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    Name:
    Control siRNA F
    Description:
    resuspend to 66 µl 10 µM sufficient for 10 20 transfections suitable as a negative control for experiments using targeted siRNA transfection consists of a scrambled sequence that will not lead to the specific degradation of any cellular message aliquot and store at 20°C
    Catalog Number:
    SC-44234
    Price:
    None
    Category:
    Gene Editing Control siRNA F
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    Structured Review

    Santa Cruz Biotechnology sirna
    <t>FoxO3a</t> mediates idelalisib induced Bim induction. a HepG2 cells were treated with 5 μmol/L idelalisib at indicated time point. E2F1, Egr-1 and myc expression was analyzed by western blotting and normalized to β-actin. The data represent the mean ± SD of three independent experiments. b HepG2 cells were transfected with either a control scrambled <t>siRNA</t> or a FoxO3a siRNA for 24 h, and then treated with 5 μmol/L idelalisib for 24 h. FoxO3a and Bim expression was analyzed by western blotting and normalized to β-actin. Data represent the mean ± SD of three independent experiments. ** P
    resuspend to 66 µl 10 µM sufficient for 10 20 transfections suitable as a negative control for experiments using targeted siRNA transfection consists of a scrambled sequence that will not lead to the specific degradation of any cellular message aliquot and store at 20°C
    https://www.bioz.com/result/sirna/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sirna - by Bioz Stars, 2021-09
    86/100 stars

    Images

    1) Product Images from "Idelalisib promotes Bim-dependent apoptosis through AKT/FoxO3a in hepatocellular carcinoma"

    Article Title: Idelalisib promotes Bim-dependent apoptosis through AKT/FoxO3a in hepatocellular carcinoma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0960-8

    FoxO3a mediates idelalisib induced Bim induction. a HepG2 cells were treated with 5 μmol/L idelalisib at indicated time point. E2F1, Egr-1 and myc expression was analyzed by western blotting and normalized to β-actin. The data represent the mean ± SD of three independent experiments. b HepG2 cells were transfected with either a control scrambled siRNA or a FoxO3a siRNA for 24 h, and then treated with 5 μmol/L idelalisib for 24 h. FoxO3a and Bim expression was analyzed by western blotting and normalized to β-actin. Data represent the mean ± SD of three independent experiments. ** P
    Figure Legend Snippet: FoxO3a mediates idelalisib induced Bim induction. a HepG2 cells were treated with 5 μmol/L idelalisib at indicated time point. E2F1, Egr-1 and myc expression was analyzed by western blotting and normalized to β-actin. The data represent the mean ± SD of three independent experiments. b HepG2 cells were transfected with either a control scrambled siRNA or a FoxO3a siRNA for 24 h, and then treated with 5 μmol/L idelalisib for 24 h. FoxO3a and Bim expression was analyzed by western blotting and normalized to β-actin. Data represent the mean ± SD of three independent experiments. ** P

    Techniques Used: Expressing, Western Blot, Transfection

    2) Product Images from "Muscle‐specific sirtuin1 gain‐of‐function ameliorates skeletal muscle atrophy in a pre‐clinical mouse model of cerebral ischemic stroke. Muscle‐specific sirtuin1 gain‐of‐function ameliorates skeletal muscle atrophy in a pre‐clinical mouse model of cerebral ischemic stroke"

    Article Title: Muscle‐specific sirtuin1 gain‐of‐function ameliorates skeletal muscle atrophy in a pre‐clinical mouse model of cerebral ischemic stroke. Muscle‐specific sirtuin1 gain‐of‐function ameliorates skeletal muscle atrophy in a pre‐clinical mouse model of cerebral ischemic stroke

    Journal: FASEB BioAdvances

    doi: 10.1096/fba.2020-00017

    PARP‐1 binds on ZNF216 promoter. Male, ~20‐w old, SirT1 +/+ and their age‐matched WT (C57BL/6J) control mice were subjected to 60’MCAO or sham surgery followed by 3 d of reperfusion. B‐C, Chromatin was isolated from paretic tibialis anterior (PTA) or the corresponding TA (CTA) muscle of sham mice from WT and SirT1 +/+ mice (B) or PARP‐1 siRNA transfected myotubes treated with 125 μmol/L of H 2 O 2 with or without resveratrol (C) and precipitated with anti‐PARP‐1, anti‐RNA Poly II, or nonspecific IgG. qPCRs were performed with primers specific for ZNF216 or GAPDH. Values in each graph indicate the mean ± SEM (n = 3)
    Figure Legend Snippet: PARP‐1 binds on ZNF216 promoter. Male, ~20‐w old, SirT1 +/+ and their age‐matched WT (C57BL/6J) control mice were subjected to 60’MCAO or sham surgery followed by 3 d of reperfusion. B‐C, Chromatin was isolated from paretic tibialis anterior (PTA) or the corresponding TA (CTA) muscle of sham mice from WT and SirT1 +/+ mice (B) or PARP‐1 siRNA transfected myotubes treated with 125 μmol/L of H 2 O 2 with or without resveratrol (C) and precipitated with anti‐PARP‐1, anti‐RNA Poly II, or nonspecific IgG. qPCRs were performed with primers specific for ZNF216 or GAPDH. Values in each graph indicate the mean ± SEM (n = 3)

    Techniques Used: Mouse Assay, Isolation, Transfection

    PARP‐1 regulates the ZNF216 gene. Male, ~20‐ w old, SirT1 +/+ mice and their age‐matched WT (C57BL/6J) control mice were subjected to 60’MCAO or sham surgery followed by 3 d of reperfusion. RNA or cell lysate were extracted from paretic tibialis anterior (PTA) or the corresponding TA (CTA) muscle of sham mice in WT and SirT1 +/+ mice (n = 4). A, qPCR data showed PARP‐1 mRNA levels in the PTA and CTA muscles of WT and SirT1 +/+ mice (n = 4). B, Representative western blot images showed global protein parylation (PARP activity) in the TA muscles of WT and SirT1 +/+ mice (n = 3). C, Myotubes generatred from primary myoblasts were transfected with PARP‐1 siRNAs or control siRNAs for 48 h and total RNA was isolated and used in qPCR data to measure PARP‐1 mRNA levels. D, PARP‐1 siRNA transfected myotubes were treated with 125 μmol/L of H 2 O 2 with or without resveratrol for 4 h to measure ZNF216 mRNA levels in the total RNA using qPCR (n = 4). Values in each graph indicate the mean ± SEM. * indicates comparison vs control. φ and δ indicate the comparison of the specific gene in the H 2 O 2 ‐treated group
    Figure Legend Snippet: PARP‐1 regulates the ZNF216 gene. Male, ~20‐ w old, SirT1 +/+ mice and their age‐matched WT (C57BL/6J) control mice were subjected to 60’MCAO or sham surgery followed by 3 d of reperfusion. RNA or cell lysate were extracted from paretic tibialis anterior (PTA) or the corresponding TA (CTA) muscle of sham mice in WT and SirT1 +/+ mice (n = 4). A, qPCR data showed PARP‐1 mRNA levels in the PTA and CTA muscles of WT and SirT1 +/+ mice (n = 4). B, Representative western blot images showed global protein parylation (PARP activity) in the TA muscles of WT and SirT1 +/+ mice (n = 3). C, Myotubes generatred from primary myoblasts were transfected with PARP‐1 siRNAs or control siRNAs for 48 h and total RNA was isolated and used in qPCR data to measure PARP‐1 mRNA levels. D, PARP‐1 siRNA transfected myotubes were treated with 125 μmol/L of H 2 O 2 with or without resveratrol for 4 h to measure ZNF216 mRNA levels in the total RNA using qPCR (n = 4). Values in each graph indicate the mean ± SEM. * indicates comparison vs control. φ and δ indicate the comparison of the specific gene in the H 2 O 2 ‐treated group

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Transfection, Isolation

    3) Product Images from "AKT regulates NLRP3 inflammasome activation by phosphorylating NLRP3 serine 5"

    Article Title: AKT regulates NLRP3 inflammasome activation by phosphorylating NLRP3 serine 5

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.2000649

    AKT is involved in NLRP3-mediated inflammasome activation. (A) Immunoblots of culture supernatants and the whole cell lysates of LPS-primed THP-1 cells treated with indicated doses of MK2206 (AKT inhibitor) or SC-79 (AKT activator) for 1 h, followed by LPS (100 ng/ml, 6 hrs), and Nigericin treatment (5 μM, 45 min). (B) Immunoblots of the culture supernatants (Sup) and whole cell lysates (WCL) of BMDMs treated with indicated stimuli: MK2206 (AKT inhibitor, 100 nM, pre-treated 1 h), SC-79 (AKT activator, 1 μg/ml, 1.5 hrs), and/or LPS (100 ng/ml, 6 hrs), Nigericin (5 μM, 45 min). ( C ) Immunoblots of the culture supernatants (Sup) and whole cell lysates (WCL) of BMDMs transfected with poly (dA:dT) (2 μg/ml, 6 hrs), and/or MK2206 (AKT inhibitor, 100 nM, pre-treated 1 h). (D) Immunoblots of the supernatant and the whole cell lysates of the control-siRNA or Akt1/2-siRNA transfected THP-1 cells for 24 hours, followed by LPS (100 ng/ml, 6 h) and/or Nigericin (5 μM, 45 min) treatment. ( E ) Quantification of the p-AKT and total-AKT protein levels in the control-siRNA or Akt1/2-siRNA transfected THP-1 cells. (F) Elisa assays of IL-1β (left), IL-6 (middle), and TNF-α (right) in supernatants from the BMDMs treated with the indicated stimuli: MK2206 (AKT inhibitor, 100 nM, pre-treated 1 h); LPS (100 ng/ml, 6 h), following by Nigericin (5 μM, 45 min) or ATP (2 mM, 1 h); 2μg/ml poly (dA:dT) transfected for 6 hours. UT- untreated. Immunoblots shown are representative of at least 3 independent experiments and band quantification is shown for the representative blot. All experiments were repeated at least three times, and bars represent the means ± SEM. *p
    Figure Legend Snippet: AKT is involved in NLRP3-mediated inflammasome activation. (A) Immunoblots of culture supernatants and the whole cell lysates of LPS-primed THP-1 cells treated with indicated doses of MK2206 (AKT inhibitor) or SC-79 (AKT activator) for 1 h, followed by LPS (100 ng/ml, 6 hrs), and Nigericin treatment (5 μM, 45 min). (B) Immunoblots of the culture supernatants (Sup) and whole cell lysates (WCL) of BMDMs treated with indicated stimuli: MK2206 (AKT inhibitor, 100 nM, pre-treated 1 h), SC-79 (AKT activator, 1 μg/ml, 1.5 hrs), and/or LPS (100 ng/ml, 6 hrs), Nigericin (5 μM, 45 min). ( C ) Immunoblots of the culture supernatants (Sup) and whole cell lysates (WCL) of BMDMs transfected with poly (dA:dT) (2 μg/ml, 6 hrs), and/or MK2206 (AKT inhibitor, 100 nM, pre-treated 1 h). (D) Immunoblots of the supernatant and the whole cell lysates of the control-siRNA or Akt1/2-siRNA transfected THP-1 cells for 24 hours, followed by LPS (100 ng/ml, 6 h) and/or Nigericin (5 μM, 45 min) treatment. ( E ) Quantification of the p-AKT and total-AKT protein levels in the control-siRNA or Akt1/2-siRNA transfected THP-1 cells. (F) Elisa assays of IL-1β (left), IL-6 (middle), and TNF-α (right) in supernatants from the BMDMs treated with the indicated stimuli: MK2206 (AKT inhibitor, 100 nM, pre-treated 1 h); LPS (100 ng/ml, 6 h), following by Nigericin (5 μM, 45 min) or ATP (2 mM, 1 h); 2μg/ml poly (dA:dT) transfected for 6 hours. UT- untreated. Immunoblots shown are representative of at least 3 independent experiments and band quantification is shown for the representative blot. All experiments were repeated at least three times, and bars represent the means ± SEM. *p

    Techniques Used: Activation Assay, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

    S5 phosphorylation prevents Trim31-mediated ubiquitination of NLRP3 at K496 site. (A) Immunoblots of the whole cell lysates of control-siRNA-, AKT1/2-siRNA-, or/and Trim31-siRNA-transfected LPS-primed THP-1 cells. The endogenous NLRP3 protein levels were normalized to control-siRNA transfected untreated sample (showed in fold). (B) Quantification of the Trim31 mRNA level in control-siRNA- or Trim31-siRNA-treated THP-1 cells. Control-siRNA or Trim31-siRNA was transfected into THP-1 cells for 48 hrs, and the trim31 mRNA level was tested from real-time PCR. Experiments were repeated three times, and bars represent the means ± SEM. *p
    Figure Legend Snippet: S5 phosphorylation prevents Trim31-mediated ubiquitination of NLRP3 at K496 site. (A) Immunoblots of the whole cell lysates of control-siRNA-, AKT1/2-siRNA-, or/and Trim31-siRNA-transfected LPS-primed THP-1 cells. The endogenous NLRP3 protein levels were normalized to control-siRNA transfected untreated sample (showed in fold). (B) Quantification of the Trim31 mRNA level in control-siRNA- or Trim31-siRNA-treated THP-1 cells. Control-siRNA or Trim31-siRNA was transfected into THP-1 cells for 48 hrs, and the trim31 mRNA level was tested from real-time PCR. Experiments were repeated three times, and bars represent the means ± SEM. *p

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

    4) Product Images from "Gβγ translocation to the Golgi apparatus activates ARF1 to spatiotemporally regulate G protein–coupled receptor signaling to MAPK"

    Article Title: Gβγ translocation to the Golgi apparatus activates ARF1 to spatiotemporally regulate G protein–coupled receptor signaling to MAPK

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.100805

    Inhibition and depletion of ARF1 suppress PC3 migration and invasion induced by SDF1α. A , inhibition of PC3 migration and invasion by ARF1 and PI3Kγ inhibitors as measured in transwell assays. PC3 cells were treated with SDF1α at 1 μg/ml together with secinH3 (100 μM), GCA (30 μM), Exo2 (60 μM), or AA-604850 (2.5 μM) for 48 h. Stimulation with FBS at 10% was used as a positive control. B , inhibition of PC3 migration and invasion by siRNA-mediated ARF1 knockdown. PC3 cells were transfected with control or ARF1 siRNA and then treated with SDF1α for 48 h. C , inhibition of PC3 migration and invasion by CRISPR–Cas9-mediated depletion of ARF1. PC3 cells were transfected with control or ARF1 knockout plasmids and then treated with SDF1α for 48 h. The quantitative data are presented as means ± SD (n = 10). ∗ and ∗∗ p
    Figure Legend Snippet: Inhibition and depletion of ARF1 suppress PC3 migration and invasion induced by SDF1α. A , inhibition of PC3 migration and invasion by ARF1 and PI3Kγ inhibitors as measured in transwell assays. PC3 cells were treated with SDF1α at 1 μg/ml together with secinH3 (100 μM), GCA (30 μM), Exo2 (60 μM), or AA-604850 (2.5 μM) for 48 h. Stimulation with FBS at 10% was used as a positive control. B , inhibition of PC3 migration and invasion by siRNA-mediated ARF1 knockdown. PC3 cells were transfected with control or ARF1 siRNA and then treated with SDF1α for 48 h. C , inhibition of PC3 migration and invasion by CRISPR–Cas9-mediated depletion of ARF1. PC3 cells were transfected with control or ARF1 knockout plasmids and then treated with SDF1α for 48 h. The quantitative data are presented as means ± SD (n = 10). ∗ and ∗∗ p

    Techniques Used: Inhibition, Migration, Positive Control, Transfection, CRISPR, Knock-Out

    Depletion of ARF1 by siRNA and CRISPR–Cas9 abolishes ERK1/2 activation by SDF1α and Golgi-Gγ9. A , effect of siRNA-mediated ARF1 knockdown on ERK1/2 activation by SDF1α stimulation at 200 ng/ml for 5 min. B , effect of ARF1 knockdown by siRNA on ERK1/2 activation by Golgi-Gγ9. The cells were transfected with FRB-γ9, Gβ1, and Golgi-FKBP (500 ng each) and then incubated with rapamycin for 30 min. C , effect of CRISPR–Cas9-mediated depletion of ARF1 on ERK1/2 activation by SDF1α. D , effect of ARF1 depletion by CRISPR–Cas9 on ERK1/2 activation by Golgi-Gγ9. The Western blots shown in each panel are representatives of at least three experiments. ARF1, ADP-ribosylation factor 1; ERK1/2, extracellular signal–regulated protein kinases 1 and 2; FKBP, FK506-binding protein; FRB, FKBP–rapamycin binding; SDF1α, stromal cell–derived factor 1α.
    Figure Legend Snippet: Depletion of ARF1 by siRNA and CRISPR–Cas9 abolishes ERK1/2 activation by SDF1α and Golgi-Gγ9. A , effect of siRNA-mediated ARF1 knockdown on ERK1/2 activation by SDF1α stimulation at 200 ng/ml for 5 min. B , effect of ARF1 knockdown by siRNA on ERK1/2 activation by Golgi-Gγ9. The cells were transfected with FRB-γ9, Gβ1, and Golgi-FKBP (500 ng each) and then incubated with rapamycin for 30 min. C , effect of CRISPR–Cas9-mediated depletion of ARF1 on ERK1/2 activation by SDF1α. D , effect of ARF1 depletion by CRISPR–Cas9 on ERK1/2 activation by Golgi-Gγ9. The Western blots shown in each panel are representatives of at least three experiments. ARF1, ADP-ribosylation factor 1; ERK1/2, extracellular signal–regulated protein kinases 1 and 2; FKBP, FK506-binding protein; FRB, FKBP–rapamycin binding; SDF1α, stromal cell–derived factor 1α.

    Techniques Used: CRISPR, Activation Assay, Transfection, Incubation, Western Blot, Binding Assay, Derivative Assay

    5) Product Images from "Formononetin attenuates atherosclerosis via regulating interaction between KLF4 and SRA in apoE-/- mice"

    Article Title: Formononetin attenuates atherosclerosis via regulating interaction between KLF4 and SRA in apoE-/- mice

    Journal: Theranostics

    doi: 10.7150/thno.38115

    Function of SRA in formononetin-inhibited lipid overload in HASMCs and macrophages. ( A, B ) Expression of SRA, CD36, SRBI, LOX1, ABCA1, and ABCG1 in protein of PMs after formononetin or siSRA treatment by Western blot, followed by quantification, n=5. ( C, D ) After knockdown of SRA by siRNA, PMs and HASMCs were treated with oxLDL (100 μg mL -1 ) and followed by ORO staining (left) and corresponding quantitative analysis (right), n=5. ( E, F ) Cholesterol uptake of PMs and HASMCs were determined by Dil-oxLDL after treatment as indicated, n=5. ( G, H ) Aortic root cross sections from mice used in Figure 1 were conducted co-immunofluorescent staining with anti-SRA and CD68 or αSMA antibodies with quantitative analysis of CD68 + SRA + or αSMA + SRA + area, n=5. ( I ) CD68, SRA and αSMA in HASMCs were evaluated by Western blot after treatment as indicated, n=5. ( J ) Cholesterol efflux of PMs and HASMCs were assessed after treatment as indicated, n=5. Data are presented as mean ± SEM, * P
    Figure Legend Snippet: Function of SRA in formononetin-inhibited lipid overload in HASMCs and macrophages. ( A, B ) Expression of SRA, CD36, SRBI, LOX1, ABCA1, and ABCG1 in protein of PMs after formononetin or siSRA treatment by Western blot, followed by quantification, n=5. ( C, D ) After knockdown of SRA by siRNA, PMs and HASMCs were treated with oxLDL (100 μg mL -1 ) and followed by ORO staining (left) and corresponding quantitative analysis (right), n=5. ( E, F ) Cholesterol uptake of PMs and HASMCs were determined by Dil-oxLDL after treatment as indicated, n=5. ( G, H ) Aortic root cross sections from mice used in Figure 1 were conducted co-immunofluorescent staining with anti-SRA and CD68 or αSMA antibodies with quantitative analysis of CD68 + SRA + or αSMA + SRA + area, n=5. ( I ) CD68, SRA and αSMA in HASMCs were evaluated by Western blot after treatment as indicated, n=5. ( J ) Cholesterol efflux of PMs and HASMCs were assessed after treatment as indicated, n=5. Data are presented as mean ± SEM, * P

    Techniques Used: Expressing, Western Blot, Staining, Mouse Assay

    Function of SRA in formononetin-inhibited lipid overload in HASMCs and macrophages. ( A, B ) Expression of SRA, CD36, SRBI, LOX1, ABCA1, and ABCG1 in protein of PMs after formononetin or siSRA treatment by Western blot, followed by quantification, n=5. ( C, D ) After knockdown of SRA by siRNA, PMs and HASMCs were treated with oxLDL (100 μg mL -1 ) and followed by ORO staining (left) and corresponding quantitative analysis (right), n=5. ( E, F ) Cholesterol uptake of PMs and HASMCs were determined by Dil-oxLDL after treatment as indicated, n=5. ( G, H ) Aortic root cross sections from mice used in Figure 1 were conducted co-immunofluorescent staining with anti-SRA and CD68 or αSMA antibodies with quantitative analysis of CD68 + SRA + or αSMA + SRA + area, n=5. ( I ) CD68, SRA and αSMA in HASMCs were evaluated by Western blot after treatment as indicated, n=5. ( J ) Cholesterol efflux of PMs and HASMCs were assessed after treatment as indicated, n=5. Data are presented as mean ± SEM, * P
    Figure Legend Snippet: Function of SRA in formononetin-inhibited lipid overload in HASMCs and macrophages. ( A, B ) Expression of SRA, CD36, SRBI, LOX1, ABCA1, and ABCG1 in protein of PMs after formononetin or siSRA treatment by Western blot, followed by quantification, n=5. ( C, D ) After knockdown of SRA by siRNA, PMs and HASMCs were treated with oxLDL (100 μg mL -1 ) and followed by ORO staining (left) and corresponding quantitative analysis (right), n=5. ( E, F ) Cholesterol uptake of PMs and HASMCs were determined by Dil-oxLDL after treatment as indicated, n=5. ( G, H ) Aortic root cross sections from mice used in Figure 1 were conducted co-immunofluorescent staining with anti-SRA and CD68 or αSMA antibodies with quantitative analysis of CD68 + SRA + or αSMA + SRA + area, n=5. ( I ) CD68, SRA and αSMA in HASMCs were evaluated by Western blot after treatment as indicated, n=5. ( J ) Cholesterol efflux of PMs and HASMCs were assessed after treatment as indicated, n=5. Data are presented as mean ± SEM, * P

    Techniques Used: Expressing, Western Blot, Staining, Mouse Assay

    6) Product Images from "Mitochondrion-associated protein peroxiredoxin 3 promotes benign prostatic hyperplasia through autophagy suppression and pyroptosis activation"

    Article Title: Mitochondrion-associated protein peroxiredoxin 3 promotes benign prostatic hyperplasia through autophagy suppression and pyroptosis activation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17927

    Impacts of PRDX3 on pyroptosis ( A – C ) Representative immunoblot (A) and quantification (B–C) showing the levels of caspase 1 (P45) (B) and caspase 1 (P20) (C) in BPH-1 cells treated with random (−) or PRDX3-specific siRNA (+). ( D ) Plots of lactate dehydrogenase ( LDH ) activity released in medium from cultured BPH-1 cells treated with random (−) or PRDX3-specific siRNA (+). Data are mean and standard deviation of three repeats and differences are tested with Student's T -test. * P ≤ 0.05; ** P ≤ 0.01.
    Figure Legend Snippet: Impacts of PRDX3 on pyroptosis ( A – C ) Representative immunoblot (A) and quantification (B–C) showing the levels of caspase 1 (P45) (B) and caspase 1 (P20) (C) in BPH-1 cells treated with random (−) or PRDX3-specific siRNA (+). ( D ) Plots of lactate dehydrogenase ( LDH ) activity released in medium from cultured BPH-1 cells treated with random (−) or PRDX3-specific siRNA (+). Data are mean and standard deviation of three repeats and differences are tested with Student's T -test. * P ≤ 0.05; ** P ≤ 0.01.

