MDC (upper) or 10 μM acridine orange (lower). Note the formation of green (upper) or red particles (lower)–accumulating autophagic vacuoles in I/R treatment cells (Confocal microscopy, Bar, 8 μm.). ( B ) The percentage of cells with MDC-stained dots and acridine orange–accumulating autophagic cells was quantified. Representative results of three independent experiments. *P
    Figure Legend Snippet: Suppression of NF-κB p65 by PDTC or p65 siRNA diminished sI/R-induced development of autophagic vacuoles. ( A ) HUVECs subjected to normal media (control) or I/R for 4 h after either transfected with a scramble or p65 siRNA for 48 h or treated with 0.5 mM PDTC for 1 h, were labeled either with 50 μM MDC (upper) or 10 μM acridine orange (lower). Note the formation of green (upper) or red particles (lower)–accumulating autophagic vacuoles in I/R treatment cells (Confocal microscopy, Bar, 8 μm.). ( B ) The percentage of cells with MDC-stained dots and acridine orange–accumulating autophagic cells was quantified. Representative results of three independent experiments. *P

    Techniques Used: Transfection, Labeling, Confocal Microscopy, Staining

    14) Product Images from "Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O2ˉ· production in cancer cells"

    Article Title: Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O2ˉ· production in cancer cells

    Journal: British Journal of Cancer

    doi: 10.1038/s41416-018-0288-2

    Mitochondrial superoxide production is due to mutp53-dependent UCP2 inhibition. a Panc1 mutR273H-p53 and AsPC1-p53 null cells were transfected with pRSuper-p53 and vector for mutant p53 ectopic expression, respectively, and their relative controls. In addition, Panc1 cells were co-transfected with siRNA-UCP2, or treated with 150 μM genipin for 24 h; while AsPC1 cells were co-transfected with the UCP2 vector. ROS production, corresponding to DCF fluorescence intensity, was analyzed by a multimode plate reader. * p
    Figure Legend Snippet: Mitochondrial superoxide production is due to mutp53-dependent UCP2 inhibition. a Panc1 mutR273H-p53 and AsPC1-p53 null cells were transfected with pRSuper-p53 and vector for mutant p53 ectopic expression, respectively, and their relative controls. In addition, Panc1 cells were co-transfected with siRNA-UCP2, or treated with 150 μM genipin for 24 h; while AsPC1 cells were co-transfected with the UCP2 vector. ROS production, corresponding to DCF fluorescence intensity, was analyzed by a multimode plate reader. * p

    Techniques Used: Inhibition, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Fluorescence

    Mutant p53 increases ΔΨm without damaging mtDNA. a Melanoma cell lines A375 (WTp53) and MeWo (mutant p53) were transfected with control or siRNA-p53. H 2 O 2 production was evaluated with the Amplex Red Kit, whereas UCP2 and p53 levels were determined by RT-qPCR. * p
    Figure Legend Snippet: Mutant p53 increases ΔΨm without damaging mtDNA. a Melanoma cell lines A375 (WTp53) and MeWo (mutant p53) were transfected with control or siRNA-p53. H 2 O 2 production was evaluated with the Amplex Red Kit, whereas UCP2 and p53 levels were determined by RT-qPCR. * p

    Techniques Used: Mutagenesis, Transfection, Quantitative RT-PCR

    15) Product Images from "Rapid non-uniform adaptation to conformation-specific KRASG12C inhibition"

    Article Title: Rapid non-uniform adaptation to conformation-specific KRASG12C inhibition

    Journal: Nature

    doi: 10.1038/s41586-019-1884-x

    Newly-synthesized KRAS G12C is reactivated to escape trapping by the drug. a , Quiescence biosensor-expressing cells (H358/p27K-, KRAS G12C +/− ) were transfected with KRAS- specific siRNAs targeting both wild-type and G12C alleles or only G12C for 72h and analyzed by FACS. The effect of a 72h G12Ci-treatment is shown. Inset: Cell extracts were immunoblotted and quantified to determine the intensity of KRAS expression and ERK phosphorylation. b , Effect of the indicated treatments on KRAS mRNA . FC: fold change, AcD: actinomycin D. c , Inhibitortreated cell extracts were analyzed by immunoblotting. d , e , Normalized KRAS expression across single-cells as a function of KRAS G12C -output score (d) or in quiescent vs. proliferating cells (e). f , H358 cells engineered to express HA-KRAS G12C under a dox-inducible promoter were treated with the G12Ci in the presence of dox (0-2 μg/mL). g , Biosensor expressing cells, engineered to stably express dox-inducible siRNA-resistant (siRes) KRAS G12C , were transfected with G12C-specific siRNA followed by dox treatment (100 ng/mL). A representative of three independent experiments are shown in a-c, f, g.
    Figure Legend Snippet: Newly-synthesized KRAS G12C is reactivated to escape trapping by the drug. a , Quiescence biosensor-expressing cells (H358/p27K-, KRAS G12C +/− ) were transfected with KRAS- specific siRNAs targeting both wild-type and G12C alleles or only G12C for 72h and analyzed by FACS. The effect of a 72h G12Ci-treatment is shown. Inset: Cell extracts were immunoblotted and quantified to determine the intensity of KRAS expression and ERK phosphorylation. b , Effect of the indicated treatments on KRAS mRNA . FC: fold change, AcD: actinomycin D. c , Inhibitortreated cell extracts were analyzed by immunoblotting. d , e , Normalized KRAS expression across single-cells as a function of KRAS G12C -output score (d) or in quiescent vs. proliferating cells (e). f , H358 cells engineered to express HA-KRAS G12C under a dox-inducible promoter were treated with the G12Ci in the presence of dox (0-2 μg/mL). g , Biosensor expressing cells, engineered to stably express dox-inducible siRNA-resistant (siRes) KRAS G12C , were transfected with G12C-specific siRNA followed by dox treatment (100 ng/mL). A representative of three independent experiments are shown in a-c, f, g.

    Techniques Used: Synthesized, Expressing, Transfection, FACS, Stable Transfection

    Inhibition of MAPK signaling stimulates new KRAS synthesis. a, The cells were treated with the indicated inhibitors and analyzed to determine the level of KRAS mRNA or protein expression (mean ± s.e.m., n=3) LFC: log 2 fold change relative to 0h. The indicated p values were determined by ANOVA (p=0.001) followed by pairwise comparisons vs. baseline, while correcting for multiple hypotheses (using Dunnett’s test in Prism). b , SW1573 (KRAS G12C+/+ ) cells were transfected with non-targeting (NT) or KRAS -specific siRNAs followed by treatment with the G12Ci and immunoblotting. c , H358 cells engineered to express HA-KRAS G12C under a dox-inducible promoter were treated with the G12Ci, alone or in the presence of dox, to determine the effect on cell viability at 72h (mean ± s.e.m., n=3). d , H358 p27K- cells were stably transfected with dox-inducible siRNA-resistant KRAS G12C (siRes-G12C). The cells were transfected with KRAS G12C -specific siRNA (siG12C) followed by dox (2 μg/mL) induction. The effect on cell viability is shown as mean ± s.e.m (n=5 for −dox and n=4 for +dox). A two-tailed t test p value is shown. e , H358 cells with dox-inducible HA-KRAS G12C were treated with dox (2 μg/mL) for 24h in serumfree medium. Then, the cells were exposed to either EGF (200 ng/mL) followed by the G12Ci (10 μM), or vice versa. Cell extracts were analyzed by RBD pull-down and immunoblotting. The specific effect on KRAS G12C was determined by the HA-tag. A representative of at least two independent experiments is shown in b, d, e. Unless otherwise indicated n denotes biological replicates.
    Figure Legend Snippet: Inhibition of MAPK signaling stimulates new KRAS synthesis. a, The cells were treated with the indicated inhibitors and analyzed to determine the level of KRAS mRNA or protein expression (mean ± s.e.m., n=3) LFC: log 2 fold change relative to 0h. The indicated p values were determined by ANOVA (p=0.001) followed by pairwise comparisons vs. baseline, while correcting for multiple hypotheses (using Dunnett’s test in Prism). b , SW1573 (KRAS G12C+/+ ) cells were transfected with non-targeting (NT) or KRAS -specific siRNAs followed by treatment with the G12Ci and immunoblotting. c , H358 cells engineered to express HA-KRAS G12C under a dox-inducible promoter were treated with the G12Ci, alone or in the presence of dox, to determine the effect on cell viability at 72h (mean ± s.e.m., n=3). d , H358 p27K- cells were stably transfected with dox-inducible siRNA-resistant KRAS G12C (siRes-G12C). The cells were transfected with KRAS G12C -specific siRNA (siG12C) followed by dox (2 μg/mL) induction. The effect on cell viability is shown as mean ± s.e.m (n=5 for −dox and n=4 for +dox). A two-tailed t test p value is shown. e , H358 cells with dox-inducible HA-KRAS G12C were treated with dox (2 μg/mL) for 24h in serumfree medium. Then, the cells were exposed to either EGF (200 ng/mL) followed by the G12Ci (10 μM), or vice versa. Cell extracts were analyzed by RBD pull-down and immunoblotting. The specific effect on KRAS G12C was determined by the HA-tag. A representative of at least two independent experiments is shown in b, d, e. Unless otherwise indicated n denotes biological replicates.