    Techniques Used: Activity Assay, Cell Culture, Standard Deviation

    Mitochondrial association and impacts on oxidative stress of PRDX3 ( A ) Representative images showing the colocalization of PRDX3 (green) with TOM20 (red) in BPH-1 cells. Bar = 10 μm. ( B ) A image showing a part of the merge showing in (A). ( C ) A representative immunoblot showing the levels of PRDX3 in BPH-1 cells treated with random (MOCK) or PRDX3-specific siRNA (PRDX3). ( D , E ) Representative images (D) and quantification (E) oxidative stress as indicated by the intensities of red fluorescence after staining with dihydroethidine hydrochloride. Bar = 200 μm. Data are mean and standard deviation of three repeats and differences are tested with Student's T -test. *** P ≤ 0.001.
    Figure Legend Snippet: Mitochondrial association and impacts on oxidative stress of PRDX3 ( A ) Representative images showing the colocalization of PRDX3 (green) with TOM20 (red) in BPH-1 cells. Bar = 10 μm. ( B ) A image showing a part of the merge showing in (A). ( C ) A representative immunoblot showing the levels of PRDX3 in BPH-1 cells treated with random (MOCK) or PRDX3-specific siRNA (PRDX3). ( D , E ) Representative images (D) and quantification (E) oxidative stress as indicated by the intensities of red fluorescence after staining with dihydroethidine hydrochloride. Bar = 200 μm. Data are mean and standard deviation of three repeats and differences are tested with Student's T -test. *** P ≤ 0.001.

    Techniques Used: Fluorescence, Staining, Standard Deviation

    Impacts of PRDX3 protein on autophagy flux ( A – D ) Representative immunoblot (A, C) and quantification (B, D) showing the levels of LC3-II in BPH-1 cells treated with random (MOCK) or PRDX3-specific siRNA (PRDX3) (A, B) or RWPE-1 cells transiently expressing different amount of PRDX3 (C, D) in the absence (Ctrl) or presence of bafilomycin A1 (BAF). Data are mean and standard deviation of three repeats and differences are tested with Student's T -test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. ( E – G ) Representative immunoblot (E) and quantification (F, G) showing the levels of Beclin 1 (F) and PI3KCIII (G) in BPH-1 cells treated with random (MOCK) or PRDX3-specific siRNA (PRDX3). Ns, not significant; * P ≤ 0.05.
    Figure Legend Snippet: Impacts of PRDX3 protein on autophagy flux ( A – D ) Representative immunoblot (A, C) and quantification (B, D) showing the levels of LC3-II in BPH-1 cells treated with random (MOCK) or PRDX3-specific siRNA (PRDX3) (A, B) or RWPE-1 cells transiently expressing different amount of PRDX3 (C, D) in the absence (Ctrl) or presence of bafilomycin A1 (BAF). Data are mean and standard deviation of three repeats and differences are tested with Student's T -test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. ( E – G ) Representative immunoblot (E) and quantification (F, G) showing the levels of Beclin 1 (F) and PI3KCIII (G) in BPH-1 cells treated with random (MOCK) or PRDX3-specific siRNA (PRDX3). Ns, not significant; * P ≤ 0.05.

    Techniques Used: Expressing, Standard Deviation

    7) Product Images from "Decrease in invasion of HTR-8/SVneo trophoblastic cells by interferon gamma involves cross-communication of STAT1 and BATF2 that regulates the expression of JUN"

    Article Title: Decrease in invasion of HTR-8/SVneo trophoblastic cells by interferon gamma involves cross-communication of STAT1 and BATF2 that regulates the expression of JUN

    Journal: Cell Adhesion & Migration

    doi: 10.1080/19336918.2018.1434030

    Effect of BATF2 silencing on the expression of STAT1 in HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and BATF2 siRNA and subsequently used to study the expression of total STAT1 in the presence or absence of treatment with IFNG (10 ng/mL) by qRT-PCR and Western blotting as described in Materials and Methods . Panels A and B show the transcript and the protein levels of STAT1 in control siRNA and BATF2 siRNA transfected HTR-8/SVneo cells, with and without IFNG treatment respectively. Each bar represents relative expression after normalization with 18S rRNA at transcript level and GAPDH at the protein level. Results are expressed as mean ± S.E.M. of three independent experiments. Representative blots of STAT1 expression in BATF2 silenced cells is appended in Panel B.
    Figure Legend Snippet: Effect of BATF2 silencing on the expression of STAT1 in HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and BATF2 siRNA and subsequently used to study the expression of total STAT1 in the presence or absence of treatment with IFNG (10 ng/mL) by qRT-PCR and Western blotting as described in Materials and Methods . Panels A and B show the transcript and the protein levels of STAT1 in control siRNA and BATF2 siRNA transfected HTR-8/SVneo cells, with and without IFNG treatment respectively. Each bar represents relative expression after normalization with 18S rRNA at transcript level and GAPDH at the protein level. Results are expressed as mean ± S.E.M. of three independent experiments. Representative blots of STAT1 expression in BATF2 silenced cells is appended in Panel B.

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

    Expression of JUN in STAT1 silenced HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and STAT1 siRNA and subsequently used to study the expression of phosphorylated JUN ser 63 and JUN ser 73 in the presence or absence of IFNG (10 ng/mL) by Western blotting. Panels A, B and C show protein expression of p-JUN ser 63, p-JUN ser 73, and total JUN in control siRNA transfected and STAT1 silenced cells, respectively, on treatment with or without IFNG. Each bar represents relative expression after normalization with total JUN and GAPDH respectively with respect to untreated control siRNA transfected cells. Values are expressed as mean ± S.E.M. of three independent experiments. Panel D shows the representative blots of p-JUN ser 63, p-JUN ser 73, total JUN and GAPDH from one of the three experiments.
    Figure Legend Snippet: Expression of JUN in STAT1 silenced HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and STAT1 siRNA and subsequently used to study the expression of phosphorylated JUN ser 63 and JUN ser 73 in the presence or absence of IFNG (10 ng/mL) by Western blotting. Panels A, B and C show protein expression of p-JUN ser 63, p-JUN ser 73, and total JUN in control siRNA transfected and STAT1 silenced cells, respectively, on treatment with or without IFNG. Each bar represents relative expression after normalization with total JUN and GAPDH respectively with respect to untreated control siRNA transfected cells. Values are expressed as mean ± S.E.M. of three independent experiments. Panel D shows the representative blots of p-JUN ser 63, p-JUN ser 73, total JUN and GAPDH from one of the three experiments.

    Techniques Used: Expressing, Transfection, Western Blot

    Expression of BATF2 in STAT1 silenced HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and STAT1 siRNA followed by treatment with or without IFNG (10 ng/mL) for 24 h and subsequently used to analyze expression of BATF2 both at the transcript and protein levels by qRT-PCR and Western blotting. Panel A shows the transcript level of BATF2 in control siRNA and STAT1 siRNA transfected cells with and without IFNG treatment. Panel B represents the densitometric analysis and representative blots of BATF2 expression in control siRNA and STAT1 siRNA transfected cells with and without IFNG treatment. Each bar represents relative expression after normalization with 18S rRNA at the transcript level and GAPDH at the protein level. Values are expressed as mean ± S.E.M. of three independent experiments.
    Figure Legend Snippet: Expression of BATF2 in STAT1 silenced HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and STAT1 siRNA followed by treatment with or without IFNG (10 ng/mL) for 24 h and subsequently used to analyze expression of BATF2 both at the transcript and protein levels by qRT-PCR and Western blotting. Panel A shows the transcript level of BATF2 in control siRNA and STAT1 siRNA transfected cells with and without IFNG treatment. Panel B represents the densitometric analysis and representative blots of BATF2 expression in control siRNA and STAT1 siRNA transfected cells with and without IFNG treatment. Each bar represents relative expression after normalization with 18S rRNA at the transcript level and GAPDH at the protein level. Values are expressed as mean ± S.E.M. of three independent experiments.

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

    Role of STAT1 in IFNG-mediated decrease in invasion of HTR-8/SVneo cells. To study the importance of STAT1 in the invasion of HTR-8/SVneo cells after treatment with IFNG, silencing of STAT1 was done using siRNA as described in Materials and Methods . Silencing of STAT1 was confirmed by qRT-PCR and Western blotting. Panels A and B show expression profile of STAT1 at the transcript and protein levels in control siRNA and STAT1 silenced cells, respectively, on treatment with and without IFNG (10 ng/mL) for 24 h. Each bar represents relative expression after normalization with 18S rRNA and GAPDH and values expressed as mean ± S.E.M. of three independent experiments. Representative blots showing expression profile of STAT1 siRNA transfected cells is appended as part of Panel B. Panel C shows fold change in the invasion of control siRNA and STAT1 siRNA transfected cells on treatment with and without IFNG for 24 h. The results are expressed as mean ± S.E.M. of fold change in the invasion with respect to control siRNA transfected cells without IFNG (10 ng/mL) treatment, observed in three independent experiments.
    Figure Legend Snippet: Role of STAT1 in IFNG-mediated decrease in invasion of HTR-8/SVneo cells. To study the importance of STAT1 in the invasion of HTR-8/SVneo cells after treatment with IFNG, silencing of STAT1 was done using siRNA as described in Materials and Methods . Silencing of STAT1 was confirmed by qRT-PCR and Western blotting. Panels A and B show expression profile of STAT1 at the transcript and protein levels in control siRNA and STAT1 silenced cells, respectively, on treatment with and without IFNG (10 ng/mL) for 24 h. Each bar represents relative expression after normalization with 18S rRNA and GAPDH and values expressed as mean ± S.E.M. of three independent experiments. Representative blots showing expression profile of STAT1 siRNA transfected cells is appended as part of Panel B. Panel C shows fold change in the invasion of control siRNA and STAT1 siRNA transfected cells on treatment with and without IFNG for 24 h. The results are expressed as mean ± S.E.M. of fold change in the invasion with respect to control siRNA transfected cells without IFNG (10 ng/mL) treatment, observed in three independent experiments.

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

    Expression of JUN in BATF2 silenced HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and BATF2 siRNA and subsequently used to study the expression of phosphorylated JUN ser 63 and JUN ser 73 in the presence or absence of IFNG (10 ng/mL) by Western blotting as described in Materials and Methods . Panel A, B and C show protein expression of p-JUN ser 63, p-JUN ser 73 and total JUN in control siRNA transfected and BATF2 silenced cells, respectively, on treatment with and without IFNG. Each bar represents relative expression after normalization with GAPDH with respect to untreated control siRNA transfected cells. Values are expressed as mean ± S.E.M. of three independent experiments. Panel D shows the representative blots of p-JUN ser 63, p-JUN ser 73, total JUN and GAPDH from one of the three experiments.
    Figure Legend Snippet: Expression of JUN in BATF2 silenced HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and BATF2 siRNA and subsequently used to study the expression of phosphorylated JUN ser 63 and JUN ser 73 in the presence or absence of IFNG (10 ng/mL) by Western blotting as described in Materials and Methods . Panel A, B and C show protein expression of p-JUN ser 63, p-JUN ser 73 and total JUN in control siRNA transfected and BATF2 silenced cells, respectively, on treatment with and without IFNG. Each bar represents relative expression after normalization with GAPDH with respect to untreated control siRNA transfected cells. Values are expressed as mean ± S.E.M. of three independent experiments. Panel D shows the representative blots of p-JUN ser 63, p-JUN ser 73, total JUN and GAPDH from one of the three experiments.

    Techniques Used: Expressing, Transfection, Western Blot

    Effect of BATF2 silencing on the invasion of HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and BATF2 siRNA and subsequently used to study their invasion by transwell invasion assay as described in Materials and Methods . Silencing of BATF2 was confirmed by qRT-PCR and Western blotting. Panels A and B show transcript and protein levels of BATF2 in control siRNA and BATF2 silenced cells, respectively, on treatment with and without IFNG (10 ng/mL). Each bar represents relative expression after normalization with 18S rRNA or GAPDH, expressed as mean ± S.E.M. of three independent experiments. Panel C shows the fold change in the invasion of cells transfected with control or BATF2 siRNA respectively, subsequent to treatment with and without IFNG for 24 h. The results are expressed as mean ± S.E.M. of fold change in invasion, as compared to control siRNA transfected cells without treatment with IFNG, observed in three independent experiments.
    Figure Legend Snippet: Effect of BATF2 silencing on the invasion of HTR-8/SVneo cells treated with IFNG. HTR-8/SVneo cells (0.1 × 10 6 ) were transfected with control siRNA and BATF2 siRNA and subsequently used to study their invasion by transwell invasion assay as described in Materials and Methods . Silencing of BATF2 was confirmed by qRT-PCR and Western blotting. Panels A and B show transcript and protein levels of BATF2 in control siRNA and BATF2 silenced cells, respectively, on treatment with and without IFNG (10 ng/mL). Each bar represents relative expression after normalization with 18S rRNA or GAPDH, expressed as mean ± S.E.M. of three independent experiments. Panel C shows the fold change in the invasion of cells transfected with control or BATF2 siRNA respectively, subsequent to treatment with and without IFNG for 24 h. The results are expressed as mean ± S.E.M. of fold change in invasion, as compared to control siRNA transfected cells without treatment with IFNG, observed in three independent experiments.

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

    8) Product Images from "DARPP-32 Increases Interactions Between Epidermal Growth Factor Receptor and ERBB3 to Promote Tumor Resistance to Gefitinib"

    Article Title: DARPP-32 Increases Interactions Between Epidermal Growth Factor Receptor and ERBB3 to Promote Tumor Resistance to Gefitinib

    Journal: Gastroenterology

    doi: 10.1053/j.gastro.2011.06.070

    DARPP-32 promotes EGFR protein stability and tyrosine phosphorylation A) MKN-28 cells stably expressing DARPP-32 or empty vector were treated with EGF (100 ng/ml) for the indicated time points, and total EGFR protein levels were determined by Western blot analysis. Relative EGFR expression levels are presented, time=0 is shown as 1.0. B ) Western blot analysis using lysates from MKN-28/DARPP-32 stable cells (left panel) or infected with adenoviral DARPP-32 (right panel), following treatment with gefitinib (25μM) or vehicle (DMSO) overnight. C) Western blot analysis as in (B), following lentiviral shRNA DARPP-32 knockdown in SNU-16 cells. D-F) Immunoprecipitation with anti-p-Tyr(102) antibody followed by Western blot analysis of EGFR protein in MKN-28/DARPP-32 stable cells, treated with EGF (100 ng/ml) for 1h (D) or following lentiviral shRNA DARPP-32 knockdown in SNU-16 cells and treatment with EGF (E) or gefitinib (25μM) overnight (F).
    Figure Legend Snippet: DARPP-32 promotes EGFR protein stability and tyrosine phosphorylation A) MKN-28 cells stably expressing DARPP-32 or empty vector were treated with EGF (100 ng/ml) for the indicated time points, and total EGFR protein levels were determined by Western blot analysis. Relative EGFR expression levels are presented, time=0 is shown as 1.0. B ) Western blot analysis using lysates from MKN-28/DARPP-32 stable cells (left panel) or infected with adenoviral DARPP-32 (right panel), following treatment with gefitinib (25μM) or vehicle (DMSO) overnight. C) Western blot analysis as in (B), following lentiviral shRNA DARPP-32 knockdown in SNU-16 cells. D-F) Immunoprecipitation with anti-p-Tyr(102) antibody followed by Western blot analysis of EGFR protein in MKN-28/DARPP-32 stable cells, treated with EGF (100 ng/ml) for 1h (D) or following lentiviral shRNA DARPP-32 knockdown in SNU-16 cells and treatment with EGF (E) or gefitinib (25μM) overnight (F).

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Infection, shRNA, Immunoprecipitation

    DARPP-32 regulates EGFR-mediated AKT survival pathway in gastric cancer cells A) Western blot analysis of AKT, pAKT(S473), GSK-3β, and pGSK-3β(S9) in MKN-28 stably expressing DARPP-32 (DP01 and DP02) or empty vector. B) The same as in (A) following transient infection with different titers of adenoviruses expressing DARPP-32 or control. C) The same as in (A) following lentiviral shRNA knockdown of DARPP-32 or control shRNA in SNU-16. D) Human EGFR phosphorylation antibody array using lysates from MKN-28 cells stably expressing DARPP-32 or empty vector, cultured in the presence of gefitinib (25 μM) overnight, demonstrates up-regulation of total and phosphor-EGFR protein in DARPP-32 expressing cells. Quantitative data are shown on the right panel. E–F) Western blot analysis, following overexpression (E) or knockdown of DARPP-32 (F), confirms that results in (D).
    Figure Legend Snippet: DARPP-32 regulates EGFR-mediated AKT survival pathway in gastric cancer cells A) Western blot analysis of AKT, pAKT(S473), GSK-3β, and pGSK-3β(S9) in MKN-28 stably expressing DARPP-32 (DP01 and DP02) or empty vector. B) The same as in (A) following transient infection with different titers of adenoviruses expressing DARPP-32 or control. C) The same as in (A) following lentiviral shRNA knockdown of DARPP-32 or control shRNA in SNU-16. D) Human EGFR phosphorylation antibody array using lysates from MKN-28 cells stably expressing DARPP-32 or empty vector, cultured in the presence of gefitinib (25 μM) overnight, demonstrates up-regulation of total and phosphor-EGFR protein in DARPP-32 expressing cells. Quantitative data are shown on the right panel. E–F) Western blot analysis, following overexpression (E) or knockdown of DARPP-32 (F), confirms that results in (D).

    Techniques Used: Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Infection, shRNA, Ab Array, Cell Culture, Over Expression

    DARPP-32 associates with EGFR/ERBB3 and enhances their interaction A) HEK-293 cells were transiently transfected with the indicated constructs, and the interaction between DARPP-32 and ERBB3 or DARPP-32 and EGFR was examined by co-immunoprecipitation using specific antibodies. B) The protein interaction of endogenous DARPP-32, EGFR, and ERBB3 was evaluated by a three-way co-immunoprecipitation in MKN-45 cells. C) Immunofluorescence analysis using MKN-28 cells, following adenoviral infection with DARPP-32 (5 MOI), demonstrates co-localization of EGFR and DARPP-32 proteins on the cell membrane. D) Using immunoprecipitation of EGFR (left panel) or ERBB3 (right panel), the EGFR-ERBB3 interaction was examined in MKN-28 cells stably expressing DARPP-32 or empty vector. E) The same as in (D), using adenoviral infection of DARPP-32. F ) The same as in (D), following lentiviral shRNA DARPP-32 knockdown in SNU-16 cells.
    Figure Legend Snippet: DARPP-32 associates with EGFR/ERBB3 and enhances their interaction A) HEK-293 cells were transiently transfected with the indicated constructs, and the interaction between DARPP-32 and ERBB3 or DARPP-32 and EGFR was examined by co-immunoprecipitation using specific antibodies. B) The protein interaction of endogenous DARPP-32, EGFR, and ERBB3 was evaluated by a three-way co-immunoprecipitation in MKN-45 cells. C) Immunofluorescence analysis using MKN-28 cells, following adenoviral infection with DARPP-32 (5 MOI), demonstrates co-localization of EGFR and DARPP-32 proteins on the cell membrane. D) Using immunoprecipitation of EGFR (left panel) or ERBB3 (right panel), the EGFR-ERBB3 interaction was examined in MKN-28 cells stably expressing DARPP-32 or empty vector. E) The same as in (D), using adenoviral infection of DARPP-32. F ) The same as in (D), following lentiviral shRNA DARPP-32 knockdown in SNU-16 cells.