    Techniques Used: Inhibition, Expressing, Transfection, Stable Transfection, Two Tailed Test

    16) Product Images from "Synergistic antitumor effect of 5-fluorouracil and withaferin-A induces endoplasmic reticulum stress-mediated autophagy and apoptosis in colorectal cancer cells"

    Article Title: Synergistic antitumor effect of 5-fluorouracil and withaferin-A induces endoplasmic reticulum stress-mediated autophagy and apoptosis in colorectal cancer cells

    Journal: American Journal of Cancer Research

    doi:

    Combination induced apoptosis in CRC cells is dependent on PERK axis and is independent on IRE1α or ATF-6 axis of ER stress: (A) Immunoblotting analysis of knockdown CRC cells with PERK, IRE1α and ATF-6 siRNAs for 36 h, followed by combination treatment for 24 h, along with control siRNA and combination treatment alone shows the expression of ER stress proteins (CHOP, p-PERK, IRE1α, and ATF-6), and apoptosis (PARP1 and caspase-3) markers along β-actin as internal control. (B) CRC (HCT116) cells were transiently transfected with siRNAs to knockdown all the major three axis of ER stress sensors: PERK, IRE1α and ATF-6 and followed by combination treatment for 24 h, along with control siRNA and combination treatment alone. The cells were processed, subjected to Annexin V FITC and propidium iodide staining and analyzed by FACS. (C) Bar diagram showing quantification of Annexin V FITC positive cells analyzed by FACS under different settings. The data represent the mean value ± SE of 3 independent experiments. ** P
    Figure Legend Snippet: Combination induced apoptosis in CRC cells is dependent on PERK axis and is independent on IRE1α or ATF-6 axis of ER stress: (A) Immunoblotting analysis of knockdown CRC cells with PERK, IRE1α and ATF-6 siRNAs for 36 h, followed by combination treatment for 24 h, along with control siRNA and combination treatment alone shows the expression of ER stress proteins (CHOP, p-PERK, IRE1α, and ATF-6), and apoptosis (PARP1 and caspase-3) markers along β-actin as internal control. (B) CRC (HCT116) cells were transiently transfected with siRNAs to knockdown all the major three axis of ER stress sensors: PERK, IRE1α and ATF-6 and followed by combination treatment for 24 h, along with control siRNA and combination treatment alone. The cells were processed, subjected to Annexin V FITC and propidium iodide staining and analyzed by FACS. (C) Bar diagram showing quantification of Annexin V FITC positive cells analyzed by FACS under different settings. The data represent the mean value ± SE of 3 independent experiments. ** P

    Techniques Used: Expressing, Transfection, Staining, FACS

    17) Product Images from "Histone Deacetylase HDAC4 Promotes Gastric Cancer SGC-7901 Cells Progression via p21 Repression"

    Article Title: Histone Deacetylase HDAC4 Promotes Gastric Cancer SGC-7901 Cells Progression via p21 Repression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098894

    The expression of HDAC4 in transfected SGC-7901 cells. HDAC4 expression was determined in SGC-7901 cells transfected with empty vector (NC) or HDAC4 ( A ) and ( B ). HDAC4 expression was determined in SGC-7901 cells transfected with siRNA oligos targeting HDAC4 (si-HDAC4) or scrambled siRNA (si-NC) by qRT-PCR and western blot ( C ) and ( D ) (n = 4). Data were expressed as mean ± S.E.M. ***P
    Figure Legend Snippet: The expression of HDAC4 in transfected SGC-7901 cells. HDAC4 expression was determined in SGC-7901 cells transfected with empty vector (NC) or HDAC4 ( A ) and ( B ). HDAC4 expression was determined in SGC-7901 cells transfected with siRNA oligos targeting HDAC4 (si-HDAC4) or scrambled siRNA (si-NC) by qRT-PCR and western blot ( C ) and ( D ) (n = 4). Data were expressed as mean ± S.E.M. ***P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

    Roles of HDAC4 knockdown on SGC-7901 cell cycle, apoptosis and autophagy. Flow cytometry analysis depicted cell cycle progression of SGC-7901 cells after knockdown of HDAC4 ( A ) and the cell cycle profiles were analyzed to quantitate cell cycle distribution ( B ). The SGC-7901 cells transfected with scrambled control (si-NC) or HDAC4 siRNA oligos (si-HDAC4) apoptosis was evaluated by flow cytometry using Annexin V-FITC/PI ( C ). Expression of pro- and anti-apoptotic proteins and caspases 3 and 9 were assayed by western blot ( D ). The cells were immunostained with anti-LC3 antibodies (FITC, green) and nuclei were stained with DAPI (blue) and analyzed by confocal microscopy ( E ). Western blot analysis of Atg7, Beclin 1 and LC3 protein expression levels in SGC-7901 cells transfected with scrambled control (si-NC) or HDAC4 siRNA oligos (si-HDAC4) treated with or not with 3-MA ( F ). Data were expressed as mean ± S.E.M. *P
    Figure Legend Snippet: Roles of HDAC4 knockdown on SGC-7901 cell cycle, apoptosis and autophagy. Flow cytometry analysis depicted cell cycle progression of SGC-7901 cells after knockdown of HDAC4 ( A ) and the cell cycle profiles were analyzed to quantitate cell cycle distribution ( B ). The SGC-7901 cells transfected with scrambled control (si-NC) or HDAC4 siRNA oligos (si-HDAC4) apoptosis was evaluated by flow cytometry using Annexin V-FITC/PI ( C ). Expression of pro- and anti-apoptotic proteins and caspases 3 and 9 were assayed by western blot ( D ). The cells were immunostained with anti-LC3 antibodies (FITC, green) and nuclei were stained with DAPI (blue) and analyzed by confocal microscopy ( E ). Western blot analysis of Atg7, Beclin 1 and LC3 protein expression levels in SGC-7901 cells transfected with scrambled control (si-NC) or HDAC4 siRNA oligos (si-HDAC4) treated with or not with 3-MA ( F ). Data were expressed as mean ± S.E.M. *P

    Techniques Used: Flow Cytometry, Cytometry, Transfection, Expressing, Western Blot, Staining, Confocal Microscopy

    p21 knockdown reversed the effect of down-regulated HDAC4 on the inhibition of SGC-7901 cell growth. Expression of p21 was analyzed by western blot in SGC-7901 cells transfected with empty pcDNA3.1(+)-vector (NC) or HDAC4 and scrambled siRNA control (si-NC) or HDAC4 siRNA oligos (si-HDAC4) respectively ( A ). The p21 mRNA level was analyzed by qRT-PCR in SGC-7901 cells transfected with si-NC, si-HDAC4 alone or combination with siRNA p21 (si-p21) ( B ). The cell growth curve was measured by CCK-8 assay ( *P
    Figure Legend Snippet: p21 knockdown reversed the effect of down-regulated HDAC4 on the inhibition of SGC-7901 cell growth. Expression of p21 was analyzed by western blot in SGC-7901 cells transfected with empty pcDNA3.1(+)-vector (NC) or HDAC4 and scrambled siRNA control (si-NC) or HDAC4 siRNA oligos (si-HDAC4) respectively ( A ). The p21 mRNA level was analyzed by qRT-PCR in SGC-7901 cells transfected with si-NC, si-HDAC4 alone or combination with siRNA p21 (si-p21) ( B ). The cell growth curve was measured by CCK-8 assay ( *P

    Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR, CCK-8 Assay

    18) Product Images from "Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin-related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats"

    Article Title: Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin-related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats

    Journal: Journal of Biomedical Science

    doi: 10.1186/s12929-016-0262-3

    Successful delivery of siRNA into hippocampal CA1 subfield. Fluorescent double staining of FITC-siRNA (green) and DAPI (blue) were observed in the hippocampal CA1 subfield 24 h after injection of 400 nl FITC-siRNA (10 μM), which was distributed in the cytosol of hippocampal CA1 in both sham-control ( a ) and in the rats subjected to TGI-reperfusion for 4 h ( b ). Scale bar: 10 μm. I/R: ischemia/reperfusion
    Figure Legend Snippet: Successful delivery of siRNA into hippocampal CA1 subfield. Fluorescent double staining of FITC-siRNA (green) and DAPI (blue) were observed in the hippocampal CA1 subfield 24 h after injection of 400 nl FITC-siRNA (10 μM), which was distributed in the cytosol of hippocampal CA1 in both sham-control ( a ) and in the rats subjected to TGI-reperfusion for 4 h ( b ). Scale bar: 10 μm. I/R: ischemia/reperfusion

    Techniques Used: Double Staining, Injection

    19) Product Images from "Protein kinase C? mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expression and cell invasion in various human cancer cell lines via integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2)"

    Article Title: Protein kinase C? mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expression and cell invasion in various human cancer cell lines via integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2)

    Journal: Oncogene

    doi: 10.1038/onc.2010.63

    PKCε integrates with ERK1/2 to phosphorylate Stat3Ser727 DU145 cells were untransfected (lane 1), or transfected with nontargeting ERK1/2 siRNA (lane 2), or ERK1/2 specific siRNA (lane 3), or nontargeting PKCε siRNA (lane 4), or PKCε-specific siRNA (lane 5) (ERK1/2 specific siRNA from Santa Cruz Biotechnology, Santa Cruz, CA and PKCε-specific siRNA from Dharmacon Inc., Lafayette, CO), for 48hr and whole-cell lysates were prepared as described in Materials and Methods. a : The protein extracts (25 µg protein) were immunoblotted and indicated protein expression levels were detected with the appropriate antibodies. β-actin was used as a control for gel loading variations. b : The quantification of proteins (normalized to β-actin) was done as described in Materials and Methods. i : % of control not treated with PKCε-siRNA, ii : % of control not treated with ERK1/2-siRNA.
    Figure Legend Snippet: PKCε integrates with ERK1/2 to phosphorylate Stat3Ser727 DU145 cells were untransfected (lane 1), or transfected with nontargeting ERK1/2 siRNA (lane 2), or ERK1/2 specific siRNA (lane 3), or nontargeting PKCε siRNA (lane 4), or PKCε-specific siRNA (lane 5) (ERK1/2 specific siRNA from Santa Cruz Biotechnology, Santa Cruz, CA and PKCε-specific siRNA from Dharmacon Inc., Lafayette, CO), for 48hr and whole-cell lysates were prepared as described in Materials and Methods. a : The protein extracts (25 µg protein) were immunoblotted and indicated protein expression levels were detected with the appropriate antibodies. β-actin was used as a control for gel loading variations. b : The quantification of proteins (normalized to β-actin) was done as described in Materials and Methods. i : % of control not treated with PKCε-siRNA, ii : % of control not treated with ERK1/2-siRNA.

    Techniques Used: Transfection, Expressing

    PKCε integrates with MAPK cascade to phosphorylate Stat3Ser727 a : Tissue extracts of prostate cancer from TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice (50 µg protein) were used for reciprocal IP experiments with antibodies specific to PKCε, Stat3, Raf-1, MEK1/2, and ERK1/2. The immunoprecipitates were subjected to western blot analysis using the indicated antibodies. b, c and d : DU145 cells were transfected with non-targeting siRNA (Lanes 1 and 2) or PKCε specific siRNA (Lanes 3 and 4) (from Dharmacon Inc., Lafayette, CO), and whole cell lysates were prepared as described before (3). The lysates (25 µg protein) were immunoblotted and indicated protein expression levels were detected with the appropriate antibodies. β-actin was used as a control for gel loading variations. Protein quantification (normalized to β-actin) was performed as described in Materials and Methods. Each value is the mean ± S.E. of three independent experiments. e : EMSA . DU145 total cells were suspended in buffer A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MagCl 2 , 10 mmol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L PMSF]. After 15 min of incubation on ice, the cells were pelleted and resuspended in buffer B [20 mmol/L HEPES (pH 7.9), 20 mmol/L NaF, 1.5 mmol/L MgCl 2 , 1 mmol/L Na 3 VO 4 , 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, 420 mmol/L NaCl, 20% glycerol, 1 µg/mL leupeptin, 1 µg/mL aprotinin]. The samples were then centrifuged and the clear supernatant was used for EMSA as described in Materials and Methods. Lane 1, free probe only, Lane 2, nontargeting; lane 3, PKCε siRNA and Lane 4, mutant probe.
    Figure Legend Snippet: PKCε integrates with MAPK cascade to phosphorylate Stat3Ser727 a : Tissue extracts of prostate cancer from TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice (50 µg protein) were used for reciprocal IP experiments with antibodies specific to PKCε, Stat3, Raf-1, MEK1/2, and ERK1/2. The immunoprecipitates were subjected to western blot analysis using the indicated antibodies. b, c and d : DU145 cells were transfected with non-targeting siRNA (Lanes 1 and 2) or PKCε specific siRNA (Lanes 3 and 4) (from Dharmacon Inc., Lafayette, CO), and whole cell lysates were prepared as described before (3). The lysates (25 µg protein) were immunoblotted and indicated protein expression levels were detected with the appropriate antibodies. β-actin was used as a control for gel loading variations. Protein quantification (normalized to β-actin) was performed as described in Materials and Methods. Each value is the mean ± S.E. of three independent experiments. e : EMSA . DU145 total cells were suspended in buffer A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MagCl 2 , 10 mmol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L PMSF]. After 15 min of incubation on ice, the cells were pelleted and resuspended in buffer B [20 mmol/L HEPES (pH 7.9), 20 mmol/L NaF, 1.5 mmol/L MgCl 2 , 1 mmol/L Na 3 VO 4 , 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, 420 mmol/L NaCl, 20% glycerol, 1 µg/mL leupeptin, 1 µg/mL aprotinin]. The samples were then centrifuged and the clear supernatant was used for EMSA as described in Materials and Methods. Lane 1, free probe only, Lane 2, nontargeting; lane 3, PKCε siRNA and Lane 4, mutant probe.

    Techniques Used: Transgenic Assay, Mouse Assay, Western Blot, Transfection, Expressing, Incubation, Mutagenesis

    20) Product Images from "Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway"