    Techniques Used: Transfection, Construct, Immunoprecipitation, Immunofluorescence, Infection, Stable Transfection, Expressing, Plasmid Preparation, shRNA

    9) Product Images from "Stimulatory Effects of Insulin-like Growth Factor-I on Growth Plate Chondrogenesis Are Mediated by Nuclear Factor-?B p65 *"

    Article Title: Stimulatory Effects of Insulin-like Growth Factor-I on Growth Plate Chondrogenesis Are Mediated by Nuclear Factor-?B p65 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M803754200

    Effects of IGF-I, PDTC, and p65 siRNA on chondrocyte proliferation and differentiation. A and B, chondrocytes isolated from fetal rat metatarsal growth plates were washed with fresh serum-free DMEM, seeded in 24-well plate, and transfected with p65
    Figure Legend Snippet: Effects of IGF-I, PDTC, and p65 siRNA on chondrocyte proliferation and differentiation. A and B, chondrocytes isolated from fetal rat metatarsal growth plates were washed with fresh serum-free DMEM, seeded in 24-well plate, and transfected with p65

    Techniques Used: Isolation, Transfection

    10) Product Images from "Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II"

    Article Title: Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II

    Journal: Veterinary Research

    doi: 10.1186/s13567-018-0584-0

    PSaV entry and RNA release depend on clathrin-, dynamin-, and cholesterol-mediated endocytosis. A LLC-PK cells grown in 6-well plates were incubated with neutral red (NR)-labeled PSaV Cowden strain for the indicated times and then exposed or not to UV light. The supernatants of each cell lysate were inoculated into fresh LLC-PK monolayers in 6-well plates, overlaid with agar, and incubated for 4 days. Results are shown as percentages to the number of plaques in the unilluminated control. The NR infectious center assay was performed in cells pretreated with DMSO vehicle or 20 μM chlorpromazine (CPZ), or transfected with scrambled siRNA or CHC siRNA ( B ); pretreated with DMSO vehicle or 100 μM dynasore (Dyna), or transfected with scrambled siRNA or dynamin II (Dyn II) siRNA ( C ); or pretreated with DMSO vehicle, 20 mM MβCD for 1 h, or MβCD followed by incubation with 200 μM soluble cholesterol for 30 min ( D ). The cells were then incubated with NR-labeled PSaV for 120 min. The number of plaques was normalized to those obtained with unilluminated controls exposed to the above conditions. Data for panels A – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one - way ANOVA. * P
    Figure Legend Snippet: PSaV entry and RNA release depend on clathrin-, dynamin-, and cholesterol-mediated endocytosis. A LLC-PK cells grown in 6-well plates were incubated with neutral red (NR)-labeled PSaV Cowden strain for the indicated times and then exposed or not to UV light. The supernatants of each cell lysate were inoculated into fresh LLC-PK monolayers in 6-well plates, overlaid with agar, and incubated for 4 days. Results are shown as percentages to the number of plaques in the unilluminated control. The NR infectious center assay was performed in cells pretreated with DMSO vehicle or 20 μM chlorpromazine (CPZ), or transfected with scrambled siRNA or CHC siRNA ( B ); pretreated with DMSO vehicle or 100 μM dynasore (Dyna), or transfected with scrambled siRNA or dynamin II (Dyn II) siRNA ( C ); or pretreated with DMSO vehicle, 20 mM MβCD for 1 h, or MβCD followed by incubation with 200 μM soluble cholesterol for 30 min ( D ). The cells were then incubated with NR-labeled PSaV for 120 min. The number of plaques was normalized to those obtained with unilluminated controls exposed to the above conditions. Data for panels A – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one - way ANOVA. * P

    Techniques Used: Incubation, Labeling, Transfection, Standard Deviation

    Small interfering RNAs (siRNAs) against clathrin- and dynamin-mediated endocytosis reduce PSaV infection. A LLC-PK cells transfected with scrambled siRNA (Scram) or siRNAs against clathrin heavy chain (CHC), dynamin II, or caveolin-1 were harvested at 24 and 48 h post-transfection. The down-regulation of each protein by siRNA knock-down was evaluated by western blotting analysis using antibodies specific for each protein. The intensity of each target protein relative to GAPDH was determined by densitometric analysis and is indicated above each lane. The cells transfected with each siRNA and then incubated were infected with PSaV Cowden ( B ), CVB3 Nancy ( C ), or rotavirus Wa ( D ) strains. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, and normalized to the results obtained in the scrambled siRNA-transfected cells. Data for panels B – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. * P
    Figure Legend Snippet: Small interfering RNAs (siRNAs) against clathrin- and dynamin-mediated endocytosis reduce PSaV infection. A LLC-PK cells transfected with scrambled siRNA (Scram) or siRNAs against clathrin heavy chain (CHC), dynamin II, or caveolin-1 were harvested at 24 and 48 h post-transfection. The down-regulation of each protein by siRNA knock-down was evaluated by western blotting analysis using antibodies specific for each protein. The intensity of each target protein relative to GAPDH was determined by densitometric analysis and is indicated above each lane. The cells transfected with each siRNA and then incubated were infected with PSaV Cowden ( B ), CVB3 Nancy ( C ), or rotavirus Wa ( D ) strains. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, and normalized to the results obtained in the scrambled siRNA-transfected cells. Data for panels B – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. * P

    Techniques Used: Infection, Transfection, Western Blot, Incubation, Staining, Standard Deviation

    PSaV entry depends on Rab5 and Rab7. A , B LLC-PK cells were transfected with scrambled siRNA or siRNAs against Rab5 ( A ) or Rab7 ( B ). Next, cells were exposed to AF594-labeled PSaV particles (approximately 415 particles per cell) for EEA1 colocalization or for LAMP2 colocalization. This experiment was done in triplicate and representative images are shown. The scale bars correspond to 10 μm. The bottom portion displays immunoblots to confirm silencing levels of Rab5 and Rab7 by transfection of each corresponding siRNA. The intensity of each target protein relative to GAPDH was determined by densitometric analysis and is indicated above each lane.
    Figure Legend Snippet: PSaV entry depends on Rab5 and Rab7. A , B LLC-PK cells were transfected with scrambled siRNA or siRNAs against Rab5 ( A ) or Rab7 ( B ). Next, cells were exposed to AF594-labeled PSaV particles (approximately 415 particles per cell) for EEA1 colocalization or for LAMP2 colocalization. This experiment was done in triplicate and representative images are shown. The scale bars correspond to 10 μm. The bottom portion displays immunoblots to confirm silencing levels of Rab5 and Rab7 by transfection of each corresponding siRNA. The intensity of each target protein relative to GAPDH was determined by densitometric analysis and is indicated above each lane.

    Techniques Used: Transfection, Labeling, Western Blot

    PSaV infection depends on Rab5 and Rab7. A – D LLC-PK, Caco-2, and MA104 cells were transfected with scrambled siRNA or siRNAs against Rab5 or Rab7, and then incubated with PSaV Cowden ( A ), CVB3 Nancy ( B ), or rotavirus Wa ( C ) strains, respectively. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, normalized to the results obtained in the scrambled siRNA-transfected cells. D The virus titer was determined by TCID 50 using the cell lysates harvested from the cells in the above experimental conditions. Data for panels A – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using the one-way ANOVA. * P
    Figure Legend Snippet: PSaV infection depends on Rab5 and Rab7. A – D LLC-PK, Caco-2, and MA104 cells were transfected with scrambled siRNA or siRNAs against Rab5 or Rab7, and then incubated with PSaV Cowden ( A ), CVB3 Nancy ( B ), or rotavirus Wa ( C ) strains, respectively. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, normalized to the results obtained in the scrambled siRNA-transfected cells. D The virus titer was determined by TCID 50 using the cell lysates harvested from the cells in the above experimental conditions. Data for panels A – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using the one-way ANOVA. * P

    Techniques Used: Infection, Transfection, Incubation, Staining, Standard Deviation

    11) Product Images from "Infected erythrocytes expressing DC13 PfEMP1 differ from recombinant proteins in EPCR-binding function"

    Article Title: Infected erythrocytes expressing DC13 PfEMP1 differ from recombinant proteins in EPCR-binding function

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

    doi: 10.1073/pnas.1712879115

    DC8 IT4var19, but not DC13 HB3var03 and IT4var07, IE binding to the HBEC line HBEC-5i is reduced by EPCR siRNA knockdown. ( A ) Expression of EPCR on HBEC-5i by immunostaining and flow cytometry. HBEC-5i was transfected with EPCR siRNA (orange) or a negative control siRNA (blue). Forty-eight hours after transfection, HBEC-5i was stained with 1 μg/mL goat polyclonal antibody to EPCR (orange/blue) or goat IgG-negative control (red), followed by Alexa Fluor 488 donkey anti-goat IgG at a 1:1,500 dilution. Data shown are representative of four similar experiments. ( B – D ) Binding of IEs to HBEC-5i transfected with control siRNA or EPCR siRNA under static conditions. The data shown are the mean and SEM from four independent experiments. The difference in mean values compared with the siRNA control was analyzed by a two-tailed paired t test. * P
    Figure Legend Snippet: DC8 IT4var19, but not DC13 HB3var03 and IT4var07, IE binding to the HBEC line HBEC-5i is reduced by EPCR siRNA knockdown. ( A ) Expression of EPCR on HBEC-5i by immunostaining and flow cytometry. HBEC-5i was transfected with EPCR siRNA (orange) or a negative control siRNA (blue). Forty-eight hours after transfection, HBEC-5i was stained with 1 μg/mL goat polyclonal antibody to EPCR (orange/blue) or goat IgG-negative control (red), followed by Alexa Fluor 488 donkey anti-goat IgG at a 1:1,500 dilution. Data shown are representative of four similar experiments. ( B – D ) Binding of IEs to HBEC-5i transfected with control siRNA or EPCR siRNA under static conditions. The data shown are the mean and SEM from four independent experiments. The difference in mean values compared with the siRNA control was analyzed by a two-tailed paired t test. * P

    Techniques Used: Binding Assay, Expressing, Immunostaining, Flow Cytometry, Cytometry, Transfection, Negative Control, Staining, Two Tailed Test

    12) Product Images from "Molecular interplay between cdk4 and p21 dictates G0/G1 cell cycle arrest in prostate cancer cells"

    Article Title: Molecular interplay between cdk4 and p21 dictates G0/G1 cell cycle arrest in prostate cancer cells

    Journal: Cancer letters

    doi: 10.1016/j.canlet.2013.05.014

    SiRNA knockdown of p21 and p27 in PC-3 cells, attenuates pso-induced G0/G1 phase arrest. ( A ). PC3 cells were transiently transfected with scrambled (SCR-siRNA), p21 siRNA, or p27 siRNA and treated with pso for 24h and cell lysates were subjected to Western
    Figure Legend Snippet: SiRNA knockdown of p21 and p27 in PC-3 cells, attenuates pso-induced G0/G1 phase arrest. ( A ). PC3 cells were transiently transfected with scrambled (SCR-siRNA), p21 siRNA, or p27 siRNA and treated with pso for 24h and cell lysates were subjected to Western

    Techniques Used: Transfection, Western Blot

    13) Product Images from "Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins *"

    Article Title: Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.538587

    siRNA-targeted silencing of CMG2 and evaluation of anthrax LeTx toxicity in Raw 264.7 cells. A , Raw 264.7 cells were cultured in 24-well plates and treated as follows: 1) untransfected (−); 2) RNAiMAX alone ( L ), 3) siGFP, 10 and 20 pmol; 4) si-mTEM8,
    Figure Legend Snippet: siRNA-targeted silencing of CMG2 and evaluation of anthrax LeTx toxicity in Raw 264.7 cells. A , Raw 264.7 cells were cultured in 24-well plates and treated as follows: 1) untransfected (−); 2) RNAiMAX alone ( L ), 3) siGFP, 10 and 20 pmol; 4) si-mTEM8,

    Techniques Used: Cell Culture

    14) Product Images from "Sildenafil Ameliorates Advanced Glycation End Products-Induced Mitochondrial Dysfunction in HT-22 Hippocampal Neuronal Cells"

    Article Title: Sildenafil Ameliorates Advanced Glycation End Products-Induced Mitochondrial Dysfunction in HT-22 Hippocampal Neuronal Cells

    Journal: Journal of Korean Neurosurgical Society

    doi: 10.3340/jkns.2016.59.3.259

    A : Effect of siRNA transfection on HO-1 protein expression. Cells were transfected with 30 picomoles of HO-1 siRNA or scrambled siRNA (siRNA control) and incubated for 24 hr with vehicle (Veh) or each 20 µM of sildenafil citrate (SC) and cobalt protophorphyrin (CoPP). HO-1 protein was determined by Western blot analysis of the cell extracts. B : MTT reduction was determined by colorimetric analysis. C : Confocal microscopic analysis was performed in cells double-stained with TMRM and calcein/AM. Data were represented as the mean±SEM of 4 experiments. * p
    Figure Legend Snippet: A : Effect of siRNA transfection on HO-1 protein expression. Cells were transfected with 30 picomoles of HO-1 siRNA or scrambled siRNA (siRNA control) and incubated for 24 hr with vehicle (Veh) or each 20 µM of sildenafil citrate (SC) and cobalt protophorphyrin (CoPP). HO-1 protein was determined by Western blot analysis of the cell extracts. B : MTT reduction was determined by colorimetric analysis. C : Confocal microscopic analysis was performed in cells double-stained with TMRM and calcein/AM. Data were represented as the mean±SEM of 4 experiments. * p

    Techniques Used: Transfection, Expressing, Incubation, Western Blot, MTT Assay, Staining

    15) Product Images from "CD147 promotes Src-dependent activation of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular carcinoma cells"

    Article Title: CD147 promotes Src-dependent activation of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular carcinoma cells

    Journal: Oncotarget

    doi:

    CD147 promotes Src activation through FAK (A) Src activity in CD147-pcDNA3.1-transfected 7721 cells was blocked with Src I-1. Left panel: representative image. Right panel: quantification. (B) Src activity in 7721 or CD147-pcDNA3.1-transfected 7721 cells was blocked with FAK inhibitor (PF573,228). Left panel: representative image. Right panel: quantification. (C) Src activity in CD147-pcDNA3.1-transfected 7721 cells was blocked with FAK inhibitor-14 (Y-15). Left panel: representative image. (D) Src activity in CD147-pcDNA3.1-transfected 7721 cells was blocked with FAK siRNA (si-FAK). Left panel: representative image. Right panel: quantification. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. ** P
    Figure Legend Snippet: CD147 promotes Src activation through FAK (A) Src activity in CD147-pcDNA3.1-transfected 7721 cells was blocked with Src I-1. Left panel: representative image. Right panel: quantification. (B) Src activity in 7721 or CD147-pcDNA3.1-transfected 7721 cells was blocked with FAK inhibitor (PF573,228). Left panel: representative image. Right panel: quantification. (C) Src activity in CD147-pcDNA3.1-transfected 7721 cells was blocked with FAK inhibitor-14 (Y-15). Left panel: representative image. (D) Src activity in CD147-pcDNA3.1-transfected 7721 cells was blocked with FAK siRNA (si-FAK). Left panel: representative image. Right panel: quantification. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. ** P

    Techniques Used: Activation Assay, Activity Assay, Transfection

    Src promotes DOCK8 expression via enhancing STAT3 phosphorylation (A) DOCK8 expression was detected using western blotting after treatment of 7721 cells with Src I-1. (B) Confocal microscopy images of 7721, 7721 treated with Src I-1 and K7721 cells. Red: CD147; Green: DOCK8; Blue: DAPI. Scale bar = 20μm. (C) DOCK8 expression and Src activity were examined in total lysates of 7721, K7721, K7721 transfected with Src Y530F and K7721-pcDNA3.1 cells (K7721 cells were transfected with pcDNA3.1 vector as a mock control for R7721) using western blotting. Phosphorylation of Src at Tyr416 can also be seen in the overexposed panel (right).(D) Phosphorylation level of STAT3 and DOCK8 expression were detected using western blotting after treatment of 7721 cells with WP1066. (E) The DOCK8 mRNA level was detected using real-time PCR after treatment of 7721 cells with WP1066. (F) Phosphorylation level of STAT3 was detected using western blotting after treatment of 7721 cells with Src I-1. (G) Phosphorylation level of STAT3 and CD147 expression were determined in 7721 cells overexpressing CD147 and/or transfected with STAT3 siRNA. (H) The effects of CD147 overexpression and/or STAT3 silencing on cell motility of 7721 cells. (I) CD147 expression using normalized microarray gene expression data. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. * P
    Figure Legend Snippet: Src promotes DOCK8 expression via enhancing STAT3 phosphorylation (A) DOCK8 expression was detected using western blotting after treatment of 7721 cells with Src I-1. (B) Confocal microscopy images of 7721, 7721 treated with Src I-1 and K7721 cells. Red: CD147; Green: DOCK8; Blue: DAPI. Scale bar = 20μm. (C) DOCK8 expression and Src activity were examined in total lysates of 7721, K7721, K7721 transfected with Src Y530F and K7721-pcDNA3.1 cells (K7721 cells were transfected with pcDNA3.1 vector as a mock control for R7721) using western blotting. Phosphorylation of Src at Tyr416 can also be seen in the overexposed panel (right).(D) Phosphorylation level of STAT3 and DOCK8 expression were detected using western blotting after treatment of 7721 cells with WP1066. (E) The DOCK8 mRNA level was detected using real-time PCR after treatment of 7721 cells with WP1066. (F) Phosphorylation level of STAT3 was detected using western blotting after treatment of 7721 cells with Src I-1. (G) Phosphorylation level of STAT3 and CD147 expression were determined in 7721 cells overexpressing CD147 and/or transfected with STAT3 siRNA. (H) The effects of CD147 overexpression and/or STAT3 silencing on cell motility of 7721 cells. (I) CD147 expression using normalized microarray gene expression data. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. * P

    Techniques Used: Expressing, Western Blot, Confocal Microscopy, Activity Assay, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Over Expression, Microarray

    16) Product Images from "Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells, et al. Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells"

    Article Title: Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells, et al. Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13523

    Effects of CB1R on gene expression depends on insulin receptor (IR). A, Western blot analysis of IR β‐subunit (IRβ) and GAPDH in βIRWT and βIRKO cells. B, Quantitative real‐time PCR analysis of CB1R expression in βIRWT and βIRKO cells. Data were normalized to 18S ribosomal RNA levels. C, Quantitative real‐time PCR analysis of CB1R, insulin, GCK and GLUT2 expression in βIRWT and βIRKO cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. D, Schematic unifying the regulation of β‐cell function by ECs and CB1Rs. Data are shown as the mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: Effects of CB1R on gene expression depends on insulin receptor (IR). A, Western blot analysis of IR β‐subunit (IRβ) and GAPDH in βIRWT and βIRKO cells. B, Quantitative real‐time PCR analysis of CB1R expression in βIRWT and βIRKO cells. Data were normalized to 18S ribosomal RNA levels. C, Quantitative real‐time PCR analysis of CB1R, insulin, GCK and GLUT2 expression in βIRWT and βIRKO cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. D, Schematic unifying the regulation of β‐cell function by ECs and CB1Rs. Data are shown as the mean ± SEM from three independent experiments. * P

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Cell Function Assay

    Effects of blocking CB1R on intra‐β‐cell insulin content and glucokinase (GCK) and glucose transporter 2 (GLUT2) expressions. A, CB1R and Insulin ( Ins2 ) mRNA levels in βTC6 cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. B, Intra‐islet insulin content in islets isolated from CB1R +/+ and CB1R −/− mice. Insulin was extracted from islets freshly isolated from CB1R +/+ and CB1R −/− mice using acid‐alcohol (n = 3 separate isolates). Size‐matched 10 islets per tube were analysed, and data were normalized to protein concentration. C, Insulin ( Ins1 and Ins2 ) mRNA levels in islets isolated from CB1R +/+ and CB1R −/− mice (n = 4 separate isolates). Data were normalized to 18S ribosomal RNA levels. D, Western blot analysis of preproinsulin, GCK and GLUT2 expressions in total lysates prepared from whole pancreata of overnight‐fasted CB1R +/+ and CB1R −/− (n = 4 per genotype) mice. Signals on Western blots were quantified by densitometry and are shown on the right. E and F, Immunofluorescent staining for GCK (E) and GLUT2 (F) in islets of overnight‐fasted CB1R +/+ and CB1R −/− mice. Scale bar, 50 μm. Relative signal intensity for the indicated proteins in islets is shown on the right (n = 3 per genotype). Quantification of GCK and GLUT2 intensities was shown on the right. Data are shown as the mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: Effects of blocking CB1R on intra‐β‐cell insulin content and glucokinase (GCK) and glucose transporter 2 (GLUT2) expressions. A, CB1R and Insulin ( Ins2 ) mRNA levels in βTC6 cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. B, Intra‐islet insulin content in islets isolated from CB1R +/+ and CB1R −/− mice. Insulin was extracted from islets freshly isolated from CB1R +/+ and CB1R −/− mice using acid‐alcohol (n = 3 separate isolates). Size‐matched 10 islets per tube were analysed, and data were normalized to protein concentration. C, Insulin ( Ins1 and Ins2 ) mRNA levels in islets isolated from CB1R +/+ and CB1R −/− mice (n = 4 separate isolates). Data were normalized to 18S ribosomal RNA levels. D, Western blot analysis of preproinsulin, GCK and GLUT2 expressions in total lysates prepared from whole pancreata of overnight‐fasted CB1R +/+ and CB1R −/− (n = 4 per genotype) mice. Signals on Western blots were quantified by densitometry and are shown on the right. E and F, Immunofluorescent staining for GCK (E) and GLUT2 (F) in islets of overnight‐fasted CB1R +/+ and CB1R −/− mice. Scale bar, 50 μm. Relative signal intensity for the indicated proteins in islets is shown on the right (n = 3 per genotype). Quantification of GCK and GLUT2 intensities was shown on the right. Data are shown as the mean ± SEM from three independent experiments. * P

    Techniques Used: Blocking Assay, Transfection, Isolation, Mouse Assay, Protein Concentration, Western Blot, Staining

    17) Product Images from "Loss of endogenous thymosin β4 accelerates glomerular disease"

    Article Title: Loss of endogenous thymosin β4 accelerates glomerular disease

    Journal: Kidney International

    doi: 10.1016/j.kint.2016.06.032

    Effects of downregulating endogenous Tmsb4x expression in podocytes in vitro . ( a ) Podocytes grown in vitro under permissive conditions were differentiated for 14 days before transfecting them with control siRNA or siRNA targeting Tmsb4x . ( b ) Quantification of Tmsb4x mRNA levels in podocytes 48 hours after transfection. ( c ) Cell viability after knockdown of endogenous Tmsb4x was assessed by MTT assay. ( d ) Podocyte migration after knockdown of endogenous Tmsb4x was assessed by a wound-healing assay, and the number of cells that migrated into the wound area was counted. ( e ) Representative images of podocytes transfected with control or Tmsb4x siRNA 0, 6, and 24 hours after wound formation. Representative images showing a podocyte with cytoplasmic stress fiber F-actin distribution ( f ) or cortical F-actin distribution ( g ). The percentage of cells with predominantly cytoplasmic stress fibers or cortical actin formation was quantified 48 hours after transfection ( h ). Quantification of active RhoA ( i ) and active Cdc42 ( j ) 48 hours after transfection. All experiments were repeated 3 to 4 times, and the data are presented as mean ± SEM. * P ≤ 0.05, *** P ≤ 0.001. IFN-γ, interferon-γ; Lip, lipofectamine; OD, optical density; siRNA, small, interfering RNA; TB, Tmsb4x, thymosin β 4 .
    Figure Legend Snippet: Effects of downregulating endogenous Tmsb4x expression in podocytes in vitro . ( a ) Podocytes grown in vitro under permissive conditions were differentiated for 14 days before transfecting them with control siRNA or siRNA targeting Tmsb4x . ( b ) Quantification of Tmsb4x mRNA levels in podocytes 48 hours after transfection. ( c ) Cell viability after knockdown of endogenous Tmsb4x was assessed by MTT assay. ( d ) Podocyte migration after knockdown of endogenous Tmsb4x was assessed by a wound-healing assay, and the number of cells that migrated into the wound area was counted. ( e ) Representative images of podocytes transfected with control or Tmsb4x siRNA 0, 6, and 24 hours after wound formation. Representative images showing a podocyte with cytoplasmic stress fiber F-actin distribution ( f ) or cortical F-actin distribution ( g ). The percentage of cells with predominantly cytoplasmic stress fibers or cortical actin formation was quantified 48 hours after transfection ( h ). Quantification of active RhoA ( i ) and active Cdc42 ( j ) 48 hours after transfection. All experiments were repeated 3 to 4 times, and the data are presented as mean ± SEM. * P ≤ 0.05, *** P ≤ 0.001. IFN-γ, interferon-γ; Lip, lipofectamine; OD, optical density; siRNA, small, interfering RNA; TB, Tmsb4x, thymosin β 4 .

    Techniques Used: Expressing, In Vitro, Transfection, MTT Assay, Migration, Wound Healing Assay, Small Interfering RNA

    18) Product Images from "Ubiquitin-specific protease 4 controls metastatic potential through β-catenin stabilization in brain metastatic lung adenocarcinoma"

    Article Title: Ubiquitin-specific protease 4 controls metastatic potential through β-catenin stabilization in brain metastatic lung adenocarcinoma

    Journal: Scientific Reports

    doi: 10.1038/srep21596

    USP4 regulated the expression of β-catenin by controlling its protein stability. ( A ) To compare the β-catenin protein stability, PC14PE6 and PC14PE6/LvBr4 cells were treated with cycloheximide (20 μg/mL) and harvested at the indicated times. Whole cell lysates were prepared, and the level of β-catenin protein was determined by western blotting. The stability of β-catenin was assessed by image analysis. ( B ) The expression level of USP4 in PC14PE6 and PC14PE6/LvBr4 cells was examined by western blotting (left panel) and RT-qPCR (right panel). ( C ) To determine whether USP regulates the expression of β-catenin, brain metastatic PC14PE6/LvBr4 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. The whole cell extract was prepared and the expression level of USP4 and β-catenin was determined by western blotting. ( D,E ) To evaluate the direct interaction between USP4 and β-catenin, whole cell lysates were prepared using RIPA buffer, and equal amounts of protein were incubated with appropriate control IgG or an antibody against USP4 ( D ) or β-catenin ( E ). The level of β-catenin and USP4 in IP materials was assessed by western blotting. ( F ) To check the effect of USP4 silencing on the ubiquitination of β-catenin, PC14PE6 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. Whole cell lysates were prepared using RIPA buffer and incubated with appropriate control IgG and β-catenin antibody. The levels of β-catenin and ubiquitin were assessed by western blotting. ( G ) To compare the protein stability of β-catenin, parental PC14PE6 and brain metastatic PC14PE6/LvBr4 cells were treated with cycloheximide (40 μg/mL) and harvested at the indicated times. The stability of β-catenin was determined as described above. Data are means and standard deviation from more than three independent experiments. * p
    Figure Legend Snippet: USP4 regulated the expression of β-catenin by controlling its protein stability. ( A ) To compare the β-catenin protein stability, PC14PE6 and PC14PE6/LvBr4 cells were treated with cycloheximide (20 μg/mL) and harvested at the indicated times. Whole cell lysates were prepared, and the level of β-catenin protein was determined by western blotting. The stability of β-catenin was assessed by image analysis. ( B ) The expression level of USP4 in PC14PE6 and PC14PE6/LvBr4 cells was examined by western blotting (left panel) and RT-qPCR (right panel). ( C ) To determine whether USP regulates the expression of β-catenin, brain metastatic PC14PE6/LvBr4 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. The whole cell extract was prepared and the expression level of USP4 and β-catenin was determined by western blotting. ( D,E ) To evaluate the direct interaction between USP4 and β-catenin, whole cell lysates were prepared using RIPA buffer, and equal amounts of protein were incubated with appropriate control IgG or an antibody against USP4 ( D ) or β-catenin ( E ). The level of β-catenin and USP4 in IP materials was assessed by western blotting. ( F ) To check the effect of USP4 silencing on the ubiquitination of β-catenin, PC14PE6 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. Whole cell lysates were prepared using RIPA buffer and incubated with appropriate control IgG and β-catenin antibody. The levels of β-catenin and ubiquitin were assessed by western blotting. ( G ) To compare the protein stability of β-catenin, parental PC14PE6 and brain metastatic PC14PE6/LvBr4 cells were treated with cycloheximide (40 μg/mL) and harvested at the indicated times. The stability of β-catenin was determined as described above. Data are means and standard deviation from more than three independent experiments. * p

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Incubation, Standard Deviation

    Knockdown of β-catenin suppressed metastatic potential of brain metastatic PC14PE6/LvBr4 cells. ( A ) To evaluate the effect of β-catenin silencing on metastatic potential, brain metastatic PC14PE6/LvBr4 cells were transfected with control (CTRL) or β-catenin-specific siRNA for 48 h. The expression level of β-catenin was determined by western blotting. ( B–D ) Metast atic potential including invasion ( B ) and migration (C: Transwell, D: wound closure) was determined as described in the Materials and Methods. Data are means and standard deviation from more than three independent experiments. * p
    Figure Legend Snippet: Knockdown of β-catenin suppressed metastatic potential of brain metastatic PC14PE6/LvBr4 cells. ( A ) To evaluate the effect of β-catenin silencing on metastatic potential, brain metastatic PC14PE6/LvBr4 cells were transfected with control (CTRL) or β-catenin-specific siRNA for 48 h. The expression level of β-catenin was determined by western blotting. ( B–D ) Metast atic potential including invasion ( B ) and migration (C: Transwell, D: wound closure) was determined as described in the Materials and Methods. Data are means and standard deviation from more than three independent experiments. * p

    Techniques Used: Transfection, Expressing, Western Blot, Migration, Standard Deviation

    Knockdown of USP4 inhibited metastatic potential of brain metastatic PC14PE6/LvBr4 cells. ( A ) To assess the function of USP4 in metastatic potential, PC14PE6/LvBr4 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. The expression level of USP4 was determined by western blotting. ( B–D ) Metastatic potential including invasion ( B ) and migration ( C : Transwell, D : wound closure) was determined as described in the Materials and Methods. ( E,F ) To check whether USP4 overexpression upregulates β-catenin expression and increases invasiveness, PC14PE6 cells were transfected with blank (FLAG) or USP4-overexpressing vector (FLAG-USP4). The level of USP4 and β-catenin was determined by Western blot ( E ) and invasiveness was assessed using a Matrigel Invasion Chamber ( F ). Data are means and standard deviation from more than three independent experiments. * p
    Figure Legend Snippet: Knockdown of USP4 inhibited metastatic potential of brain metastatic PC14PE6/LvBr4 cells. ( A ) To assess the function of USP4 in metastatic potential, PC14PE6/LvBr4 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. The expression level of USP4 was determined by western blotting. ( B–D ) Metastatic potential including invasion ( B ) and migration ( C : Transwell, D : wound closure) was determined as described in the Materials and Methods. ( E,F ) To check whether USP4 overexpression upregulates β-catenin expression and increases invasiveness, PC14PE6 cells were transfected with blank (FLAG) or USP4-overexpressing vector (FLAG-USP4). The level of USP4 and β-catenin was determined by Western blot ( E ) and invasiveness was assessed using a Matrigel Invasion Chamber ( F ). Data are means and standard deviation from more than three independent experiments. * p

    Techniques Used: Transfection, Expressing, Western Blot, Migration, Over Expression, Plasmid Preparation, Standard Deviation

    USP4/β-catenin axis regulated the clonogenicity and epithelial-mesenchymal transition in brain metastatic PC14PE6/LvBr4 cells. ( A ) For the colony forming assay, PC14PE6/LvBr4 cells were transfected with control (CTRL) or siRNAs targeting USP4 or β-catenin for 48 h. The level of USP4 and β-catenin was determined by western blotting. ( B ) Transfected cells were seeded into 6-well plates and cultured with complete medium for 2 weeks. After cells were stained with 0.2% crystal violet, the number of stained colonies was counted. ( C,D ) To compare the EMT characteristics between PC14PE6 and PC14PE6/LvBr4 cells, the expression level of E-cadherin and ZEB1 was determined by western blotting ( C ) and RT-qPCR ( D ). ( E ) To examine the effect of USP4 and β-catenin on ZEB1 expression, PC14PE6/LvBr4 cells were transfected with control (CTRL) siRNA or siRNAs targeting USP4 or β-catenin for 48 h. The levels of USP4, β-catenin, and ZEB1 were determined by western blotting. ( F,G ) To investigate the effect of ZEB1 silencing on EMT and invasiveness, PC14PE6/LvBr4 cells were transfected with control (CTRL) or ZEB1-specific siRNA for 48 h. The levels of ZEB1 and E-cadherin were determined by western blotting ( F ) and the invasiveness of transfected cells was assessed using a Matrigel Invasion Chamber ( G ). Data are means and standard deviation from more than three independent experiments. * p
    Figure Legend Snippet: USP4/β-catenin axis regulated the clonogenicity and epithelial-mesenchymal transition in brain metastatic PC14PE6/LvBr4 cells. ( A ) For the colony forming assay, PC14PE6/LvBr4 cells were transfected with control (CTRL) or siRNAs targeting USP4 or β-catenin for 48 h. The level of USP4 and β-catenin was determined by western blotting. ( B ) Transfected cells were seeded into 6-well plates and cultured with complete medium for 2 weeks. After cells were stained with 0.2% crystal violet, the number of stained colonies was counted. ( C,D ) To compare the EMT characteristics between PC14PE6 and PC14PE6/LvBr4 cells, the expression level of E-cadherin and ZEB1 was determined by western blotting ( C ) and RT-qPCR ( D ). ( E ) To examine the effect of USP4 and β-catenin on ZEB1 expression, PC14PE6/LvBr4 cells were transfected with control (CTRL) siRNA or siRNAs targeting USP4 or β-catenin for 48 h. The levels of USP4, β-catenin, and ZEB1 were determined by western blotting. ( F,G ) To investigate the effect of ZEB1 silencing on EMT and invasiveness, PC14PE6/LvBr4 cells were transfected with control (CTRL) or ZEB1-specific siRNA for 48 h. The levels of ZEB1 and E-cadherin were determined by western blotting ( F ) and the invasiveness of transfected cells was assessed using a Matrigel Invasion Chamber ( G ). Data are means and standard deviation from more than three independent experiments. * p

    Techniques Used: Transfection, Western Blot, Cell Culture, Staining, Expressing, Quantitative RT-PCR, Standard Deviation

    Knockdown of USP4 inhibited brain metastasis and promoted overall survival (OS) and brain metastasis-free survival (BMFS). ( A ) PC14PE6/LvBr5-Luc cells were transfected with control (CTRL) siRNA or USP4-specific siRNA for 48 h. The levels of USP4, β-catenin, E-cadherin, and ZEB1 were determined by western blotting. ( B ) For the invasion assay, equal numbers of transfected cells were inoculated into a Transwell and invaded cells were stained and counted under a microscope. Data are means and standard deviation from more than three independent experiments. ( C ) To investigate the effect of USP4 silencing on brain metastasis in vivo , transfected PC14PE6/LvBr5-Luc cells were directly injected into the left ventricle (LV) of the heart. Bioluminescence images were acquired at 22 days post-injection. ( D , E ) Incidence of whole body ( D ) and brain metastasis ( E ) were quantified by the luminescent signal at a given time point. ( F,G ) Kaplan–Meier curves and p -values for overall survival (OS) and brain metastasis-free survival (BMFS) were analyzed in systemic metastasis models. * p
    Figure Legend Snippet: Knockdown of USP4 inhibited brain metastasis and promoted overall survival (OS) and brain metastasis-free survival (BMFS). ( A ) PC14PE6/LvBr5-Luc cells were transfected with control (CTRL) siRNA or USP4-specific siRNA for 48 h. The levels of USP4, β-catenin, E-cadherin, and ZEB1 were determined by western blotting. ( B ) For the invasion assay, equal numbers of transfected cells were inoculated into a Transwell and invaded cells were stained and counted under a microscope. Data are means and standard deviation from more than three independent experiments. ( C ) To investigate the effect of USP4 silencing on brain metastasis in vivo , transfected PC14PE6/LvBr5-Luc cells were directly injected into the left ventricle (LV) of the heart. Bioluminescence images were acquired at 22 days post-injection. ( D , E ) Incidence of whole body ( D ) and brain metastasis ( E ) were quantified by the luminescent signal at a given time point. ( F,G ) Kaplan–Meier curves and p -values for overall survival (OS) and brain metastasis-free survival (BMFS) were analyzed in systemic metastasis models. * p

    Techniques Used: Transfection, Western Blot, Invasion Assay, Staining, Microscopy, Standard Deviation, In Vivo, Injection

    19) Product Images from "IFN-α Induces Transcription of Hypoxia-Inducible Factor-1α to Inhibit Proliferation of Human Endothelial Cells 1"

    Article Title: IFN-α Induces Transcription of Hypoxia-Inducible Factor-1α to Inhibit Proliferation of Human Endothelial Cells 1

    Journal:

    doi:

    Analysis of IRF9 on IFN- α -mediated induction of HIF-1 α . HUVECs were transfected with siRNA against IRF9 or control and stimulated with 100 ng/ml IFN- α for the indicated times. To assess IRF9 knockdown, cell lysates were collected
    Figure Legend Snippet: Analysis of IRF9 on IFN- α -mediated induction of HIF-1 α . HUVECs were transfected with siRNA against IRF9 or control and stimulated with 100 ng/ml IFN- α for the indicated times. To assess IRF9 knockdown, cell lysates were collected

    Techniques Used: Transfection

    20) Product Images from "MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signaling in cancer"

    Article Title: MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signaling in cancer

    Journal: Nature communications

    doi: 10.1038/ncomms7053

    Comparative MnSOD expression and effects in non tumorigenic (MCF10A), tumorigenic (MCF7) and metastatic (MDA-MB-231 and U2OS) cells (A) Western blot analysis of MnSOD expression levels in non tumorigenic (non-induced MCF10AErSrc), tumorigenic (TMX-treated MCF10A-Er-Src, MCF7) and metastatic (MDA-MB-231 and U2OS) cell lines. (B) Levels of MnSOD in MDA-MB231 cells manipulated with MnSOD targeted siRNA and respective diminishment in AMPK phosphorylation levels as analyzed by Western blot. (C) Quantification of MnSOD expression by Western blot before and after treatment of MDA-MB231 cells with silencing RNA. (D) Quantification of AMPK phosphorylation before and after MnSOD silencing in MDA-MB231. (E) Effect of MnSOD silencing on the glycolytic rate of MDA-MB-231 cells measured by extracellular flow analysis using Seahorse Biosciences XF analyser. (F) Effect of MnSOD silencing on steady state ATP levels of MDA-MB-231 cells. Cells were harvested and analysis was performed 72h after the delivery of siRNA. Statistical analysis was performed using one-way ANOVA with post-hoc t-test (GraphPad InStat) *p
    Figure Legend Snippet: Comparative MnSOD expression and effects in non tumorigenic (MCF10A), tumorigenic (MCF7) and metastatic (MDA-MB-231 and U2OS) cells (A) Western blot analysis of MnSOD expression levels in non tumorigenic (non-induced MCF10AErSrc), tumorigenic (TMX-treated MCF10A-Er-Src, MCF7) and metastatic (MDA-MB-231 and U2OS) cell lines. (B) Levels of MnSOD in MDA-MB231 cells manipulated with MnSOD targeted siRNA and respective diminishment in AMPK phosphorylation levels as analyzed by Western blot. (C) Quantification of MnSOD expression by Western blot before and after treatment of MDA-MB231 cells with silencing RNA. (D) Quantification of AMPK phosphorylation before and after MnSOD silencing in MDA-MB231. (E) Effect of MnSOD silencing on the glycolytic rate of MDA-MB-231 cells measured by extracellular flow analysis using Seahorse Biosciences XF analyser. (F) Effect of MnSOD silencing on steady state ATP levels of MDA-MB-231 cells. Cells were harvested and analysis was performed 72h after the delivery of siRNA. Statistical analysis was performed using one-way ANOVA with post-hoc t-test (GraphPad InStat) *p

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot, Flow Cytometry

    Suppression of MnSOD or inhibition of AMPK impair the shift to glycolysis in cells overexpressing MnSOD MnSOD siRNA was introduced into cells overexpressing MnSOD by electroporation. Cells were harvested 48 h after siRNA delivery. (A) Western blot of protein lysates showed the decrease of MnSOD expression in Mn44 and Mn11 to levels compared to undisturbed neo. The partial downregulation of MnSOD in Mn44 and Mn11 corresponded to a decrease in activated AMPK (Thr172p) analyzed by Western blot. (B) MnSOD siRNA transfection resulted in decreased glycolytic rate compared to untreated cells. The glycolytic rate was assessed by monitoring lactate production between 48 and 52 h post MnSOD silencing. (C) AMPK inhibition with compound C (for 24 h, 25 μM) decreased glycolysis. (D) ATP steady state levels were slightly and markedly decreased in Mn44 and Mn11, respectively by the inhibition of AMPK with compound C. (E) Cells constitutively expressing AMPKα1 siRNA were transfected with MnSOD-GFP or GFP carrying constructs using lipofectamine. Western blot analysis of MnSOD expression levels was performed by Western blot 48h after transfection. (F) Cells overexpressing MnSOD on an AMPK- competent and AMPK-depleted background were analyzed by extracellular flow analysis (seahorse). MnSOD overexpression led to the metabolic shift to glycolysis only when expressed in AMPK-competent cells. *p
    Figure Legend Snippet: Suppression of MnSOD or inhibition of AMPK impair the shift to glycolysis in cells overexpressing MnSOD MnSOD siRNA was introduced into cells overexpressing MnSOD by electroporation. Cells were harvested 48 h after siRNA delivery. (A) Western blot of protein lysates showed the decrease of MnSOD expression in Mn44 and Mn11 to levels compared to undisturbed neo. The partial downregulation of MnSOD in Mn44 and Mn11 corresponded to a decrease in activated AMPK (Thr172p) analyzed by Western blot. (B) MnSOD siRNA transfection resulted in decreased glycolytic rate compared to untreated cells. The glycolytic rate was assessed by monitoring lactate production between 48 and 52 h post MnSOD silencing. (C) AMPK inhibition with compound C (for 24 h, 25 μM) decreased glycolysis. (D) ATP steady state levels were slightly and markedly decreased in Mn44 and Mn11, respectively by the inhibition of AMPK with compound C. (E) Cells constitutively expressing AMPKα1 siRNA were transfected with MnSOD-GFP or GFP carrying constructs using lipofectamine. Western blot analysis of MnSOD expression levels was performed by Western blot 48h after transfection. (F) Cells overexpressing MnSOD on an AMPK- competent and AMPK-depleted background were analyzed by extracellular flow analysis (seahorse). MnSOD overexpression led to the metabolic shift to glycolysis only when expressed in AMPK-competent cells. *p

    Techniques Used: Inhibition, Electroporation, Western Blot, Expressing, Transfection, Construct, Flow Cytometry, Over Expression

    MnSOD induces the expression of survival proteins in an AMPK-dependent manner (A) Survivin and Bcl-2 protein expression in neo, Mn44 and Mn11 cells were measured by confocal microscopy. (B) same as (A) but protein expression was analyzed by Western blot in neo, Mn1, Mn44, Mn11 and Mn28 cells .(C). Silencing of AMPK by siRNA transfection with lipofectamine decreases Bcl-2 and survivin expression in Mn44 and Mn11 (results from the analysis of Mn44 and Mn11 are from different sections of the same nitrocellulose membrane). AMPK siRNA was transfected with lipofectamine 72 h prior to harvesting (D) Immunohistochemical analysis shows increased expression of anti-apoptotic proteins survivin and Bcl-2 in human breast cancer tissues in parallel with increased MnSOD expression. Representative images of at least 15 different cases (Stage III, ductal invasive carcinoma) are shown.
    Figure Legend Snippet: MnSOD induces the expression of survival proteins in an AMPK-dependent manner (A) Survivin and Bcl-2 protein expression in neo, Mn44 and Mn11 cells were measured by confocal microscopy. (B) same as (A) but protein expression was analyzed by Western blot in neo, Mn1, Mn44, Mn11 and Mn28 cells .(C). Silencing of AMPK by siRNA transfection with lipofectamine decreases Bcl-2 and survivin expression in Mn44 and Mn11 (results from the analysis of Mn44 and Mn11 are from different sections of the same nitrocellulose membrane). AMPK siRNA was transfected with lipofectamine 72 h prior to harvesting (D) Immunohistochemical analysis shows increased expression of anti-apoptotic proteins survivin and Bcl-2 in human breast cancer tissues in parallel with increased MnSOD expression. Representative images of at least 15 different cases (Stage III, ductal invasive carcinoma) are shown.