    Article Title: Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0147509

    The non-canonical NF-κB pathway is required for CTB-INS-induced IDO1 expression in monocyte-derived DCs. Panel (A) shows NIK mRNA levels in monocyte-derived DCs transfected with NIK-specific siRNA (siNIK) or negative control (siC) examined by RT-PCR using NIK specific primers. The β-actin gene was used as an internal standard in RT-PCR. This image is representative of two independent experiments. Panel (B) graphic representation of NIK mRNA levels in CTB-INS vaccinated and unvaccinated DCs. Panel (C) show the expression of IDO1 protein and phosphorylated IKKα in mDCs transfected with NIK-specific siRNA (siNIK) or negative control (siC) examined by Western blot analysis using anti-IDO1 as the primary antibody. This image is representative of three independent experiments. Panel (D) shows graphic representation of the cell viability of non-transfected or transfected with NIK-specific siRNA (siNIK) or negative control (siC) in mDCs. Dendritic cell viability was measured by determination of the percentage of vaccinated DCs negative for annexin V and propidium iodide. NT means non-siRNA CTB-INS transfected DCs, and–or + means without CTB-proINS and with CTB-INS. Samples were assayed in triplicates and the results represent the mean ± SD of three independent experiments p = 0.002. Statistical analysis was performed using one way analysis of variance (ANOVA).
    Figure Legend Snippet: The non-canonical NF-κB pathway is required for CTB-INS-induced IDO1 expression in monocyte-derived DCs. Panel (A) shows NIK mRNA levels in monocyte-derived DCs transfected with NIK-specific siRNA (siNIK) or negative control (siC) examined by RT-PCR using NIK specific primers. The β-actin gene was used as an internal standard in RT-PCR. This image is representative of two independent experiments. Panel (B) graphic representation of NIK mRNA levels in CTB-INS vaccinated and unvaccinated DCs. Panel (C) show the expression of IDO1 protein and phosphorylated IKKα in mDCs transfected with NIK-specific siRNA (siNIK) or negative control (siC) examined by Western blot analysis using anti-IDO1 as the primary antibody. This image is representative of three independent experiments. Panel (D) shows graphic representation of the cell viability of non-transfected or transfected with NIK-specific siRNA (siNIK) or negative control (siC) in mDCs. Dendritic cell viability was measured by determination of the percentage of vaccinated DCs negative for annexin V and propidium iodide. NT means non-siRNA CTB-INS transfected DCs, and–or + means without CTB-proINS and with CTB-INS. Samples were assayed in triplicates and the results represent the mean ± SD of three independent experiments p = 0.002. Statistical analysis was performed using one way analysis of variance (ANOVA).

    Techniques Used: CtB Assay, Expressing, Derivative Assay, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

    21) Product Images from "Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation"

    Article Title: Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation

    Journal: Scientific Reports

    doi: 10.1038/srep36266

    ILK/β-catenin/p63 signaling cascade expressed by human OMECs isolated by dispase II/trypsin-EDTA (D/T) or collagenase (Col). ( A , B ) Upon OMEC attachment to the basement membrane via integrin receptors, ILK is phosphorylated and activated. This not only phosphorylates and activates AKT but also phosphorylates and inactivates GSK-3β, such that β-catenin can avoid degradation and translocate to the nucleus. In the nucleus, β-catenin works with other transcription factors, such as TCF4, to turn on Wnt-associated genes, such as ΔNp63 and cyclin D1, to promote cell proliferation (activated ΔNp63 down-regulates p27 KIP1 ). ( C ) Effect of ILK silencing on the signaling pathway. The causal relationship of the ILK/β-catenin/p63 pathway was confirmed by transfecting ILK siRNA into OMECs isolated by collagenase. Consequently, when the ILK pathway was blocked, nuclear β-catenin translocation, p63 expression, and cell cycle mediators were all significantly suppressed. ( D ) The immunoreactive band density of each experiment was quantified, first by normalization by the density of internal control (GAPDH or histone H3), then the relative signal intensity was expressed as D/T group divided by Col. group (with or without ILK silencing). Data are expressed as mean ± SD from three independent experiments. * p
    Figure Legend Snippet: ILK/β-catenin/p63 signaling cascade expressed by human OMECs isolated by dispase II/trypsin-EDTA (D/T) or collagenase (Col). ( A , B ) Upon OMEC attachment to the basement membrane via integrin receptors, ILK is phosphorylated and activated. This not only phosphorylates and activates AKT but also phosphorylates and inactivates GSK-3β, such that β-catenin can avoid degradation and translocate to the nucleus. In the nucleus, β-catenin works with other transcription factors, such as TCF4, to turn on Wnt-associated genes, such as ΔNp63 and cyclin D1, to promote cell proliferation (activated ΔNp63 down-regulates p27 KIP1 ). ( C ) Effect of ILK silencing on the signaling pathway. The causal relationship of the ILK/β-catenin/p63 pathway was confirmed by transfecting ILK siRNA into OMECs isolated by collagenase. Consequently, when the ILK pathway was blocked, nuclear β-catenin translocation, p63 expression, and cell cycle mediators were all significantly suppressed. ( D ) The immunoreactive band density of each experiment was quantified, first by normalization by the density of internal control (GAPDH or histone H3), then the relative signal intensity was expressed as D/T group divided by Col. group (with or without ILK silencing). Data are expressed as mean ± SD from three independent experiments. * p

    Techniques Used: Isolation, Translocation Assay, Expressing

    22) Product Images from "SENP3 grants tight junction integrity and cytoskeleton architecture in mouse Sertoli cells"

    Article Title: SENP3 grants tight junction integrity and cytoskeleton architecture in mouse Sertoli cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16915

    SENP3 knockdown disrupts actin microfilaments reorganization in Sertoli cells by dysfunction of Arp3 and Profilin-1 (A) Effects of SENP3 on the actin cytoskeleton in Sertoli cells. Sertoli cells cultured for 2 d were transfected with control or Senp3 siRNA. Two days thereafter, Sertoli cells were fixed and processed for FITC-phalloidin (F-actin, green) staining or immunofluorescence staining of SENP3 (red). DNA was visualized with DAPI (blue) staining. Significantly, Sertoli cells depleted for SENP3 were prominently featured by disorganized actin microfilaments with dense clusters (annotated by white asterisk). (B) Immunoblot analysis of selected regulators of actin dynamics as indicated, and the relative bands intensities normalized to GAPDH were shown in (C) . Notably, up-regulation of Arp3 and down-regulation of Rac1, CDC42, N-WASP, and Profilin-1 were observed by SENP3 depletion. Scale bars: 20 μm. Each bar in the histogram is a mean ±SD of n=3 experiments. *, p
    Figure Legend Snippet: SENP3 knockdown disrupts actin microfilaments reorganization in Sertoli cells by dysfunction of Arp3 and Profilin-1 (A) Effects of SENP3 on the actin cytoskeleton in Sertoli cells. Sertoli cells cultured for 2 d were transfected with control or Senp3 siRNA. Two days thereafter, Sertoli cells were fixed and processed for FITC-phalloidin (F-actin, green) staining or immunofluorescence staining of SENP3 (red). DNA was visualized with DAPI (blue) staining. Significantly, Sertoli cells depleted for SENP3 were prominently featured by disorganized actin microfilaments with dense clusters (annotated by white asterisk). (B) Immunoblot analysis of selected regulators of actin dynamics as indicated, and the relative bands intensities normalized to GAPDH were shown in (C) . Notably, up-regulation of Arp3 and down-regulation of Rac1, CDC42, N-WASP, and Profilin-1 were observed by SENP3 depletion. Scale bars: 20 μm. Each bar in the histogram is a mean ±SD of n=3 experiments. *, p

    Techniques Used: Cell Culture, Transfection, Staining, Immunofluorescence

    SENP3 depletion intervene SUMO-2/3-ylation profile in Sertoli cells Sertoli cells cultured for 3 d were processed to introduction of SENP3 siRNA or control siRNA at 100 nM, respectively, for 48 h prior to further assays. (A) Expression of SENP3 in Sertoli cells determined by RT-PCR analysis using primer specific to SENP3 with S16 served as the loading control. (B) Sertoli cells were lysed for determination of SENP3 expression by immunoblot analysis with anti-SENP3 antibody. Relative band intensity normalized to β-actin was shown in the below panel. SUMO2/3-ylation (C) and SUMO1-ylation (D) dynamics were detected in Sertoli cells. Each bar in the graph was presented as mean ±SD of at least three independent experiments. *, p
    Figure Legend Snippet: SENP3 depletion intervene SUMO-2/3-ylation profile in Sertoli cells Sertoli cells cultured for 3 d were processed to introduction of SENP3 siRNA or control siRNA at 100 nM, respectively, for 48 h prior to further assays. (A) Expression of SENP3 in Sertoli cells determined by RT-PCR analysis using primer specific to SENP3 with S16 served as the loading control. (B) Sertoli cells were lysed for determination of SENP3 expression by immunoblot analysis with anti-SENP3 antibody. Relative band intensity normalized to β-actin was shown in the below panel. SUMO2/3-ylation (C) and SUMO1-ylation (D) dynamics were detected in Sertoli cells. Each bar in the graph was presented as mean ±SD of at least three independent experiments. *, p

    Techniques Used: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

    SENP3 depletion compromises Sertoli cell BTB function (A) Schematic representation of the regime used in vitro assessment of BTB function. (B) Graph showing the integrity of TJ permeability barrier. Control and SENP3-depleted Sertoli cells processed as in A were prepared prior to determination of TJ permeability barrier function by trans-epithelial electrical resistance (TER) detection. The TER was detected up to 7 d. Date was presented as mean ± SD of 5 independent replicates. (C) Sertoli cells depleted for SENP3 were prepared for immunoblot analysis of those factors required for BTB function, such as TJ proteins (ZO-1 and occludin), basal ES protein (β-catenin and N-cadherin), GJ protein (Connexin 43 and p-Connexin 43), and Hemidesome protein (β1-integrin). The relative abundances normalized to GAPDH was shown in the below panel. Of note, SENP3 depletion (~80%) by introducing 100 nM senp3 siRNA incites significant decrease in TJ protein as well as in basal ES protein, while the abundances of GJ protein remain unchanged. (D) Representative confocal Z-projections showing the distribution of TJ protein at cell-cell interface. Sertoli cells cultured for 2 d were transfected with control or senp3 siRNA. Two days thereafter, Sertoli cells were fixed and processed for immunofluorescence staining of ZO-1/occludin (red) and DAPI (DNA, blue). Notably, disruption of either ZO-1 or occludin (indicated by yellow triangle) at cell-cell interface was witnessed by SENP3 depletion. Scale bars: 20 μm. *, p
    Figure Legend Snippet: SENP3 depletion compromises Sertoli cell BTB function (A) Schematic representation of the regime used in vitro assessment of BTB function. (B) Graph showing the integrity of TJ permeability barrier. Control and SENP3-depleted Sertoli cells processed as in A were prepared prior to determination of TJ permeability barrier function by trans-epithelial electrical resistance (TER) detection. The TER was detected up to 7 d. Date was presented as mean ± SD of 5 independent replicates. (C) Sertoli cells depleted for SENP3 were prepared for immunoblot analysis of those factors required for BTB function, such as TJ proteins (ZO-1 and occludin), basal ES protein (β-catenin and N-cadherin), GJ protein (Connexin 43 and p-Connexin 43), and Hemidesome protein (β1-integrin). The relative abundances normalized to GAPDH was shown in the below panel. Of note, SENP3 depletion (~80%) by introducing 100 nM senp3 siRNA incites significant decrease in TJ protein as well as in basal ES protein, while the abundances of GJ protein remain unchanged. (D) Representative confocal Z-projections showing the distribution of TJ protein at cell-cell interface. Sertoli cells cultured for 2 d were transfected with control or senp3 siRNA. Two days thereafter, Sertoli cells were fixed and processed for immunofluorescence staining of ZO-1/occludin (red) and DAPI (DNA, blue). Notably, disruption of either ZO-1 or occludin (indicated by yellow triangle) at cell-cell interface was witnessed by SENP3 depletion. Scale bars: 20 μm. *, p

    Techniques Used: In Vitro, Permeability, Cell Culture, Transfection, Immunofluorescence, Staining

    SENP3 involves in MAPK signaling pathway in Sertoli cells Sertoli cells cultured for 3 d were transfected with 100 nM senp3 siRNA or control siRNA for 48 h followed by further analysis. ( A and B ) Immunoblot analysis of the well-known components in MAPK signaling pathways were indicated (A) , and their relative abundances normalized to GAPDH were shown in (B) . (C) Graphs shown were the relative mRNA levels of p-Elk1 target gene c-fos , Mmp2 and profilin1 by qRT-PCR analysis at 48 h post transfection, and normalized to S16 by 2 −ΔΔCT method. Each bar was presented as mean ±SD of n=3 experiments. *, p
    Figure Legend Snippet: SENP3 involves in MAPK signaling pathway in Sertoli cells Sertoli cells cultured for 3 d were transfected with 100 nM senp3 siRNA or control siRNA for 48 h followed by further analysis. ( A and B ) Immunoblot analysis of the well-known components in MAPK signaling pathways were indicated (A) , and their relative abundances normalized to GAPDH were shown in (B) . (C) Graphs shown were the relative mRNA levels of p-Elk1 target gene c-fos , Mmp2 and profilin1 by qRT-PCR analysis at 48 h post transfection, and normalized to S16 by 2 −ΔΔCT method. Each bar was presented as mean ±SD of n=3 experiments. *, p

    Techniques Used: Cell Culture, Transfection, Quantitative RT-PCR

    23) Product Images from "Autophagic degradation of SQSTM1 inhibits ovarian cancer motility by decreasing DICER1 and AGO2 to induce MIRLET7A-3P"

    Article Title: Autophagic degradation of SQSTM1 inhibits ovarian cancer motility by decreasing DICER1 and AGO2 to induce MIRLET7A-3P

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1501135

    Induction of autophagy increases MIRLET7A-3P miRNA expression in ovarian cancer cells. SKOV3 and KURAMOCHI cells were serum-starved or incubated with 3 μM rVP1 for various periods of time as indicated. RNA samples were collected and relative expression ratios of (A) MIRLET7A-3P (B) MIRLET7A-5P miRNA were determined using qPCR analysis. SKOV3 and KURAMOCHI cells were transfected with 10 nM (C) non-targeting (si-Cont) siRNA or siRNA targeting WIPI1, WIPI2, WIPI1 + 2 or (D) siRNA targeting ATG5, ATG7 and LC3B , as indicated. After transfection, SKOV3 cells were serum-starved for 6 h or incubated with 3 μM rVP1 for 24 h and KURAMOCHI cells were serum-starved or incubated with 3 μM rVP1 for 24 h. RNA samples were then collected and relative expression ratios of MIRLET7A-3P miRNA were determined using qPCR analysis. Data represent means ± SEM of 3 independent experiments. * P
    Figure Legend Snippet: Induction of autophagy increases MIRLET7A-3P miRNA expression in ovarian cancer cells. SKOV3 and KURAMOCHI cells were serum-starved or incubated with 3 μM rVP1 for various periods of time as indicated. RNA samples were collected and relative expression ratios of (A) MIRLET7A-3P (B) MIRLET7A-5P miRNA were determined using qPCR analysis. SKOV3 and KURAMOCHI cells were transfected with 10 nM (C) non-targeting (si-Cont) siRNA or siRNA targeting WIPI1, WIPI2, WIPI1 + 2 or (D) siRNA targeting ATG5, ATG7 and LC3B , as indicated. After transfection, SKOV3 cells were serum-starved for 6 h or incubated with 3 μM rVP1 for 24 h and KURAMOCHI cells were serum-starved or incubated with 3 μM rVP1 for 24 h. RNA samples were then collected and relative expression ratios of MIRLET7A-3P miRNA were determined using qPCR analysis. Data represent means ± SEM of 3 independent experiments. * P

    Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction, Transfection

    Nutrient depletion and rVP1 induce autophagy in KURAMOCHI ovarian cancer cells. (A) Cells were treated with Earle balanced salt solution (EBSS) or 3 μM rVP1 for 4 h in the presence or absence of 30 μM chloroquine (CQ) as indicated. Cells were then immunolabeled with anti-LC3B antibodies, followed by Alexa Fluor 488-conjugated anti-IgG (green). Nuclei were stained with DAPI (blue). Fluorescent images were acquired by confocal microscopy. Scale bar: 20 μm. LC3B puncta per cell are represented as means ± SEM in 50 to 100 cells/experiment in 3 independent experiments. (B) Cells were incubated with or without 30 μM CQ for 30 min and then treated with EBSS or 3 μM rVP1 for 2 to 4 h as indicated. Cell lysates were collected and analyzed by immunoblotting using anti-LC3B and anti-SQSTM1 antibodies. SQSTM1 was used as an autophagic flux marker. (C) Cells were transfected with 10 nM non-targeting (si-Cont) siRNA or siRNA targeting BECN1, LC3B, WIPI1 and WIPI2 as indicated and then treated with or without EBSS or 3 μM rVP1 for 6 h in the presence of 30 μM CQ. Cell lysates were collected and analyzed by immunoblotting using anti-BECN1, anti-WIPI1, anti-WIPI2 and anti-LC3B antibodies as indicated. ACTB was used as a loading control. Data represent means ± SEM of densitometric measurement of LC3B-II:LC3B-I and LC3B-II:ACTB from 3 independent experiments. (D) Cells were transfected with 10 nM of si-Cont siRNA or siRNA targeting BECN1 as indicated then treated with or without EBSS or 3 μM rVP1 for 4 h. Cells were collected and double-membrane autophagosome structures were observed with transmission electron microscopy and indicated by arrows. Scale bar: 0.5 μm. The autophagosome pointed by red arrows were shown in insets with scale 0.2 μm. Data represent means ± SEM of volume fraction of autophagic compartments. N.S., not significant, * P
    Figure Legend Snippet: Nutrient depletion and rVP1 induce autophagy in KURAMOCHI ovarian cancer cells. (A) Cells were treated with Earle balanced salt solution (EBSS) or 3 μM rVP1 for 4 h in the presence or absence of 30 μM chloroquine (CQ) as indicated. Cells were then immunolabeled with anti-LC3B antibodies, followed by Alexa Fluor 488-conjugated anti-IgG (green). Nuclei were stained with DAPI (blue). Fluorescent images were acquired by confocal microscopy. Scale bar: 20 μm. LC3B puncta per cell are represented as means ± SEM in 50 to 100 cells/experiment in 3 independent experiments. (B) Cells were incubated with or without 30 μM CQ for 30 min and then treated with EBSS or 3 μM rVP1 for 2 to 4 h as indicated. Cell lysates were collected and analyzed by immunoblotting using anti-LC3B and anti-SQSTM1 antibodies. SQSTM1 was used as an autophagic flux marker. (C) Cells were transfected with 10 nM non-targeting (si-Cont) siRNA or siRNA targeting BECN1, LC3B, WIPI1 and WIPI2 as indicated and then treated with or without EBSS or 3 μM rVP1 for 6 h in the presence of 30 μM CQ. Cell lysates were collected and analyzed by immunoblotting using anti-BECN1, anti-WIPI1, anti-WIPI2 and anti-LC3B antibodies as indicated. ACTB was used as a loading control. Data represent means ± SEM of densitometric measurement of LC3B-II:LC3B-I and LC3B-II:ACTB from 3 independent experiments. (D) Cells were transfected with 10 nM of si-Cont siRNA or siRNA targeting BECN1 as indicated then treated with or without EBSS or 3 μM rVP1 for 4 h. Cells were collected and double-membrane autophagosome structures were observed with transmission electron microscopy and indicated by arrows. Scale bar: 0.5 μm. The autophagosome pointed by red arrows were shown in insets with scale 0.2 μm. Data represent means ± SEM of volume fraction of autophagic compartments. N.S., not significant, * P

    Techniques Used: Immunolabeling, Staining, Confocal Microscopy, Incubation, Marker, Transfection, Transmission Assay, Electron Microscopy

    Induction of autophagy inhibits ovarian cancer cell migration. (A) SKOV3 cells were transfected for 48 h with 10 nM non-targeting (si-Cont) siRNA or siRNA targeting WIPI1, WIPI2, WIPI1 + 2, ATG5, ATG7 and LC3B , as indicated. After transfection, migration trajectories of cells in response to 3 μM rVP1 or serum starvation were observed by time-lapse microscopy for 24 h and displayed in diagrams drawn with the initial point of each trajectory placed at the origin of the plot. (B) Migration velocities of SKOV3 cells were counted according to serial phase-contrast images every 15 min for 24 h as described in Materials and Methods. Data represent means ± SEM of migration velocities of 20 cells with different treatments. (C) KURAMOCHI cells were transfected with 10 nM of si-Cont or siRNA targeting WIPI1, WIPI2, WIPI1 + 2, ATG5, ATG7 and LC3B for 72 h and then incubated with or without EBSS medium or 3 μM rVP1 for 24 h, as indicated. After incubation, cells were collected and their migration ability was determined by transwell migration assay. Data represent means ± SEM of 3 independent experiments. * P
    Figure Legend Snippet: Induction of autophagy inhibits ovarian cancer cell migration. (A) SKOV3 cells were transfected for 48 h with 10 nM non-targeting (si-Cont) siRNA or siRNA targeting WIPI1, WIPI2, WIPI1 + 2, ATG5, ATG7 and LC3B , as indicated. After transfection, migration trajectories of cells in response to 3 μM rVP1 or serum starvation were observed by time-lapse microscopy for 24 h and displayed in diagrams drawn with the initial point of each trajectory placed at the origin of the plot. (B) Migration velocities of SKOV3 cells were counted according to serial phase-contrast images every 15 min for 24 h as described in Materials and Methods. Data represent means ± SEM of migration velocities of 20 cells with different treatments. (C) KURAMOCHI cells were transfected with 10 nM of si-Cont or siRNA targeting WIPI1, WIPI2, WIPI1 + 2, ATG5, ATG7 and LC3B for 72 h and then incubated with or without EBSS medium or 3 μM rVP1 for 24 h, as indicated. After incubation, cells were collected and their migration ability was determined by transwell migration assay. Data represent means ± SEM of 3 independent experiments. * P

    Techniques Used: Migration, Transfection, Time-lapse Microscopy, Incubation, Transwell Migration Assay

    24) Product Images from "Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation"

    Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05421-x

    Ssu72 controls inflammatory responses in vitro . ( A ) NIH-3T3 cells were transfected with a control siRNA or Ssu72 siRNA and stimulated with IL-6 (20 ng/ml) for 0.5 h. Cells were used to examine the p-STAT3 Tyr705 and p-STAT3 Ser727 levels. ( B ) Expression levels of the Ssu72 mRNA in cells transfected with the siRNAs were measured by real-time PCR. ( C ) NIH-3T3 cells were transfected with the Il17a promoter construct and either the siRNA control or siRNA Ssu72 to detect luciferase activity. ( D ) NIH-3T3 cells were transfected with siRNAs and stimulated with IL-6 (20 ng/ml) for 0.5 h. Real-time PCR was performed to measure the expression levels of the IL-1β , IL-17A , IL-21 , TBK1 , and IKBKE mRNAs. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P
    Figure Legend Snippet: Ssu72 controls inflammatory responses in vitro . ( A ) NIH-3T3 cells were transfected with a control siRNA or Ssu72 siRNA and stimulated with IL-6 (20 ng/ml) for 0.5 h. Cells were used to examine the p-STAT3 Tyr705 and p-STAT3 Ser727 levels. ( B ) Expression levels of the Ssu72 mRNA in cells transfected with the siRNAs were measured by real-time PCR. ( C ) NIH-3T3 cells were transfected with the Il17a promoter construct and either the siRNA control or siRNA Ssu72 to detect luciferase activity. ( D ) NIH-3T3 cells were transfected with siRNAs and stimulated with IL-6 (20 ng/ml) for 0.5 h. Real-time PCR was performed to measure the expression levels of the IL-1β , IL-17A , IL-21 , TBK1 , and IKBKE mRNAs. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P

    Techniques Used: In Vitro, Transfection, Expressing, Real-time Polymerase Chain Reaction, Construct, Luciferase, Activity Assay, MANN-WHITNEY

    25) Product Images from "Heterogeneous nuclear ribonucleoprotein K and nucleolin as transcriptional activators of the vascular endothelial growth factor promoter through interaction with secondary DNA structures"

    Article Title: Heterogeneous nuclear ribonucleoprotein K and nucleolin as transcriptional activators of the vascular endothelial growth factor promoter through interaction with secondary DNA structures

    Journal: Biochemistry

    doi: 10.1021/bi101633b

    siRNA KO of nucleolin and hnRNP K decreases VEGF mRNA levels
    Figure Legend Snippet: siRNA KO of nucleolin and hnRNP K decreases VEGF mRNA levels

    Techniques Used:

    26) Product Images from "Impact of Brachyury on epithelial-mesenchymal transitions and chemosensitivity in non-small cell lung cancer"

    Article Title: Impact of Brachyury on epithelial-mesenchymal transitions and chemosensitivity in non-small cell lung cancer