    Techniques Used: Expressing, Confocal Microscopy, Western Blot, Transfection, Immunohistochemistry

    21) Product Images from "Activation of the Nrf2/HO-1 signaling pathway contributes to the protective effects of baicalein against oxidative stress-induced DNA damage and apoptosis in HEI193 Schwann cells"

    Article Title: Activation of the Nrf2/HO-1 signaling pathway contributes to the protective effects of baicalein against oxidative stress-induced DNA damage and apoptosis in HEI193 Schwann cells

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.27005

    Attenuation of H 2 O 2 -induced mitochondrial dysfunction and changes of apoptosis-related proteins by baicalein in HEI193 cells. After transfection with or without Nrf2 siRNA, the cells were treated with 100 μM baicalein for 1 h and then stimulated with or without 1 mM H 2 O 2 for 24 h. (A) The cells were collected and incubated with 10 μM JC-1 for 20 min at 37°C in the dark. The values of MMP were evaluated using a flow cytometer. The data are the means of the two different experiments. (B) The cellular proteins were separated by SDS-polyacrylamide gel electrophoresis, and then transferred to the membranes. The membranes were probed with the indicated antibodies. The proteins were visualized using an ECL detection system. Actin was used as an internal control. The relative ratios of expression in the results of Western blotting are presented at the bottom of each result as relative value of actin expression.
    Figure Legend Snippet: Attenuation of H 2 O 2 -induced mitochondrial dysfunction and changes of apoptosis-related proteins by baicalein in HEI193 cells. After transfection with or without Nrf2 siRNA, the cells were treated with 100 μM baicalein for 1 h and then stimulated with or without 1 mM H 2 O 2 for 24 h. (A) The cells were collected and incubated with 10 μM JC-1 for 20 min at 37°C in the dark. The values of MMP were evaluated using a flow cytometer. The data are the means of the two different experiments. (B) The cellular proteins were separated by SDS-polyacrylamide gel electrophoresis, and then transferred to the membranes. The membranes were probed with the indicated antibodies. The proteins were visualized using an ECL detection system. Actin was used as an internal control. The relative ratios of expression in the results of Western blotting are presented at the bottom of each result as relative value of actin expression.

    Techniques Used: Transfection, Incubation, Flow Cytometry, Cytometry, Polyacrylamide Gel Electrophoresis, Expressing, Western Blot

    Involvement of Nrf2/HO-1 signaling in the protective effect of baicalein on H 2 O 2 -induced HEI193 cell cytotoxicity. Cells transfected with or without Nrf2-siRNA were pretreated with 100 μM baicalein for 1 h and then stimulated with or without 1 mM H 2 O 2 for 24 h. (A) The cells were lysed and then equal amounts of cell lysates were separated on SDS-polyacrylamide gels and transferred to the membranes. The membranes were probed with specific antibodies against Nrf2 and HO-1, and the proteins were visualized using an ECL detection system. Actin was used as an internal control. The relative ratios of expression in the results of Western blotting are presented at the bottom of each result as relative value of actin expression. (B) The cell viability was estimated using an MTT assay. The results are the mean ± SD values obtained from three independent experiments ( * p
    Figure Legend Snippet: Involvement of Nrf2/HO-1 signaling in the protective effect of baicalein on H 2 O 2 -induced HEI193 cell cytotoxicity. Cells transfected with or without Nrf2-siRNA were pretreated with 100 μM baicalein for 1 h and then stimulated with or without 1 mM H 2 O 2 for 24 h. (A) The cells were lysed and then equal amounts of cell lysates were separated on SDS-polyacrylamide gels and transferred to the membranes. The membranes were probed with specific antibodies against Nrf2 and HO-1, and the proteins were visualized using an ECL detection system. Actin was used as an internal control. The relative ratios of expression in the results of Western blotting are presented at the bottom of each result as relative value of actin expression. (B) The cell viability was estimated using an MTT assay. The results are the mean ± SD values obtained from three independent experiments ( * p

    Techniques Used: Transfection, Expressing, Western Blot, MTT Assay

    Protection of H 2 O 2 -induced ROS generation and DNA damage by baicalein in HEI193 cells. Cells transfected with or without Nrf2-siRNA were pretreated with 100 μM baicalein for 1 h and then stimulated with or without 1 mM H 2 O 2 for 1 h (A) or 24 h (B and C). (A) The cells were incubated with culture medium containing 10 μM DCF-DA to monitor ROS production. The degree of ROS production was measured with a flow cytometer. The data are the means of the two different experiments. (B) To detect cellular DNA damage, a comet assay was performed, and representative photographs of the comets were captured using a fluorescence microscope (original magnification, 200×). (C) Equal amounts of cell lysates were separated on SDS-polyacrylamide gels and transferred to the membranes. The membranes were probed with specific antibodies against γH2A.X and p-γH2A.X, and the proteins were visualized using an ECL detection system. Actin was used as an internal control. The relative ratios of expression in the results of Western blotting are presented at the bottom of each result as relative value of actin expression.
    Figure Legend Snippet: Protection of H 2 O 2 -induced ROS generation and DNA damage by baicalein in HEI193 cells. Cells transfected with or without Nrf2-siRNA were pretreated with 100 μM baicalein for 1 h and then stimulated with or without 1 mM H 2 O 2 for 1 h (A) or 24 h (B and C). (A) The cells were incubated with culture medium containing 10 μM DCF-DA to monitor ROS production. The degree of ROS production was measured with a flow cytometer. The data are the means of the two different experiments. (B) To detect cellular DNA damage, a comet assay was performed, and representative photographs of the comets were captured using a fluorescence microscope (original magnification, 200×). (C) Equal amounts of cell lysates were separated on SDS-polyacrylamide gels and transferred to the membranes. The membranes were probed with specific antibodies against γH2A.X and p-γH2A.X, and the proteins were visualized using an ECL detection system. Actin was used as an internal control. The relative ratios of expression in the results of Western blotting are presented at the bottom of each result as relative value of actin expression.

    Techniques Used: Transfection, Incubation, Flow Cytometry, Cytometry, Single Cell Gel Electrophoresis, Fluorescence, Microscopy, Expressing, Western Blot

    Suppression of H 2 O 2 -induced apoptosis by baicalein in HEI193 cells. After transfection with or without Nrf2 siRNA, the cells were treated with 100 μM baicalein for 1 h and then stimulated with or without 1 mM H 2 O 2 for 24 h. (A) The cells were fixed and stained with DAPI solution. The stained nuclei were observed using a fluorescence microscope (original magnification, ×400). (B) DNA fragmentation was analyzed by extracting genomic DNA, electrophoresis in a 1.5% agarose gel, and then visualizing by EtBr staining. (C) The cells were collected and stained with annexin-V and PI, and the percentages of apoptotic cells were then analyzed using flow cytometric analysis. The results are the means of two independent experiments.
    Figure Legend Snippet: Suppression of H 2 O 2 -induced apoptosis by baicalein in HEI193 cells. After transfection with or without Nrf2 siRNA, the cells were treated with 100 μM baicalein for 1 h and then stimulated with or without 1 mM H 2 O 2 for 24 h. (A) The cells were fixed and stained with DAPI solution. The stained nuclei were observed using a fluorescence microscope (original magnification, ×400). (B) DNA fragmentation was analyzed by extracting genomic DNA, electrophoresis in a 1.5% agarose gel, and then visualizing by EtBr staining. (C) The cells were collected and stained with annexin-V and PI, and the percentages of apoptotic cells were then analyzed using flow cytometric analysis. The results are the means of two independent experiments.

    Techniques Used: Transfection, Staining, Fluorescence, Microscopy, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Flow Cytometry

    22) Product Images from "Differentiation of Human Skeletal Muscle Stem Cells into Odontoblasts Is Dependent on Induction of α1 Integrin Expression *"

    Article Title: Differentiation of Human Skeletal Muscle Stem Cells into Odontoblasts Is Dependent on Induction of α1 Integrin Expression *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.526772

    Induction of α1 integrin is required for odontoblastic differentiation. A , α7 + hSMSCs were transiently transfected with α1 integrin siRNA or a nonrelevant siRNA (control ( Cont )). After 24 h, α1 integrin silencing was determined for both mRNA ( upper ; RT-PCR analysis) and protein ( lower ; immunoblot analysis) levels, respectively. GAPDH is provided as a loading control for RT-PCR, and for Western blotting, β1 integrin and β-tubulin are shown. B , the effect of transfection of α7 + hSMSCs with α1 integrin siRNA as above on levels of DSPP, MMP-20, and MYOD transcripts following processing for differentiation with the GS/BMP-4 protocol measured by RT-PCR ( upper ) and Western blotting ( lower ). C , silencing α1 integrin abolished ALPase activity. Control α7 + hSMSC cultures were negative for enzyme activity ( panel a ) but following treatment with GS/BMP-4 showed strong induction of ALPase ( panel b ) that was abolished with α1 integrin siRNA ( panel c ). D , mineralization of α7 + hSMSCs in the absence ( panel a ) or presence ( panel b ) of 15% GS/BMP-4 for 7 days as described under “Experimental Procedures” and assessed by ARS staining. The addition of α1 integrin siRNA with GS/BMP-4 suppressed the ARS staining of positive cells ( panel c ). E , ALPase activity ( top panel ) and mineralization ( bottom panel ) of α7 + hSMSCs in the absence or presence of GS/BMP-4 for 7 days was assessed. The addition of α1 integrin siRNA with GS/BMP-4 suppressed both the up-regulation of ALPase activity (measured at 405 nm) and mineralization (measured by quantification of ARS at 405 nm). Error bars represent S.D. (control versus **, p
    Figure Legend Snippet: Induction of α1 integrin is required for odontoblastic differentiation. A , α7 + hSMSCs were transiently transfected with α1 integrin siRNA or a nonrelevant siRNA (control ( Cont )). After 24 h, α1 integrin silencing was determined for both mRNA ( upper ; RT-PCR analysis) and protein ( lower ; immunoblot analysis) levels, respectively. GAPDH is provided as a loading control for RT-PCR, and for Western blotting, β1 integrin and β-tubulin are shown. B , the effect of transfection of α7 + hSMSCs with α1 integrin siRNA as above on levels of DSPP, MMP-20, and MYOD transcripts following processing for differentiation with the GS/BMP-4 protocol measured by RT-PCR ( upper ) and Western blotting ( lower ). C , silencing α1 integrin abolished ALPase activity. Control α7 + hSMSC cultures were negative for enzyme activity ( panel a ) but following treatment with GS/BMP-4 showed strong induction of ALPase ( panel b ) that was abolished with α1 integrin siRNA ( panel c ). D , mineralization of α7 + hSMSCs in the absence ( panel a ) or presence ( panel b ) of 15% GS/BMP-4 for 7 days as described under “Experimental Procedures” and assessed by ARS staining. The addition of α1 integrin siRNA with GS/BMP-4 suppressed the ARS staining of positive cells ( panel c ). E , ALPase activity ( top panel ) and mineralization ( bottom panel ) of α7 + hSMSCs in the absence or presence of GS/BMP-4 for 7 days was assessed. The addition of α1 integrin siRNA with GS/BMP-4 suppressed both the up-regulation of ALPase activity (measured at 405 nm) and mineralization (measured by quantification of ARS at 405 nm). Error bars represent S.D. (control versus **, p

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Staining

    23) Product Images from "Calnexin, an ER-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer"

    Article Title: Calnexin, an ER-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-016-0948-z

    Gene silencing of calnexin attenuates HCT116 colon cancer cell survival. Western blot analysis depicting decreased expression of calnexin in HCT116 colon cancer cells following transfection for 24 h with calnexin siRNA compared with control siRNA ( a ). Sample image of a clonogenic survival assay and histogram depicting total colony number following transfection of HCT116 cells with control or calnexin siRNA for 24 h and subsequent colony formation for a further 14 days ( b ; n = 3). Annexin V and PI staining of HCT116 cells transfected with control or calnexin siRNA for 24 h followed by treatment with 5FU (20 µg/ml) for 48 h as determined by flow cytometry ( c , *p
    Figure Legend Snippet: Gene silencing of calnexin attenuates HCT116 colon cancer cell survival. Western blot analysis depicting decreased expression of calnexin in HCT116 colon cancer cells following transfection for 24 h with calnexin siRNA compared with control siRNA ( a ). Sample image of a clonogenic survival assay and histogram depicting total colony number following transfection of HCT116 cells with control or calnexin siRNA for 24 h and subsequent colony formation for a further 14 days ( b ; n = 3). Annexin V and PI staining of HCT116 cells transfected with control or calnexin siRNA for 24 h followed by treatment with 5FU (20 µg/ml) for 48 h as determined by flow cytometry ( c , *p

    Techniques Used: Western Blot, Expressing, Transfection, Clonogenic Cell Survival Assay, Staining, Flow Cytometry, Cytometry

    24) Product Images from "Global Mapping of Cell Type-Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation"

    Article Title: Global Mapping of Cell Type-Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002311

    NFIA and NFIB are novel regulators of adipocyte differentiation. (A) Transcriptional regulation of NFI transcription factors during adipocyte differentiation (3T3-F442A). (B) Tissue distribution of the NFI family genes. Expression levels relative to 36B4 in various tissues were determined by qPCR. (C, D) Effects of siRNA-mediated knockdown of NFIA and NFIB on adipogenic gene expression (C) and lipid accumulation in 3T3-L1 adipocytes judged by oil red O staining (D). Knockdown of either NFIA or NFIB resulted in suppression of the induction of PPARγ, C/EBPα and the PPARγ target gene, aP2, as well as increase in lipid accumulation during adipocyte differentiation.
    Figure Legend Snippet: NFIA and NFIB are novel regulators of adipocyte differentiation. (A) Transcriptional regulation of NFI transcription factors during adipocyte differentiation (3T3-F442A). (B) Tissue distribution of the NFI family genes. Expression levels relative to 36B4 in various tissues were determined by qPCR. (C, D) Effects of siRNA-mediated knockdown of NFIA and NFIB on adipogenic gene expression (C) and lipid accumulation in 3T3-L1 adipocytes judged by oil red O staining (D). Knockdown of either NFIA or NFIB resulted in suppression of the induction of PPARγ, C/EBPα and the PPARγ target gene, aP2, as well as increase in lipid accumulation during adipocyte differentiation.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Staining

    NFIA and NFIB are novel regulators of adipocyte differentiation. (A) Transcriptional regulation of NFI transcription factors during adipocyte differentiation (3T3-F442A). (B) Tissue distribution of the NFI family genes. Expression levels relative to 36B4 in various tissues were determined by qPCR. (C, D) Effects of siRNA-mediated knockdown of NFIA and NFIB on adipogenic gene expression (C) and lipid accumulation in 3T3-L1 adipocytes judged by oil red O staining (D). Knockdown of either NFIA or NFIB resulted in suppression of the induction of PPARγ, C/EBPα and the PPARγ target gene, aP2, as well as increase in lipid accumulation during adipocyte differentiation.
    Figure Legend Snippet: NFIA and NFIB are novel regulators of adipocyte differentiation. (A) Transcriptional regulation of NFI transcription factors during adipocyte differentiation (3T3-F442A). (B) Tissue distribution of the NFI family genes. Expression levels relative to 36B4 in various tissues were determined by qPCR. (C, D) Effects of siRNA-mediated knockdown of NFIA and NFIB on adipogenic gene expression (C) and lipid accumulation in 3T3-L1 adipocytes judged by oil red O staining (D). Knockdown of either NFIA or NFIB resulted in suppression of the induction of PPARγ, C/EBPα and the PPARγ target gene, aP2, as well as increase in lipid accumulation during adipocyte differentiation.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Staining

    25) Product Images from "Augmented Osteogenic Responses in Human Aortic Valve Cells Exposed to oxLDL and TLR4 Agonist: A Mechanistic Role of Notch1 and NF-?B Interaction"

    Article Title: Augmented Osteogenic Responses in Human Aortic Valve Cells Exposed to oxLDL and TLR4 Agonist: A Mechanistic Role of Notch1 and NF-?B Interaction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095400

    Notch1 knockdown reduces the expression of BMP-2 and ALP following stimulation with LPS plus oxLDL. Human AVICs were treated with Notch1-specific siRNA and then stimulated with LPS plus oxLDL. Representative immunoblots of 2 separate experiments show that knockdown of Notch1 results in decreased expression of BMP-2 and ALP following stimulation with LPS plus oxLDL that is associated with reduced levels of NICD1.
    Figure Legend Snippet: Notch1 knockdown reduces the expression of BMP-2 and ALP following stimulation with LPS plus oxLDL. Human AVICs were treated with Notch1-specific siRNA and then stimulated with LPS plus oxLDL. Representative immunoblots of 2 separate experiments show that knockdown of Notch1 results in decreased expression of BMP-2 and ALP following stimulation with LPS plus oxLDL that is associated with reduced levels of NICD1.