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2015.3348

    Impact of Brachyury expression on cell proliferation and invasion. (A) Expression of Brachyury following knockdown with siRNA in A549 cells. (B) Cell proliferation capacity of A549 cells was reduced following Brachyury knockdown ( * P
    Figure Legend Snippet: Impact of Brachyury expression on cell proliferation and invasion. (A) Expression of Brachyury following knockdown with siRNA in A549 cells. (B) Cell proliferation capacity of A549 cells was reduced following Brachyury knockdown ( * P

    Techniques Used: Expressing

    27) Product Images from "Inhibitor of growth protein 4 interacts with Beclin 1 and represses autophagy"

    Article Title: Inhibitor of growth protein 4 interacts with Beclin 1 and represses autophagy

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19033

    ING4 silencing induces autophagy A. Representative photomicrographs of ING4 knockdown (siING4A) HCT116 cells stably expressing GFP-LC3 treated with complete (CM) or nutrient free (NF) medium for 6h, in presence or absence of 1μM bafilomycin A1 (BAFA1) for the last 3h. The non-targeting siRNA (siUNR) was used as internal control of the experiments. Hoechst 33342 (Ho, blue) represents nuclear staining. GFP-LC3B puncta (green) correspond to autophagosomes. Scale bars: 10 μm. B. - E. Quantification of GFP-LC3B dots in ING4 knockdown HCT116 cells treated in CM or NF medium for 6h in presence or absence of BAFA1 for the last 3h. To knockdown ING4, 3 different siRNAs were used (siING4A, siING4B and siING4C). Western blot proves the efficiency of siING4s. Data are means ± SD ( n = 5). Statistical analysis was performed by Student's t test in comparison with the control condition, *** p
    Figure Legend Snippet: ING4 silencing induces autophagy A. Representative photomicrographs of ING4 knockdown (siING4A) HCT116 cells stably expressing GFP-LC3 treated with complete (CM) or nutrient free (NF) medium for 6h, in presence or absence of 1μM bafilomycin A1 (BAFA1) for the last 3h. The non-targeting siRNA (siUNR) was used as internal control of the experiments. Hoechst 33342 (Ho, blue) represents nuclear staining. GFP-LC3B puncta (green) correspond to autophagosomes. Scale bars: 10 μm. B. - E. Quantification of GFP-LC3B dots in ING4 knockdown HCT116 cells treated in CM or NF medium for 6h in presence or absence of BAFA1 for the last 3h. To knockdown ING4, 3 different siRNAs were used (siING4A, siING4B and siING4C). Western blot proves the efficiency of siING4s. Data are means ± SD ( n = 5). Statistical analysis was performed by Student's t test in comparison with the control condition, *** p

    Techniques Used: Stable Transfection, Expressing, Staining, Western Blot

    Related Articles

    Transfection:

    Article Title: MiR-375 Is Epigenetically Downregulated by HPV-16 E6 Mediated DNMT1 Upregulation and Modulates EMT of Cervical Cancer Cells by Suppressing lncRNA MALAT1
    Article Snippet: .. Cell transfection and treatmentSiHa and CaSki cells were transfected with 100 nM HPV-16 E6 siRNA (sc-156008, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 100 nM DNMT1 siRNA (Ribobio, Guangzhou, China) or 100 nM MALAT1 siRNAs (QIAGEN, Hilden, Germany) or the corresponding negative control siRNA according to the manufacturer's instructions. ..

    Article Title: Human papillomavirus regulates HER3 expression in head and neck cancer: implications for targeted HER3 therapy in HPV(+) patients
    Article Snippet: .. For siRNA-mediated silencing, cells were transiently transfected with two separate HER3 siRNAs (siHER3-1, used in ; ON-TARGETplus, SMARTpool #L-003127; GE Dharmacon, or siHER3-2, used in ; Origene HER3 siRNA #6263) or non-targeting siRNA (siNT; ON-TARGETplus Non-targeting Pool, #D-001810; Dharmacon). siRNAs targeting E6 and E7 were purchased from Santa Cruz Biotechnology, sc-156008 and sc-270423. ..

    Article Title: Human Papillomavirus Oncoprotein E6 Upregulates c-Met through p53 Downregulation
    Article Snippet: .. Cells were seeded into 6-cm or 6-well plates and reached 30–50% confluency before transfection. siRNAs for HPV16 E6, p53, and c-Met (Santa Cruz Biotechnology, Santa Cruz, CA) were complexed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and applied to each plate, respectively. ..

    Article Title: MiR-375 Is Epigenetically Downregulated by HPV-16 E6 Mediated DNMT1 Upregulation and Modulates EMT of Cervical Cancer Cells by Suppressing lncRNA MALAT1
    Article Snippet: .. SiHa and CaSki cells were transfected with DNMT1 siRNA ( ).The following qRT-PCR assay showed that both HPV-16 E6 siRNA ( ) and DNMT1 siRNA ( ) significantly increased miR-375 expression. ..

    Negative Control:

    Article Title: MiR-375 Is Epigenetically Downregulated by HPV-16 E6 Mediated DNMT1 Upregulation and Modulates EMT of Cervical Cancer Cells by Suppressing lncRNA MALAT1
    Article Snippet: .. Cell transfection and treatmentSiHa and CaSki cells were transfected with 100 nM HPV-16 E6 siRNA (sc-156008, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 100 nM DNMT1 siRNA (Ribobio, Guangzhou, China) or 100 nM MALAT1 siRNAs (QIAGEN, Hilden, Germany) or the corresponding negative control siRNA according to the manufacturer's instructions. ..

    Quantitative RT-PCR:

    Article Title: MiR-375 Is Epigenetically Downregulated by HPV-16 E6 Mediated DNMT1 Upregulation and Modulates EMT of Cervical Cancer Cells by Suppressing lncRNA MALAT1
    Article Snippet: .. SiHa and CaSki cells were transfected with DNMT1 siRNA ( ).The following qRT-PCR assay showed that both HPV-16 E6 siRNA ( ) and DNMT1 siRNA ( ) significantly increased miR-375 expression. ..

    Expressing:

    Article Title: MiR-375 Is Epigenetically Downregulated by HPV-16 E6 Mediated DNMT1 Upregulation and Modulates EMT of Cervical Cancer Cells by Suppressing lncRNA MALAT1
    Article Snippet: .. SiHa and CaSki cells were transfected with DNMT1 siRNA ( ).The following qRT-PCR assay showed that both HPV-16 E6 siRNA ( ) and DNMT1 siRNA ( ) significantly increased miR-375 expression. ..

    Sequencing:

    Article Title: MicroRNA-18a targeting of the STK4/MST1 tumour suppressor is necessary for transformation in HPV positive cervical cancer
    Article Snippet: .. siRNA and antagomirThe HPV16 E6 siRNA was purchased form Santa Cruz Biotechnology (USA) and had the following sequence: 5’–UGUGUACUGCAAGCAACAG– 3’. ..

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    Santa Cruz Biotechnology scramble non targeting shrna
    Knockdown of <t>HuR</t> enhances OS cell apoptosis, and inhibits cell viability and the EMT process. Knockdown efficiency of <t>Lenti-HuR-shRNA</t> was validated via (A) RT-qPCR and (B) western blot analysis. (C) Cells with or without HuR knockdown were subjected to an MTT assay to examine cell viability. (D) The mRNA expression level of proliferation marker ki67 was measured in the cells depicted in Fig. 2C . (E) Cell apoptosis assay was performed to detect the apoptotic rate of the cells depicted in Fig. 2C . (F) The protein expression level of apoptotic executor Cleaved caspase-3 was tested in cells depicted in Fig. 2C . EMT markers (E-cadherin and Vimentin) (G) mRNA and (H) protein expression levels were measured in cells depicted in Fig. 2C . *P
    Scramble Non Targeting Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology non targeting sirna control sirna
    Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were <t>transfected</t> with PKCε <t>siRNA</t> (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0
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    Santa Cruz Biotechnology control shrna lentiviral particles
    Knockdown of Cav-1 inhibits the P2Y 2 R-mediated ERK1/2 phosphorylation. A and B, hHAP2Y 2 R 1321N1 cells were uninfected ( A ) or infected ( B ) with Cav-1 <t>shRNA</t> <t>lentiviral</t> particles, then serum-starved for 16–24 h and stimulated with 100 μ m
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    Santa Cruz Biotechnology rna sirna
    Effects of Pin1 on the clonogenicity of breast cancer cells . (A) MDA-MB-231 cells were seeded in 6-well plates and treated with ATRA (25 μM) for 24 hours. Attached cells were photographed after crystal violet staining, and the proportion of attached cells was quantified by counting the number of colonies. Data are shown as the mean ± SD of three independent experiments, and the statistical significance was determined by Student’s t-test. (B) Stable H-Ras MCF10A cells were generated with control and Pin1 Lentivirus short hairpin <t>RNA.</t> Cells were then treated with PTX (100 nM) for 4 hours, and the proportion of attached cells was quantified by counting the number of colonies. (C) Effects of Pin1 knockout on the expression of apoptotic cell death markers in H-Ras MCF10A stable cells. Pin1, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1. **P
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    Knockdown of HuR enhances OS cell apoptosis, and inhibits cell viability and the EMT process. Knockdown efficiency of Lenti-HuR-shRNA was validated via (A) RT-qPCR and (B) western blot analysis. (C) Cells with or without HuR knockdown were subjected to an MTT assay to examine cell viability. (D) The mRNA expression level of proliferation marker ki67 was measured in the cells depicted in Fig. 2C . (E) Cell apoptosis assay was performed to detect the apoptotic rate of the cells depicted in Fig. 2C . (F) The protein expression level of apoptotic executor Cleaved caspase-3 was tested in cells depicted in Fig. 2C . EMT markers (E-cadherin and Vimentin) (G) mRNA and (H) protein expression levels were measured in cells depicted in Fig. 2C . *P

    Journal: Oncology Letters

    Article Title: RNA binding protein HuR promotes osteosarcoma cell progression via suppressing the miR-142-3p/HMGA1 axis

    doi: 10.3892/ol.2018.8855

    Figure Lengend Snippet: Knockdown of HuR enhances OS cell apoptosis, and inhibits cell viability and the EMT process. Knockdown efficiency of Lenti-HuR-shRNA was validated via (A) RT-qPCR and (B) western blot analysis. (C) Cells with or without HuR knockdown were subjected to an MTT assay to examine cell viability. (D) The mRNA expression level of proliferation marker ki67 was measured in the cells depicted in Fig. 2C . (E) Cell apoptosis assay was performed to detect the apoptotic rate of the cells depicted in Fig. 2C . (F) The protein expression level of apoptotic executor Cleaved caspase-3 was tested in cells depicted in Fig. 2C . EMT markers (E-cadherin and Vimentin) (G) mRNA and (H) protein expression levels were measured in cells depicted in Fig. 2C . *P

    Article Snippet: Lentivirus package shRNAs against HuR and a scramble non-targeting shRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

    Techniques: shRNA, Quantitative RT-PCR, Western Blot, MTT Assay, Expressing, Marker, Apoptosis Assay

    Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Immunofluorescence, Transfection, Imaging

    Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection

    Changes in autophagy gene expression in U-138 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-138 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Changes in autophagy gene expression in U-138 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-138 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Transfection

    Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Activation Assay, Transfection, Construct

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection

    Changes in autophagy gene expression in U-118 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-118 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Changes in autophagy gene expression in U-118 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-118 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Transfection

    Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection, Microscopy, Expressing

    Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection

    The effect of PRKCE silencing on U-138 MG and U-118 MG cells. Cells were harvested 72 h after transfection with non-targeting siRNA (Control siRNA) or PKCε siRNA. The downregulation of PRKCE mRNA expression ( a ) and PKCε protein level ( b ) are shown. The bar graphs present average values of three independent experiments. ** P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: The effect of PRKCE silencing on U-138 MG and U-118 MG cells. Cells were harvested 72 h after transfection with non-targeting siRNA (Control siRNA) or PKCε siRNA. The downregulation of PRKCE mRNA expression ( a ) and PKCε protein level ( b ) are shown. The bar graphs present average values of three independent experiments. ** P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection, Expressing

    Knockdown of Cav-1 inhibits the P2Y 2 R-mediated ERK1/2 phosphorylation. A and B, hHAP2Y 2 R 1321N1 cells were uninfected ( A ) or infected ( B ) with Cav-1 shRNA lentiviral particles, then serum-starved for 16–24 h and stimulated with 100 μ m

    Journal: The Journal of Biological Chemistry

    Article Title: Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells *

    doi: 10.1074/jbc.M116.730226

    Figure Lengend Snippet: Knockdown of Cav-1 inhibits the P2Y 2 R-mediated ERK1/2 phosphorylation. A and B, hHAP2Y 2 R 1321N1 cells were uninfected ( A ) or infected ( B ) with Cav-1 shRNA lentiviral particles, then serum-starved for 16–24 h and stimulated with 100 μ m

    Article Snippet: Cells were infected with either control shRNA lentiviral particles (scrambled or non-targeted shRNA) or caveolin-1 shRNA-containing lentiviral particles obtained from Santa Cruz Biotechnology, following the manufacturer's recommendations.

    Techniques: Infection, shRNA

    Cav-1 is necessary for the P2Y 2 R-mediated Akt phosphorylation. A and B, hHAP2Y 2 R 1321N1 cells were uninfected ( A ) or infected ( B ) with Cav-1 shRNA lentiviral particles, serum-starved for 16–24 h, and stimulated with 100 μ m ATP in DMEM

    Journal: The Journal of Biological Chemistry

    Article Title: Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells *

    doi: 10.1074/jbc.M116.730226

    Figure Lengend Snippet: Cav-1 is necessary for the P2Y 2 R-mediated Akt phosphorylation. A and B, hHAP2Y 2 R 1321N1 cells were uninfected ( A ) or infected ( B ) with Cav-1 shRNA lentiviral particles, serum-starved for 16–24 h, and stimulated with 100 μ m ATP in DMEM

    Article Snippet: Cells were infected with either control shRNA lentiviral particles (scrambled or non-targeted shRNA) or caveolin-1 shRNA-containing lentiviral particles obtained from Santa Cruz Biotechnology, following the manufacturer's recommendations.

    Techniques: Infection, shRNA

    Effects of Pin1 on the clonogenicity of breast cancer cells . (A) MDA-MB-231 cells were seeded in 6-well plates and treated with ATRA (25 μM) for 24 hours. Attached cells were photographed after crystal violet staining, and the proportion of attached cells was quantified by counting the number of colonies. Data are shown as the mean ± SD of three independent experiments, and the statistical significance was determined by Student’s t-test. (B) Stable H-Ras MCF10A cells were generated with control and Pin1 Lentivirus short hairpin RNA. Cells were then treated with PTX (100 nM) for 4 hours, and the proportion of attached cells was quantified by counting the number of colonies. (C) Effects of Pin1 knockout on the expression of apoptotic cell death markers in H-Ras MCF10A stable cells. Pin1, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1. **P

    Journal: Journal of Cancer Prevention

    Article Title: Interaction between Peptidyl-prolyl Cis-trans Isomerase NIMA-interacting 1 and GTP-H-Ras: Implications for Aggressiveness of Human Mammary Epithelial Cells and Drug Resistance

    doi: 10.15430/JCP.2020.25.4.234

    Figure Lengend Snippet: Effects of Pin1 on the clonogenicity of breast cancer cells . (A) MDA-MB-231 cells were seeded in 6-well plates and treated with ATRA (25 μM) for 24 hours. Attached cells were photographed after crystal violet staining, and the proportion of attached cells was quantified by counting the number of colonies. Data are shown as the mean ± SD of three independent experiments, and the statistical significance was determined by Student’s t-test. (B) Stable H-Ras MCF10A cells were generated with control and Pin1 Lentivirus short hairpin RNA. Cells were then treated with PTX (100 nM) for 4 hours, and the proportion of attached cells was quantified by counting the number of colonies. (C) Effects of Pin1 knockout on the expression of apoptotic cell death markers in H-Ras MCF10A stable cells. Pin1, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1. **P

    Article Snippet: Control and Pin1 targeting small interfering RNA (siRNA) were purchased from Santa Cruz Biotechnology.

    Techniques: Multiple Displacement Amplification, Staining, Generated, shRNA, Knock-Out, Expressing