    Techniques Used: Expressing, ALP Assay, Western Blot

    26) Product Images from "Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells, et al. Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells"

    Article Title: Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells, et al. Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13523

    Effects of CB1R on gene expression depends on insulin receptor (IR). A, Western blot analysis of IR β‐subunit (IRβ) and GAPDH in βIRWT and βIRKO cells. B, Quantitative real‐time PCR analysis of CB1R expression in βIRWT and βIRKO cells. Data were normalized to 18S ribosomal RNA levels. C, Quantitative real‐time PCR analysis of CB1R, insulin, GCK and GLUT2 expression in βIRWT and βIRKO cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. D, Schematic unifying the regulation of β‐cell function by ECs and CB1Rs. Data are shown as the mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: Effects of CB1R on gene expression depends on insulin receptor (IR). A, Western blot analysis of IR β‐subunit (IRβ) and GAPDH in βIRWT and βIRKO cells. B, Quantitative real‐time PCR analysis of CB1R expression in βIRWT and βIRKO cells. Data were normalized to 18S ribosomal RNA levels. C, Quantitative real‐time PCR analysis of CB1R, insulin, GCK and GLUT2 expression in βIRWT and βIRKO cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. D, Schematic unifying the regulation of β‐cell function by ECs and CB1Rs. Data are shown as the mean ± SEM from three independent experiments. * P

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Cell Function Assay

    Effects of blocking CB1R on intra‐β‐cell insulin content and glucokinase (GCK) and glucose transporter 2 (GLUT2) expressions. A, CB1R and Insulin ( Ins2 ) mRNA levels in βTC6 cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. B, Intra‐islet insulin content in islets isolated from CB1R +/+ and CB1R −/− mice. Insulin was extracted from islets freshly isolated from CB1R +/+ and CB1R −/− mice using acid‐alcohol (n = 3 separate isolates). Size‐matched 10 islets per tube were analysed, and data were normalized to protein concentration. C, Insulin ( Ins1 and Ins2 ) mRNA levels in islets isolated from CB1R +/+ and CB1R −/− mice (n = 4 separate isolates). Data were normalized to 18S ribosomal RNA levels. D, Western blot analysis of preproinsulin, GCK and GLUT2 expressions in total lysates prepared from whole pancreata of overnight‐fasted CB1R +/+ and CB1R −/− (n = 4 per genotype) mice. Signals on Western blots were quantified by densitometry and are shown on the right. E and F, Immunofluorescent staining for GCK (E) and GLUT2 (F) in islets of overnight‐fasted CB1R +/+ and CB1R −/− mice. Scale bar, 50 μm. Relative signal intensity for the indicated proteins in islets is shown on the right (n = 3 per genotype). Quantification of GCK and GLUT2 intensities was shown on the right. Data are shown as the mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: Effects of blocking CB1R on intra‐β‐cell insulin content and glucokinase (GCK) and glucose transporter 2 (GLUT2) expressions. A, CB1R and Insulin ( Ins2 ) mRNA levels in βTC6 cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. B, Intra‐islet insulin content in islets isolated from CB1R +/+ and CB1R −/− mice. Insulin was extracted from islets freshly isolated from CB1R +/+ and CB1R −/− mice using acid‐alcohol (n = 3 separate isolates). Size‐matched 10 islets per tube were analysed, and data were normalized to protein concentration. C, Insulin ( Ins1 and Ins2 ) mRNA levels in islets isolated from CB1R +/+ and CB1R −/− mice (n = 4 separate isolates). Data were normalized to 18S ribosomal RNA levels. D, Western blot analysis of preproinsulin, GCK and GLUT2 expressions in total lysates prepared from whole pancreata of overnight‐fasted CB1R +/+ and CB1R −/− (n = 4 per genotype) mice. Signals on Western blots were quantified by densitometry and are shown on the right. E and F, Immunofluorescent staining for GCK (E) and GLUT2 (F) in islets of overnight‐fasted CB1R +/+ and CB1R −/− mice. Scale bar, 50 μm. Relative signal intensity for the indicated proteins in islets is shown on the right (n = 3 per genotype). Quantification of GCK and GLUT2 intensities was shown on the right. Data are shown as the mean ± SEM from three independent experiments. * P

    Techniques Used: Blocking Assay, Transfection, Isolation, Mouse Assay, Protein Concentration, Western Blot, Staining

    27) Product Images from "Farnesiferol c induces apoptosis via regulation of L11 and c-Myc with combinational potential with anticancer drugs in non-small-cell lung cancers"

    Article Title: Farnesiferol c induces apoptosis via regulation of L11 and c-Myc with combinational potential with anticancer drugs in non-small-cell lung cancers

    Journal: Scientific Reports

    doi: 10.1038/srep26844

    FC regulates c-Myc and L11 through a negative feedback mechanism. ( A ) FC downregulates c-Myc and NCL (C23) in H1299 and H596 cells by Western blotting. ( B ) FC attenuates mRNA expression of c-Myc and NCL in H1299 cells. H1299 cells were treated with FC for 24 h. RNA was isolated and analsis was performed to detect mRNA of c-Myc and NCL by qRT-PCR. ( C ) L11 knockdown activates c-Myc expression in H1299 cells. H1299 cells were transfected with L11 siRNA oligonucleotide (40 nM) for 48 h and then treated with FC for 24 h. Cells were prepared and subjected to Western blotting for c-Myc and L11. ( D ) FC downregulates c-Myc and L11 in Flag tagged L11 overexpression vector transfected H1299 cells. H1299 cells were transfected with Flag tagged L11 vector (1 μg) for 48 h and then treated with FC (0, 60, or 120 μM) for 24 h. Cells were prepared and subjected to Western blotting for c-Myc and L11.
    Figure Legend Snippet: FC regulates c-Myc and L11 through a negative feedback mechanism. ( A ) FC downregulates c-Myc and NCL (C23) in H1299 and H596 cells by Western blotting. ( B ) FC attenuates mRNA expression of c-Myc and NCL in H1299 cells. H1299 cells were treated with FC for 24 h. RNA was isolated and analsis was performed to detect mRNA of c-Myc and NCL by qRT-PCR. ( C ) L11 knockdown activates c-Myc expression in H1299 cells. H1299 cells were transfected with L11 siRNA oligonucleotide (40 nM) for 48 h and then treated with FC for 24 h. Cells were prepared and subjected to Western blotting for c-Myc and L11. ( D ) FC downregulates c-Myc and L11 in Flag tagged L11 overexpression vector transfected H1299 cells. H1299 cells were transfected with Flag tagged L11 vector (1 μg) for 48 h and then treated with FC (0, 60, or 120 μM) for 24 h. Cells were prepared and subjected to Western blotting for c-Myc and L11.

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

    Regulation of L11 and c-Myc is critically involved in apoptosis induced by FC in H1299 cells. ( A ) Effect of c-Myc and/or L11 depletion by siRNA on the viability of H1299 cells by MTT assay. Data represent means ± SD. *p
    Figure Legend Snippet: Regulation of L11 and c-Myc is critically involved in apoptosis induced by FC in H1299 cells. ( A ) Effect of c-Myc and/or L11 depletion by siRNA on the viability of H1299 cells by MTT assay. Data represent means ± SD. *p

    Techniques Used: MTT Assay

    28) Product Images from "AKT inhibition-mediated dephosphorylation of TFE3 promotes overactive autophagy independent of MTORC1 in cadmium-exposed bone mesenchymal stem cells"

    Article Title: AKT inhibition-mediated dephosphorylation of TFE3 promotes overactive autophagy independent of MTORC1 in cadmium-exposed bone mesenchymal stem cells

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1531198

    The TFE3 pathway in Cd-induced autophagy in MSCs. (a-b) MSCs were treated with the siRNA for Tfe3 or the control and then incubated with 14 μM Cd for another 24 h. The mRNA levels of TFE3-target genes were then measured. (c) MSCs transfected with either control siRNA or Tfe3 siRNA were grown with or without Cd (14 μM) for 24 h. The LC3B-II level was determined by western blot. ACTB was the internal standard for protein loading. (d) MSCs transfected with either control siRNA or Tfe3 siRNA were grown with or without Cd (14 μM) for 24 h. Cell death was determined by trypan blue. The results are expressed as the percentage of the control, which was set to 100%. The values are presented as the means ± SEM, **P
    Figure Legend Snippet: The TFE3 pathway in Cd-induced autophagy in MSCs. (a-b) MSCs were treated with the siRNA for Tfe3 or the control and then incubated with 14 μM Cd for another 24 h. The mRNA levels of TFE3-target genes were then measured. (c) MSCs transfected with either control siRNA or Tfe3 siRNA were grown with or without Cd (14 μM) for 24 h. The LC3B-II level was determined by western blot. ACTB was the internal standard for protein loading. (d) MSCs transfected with either control siRNA or Tfe3 siRNA were grown with or without Cd (14 μM) for 24 h. Cell death was determined by trypan blue. The results are expressed as the percentage of the control, which was set to 100%. The values are presented as the means ± SEM, **P

    Techniques Used: Incubation, Transfection, Western Blot

    Cd induces autophagic cell death in cultured MSCs. (a) The cells were treated with Cd at various concentrations (0, 3.5, 7, 14 μM) for 24 h and then analyzed for autophagy using acridine orange staining assessed with flow cytometry. (b) Electron microscopy revealed an increased number of AVs, and the lower panels are magnified portions of the upper panels. (c) A representative immunoblot and quantification analysis of SQSTM1 in MSCs treated with increasing concentrations of Cd. ACTB (actin, beta) was used as a loading control. (d) The MSCs were incubated with Cd (14 μM) in the absence or presence of Baf A1 (10 nM) for 24 h. The expression of LC3B-II was quantified by normalization of its density to that of ACTB. (e) Atg7 siRNA treatment decreased cell death by inhibiting autophagy. The results are expressed as the percentage of the control, which was set to 100 %. The values are presented as the means ±SEM, *p
    Figure Legend Snippet: Cd induces autophagic cell death in cultured MSCs. (a) The cells were treated with Cd at various concentrations (0, 3.5, 7, 14 μM) for 24 h and then analyzed for autophagy using acridine orange staining assessed with flow cytometry. (b) Electron microscopy revealed an increased number of AVs, and the lower panels are magnified portions of the upper panels. (c) A representative immunoblot and quantification analysis of SQSTM1 in MSCs treated with increasing concentrations of Cd. ACTB (actin, beta) was used as a loading control. (d) The MSCs were incubated with Cd (14 μM) in the absence or presence of Baf A1 (10 nM) for 24 h. The expression of LC3B-II was quantified by normalization of its density to that of ACTB. (e) Atg7 siRNA treatment decreased cell death by inhibiting autophagy. The results are expressed as the percentage of the control, which was set to 100 %. The values are presented as the means ±SEM, *p

    Techniques Used: Cell Culture, Staining, Flow Cytometry, Cytometry, Electron Microscopy, Incubation, Expressing

    The TFE3 pathway in Cd-induced autophagy in MSCs. (a-b) MSCs were treated with the siRNA for Tfe3 or the control and then incubated with 14 μM Cd for another 24 h. The mRNA levels of TFE3-target genes were then measured. (c) MSCs transfected with either control siRNA or Tfe3 siRNA were grown with or without Cd (14 μM) for 24 h. The LC3B-II level was determined by western blot. ACTB was the internal standard for protein loading. (d) MSCs transfected with either control siRNA or Tfe3 siRNA were grown with or without Cd (14 μM) for 24 h. Cell death was determined by trypan blue. The results are expressed as the percentage of the control, which was set to 100%. The values are presented as the means ± SEM, **P
    Figure Legend Snippet: The TFE3 pathway in Cd-induced autophagy in MSCs. (a-b) MSCs were treated with the siRNA for Tfe3 or the control and then incubated with 14 μM Cd for another 24 h. The mRNA levels of TFE3-target genes were then measured. (c) MSCs transfected with either control siRNA or Tfe3 siRNA were grown with or without Cd (14 μM) for 24 h. The LC3B-II level was determined by western blot. ACTB was the internal standard for protein loading. (d) MSCs transfected with either control siRNA or Tfe3 siRNA were grown with or without Cd (14 μM) for 24 h. Cell death was determined by trypan blue. The results are expressed as the percentage of the control, which was set to 100%. The values are presented as the means ± SEM, **P

    Techniques Used: Incubation, Transfection, Western Blot

    Cd induces autophagic cell death in cultured MSCs. (a) The cells were treated with Cd at various concentrations (0, 3.5, 7, 14 μM) for 24 h and then analyzed for autophagy using acridine orange staining assessed with flow cytometry. (b) Electron microscopy revealed an increased number of AVs, and the lower panels are magnified portions of the upper panels. (c) A representative immunoblot and quantification analysis of SQSTM1 in MSCs treated with increasing concentrations of Cd. ACTB (actin, beta) was used as a loading control. (d) The MSCs were incubated with Cd (14 μM) in the absence or presence of Baf A1 (10 nM) for 24 h. The expression of LC3B-II was quantified by normalization of its density to that of ACTB. (e) Atg7 siRNA treatment decreased cell death by inhibiting autophagy. The results are expressed as the percentage of the control, which was set to 100 %. The values are presented as the means ±SEM, *p
    Figure Legend Snippet: Cd induces autophagic cell death in cultured MSCs. (a) The cells were treated with Cd at various concentrations (0, 3.5, 7, 14 μM) for 24 h and then analyzed for autophagy using acridine orange staining assessed with flow cytometry. (b) Electron microscopy revealed an increased number of AVs, and the lower panels are magnified portions of the upper panels. (c) A representative immunoblot and quantification analysis of SQSTM1 in MSCs treated with increasing concentrations of Cd. ACTB (actin, beta) was used as a loading control. (d) The MSCs were incubated with Cd (14 μM) in the absence or presence of Baf A1 (10 nM) for 24 h. The expression of LC3B-II was quantified by normalization of its density to that of ACTB. (e) Atg7 siRNA treatment decreased cell death by inhibiting autophagy. The results are expressed as the percentage of the control, which was set to 100 %. The values are presented as the means ±SEM, *p

    Techniques Used: Cell Culture, Staining, Flow Cytometry, Cytometry, Electron Microscopy, Incubation, Expressing

    29) Product Images from "SUMO-Modification of Human Nrf2 at K110 and K533 Regulates Its Nucleocytoplasmic Localization, Stability and Transcriptional Activity"

    Article Title: SUMO-Modification of Human Nrf2 at K110 and K533 Regulates Its Nucleocytoplasmic Localization, Stability and Transcriptional Activity

    Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

    doi: 10.33594/000000351

    SUMO-2 acceptor K 110 and K 533 are needed for efficient transcriptional a ctivity of Nrf2. A, Schematic of the HO-1-ARE-Luc (firefly luciferase, experimental) and Tk-Luc (control) promoters used in the luciferase reporter assay. HEK293T cells were transfected with 100 ηM control siRNA or Nrf2 siRNA for 48 hrs. B. Quantitative RT-PCR analysis of the levels of Nrf2 mRNA in HEK293T cells after knockdown with siC: scrambled control siRNA, siN: siRNA against 3’UTR of endogenous Nrf2. Results are mean ±S.E. (n=3). ** indicate there is a statistically significant difference. C. Western blot analysis of Nrf2 protein levels after siRNA mediated knockdown (* denotes the non-specific reactivity of the antibody). D. To monitor Nrf2 mediated transcriptional activation shown is the relative luciferase activity driven by HO1-ARE promoter in HEK293T cells treated with either control siRNA (siC) or siRNA against Nrf2 (siN) without activation, or with siRNA and activation by treatment with As2O3 (siC_As2O3 and siN_As2O3). Results are mean ±S.E. (n=9). ** indicates there is a statistically significant difference in all observations compared to siC. E. To study the significance of the SUMO-acceptor lysine in Nrf2 rescue experiments were performed using plasmid expressing either wild type Flag-hNrf2 (WT) or different mutant Flag-hNrf2 (K110R, K533R or 2K, K110R/K533R double mutant) in the HEK293T cells treated with siRNA against Nrf2. pCMV-Flag vector (pFlag) alone was used as a control. Shown is the relative luciferase activity driven by HO1-ARE promoter mediated by Flag-tagged hNrf2 wild type or mutants. Results are mean ±S.E. (n=9). ****, statistically different (p
    Figure Legend Snippet: SUMO-2 acceptor K 110 and K 533 are needed for efficient transcriptional a ctivity of Nrf2. A, Schematic of the HO-1-ARE-Luc (firefly luciferase, experimental) and Tk-Luc (control) promoters used in the luciferase reporter assay. HEK293T cells were transfected with 100 ηM control siRNA or Nrf2 siRNA for 48 hrs. B. Quantitative RT-PCR analysis of the levels of Nrf2 mRNA in HEK293T cells after knockdown with siC: scrambled control siRNA, siN: siRNA against 3’UTR of endogenous Nrf2. Results are mean ±S.E. (n=3). ** indicate there is a statistically significant difference. C. Western blot analysis of Nrf2 protein levels after siRNA mediated knockdown (* denotes the non-specific reactivity of the antibody). D. To monitor Nrf2 mediated transcriptional activation shown is the relative luciferase activity driven by HO1-ARE promoter in HEK293T cells treated with either control siRNA (siC) or siRNA against Nrf2 (siN) without activation, or with siRNA and activation by treatment with As2O3 (siC_As2O3 and siN_As2O3). Results are mean ±S.E. (n=9). ** indicates there is a statistically significant difference in all observations compared to siC. E. To study the significance of the SUMO-acceptor lysine in Nrf2 rescue experiments were performed using plasmid expressing either wild type Flag-hNrf2 (WT) or different mutant Flag-hNrf2 (K110R, K533R or 2K, K110R/K533R double mutant) in the HEK293T cells treated with siRNA against Nrf2. pCMV-Flag vector (pFlag) alone was used as a control. Shown is the relative luciferase activity driven by HO1-ARE promoter mediated by Flag-tagged hNrf2 wild type or mutants. Results are mean ±S.E. (n=9). ****, statistically different (p

    Techniques Used: Luciferase, Reporter Assay, Transfection, Quantitative RT-PCR, Western Blot, Activation Assay, Activity Assay, Plasmid Preparation, Expressing, Mutagenesis

    Nrf2-dependent gene transcription of Heme Oxygenase 1 (HO-1) gene is reduced in the absence of K 110 and K 533 of Nrf2 protein. To study the significance of the SUMO-acceptor lysine in Nrf2 rescue experiments were performed using plasmid expressing either wild type Flag-hNrf2 (WT) or different mutant Flag-hNrf2 (K110R, K533R or 2K (K110R/K533R) double mutant) in the HEK293T cells treated with siRNA against Nrf2. pCMV-Flag vector (pFlag) alone was used as a control. A, shown is real-time RT-PCR analysis of the levels of Nrf2 and HO-1 mRNAs in HEK293T cells treated with control siRNA or Nrf2 siRNA. 18s rRNA mRNA was used as a normalization control. Results are the mean ± S.E. (n = 3). **** indicates statistical significance, p
    Figure Legend Snippet: Nrf2-dependent gene transcription of Heme Oxygenase 1 (HO-1) gene is reduced in the absence of K 110 and K 533 of Nrf2 protein. To study the significance of the SUMO-acceptor lysine in Nrf2 rescue experiments were performed using plasmid expressing either wild type Flag-hNrf2 (WT) or different mutant Flag-hNrf2 (K110R, K533R or 2K (K110R/K533R) double mutant) in the HEK293T cells treated with siRNA against Nrf2. pCMV-Flag vector (pFlag) alone was used as a control. A, shown is real-time RT-PCR analysis of the levels of Nrf2 and HO-1 mRNAs in HEK293T cells treated with control siRNA or Nrf2 siRNA. 18s rRNA mRNA was used as a normalization control. Results are the mean ± S.E. (n = 3). **** indicates statistical significance, p

    Techniques Used: Plasmid Preparation, Expressing, Mutagenesis, Quantitative RT-PCR

    30) Product Images from "Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells"

    Article Title: Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0147718

    The role of Akt inhibition in ramentaceone-mediated apoptosis induction. (A) Silencing of Akt by siRNA in BT474 and SKBR3 cells. Cells were transiently transfected with Akt siRNA and 24 after transfection, Akt silencing was confirmed with Western blot analysis. (B) Induction of apoptosis by ramentaceone in cells transfected with Akt siRNA and control siRNA. 24 h after transfection cells were treated with ramentaceone (5 μM) for 24 h, stained with Annexin V-PE/7-AAD, and analyzed by flow cytometry. Representative of three independent experiments. p
    Figure Legend Snippet: The role of Akt inhibition in ramentaceone-mediated apoptosis induction. (A) Silencing of Akt by siRNA in BT474 and SKBR3 cells. Cells were transiently transfected with Akt siRNA and 24 after transfection, Akt silencing was confirmed with Western blot analysis. (B) Induction of apoptosis by ramentaceone in cells transfected with Akt siRNA and control siRNA. 24 h after transfection cells were treated with ramentaceone (5 μM) for 24 h, stained with Annexin V-PE/7-AAD, and analyzed by flow cytometry. Representative of three independent experiments. p

    Techniques Used: Inhibition, Transfection, Western Blot, Staining, Flow Cytometry, Cytometry

    31) Product Images from "Extracellular Redox State Shift: A Novel Approach to Target Prostate Cancer Invasion"

    Article Title: Extracellular Redox State Shift: A Novel Approach to Target Prostate Cancer Invasion

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2018.01.023

    Concomitant overexpression of ECSOD and knockdown of xCT in prostate cancer stromal cells (WPMY1) significantly inhibits PC3 invasion and alters the redox state in the TME (A) Western blot shows overexpression of ECSOD in the media and in stromal cells by AdhSOD3 (300 MOI) and concomitant down-regulation of the xCT protein by siRNA. (B) Cell invasive ability of PC3 cells that was co-cultured with WPMY1 cells or WPMY1 cells with overexpression of ECSOD and knockdown of xCT. *p-value ≤ 0.05. (C) H 2 O 2 level and (D) redox state in the media of the co-culturing system in WPMY1 cells with overexpression of ECSOD and concomitant knockdown of xCT. *p-value ≤ 0.05 compared with the control.
    Figure Legend Snippet: Concomitant overexpression of ECSOD and knockdown of xCT in prostate cancer stromal cells (WPMY1) significantly inhibits PC3 invasion and alters the redox state in the TME (A) Western blot shows overexpression of ECSOD in the media and in stromal cells by AdhSOD3 (300 MOI) and concomitant down-regulation of the xCT protein by siRNA. (B) Cell invasive ability of PC3 cells that was co-cultured with WPMY1 cells or WPMY1 cells with overexpression of ECSOD and knockdown of xCT. *p-value ≤ 0.05. (C) H 2 O 2 level and (D) redox state in the media of the co-culturing system in WPMY1 cells with overexpression of ECSOD and concomitant knockdown of xCT. *p-value ≤ 0.05 compared with the control.

    Techniques Used: Over Expression, Western Blot, Cell Culture

    Overexpression of ECSOD and knockdown of xCT suppresses the invasive ability of aggressive PC3 (A) Western blots display comparatively lower ECSOD (~31 kDa) and greater xCT (~55 kDa) expressions in PrEC, PC3, and WPMY1 cell lines. (B). Western blots indicate an increase of ECSOD expression in PC3 and WPMY1 cells after treatment with the DNA methylation inhibitor, 5-Aza-dC, for 96 h. (C) Western blot shows an up-regulation of Nrf2 and xCT expression in PC3 and WPMY1 cells relative to the PrEC cells. (D) Cell invasive ability of PC3 after treatment with SOD and the xCT inhibitor (SASP) for 24 h. (E) Western blot indicates an overexpression of ECSOD in the media and in cell lysates with AdhSOD3 (300 MOI) treatment and a down-regulation of xCT protein by siRNA (siRNA xCT). (F) Cell invasive ability of PC3 cells after overexpression of ECSOD in the media and cells, and with concurrent inhibition of xCT expression. AdEmpty; adenovirus empty vector. 300 MOI was used for AdEmpty and AdhSOD3. *p-value ≤ 0.05 compared with the control.
    Figure Legend Snippet: Overexpression of ECSOD and knockdown of xCT suppresses the invasive ability of aggressive PC3 (A) Western blots display comparatively lower ECSOD (~31 kDa) and greater xCT (~55 kDa) expressions in PrEC, PC3, and WPMY1 cell lines. (B). Western blots indicate an increase of ECSOD expression in PC3 and WPMY1 cells after treatment with the DNA methylation inhibitor, 5-Aza-dC, for 96 h. (C) Western blot shows an up-regulation of Nrf2 and xCT expression in PC3 and WPMY1 cells relative to the PrEC cells. (D) Cell invasive ability of PC3 after treatment with SOD and the xCT inhibitor (SASP) for 24 h. (E) Western blot indicates an overexpression of ECSOD in the media and in cell lysates with AdhSOD3 (300 MOI) treatment and a down-regulation of xCT protein by siRNA (siRNA xCT). (F) Cell invasive ability of PC3 cells after overexpression of ECSOD in the media and cells, and with concurrent inhibition of xCT expression. AdEmpty; adenovirus empty vector. 300 MOI was used for AdEmpty and AdhSOD3. *p-value ≤ 0.05 compared with the control.

    Techniques Used: Over Expression, Western Blot, Expressing, DNA Methylation Assay, Inhibition, Plasmid Preparation

    32) Product Images from "Expression of Livin in Colorectal Cancer and Its Relationship to Tumor Cell Behavior and Prognosis"

    Article Title: Expression of Livin in Colorectal Cancer and Its Relationship to Tumor Cell Behavior and Prognosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073262

    The impact of Livin on apoptosis in human colorectal cancer cells. (A) The proportion of apoptotic cells induced by transfection of LS was greater than that induced by transfection of SS (6.9 vs. 19.6%) in SW480 cells but Livin knockdown had a minimal influence on apoptosis (11.3 vs. 16.7%) in DKO1 cells. Overexpression of Livin by transfection of LV inhibited the apoptosis of SW480 cells in response to 5-FU but overexpression of Livin had a minimal influence on apoptosis in DKO1 cells (B) Expression of cleaved caspase-3, -7, -9, and PARP proteins. The caspase-3, -7 and PARP expression was increased in SW480 and DKO1 cells after Livin knockdown, and decreased after overexpression of Livin (C) Expression of apoptosis regulatory proteins. Survivin protein level decreased following Livin knockdown in SW480 and DKO1 cells, but XIAP and SMAC/DIABLO protein levels were not altered in response to Livin knockdown. Additionally, Survivin, XIAP and SMAC/DIABLO protein levels were not altered after overexpression of Livin. PARP; Poly (ADP-ribose) polymerase, XIAP; X-chromosome binding IAP, SMAC/DIABLO; second mitochondria-derived activator of caspases/direct IAP binding protein with low pI, SS; scramble siRNA, LS; Livin siRNA, EV; Empty-pcDNA3.1, LV; pcDNA3.1-Livin, 5-FU; 5-fluorouracil, 7-AAD; 7-amino-actinomycin D.
    Figure Legend Snippet: The impact of Livin on apoptosis in human colorectal cancer cells. (A) The proportion of apoptotic cells induced by transfection of LS was greater than that induced by transfection of SS (6.9 vs. 19.6%) in SW480 cells but Livin knockdown had a minimal influence on apoptosis (11.3 vs. 16.7%) in DKO1 cells. Overexpression of Livin by transfection of LV inhibited the apoptosis of SW480 cells in response to 5-FU but overexpression of Livin had a minimal influence on apoptosis in DKO1 cells (B) Expression of cleaved caspase-3, -7, -9, and PARP proteins. The caspase-3, -7 and PARP expression was increased in SW480 and DKO1 cells after Livin knockdown, and decreased after overexpression of Livin (C) Expression of apoptosis regulatory proteins. Survivin protein level decreased following Livin knockdown in SW480 and DKO1 cells, but XIAP and SMAC/DIABLO protein levels were not altered in response to Livin knockdown. Additionally, Survivin, XIAP and SMAC/DIABLO protein levels were not altered after overexpression of Livin. PARP; Poly (ADP-ribose) polymerase, XIAP; X-chromosome binding IAP, SMAC/DIABLO; second mitochondria-derived activator of caspases/direct IAP binding protein with low pI, SS; scramble siRNA, LS; Livin siRNA, EV; Empty-pcDNA3.1, LV; pcDNA3.1-Livin, 5-FU; 5-fluorouracil, 7-AAD; 7-amino-actinomycin D.

    Techniques Used: Transfection, Over Expression, Expressing, Binding Assay, Derivative Assay

    The impact of Livin on invasion, migration and proliferation of human colorectal cancer cells. (A) The impact of Livin on invasion of colorectal cancer cells. The invasion assay using the siRNA or pcDNA3.1-transfected cells was performed. Stained invading cells were counted and are represented as a graph between groups. The number of LS-transfected cells that invaded was significantly lower than that of SS-transfected cells. The number of invading cells was significantly higher in LV-transfected cells compared to EV-transfected cells (mean±standard error [SE], n=6; *p
    Figure Legend Snippet: The impact of Livin on invasion, migration and proliferation of human colorectal cancer cells. (A) The impact of Livin on invasion of colorectal cancer cells. The invasion assay using the siRNA or pcDNA3.1-transfected cells was performed. Stained invading cells were counted and are represented as a graph between groups. The number of LS-transfected cells that invaded was significantly lower than that of SS-transfected cells. The number of invading cells was significantly higher in LV-transfected cells compared to EV-transfected cells (mean±standard error [SE], n=6; *p

    Techniques Used: Migration, Invasion Assay, Transfection, Staining

    The impact of Livin on cell cycle arrest in human colorectal cancer cells. (A) Livin knockdown resulted in cell cycle arrest in the G0/G1 phase of SW480 cells and the S phase of DKO1 cells. 5-FU treatment induced cell cycle arrest in the subG1 phase of SW480 and the G0/G1 phase of DKO1 cells. Overexpression of Livin inhibited 5-FU-induced cell cycle arrest in SW480 cells and had a minimal influence in DKO1 cells. One representative experiment of the three independent experiments is shown. (B) Expression of cyclin-dependent kinase (CDK) inhibitor proteins. The p27 protein level was significantly increased by Livin knockdown and decreased by overexpression of Livin in SW480 and DKO1 cells. However, p21, p57, p15 and p16 protein levels were not altered in response to knockdown or overexpression of Livin. (C) Expression of cyclins and cyclin-dependent kinase (CDKs) proteins. Cyclin D1, cyclin D3, CDK4 and CDK6 protein levels were significantly decreased by Livin knockdown and increased the cyclin D1 and CDK4 by overexpression of Livin in SW480 and DKO1 cells. SS; scramble siRNA, LS; Livin siRNA, EV; Empty-pcDNA3.1, LV; pcDNA3.1-Livin.
    Figure Legend Snippet: The impact of Livin on cell cycle arrest in human colorectal cancer cells. (A) Livin knockdown resulted in cell cycle arrest in the G0/G1 phase of SW480 cells and the S phase of DKO1 cells. 5-FU treatment induced cell cycle arrest in the subG1 phase of SW480 and the G0/G1 phase of DKO1 cells. Overexpression of Livin inhibited 5-FU-induced cell cycle arrest in SW480 cells and had a minimal influence in DKO1 cells. One representative experiment of the three independent experiments is shown. (B) Expression of cyclin-dependent kinase (CDK) inhibitor proteins. The p27 protein level was significantly increased by Livin knockdown and decreased by overexpression of Livin in SW480 and DKO1 cells. However, p21, p57, p15 and p16 protein levels were not altered in response to knockdown or overexpression of Livin. (C) Expression of cyclins and cyclin-dependent kinase (CDKs) proteins. Cyclin D1, cyclin D3, CDK4 and CDK6 protein levels were significantly decreased by Livin knockdown and increased the cyclin D1 and CDK4 by overexpression of Livin in SW480 and DKO1 cells. SS; scramble siRNA, LS; Livin siRNA, EV; Empty-pcDNA3.1, LV; pcDNA3.1-Livin.

    Techniques Used: Over Expression, Expressing

    The impact of Livin on intracellular signaling pathways involved in apoptosis and cell cycle arrest of human colorectal cancer cells. The phosphorylation levels of ERK1/2, JNK and p38 decreased following Livin knockdown of SW480 and DKO1 cells. But Akt and p65 the phosphorylation levels showed no change following Livin knockdown. Additionally, ERK1/2, JNK, p38, Akt and p65 phosphorylation levels were not changed by overexpression of Livin. SS; scramble siRNA, LS; Livin siRNA, EV; Empty-pcDNA3.1, LV; pcDNA3.1-Livin.
    Figure Legend Snippet: The impact of Livin on intracellular signaling pathways involved in apoptosis and cell cycle arrest of human colorectal cancer cells. The phosphorylation levels of ERK1/2, JNK and p38 decreased following Livin knockdown of SW480 and DKO1 cells. But Akt and p65 the phosphorylation levels showed no change following Livin knockdown. Additionally, ERK1/2, JNK, p38, Akt and p65 phosphorylation levels were not changed by overexpression of Livin. SS; scramble siRNA, LS; Livin siRNA, EV; Empty-pcDNA3.1, LV; pcDNA3.1-Livin.

    Techniques Used: Over Expression

    33) Product Images from "Estrogen receptor α protects pancreatic β-cells from apoptosis by preserving mitochondrial function and suppressing endoplasmic reticulum stress"

    Article Title: Estrogen receptor α protects pancreatic β-cells from apoptosis by preserving mitochondrial function and suppressing endoplasmic reticulum stress

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.805069

    ERα activation reduces Chop and Oma1 expression. A and B , ERα-specific activation of wildtype Min6 β-cells by PPT treatment reduced Chop ( A ) and Oma1 ( B ) expression versus vehicle control ( n = 3 group/time point in duplicate). C, chromatin immunoprecipitation studies show that ERα binds the Chop and Oma1 promoters (performed in triplicate). D and E, overexpression of HA-tagged Oma1 promotes PERK and eIF2α phosphorylation and induces CHOP protein expression in Min6 β-cells ( n = 3/condition in triplicate). F and G, confirmation studies using siRNA to achieve Oma1 knockdown confirm proof-of-concept that OMA1 expression is linked with EndoRetic stress signaling and cleaved caspase-3 abundance via CHOP, even in the basal state ( n = 3/condition in duplicate). H, schematic showing that ERα promotes cellular stress and apoptosis protection by direct repression of Chop and Oma1 expression. Our findings also support the notion that the derepression of Oma1 could contribute to the induction of CHOP1 and, in part, underlie EndoRetic stress associated with impaired ERα action. AU , arbitrary units.
    Figure Legend Snippet: ERα activation reduces Chop and Oma1 expression. A and B , ERα-specific activation of wildtype Min6 β-cells by PPT treatment reduced Chop ( A ) and Oma1 ( B ) expression versus vehicle control ( n = 3 group/time point in duplicate). C, chromatin immunoprecipitation studies show that ERα binds the Chop and Oma1 promoters (performed in triplicate). D and E, overexpression of HA-tagged Oma1 promotes PERK and eIF2α phosphorylation and induces CHOP protein expression in Min6 β-cells ( n = 3/condition in triplicate). F and G, confirmation studies using siRNA to achieve Oma1 knockdown confirm proof-of-concept that OMA1 expression is linked with EndoRetic stress signaling and cleaved caspase-3 abundance via CHOP, even in the basal state ( n = 3/condition in duplicate). H, schematic showing that ERα promotes cellular stress and apoptosis protection by direct repression of Chop and Oma1 expression. Our findings also support the notion that the derepression of Oma1 could contribute to the induction of CHOP1 and, in part, underlie EndoRetic stress associated with impaired ERα action. AU , arbitrary units.

    Techniques Used: Activation Assay, Expressing, Chromatin Immunoprecipitation, Over Expression

    34) Product Images from "Differential expression of genes in the subgranular zone and granular cell layer of the hippocampus after running"

    Article Title: Differential expression of genes in the subgranular zone and granular cell layer of the hippocampus after running

    Journal: Journal of Exercise Nutrition & Biochemistry

    doi: 10.20463/jenb.2018.0025

    (A) Heat maps representing the relative expression levels of all genes involved in neurogenesis in Neuro2A cells. Comparison of cells transfected with Tollip siRNA compared to control siRNA. Below: The list of selected genes based on fold change from PCR array (compared to si-scramble transfected cells). (B) Expression levels of Tollip and DCX in Neuro2A cells after Tollip siRNA or scramble siRNA transfection. Quantitative immunoblotting analysis was normalized by GAPDH (n=5). DCX: 45 kDa, Tollip: 30 kDa, GAPDH: 37 kDa. (C) CCK-8 analysis of the effect of knockdown of Tollip on Neuro2A survival at 72 hours after transfection. NS, not significant. Error bars show mean ± SE. *P
    Figure Legend Snippet: (A) Heat maps representing the relative expression levels of all genes involved in neurogenesis in Neuro2A cells. Comparison of cells transfected with Tollip siRNA compared to control siRNA. Below: The list of selected genes based on fold change from PCR array (compared to si-scramble transfected cells). (B) Expression levels of Tollip and DCX in Neuro2A cells after Tollip siRNA or scramble siRNA transfection. Quantitative immunoblotting analysis was normalized by GAPDH (n=5). DCX: 45 kDa, Tollip: 30 kDa, GAPDH: 37 kDa. (C) CCK-8 analysis of the effect of knockdown of Tollip on Neuro2A survival at 72 hours after transfection. NS, not significant. Error bars show mean ± SE. *P

    Techniques Used: Expressing, Transfection, Polymerase Chain Reaction, CCK-8 Assay

    35) Product Images from "Oncolytic adenoviral vectors which employ the survivin promoter induce glioma oncolysis via a process of beclin-dependent autophagy"

    Article Title: Oncolytic adenoviral vectors which employ the survivin promoter induce glioma oncolysis via a process of beclin-dependent autophagy

    Journal: International journal of oncology

    doi:

    Effect of siRNA treatment on virus-induced autophagy. No. 10 human glioma cells were transfected with 20 pMol of BECN1 (anti-beclin) siRNA and then transduced with the competent vectors as indicated: AdWT, AdRGD or CRAd-S-RGD for 72 h incubation. The cells were then examined by acridine orange staining (A) or viral E1A/beclin expression was detected by immunofluorescence (B). Inhibition of autophagosome development was measured in two independent experiments, with each experiment containing three wells per each treatment group, including controls. Data are presented as mean ± SD. * p
    Figure Legend Snippet: Effect of siRNA treatment on virus-induced autophagy. No. 10 human glioma cells were transfected with 20 pMol of BECN1 (anti-beclin) siRNA and then transduced with the competent vectors as indicated: AdWT, AdRGD or CRAd-S-RGD for 72 h incubation. The cells were then examined by acridine orange staining (A) or viral E1A/beclin expression was detected by immunofluorescence (B). Inhibition of autophagosome development was measured in two independent experiments, with each experiment containing three wells per each treatment group, including controls. Data are presented as mean ± SD. * p

    Techniques Used: Transfection, Transduction, Incubation, Staining, Expressing, Immunofluorescence, Inhibition

    36) Product Images from "Molecular functional analyses revealed essential roles of HSP90 and lamin A/C in growth, migration, and self-aggregation of dermal papilla cells"

    Article Title: Molecular functional analyses revealed essential roles of HSP90 and lamin A/C in growth, migration, and self-aggregation of dermal papilla cells

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-018-0053-6

    siRNA-mediated knockdown of HSP90 and lamin A/C in DPCs. a Western blot analysis showed decreased levels of HSP90 and lamin A/C in DPCs transfected with siRNA targeting on HSP90 (siHSP90) as compared to non-transfected and siControl-transfected cells. b Western blot analysis showed decreased levels of lamin A/C and HSP90 in DPCs transfected with siRNA targeting on lamin A/C (siLamin A/C) comparing to non-transfected and siControl-transfected cells. GAPDH served as the loading control. WB Western blotting
    Figure Legend Snippet: siRNA-mediated knockdown of HSP90 and lamin A/C in DPCs. a Western blot analysis showed decreased levels of HSP90 and lamin A/C in DPCs transfected with siRNA targeting on HSP90 (siHSP90) as compared to non-transfected and siControl-transfected cells. b Western blot analysis showed decreased levels of lamin A/C and HSP90 in DPCs transfected with siRNA targeting on lamin A/C (siLamin A/C) comparing to non-transfected and siControl-transfected cells. GAPDH served as the loading control. WB Western blotting

    Techniques Used: Western Blot, Transfection

    37) Product Images from "Egr-1 regulates RTA transcription through a cooperative involvement of transcriptional regulators"

    Article Title: Egr-1 regulates RTA transcription through a cooperative involvement of transcriptional regulators

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20648

    (A) Egr-1 silencing reduced virion production in BC-3 and BCBL-1 cells a. BC-3 and b. BCBL-1 cells were transfected with scrambled siRNA (scr siRNA)/Egr-1 siRNA and allowed to grow for 24h followed by induction with TPA for 72h. Culture supernatant containing virions was collected and concentrated through ultracentrifugation. The relative virion quantity was determined by qPCR of the DNA extracted from the virions. The expression of Egr-1 and the respective GAPDH are shown in BC-3 and BCBL-1 transfected with scr/ Egr-1 siRNA. Transfection efficiencies of scrambled siRNA (scr siRNA-FITC conjugated) (green cells) are shown in BC-3 and BCBL-1, respectively. * P
    Figure Legend Snippet: (A) Egr-1 silencing reduced virion production in BC-3 and BCBL-1 cells a. BC-3 and b. BCBL-1 cells were transfected with scrambled siRNA (scr siRNA)/Egr-1 siRNA and allowed to grow for 24h followed by induction with TPA for 72h. Culture supernatant containing virions was collected and concentrated through ultracentrifugation. The relative virion quantity was determined by qPCR of the DNA extracted from the virions. The expression of Egr-1 and the respective GAPDH are shown in BC-3 and BCBL-1 transfected with scr/ Egr-1 siRNA. Transfection efficiencies of scrambled siRNA (scr siRNA-FITC conjugated) (green cells) are shown in BC-3 and BCBL-1, respectively. * P

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

    38) Product Images from "Lysophosphatidic acid increases mesangial cell proliferation in models of diabetic nephropathy via Rac1/MAPK/KLF5 signaling"

    Article Title: Lysophosphatidic acid increases mesangial cell proliferation in models of diabetic nephropathy via Rac1/MAPK/KLF5 signaling

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-019-0217-3

    Downregulation of KLF5 inhibits the LPA-induced proliferation of SV40 MES13 cells. a Cells were transfected with a scrambled siRNA (siCon) or KLF5 siRNA (siKLF5) for 48 h and treated with LPA (10 μM) for 6 h, and the levels of the KLF5 and p27 Kip1 proteins were determined using western blotting. b , c The relative levels of the proteins were normalized to β-actin and quantified using ImageJ software ( n = 3 independent experiments). d Cell morphology was examined using light microscopy (original magnification, × 100), and ( e ) cell proliferation was analyzed using CCK-8 assay after treatment with 10 μM LPA and transfection of the KLF5 siRNA or scrambled siRNA for 24 h ( n = 3 independent experiments). * p
    Figure Legend Snippet: Downregulation of KLF5 inhibits the LPA-induced proliferation of SV40 MES13 cells. a Cells were transfected with a scrambled siRNA (siCon) or KLF5 siRNA (siKLF5) for 48 h and treated with LPA (10 μM) for 6 h, and the levels of the KLF5 and p27 Kip1 proteins were determined using western blotting. b , c The relative levels of the proteins were normalized to β-actin and quantified using ImageJ software ( n = 3 independent experiments). d Cell morphology was examined using light microscopy (original magnification, × 100), and ( e ) cell proliferation was analyzed using CCK-8 assay after treatment with 10 μM LPA and transfection of the KLF5 siRNA or scrambled siRNA for 24 h ( n = 3 independent experiments). * p

    Techniques Used: Transfection, Western Blot, Software, Light Microscopy, CCK-8 Assay

    39) Product Images from "Tridimensional infiltration of DNA viruses into the host genome shows preferential contact with active chromatin"

    Article Title: Tridimensional infiltration of DNA viruses into the host genome shows preferential contact with active chromatin

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06739-4

    Cfp1 is required for HBV transcription. a HBV transcription was analyzed by RT-qPCR in HepG2 NTCP infected or not with HBV and transfected with the indicated siRNA (left graph). The expression of Cfp1 was analyzed by RT-qPCR and western blotting (lower right and upper right panel, respectively). Error bars of nine independent experiments ( n = 9) represent standard error of the mean (SEM). *** p
    Figure Legend Snippet: Cfp1 is required for HBV transcription. a HBV transcription was analyzed by RT-qPCR in HepG2 NTCP infected or not with HBV and transfected with the indicated siRNA (left graph). The expression of Cfp1 was analyzed by RT-qPCR and western blotting (lower right and upper right panel, respectively). Error bars of nine independent experiments ( n = 9) represent standard error of the mean (SEM). *** p

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

    40) Product Images from "Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer"

    Article Title: Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3398

    PrP delays cell death induced by ER stress . (A) inset shows western blot of PrP and β-actin in siRNA transfected cells. Percentage of cell death assessed by pan-caspase FLICA (A) or by chromatin condensation (B) in MCF-7 cells transfected with control (siCtl) or PrP (siPrP) siRNAs for 24 hrs and treated with ER stressors or DMSO (control) during 6 hrs. Cell death was measured 18 hrs after the removal of ER stress. Data represent the mean ± SEM of three (A) or six (B) independent experiments. At least 150 cells were counted per experiment. * Indicates P ≤0.05 between PrP siRNA and control siRNA. (C-D) Percentage of cell death assessed by FLICA staining for active caspases (C) or by chromatin condensation in PrP +/+ and PrP-/- hippocampal cell lines (D). Data represent the mean ± SEM of four independent experiments. At least 150 cells were counted per experiment. * Indicates P ≤0.05 between PrP+/+ and PrP-/- cells. C Inset: Western blot analysis of PrP with the R155 antibody and β-actin in protein extracts from mouse PrP+/+ and PrP-/- hippocampal cell lines. PrP-/- hippocampal cell lines were treated with 2.5 μg/ml of ER stressors. These concentrations were empirically determined to induce ER stress response without initially inducing strong toxic effects. (E) Relative fluorescence units (RFU) of FLICA activity representing pan-caspase activity in MDA-MB-231 (E) or HS578T (F) cells transfected with siCtl or siPrP.
    Figure Legend Snippet: PrP delays cell death induced by ER stress . (A) inset shows western blot of PrP and β-actin in siRNA transfected cells. Percentage of cell death assessed by pan-caspase FLICA (A) or by chromatin condensation (B) in MCF-7 cells transfected with control (siCtl) or PrP (siPrP) siRNAs for 24 hrs and treated with ER stressors or DMSO (control) during 6 hrs. Cell death was measured 18 hrs after the removal of ER stress. Data represent the mean ± SEM of three (A) or six (B) independent experiments. At least 150 cells were counted per experiment. * Indicates P ≤0.05 between PrP siRNA and control siRNA. (C-D) Percentage of cell death assessed by FLICA staining for active caspases (C) or by chromatin condensation in PrP +/+ and PrP-/- hippocampal cell lines (D). Data represent the mean ± SEM of four independent experiments. At least 150 cells were counted per experiment. * Indicates P ≤0.05 between PrP+/+ and PrP-/- cells. C Inset: Western blot analysis of PrP with the R155 antibody and β-actin in protein extracts from mouse PrP+/+ and PrP-/- hippocampal cell lines. PrP-/- hippocampal cell lines were treated with 2.5 μg/ml of ER stressors. These concentrations were empirically determined to induce ER stress response without initially inducing strong toxic effects. (E) Relative fluorescence units (RFU) of FLICA activity representing pan-caspase activity in MDA-MB-231 (E) or HS578T (F) cells transfected with siCtl or siPrP.

    Techniques Used: Western Blot, Transfection, Staining, Fluorescence, Activity Assay, Multiple Displacement Amplification

    Related Articles

    Transfection:

    Article Title: Anticancer Activity of Metformin, an Antidiabetic Drug, Against Ovarian Cancer Cells Involves Inhibition of Cysteine-Rich 61 (Cyr61)/Akt/Mammalian Target of Rapamycin (mTOR) Signaling Pathway
    Article Snippet: .. Transient transfection with siCyr61 For transient knockdown of Cyr61 in OVCAR-3 cells, siCyr61 RNA (sc-39331) and scrambled siRNAs (sc-44234) were purchased from Santa Cruz and cells were transfected with 100 nM of siCyr61 RNA using Lipofectamine 2000 (Invitrogen), following the manufacturer’s protocol. ..

    Article Title: Anticancer Activity of Metformin, an Antidiabetic Drug, Against Ovarian Cancer Cells Involves Inhibition of Cysteine-Rich 61 (Cyr61)/Akt/Mammalian Target of Rapamycin (mTOR) Signaling Pathway
    Article Snippet: .. For transient knockdown of Cyr61 in OVCAR-3 cells, siCyr61 RNA (sc-39331) and scrambled siRNAs (sc-44234) were purchased from Santa Cruz and cells were transfected with 100 nM of siCyr61 RNA using Lipofectamine 2000 (Invitrogen), following the manufacturer’s protocol. ..

    Sequencing:

    Article Title: Mitochondrion-associated protein peroxiredoxin 3 promotes benign prostatic hyperplasia through autophagy suppression and pyroptosis activation
    Article Snippet: .. Primary antibodies against PRDX3 (catalog No.sc-59661), Beclin 1 (catalog no. sc-11427), and β-actin (catalog no. sc-47778), random sequence control siRNA (catalog no. sc-44234) and siRNA specific to PRDX3 (catalog no. sc-40833) were from Santa Cruz Biotechnology, Inc. HRP-conjugated secondary antibodies against mouse (catalog no. 172-1011) and rabbit (catalog no. 172-1019) were from Bio-Rad. ..

    Article Title: Potassium Bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)) Induces Apoptosis and Pyroptosis and Disrupts the P62-HDAC6 Protein Interaction to Suppress the Acetylated Microtubule-dependent Degradation of Autophagosomes *
    Article Snippet: .. The IgG control antibody from rabbit (catalog no. sc-2027), primary antibodies against PARP (catalog no. sc-7150), proliferating cell nuclear antigen, catalog no. sc-9707), α-tubulin (catalog no. sc-12462), β-actin (catalog no. sc-47778), HDAC6 (catalog no. sc-11420), acetylated α-tubulin (catalog no. sc-23950), random sequence control siRNA (catalog no. sc-44234), and siRNA specific to p62 (catalog no. sc-29679) were from Santa Cruz Biotechnology, Inc. Antibody against p62 (SQSTN1, catalog no. BWL-PW9860) was from Enzo Life Sciences International Inc. HRP-conjugated secondary antibodies against mouse (catalog no. 172-1011) and rabbit (catalog no. 172-1019) were from Bio-Rad. ..

    Immunostaining:

    Article Title: ACBD2/ECI2-Mediated Peroxisome-Mitochondria Interactions in Leydig Cell Steroid Biosynthesis
    Article Snippet: .. The results obtained showed a dramatic reduction in the immunostaining of ACBD2/ECI2 proteins in contrast to that seen after treatment of the cells with control siRNA ( , F and G). ..

    Negative Control:

    Article Title: The activation of PPARγ by 2,4,6-Octatrienoic acid protects human keratinocytes from UVR-induced damages
    Article Snippet: .. An equivalent amount of non-specific siRNA (sc-44234; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a negative control. ..

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    Santa Cruz Biotechnology smad2 sirna
    Effects of <t>Smad2</t> on Col I and Col III expression in hHSFs. hHSFs cells were transfected with Smad2-plasmid or <t>Smad2-siRNA.</t> Smad2, Col I and Col III expression were determined by (A) western blotting and (B-D) reverse transcription-quantitative polymerase chain reaction at 48 h post-transfection. Data are presented as the mean ± standard deviation. *P
    Smad2 Sirna, 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 sirna against mouse ahr
    I3C inhibits the development of intestinal organoids from crypts or Lgr5 + stem cells <t>AhR-dependently</t> Crypts, isolated from the small intestine, were resuspended with Matrigel and applied into a 24-well plate and then cultured in complete culture medium for 4 days. I3C (0.1–1 μM), α-naphthoflavone (1 μM), and DMSO (0.1% v/v) were added to the culture medium 1.5 h before the addition of growth and differentiation factors. Then, organoids were observed (A) and counted (B) under the inverted microscope, and assayed for RNA analysis (C), cell proliferation (G), or cell viability assay (H). For <t>siRNA</t> nucleofection, 1 x 10 3 crypt cells were suspended in 0.1 ml of Nucleofector solution and 5 μl of siRNA against mouse AhR or control siRNA and electroporated. Cells were cultured and harvested 24 h later and assayed for AhR expression (D). Crypt cells containing Lgr5 + cells, which were nucleofected with siRNA, were mixed with Matrigel (200 cells/25 μl) with 1 μM Jagged-1 peptide, subjected to organoid development, and observed and counted under the inverted microscope (E, F). Results are presented as mean ± SD of 5 (B, G, and H) or 3 (F) independent experiments. Statistical significance was analyzed using the paired Student’s t -test. * p
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    Santa Cruz Biotechnology control sirna
    Effects of fucoxanthin (Fx) on Smad2/Smad3 activation in NPDFs. ( A ) The NPDFs were pretreated with Fx (5–30 µM) for 30 min, followed by stimulation with TGF-β1 (1 ng/mL) for 30 min. ( B ) The <t>Smad</t> 2/3 nuclear translocation was determined via western blot analysis. ( C ) Smad 2/3 silencing inhibits the expression of α-SMA and Col-1 proteins in TGF-β1-stimulated NPDFs. The NPDFs were transfected with Smad2/3 <t>siRNA</t> (40 and 80 nM) for 24 h, followed by stimulation with TGF-β1 (1 ng/mL) for 24 h. ( D ) The NPDFs were transfected with either the control siRNA (80 nM) or the Smad 2/3 siRNA (80 nM) for 24 h, followed by stimulation with TGF-β1 (1 ng/mL) for 30 min. Each bar represents the mean ± S.E.M. from three independent experiments. Untreated cells were used as a control (CON). p-Smad 2/3, phosphor-Smad 2/3; t-Smad 2/3, total-Smad 2/3; siCon, control siRNA; siSmad, Smad 2/3 siRNA.
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    Santa Cruz Biotechnology control non silencing sirna
    KLF4 depletion increases endothelial barrier permeability A, Timeline of TER assay. B, HLMVECs plated on gold microelectrodes were left untreated (control) or transfected with a non-silencing <t>siRNA</t> or KLF4 -silencing siRNA, followed by TER assay. Note that KLF4 silencing inhibited the thrombin response relative to untreated group (no transfection) or negative control group (non-silencing siRNA; n = 5 per group). Mean value (± s.e.m.) of maximal TER responses to thrombin (50 nM) stimulation (n = 7). Thrombin-induced decrease in TER was significantly attenuated in HLMVECs transfected by KLF4-depletion compared with untreated control or negative control group transfected with a non-silencing siRNA. C-E, KLF4 knockdown increases transendothelial permeability of fluorescein isothiocyanate (FITC)-conjugated albumin by decreasing <t>VE-cadherin</t> expression and AJ integrity. C , Timeline of experiments. D , Confluent HLMVEC monolayers were grown on microporous filters for 36 h, either left alone (control) or treated with control siRNA or with KLF4 -siRNA for 12 hours. At 18 hr post transfection, transendothelial FITC-albumin permeability was measured. Control HLMVECs showed basal transendothelial FITC-albumin permeability values, while KLF4 knockdown increased transendothelial FITC-albumin permeability. Re-expression of VE-cadherin into KLF4-depleted ECs partially restored the effect of loss of KLF4. Values are mean ± s.e.m. (n= 10). *p
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    Effects of Smad2 on Col I and Col III expression in hHSFs. hHSFs cells were transfected with Smad2-plasmid or Smad2-siRNA. Smad2, Col I and Col III expression were determined by (A) western blotting and (B-D) reverse transcription-quantitative polymerase chain reaction at 48 h post-transfection. Data are presented as the mean ± standard deviation. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: MicroRNA-26a inhibits hyperplastic scar formation by targeting Smad2

    doi: 10.3892/etm.2018.5984

    Figure Lengend Snippet: Effects of Smad2 on Col I and Col III expression in hHSFs. hHSFs cells were transfected with Smad2-plasmid or Smad2-siRNA. Smad2, Col I and Col III expression were determined by (A) western blotting and (B-D) reverse transcription-quantitative polymerase chain reaction at 48 h post-transfection. Data are presented as the mean ± standard deviation. *P

    Article Snippet: To investigate the role of miR-26a in hHSFs, the 50 nM miR-26a mimic (5′-TTCAAGTAATCCAGGATAGGCT-3′), 100 nM miR-26a inhibitor (5′-AGCCTATCCTGGATTACTTGAA-3′), 50 nM negative control (NC; all Shanghai GenePharma, Co., Ltd., Shanghai, China), Smad2 plasmids, control plasmids, Smad2 siRNA, or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, CA, USA) were transfected into hHSFs using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    I3C inhibits the development of intestinal organoids from crypts or Lgr5 + stem cells AhR-dependently Crypts, isolated from the small intestine, were resuspended with Matrigel and applied into a 24-well plate and then cultured in complete culture medium for 4 days. I3C (0.1–1 μM), α-naphthoflavone (1 μM), and DMSO (0.1% v/v) were added to the culture medium 1.5 h before the addition of growth and differentiation factors. Then, organoids were observed (A) and counted (B) under the inverted microscope, and assayed for RNA analysis (C), cell proliferation (G), or cell viability assay (H). For siRNA nucleofection, 1 x 10 3 crypt cells were suspended in 0.1 ml of Nucleofector solution and 5 μl of siRNA against mouse AhR or control siRNA and electroporated. Cells were cultured and harvested 24 h later and assayed for AhR expression (D). Crypt cells containing Lgr5 + cells, which were nucleofected with siRNA, were mixed with Matrigel (200 cells/25 μl) with 1 μM Jagged-1 peptide, subjected to organoid development, and observed and counted under the inverted microscope (E, F). Results are presented as mean ± SD of 5 (B, G, and H) or 3 (F) independent experiments. Statistical significance was analyzed using the paired Student’s t -test. * p

    Journal: Molecules and Cells

    Article Title: Indole-3-Carbinol Promotes Goblet-Cell Differentiation Regulating Wnt and Notch Signaling Pathways AhR-Dependently

    doi: 10.14348/molcells.2018.2167

    Figure Lengend Snippet: I3C inhibits the development of intestinal organoids from crypts or Lgr5 + stem cells AhR-dependently Crypts, isolated from the small intestine, were resuspended with Matrigel and applied into a 24-well plate and then cultured in complete culture medium for 4 days. I3C (0.1–1 μM), α-naphthoflavone (1 μM), and DMSO (0.1% v/v) were added to the culture medium 1.5 h before the addition of growth and differentiation factors. Then, organoids were observed (A) and counted (B) under the inverted microscope, and assayed for RNA analysis (C), cell proliferation (G), or cell viability assay (H). For siRNA nucleofection, 1 x 10 3 crypt cells were suspended in 0.1 ml of Nucleofector solution and 5 μl of siRNA against mouse AhR or control siRNA and electroporated. Cells were cultured and harvested 24 h later and assayed for AhR expression (D). Crypt cells containing Lgr5 + cells, which were nucleofected with siRNA, were mixed with Matrigel (200 cells/25 μl) with 1 μM Jagged-1 peptide, subjected to organoid development, and observed and counted under the inverted microscope (E, F). Results are presented as mean ± SD of 5 (B, G, and H) or 3 (F) independent experiments. Statistical significance was analyzed using the paired Student’s t -test. * p

    Article Snippet: In brief, 1 x 103 crypt cells were resuspended in 100 μl of Nucleofector solution and 5 μl of siRNA against mouse AhR (Santa Cruz Biotechnology, USA, catalogue number sc-29655) or control siRNA (sc-37007) was added to the cell suspension at a final concentration of 0.2 μM.

    Techniques: Isolation, Cell Culture, Inverted Microscopy, Viability Assay, Expressing

    Effects of fucoxanthin (Fx) on Smad2/Smad3 activation in NPDFs. ( A ) The NPDFs were pretreated with Fx (5–30 µM) for 30 min, followed by stimulation with TGF-β1 (1 ng/mL) for 30 min. ( B ) The Smad 2/3 nuclear translocation was determined via western blot analysis. ( C ) Smad 2/3 silencing inhibits the expression of α-SMA and Col-1 proteins in TGF-β1-stimulated NPDFs. The NPDFs were transfected with Smad2/3 siRNA (40 and 80 nM) for 24 h, followed by stimulation with TGF-β1 (1 ng/mL) for 24 h. ( D ) The NPDFs were transfected with either the control siRNA (80 nM) or the Smad 2/3 siRNA (80 nM) for 24 h, followed by stimulation with TGF-β1 (1 ng/mL) for 30 min. Each bar represents the mean ± S.E.M. from three independent experiments. Untreated cells were used as a control (CON). p-Smad 2/3, phosphor-Smad 2/3; t-Smad 2/3, total-Smad 2/3; siCon, control siRNA; siSmad, Smad 2/3 siRNA.

    Journal: Marine Drugs

    Article Title: Fucoxanthin Inhibits Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts via Modulation of Smad-Dependent and Smad-Independent Signaling Pathways

    doi: 10.3390/md16090323

    Figure Lengend Snippet: Effects of fucoxanthin (Fx) on Smad2/Smad3 activation in NPDFs. ( A ) The NPDFs were pretreated with Fx (5–30 µM) for 30 min, followed by stimulation with TGF-β1 (1 ng/mL) for 30 min. ( B ) The Smad 2/3 nuclear translocation was determined via western blot analysis. ( C ) Smad 2/3 silencing inhibits the expression of α-SMA and Col-1 proteins in TGF-β1-stimulated NPDFs. The NPDFs were transfected with Smad2/3 siRNA (40 and 80 nM) for 24 h, followed by stimulation with TGF-β1 (1 ng/mL) for 24 h. ( D ) The NPDFs were transfected with either the control siRNA (80 nM) or the Smad 2/3 siRNA (80 nM) for 24 h, followed by stimulation with TGF-β1 (1 ng/mL) for 30 min. Each bar represents the mean ± S.E.M. from three independent experiments. Untreated cells were used as a control (CON). p-Smad 2/3, phosphor-Smad 2/3; t-Smad 2/3, total-Smad 2/3; siCon, control siRNA; siSmad, Smad 2/3 siRNA.

    Article Snippet: We purchased Smad 2/3-specific small interfering RNAs (siRNAs, cat. no. sc-37238) and control siRNA (cat. no. sc-37007) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Activation Assay, Translocation Assay, Western Blot, Expressing, Transfection

    KLF4 depletion increases endothelial barrier permeability A, Timeline of TER assay. B, HLMVECs plated on gold microelectrodes were left untreated (control) or transfected with a non-silencing siRNA or KLF4 -silencing siRNA, followed by TER assay. Note that KLF4 silencing inhibited the thrombin response relative to untreated group (no transfection) or negative control group (non-silencing siRNA; n = 5 per group). Mean value (± s.e.m.) of maximal TER responses to thrombin (50 nM) stimulation (n = 7). Thrombin-induced decrease in TER was significantly attenuated in HLMVECs transfected by KLF4-depletion compared with untreated control or negative control group transfected with a non-silencing siRNA. C-E, KLF4 knockdown increases transendothelial permeability of fluorescein isothiocyanate (FITC)-conjugated albumin by decreasing VE-cadherin expression and AJ integrity. C , Timeline of experiments. D , Confluent HLMVEC monolayers were grown on microporous filters for 36 h, either left alone (control) or treated with control siRNA or with KLF4 -siRNA for 12 hours. At 18 hr post transfection, transendothelial FITC-albumin permeability was measured. Control HLMVECs showed basal transendothelial FITC-albumin permeability values, while KLF4 knockdown increased transendothelial FITC-albumin permeability. Re-expression of VE-cadherin into KLF4-depleted ECs partially restored the effect of loss of KLF4. Values are mean ± s.e.m. (n= 10). *p

    Journal: Circulation research

    Article Title: Kr?ppel-Like Factor-4 Transcriptionally Regulates VE-cadherin Expression and Endothelial Barrier Function

    doi: 10.1161/CIRCRESAHA.110.219592

    Figure Lengend Snippet: KLF4 depletion increases endothelial barrier permeability A, Timeline of TER assay. B, HLMVECs plated on gold microelectrodes were left untreated (control) or transfected with a non-silencing siRNA or KLF4 -silencing siRNA, followed by TER assay. Note that KLF4 silencing inhibited the thrombin response relative to untreated group (no transfection) or negative control group (non-silencing siRNA; n = 5 per group). Mean value (± s.e.m.) of maximal TER responses to thrombin (50 nM) stimulation (n = 7). Thrombin-induced decrease in TER was significantly attenuated in HLMVECs transfected by KLF4-depletion compared with untreated control or negative control group transfected with a non-silencing siRNA. C-E, KLF4 knockdown increases transendothelial permeability of fluorescein isothiocyanate (FITC)-conjugated albumin by decreasing VE-cadherin expression and AJ integrity. C , Timeline of experiments. D , Confluent HLMVEC monolayers were grown on microporous filters for 36 h, either left alone (control) or treated with control siRNA or with KLF4 -siRNA for 12 hours. At 18 hr post transfection, transendothelial FITC-albumin permeability was measured. Control HLMVECs showed basal transendothelial FITC-albumin permeability values, while KLF4 knockdown increased transendothelial FITC-albumin permeability. Re-expression of VE-cadherin into KLF4-depleted ECs partially restored the effect of loss of KLF4. Values are mean ± s.e.m. (n= 10). *p

    Article Snippet: Goat anti-VE-cadherin (sc-6458), rabbit anti-VE-cadherin (sc-28644), and mouse anti-GAPDH (sc-51906) antibodies, control non-silencing siRNA, Klf4 -siRNA for mouse, VE-cadherin -siRNA, and KLF 4-siRNA for human were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Permeability, Transfection, Negative Control, Expressing

    Klf4 depletion worsens bacterial endotoxin LPS-induced lung inflammation and vascular leakage A, Time-line of siRNA administration, LPS challenge, myeloperoxidase (MPO) as a measure of lung neutrophil sequestration, and lung extravascular water content assays. B, Lung MPO activities were assayed in mice receiving either control siRNA or Klf4 -specific siRNA with or without LPS challenge at 20 min, 1 h, 3 h, and 6 h. C, Lung tissue extracts were prepared 18 h after siRNA administration (but prior to receiving LPS) and efficacy of Klf4 -knockdown in lung tissues was evaluated by immunoblotting with the indicated antibodies. Values are mean ± s.e.m. (n=12 per group). *p

    Journal: Circulation research

    Article Title: Kr?ppel-Like Factor-4 Transcriptionally Regulates VE-cadherin Expression and Endothelial Barrier Function

    doi: 10.1161/CIRCRESAHA.110.219592

    Figure Lengend Snippet: Klf4 depletion worsens bacterial endotoxin LPS-induced lung inflammation and vascular leakage A, Time-line of siRNA administration, LPS challenge, myeloperoxidase (MPO) as a measure of lung neutrophil sequestration, and lung extravascular water content assays. B, Lung MPO activities were assayed in mice receiving either control siRNA or Klf4 -specific siRNA with or without LPS challenge at 20 min, 1 h, 3 h, and 6 h. C, Lung tissue extracts were prepared 18 h after siRNA administration (but prior to receiving LPS) and efficacy of Klf4 -knockdown in lung tissues was evaluated by immunoblotting with the indicated antibodies. Values are mean ± s.e.m. (n=12 per group). *p

    Article Snippet: Goat anti-VE-cadherin (sc-6458), rabbit anti-VE-cadherin (sc-28644), and mouse anti-GAPDH (sc-51906) antibodies, control non-silencing siRNA, Klf4 -siRNA for mouse, VE-cadherin -siRNA, and KLF 4-siRNA for human were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Mouse Assay