control sirna  (Santa Cruz Biotechnology)

 
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    Control siRNA C
    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-44231
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    Gene Editing Control siRNA C
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    Structured Review

    Santa Cruz Biotechnology control sirna
    Effects of WTD on production of TNF-α and RANTES in MIP-1β-induced U937 cells after <t>CCR5</t> knockdown. U937 cells were transfected with or without CCR5 <t>siRNA,</t> then treated with or without WTD for 2 h, and then exposed to MIP-1β (25 nM) for 24 h. Total CCR5 and its phosphorylation form (A) were detected by western blotting. The levels of TNF-α (B) and RANTES (C) in cell supernatants were detected by ELISA. ∗∗ 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/control sirna/product/Santa Cruz Biotechnology
    Average 99 stars, based on 1 article reviews
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    Images

    1) Product Images from "Wu-Tou Decoction in Rheumatoid Arthritis: Integrating Network Pharmacology and In Vivo Pharmacological Evaluation"

    Article Title: Wu-Tou Decoction in Rheumatoid Arthritis: Integrating Network Pharmacology and In Vivo Pharmacological Evaluation

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00230

    Effects of WTD on production of TNF-α and RANTES in MIP-1β-induced U937 cells after CCR5 knockdown. U937 cells were transfected with or without CCR5 siRNA, then treated with or without WTD for 2 h, and then exposed to MIP-1β (25 nM) for 24 h. Total CCR5 and its phosphorylation form (A) were detected by western blotting. The levels of TNF-α (B) and RANTES (C) in cell supernatants were detected by ELISA. ∗∗ P
    Figure Legend Snippet: Effects of WTD on production of TNF-α and RANTES in MIP-1β-induced U937 cells after CCR5 knockdown. U937 cells were transfected with or without CCR5 siRNA, then treated with or without WTD for 2 h, and then exposed to MIP-1β (25 nM) for 24 h. Total CCR5 and its phosphorylation form (A) were detected by western blotting. The levels of TNF-α (B) and RANTES (C) in cell supernatants were detected by ELISA. ∗∗ P

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

    2) Product Images from "Importin ?1 Protein-mediated Nuclear Localization of Death Receptor 5 (DR5) Limits DR5/Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Cell Death of Human Tumor Cells *"

    Article Title: Importin ?1 Protein-mediated Nuclear Localization of Death Receptor 5 (DR5) Limits DR5/Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Cell Death of Human Tumor Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.309377

    Knockdown of importin β1 increases DR5-mediated sensitivity of HeLa cells to rTRAIL. A , importin β1 siRNA or control siRNA was transfected into HeLa cells (5 × 10 3 ) in a 96-well plate. At 24 h post-transfection, cells were treated with or without 25 ng/ml rTRAIL for 24 h and observed using a phase-contrast microscope. Scale bars indicate 50 μm. B , at 24 h after transfection with importin β1 siRNA or control siRNA, the indicated concentrations of rTRAIL were added. Cell death was quantified by WST assay as described under “Experimental Procedures.” Data are the mean ± S.D. of triplicate samples. **, p
    Figure Legend Snippet: Knockdown of importin β1 increases DR5-mediated sensitivity of HeLa cells to rTRAIL. A , importin β1 siRNA or control siRNA was transfected into HeLa cells (5 × 10 3 ) in a 96-well plate. At 24 h post-transfection, cells were treated with or without 25 ng/ml rTRAIL for 24 h and observed using a phase-contrast microscope. Scale bars indicate 50 μm. B , at 24 h after transfection with importin β1 siRNA or control siRNA, the indicated concentrations of rTRAIL were added. Cell death was quantified by WST assay as described under “Experimental Procedures.” Data are the mean ± S.D. of triplicate samples. **, p

    Techniques Used: Transfection, Microscopy, WST Assay

    3) Product Images from "A Novel Small-Molecule Inhibitor of Mcl-1 Blocks Pancreatic Cancer Growth In vitro and In vivo"

    Article Title: A Novel Small-Molecule Inhibitor of Mcl-1 Blocks Pancreatic Cancer Growth In vitro and In vivo

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-12-0767

    Down-regulation of Mcl-1 by siRNA in BxPC-3 cells blocks growth inhibition and apoptosis induced by 2 (UMI-77)
    Figure Legend Snippet: Down-regulation of Mcl-1 by siRNA in BxPC-3 cells blocks growth inhibition and apoptosis induced by 2 (UMI-77)

    Techniques Used: Inhibition

    4) Product Images from "The AKT1/NF-kappaB/Notch1/PTEN axis has an important role in chemoresistance of gastric cancer cells"

    Article Title: The AKT1/NF-kappaB/Notch1/PTEN axis has an important role in chemoresistance of gastric cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.375

    Notch1 regulated PTEN expression through CBF-1 and had a pro-apoptotic role in gastric cancer cells. ( a ) Lysate from MKN-28 cells (parental), MKN-28 cells transfected with control siRNA or Notch1 siRNA were analyzed by anti-Notch1 blotting. The MKN-28 cells transfected with control siRNA or Notch-1 siRNA were treated with PS or 3 μ M doxorubicin for 12 h, respectively. The protein and mRNA levels of AKT1, Notch1 and PTEN were detected by immunoblotting and real-time RT-PCR. Immunoblotting results of β -actin were used to show equal loading. Values are the mean±S.D. from three different experiments. ★ P
    Figure Legend Snippet: Notch1 regulated PTEN expression through CBF-1 and had a pro-apoptotic role in gastric cancer cells. ( a ) Lysate from MKN-28 cells (parental), MKN-28 cells transfected with control siRNA or Notch1 siRNA were analyzed by anti-Notch1 blotting. The MKN-28 cells transfected with control siRNA or Notch-1 siRNA were treated with PS or 3 μ M doxorubicin for 12 h, respectively. The protein and mRNA levels of AKT1, Notch1 and PTEN were detected by immunoblotting and real-time RT-PCR. Immunoblotting results of β -actin were used to show equal loading. Values are the mean±S.D. from three different experiments. ★ P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    AKT1 regulated Notch-1 and PTEN expression through NF κ B and had an anti-apoptotic role in gastric cancer cells. ( a ) Lysate from MKN-28 cells (parental), MKN-28 cells expressing lentiviral scramble shRNA (SCR shRNA), or lentiviral AKT1 shRNA were analyzed by anti-AKT1 blotting. The MKN-28 cells expressing lentiviral SCR shRNA or lentiviral AKT1 shRNA were treated with PS or 3 μ M doxorubicin for 12 h, respectively. The protein and mRNA levels of AKT1, Notch1 and PTEN were detected by immunoblotting and real-time RT-PCR. Immunoblotting results of β -actin were used to show equal loading. Values are the mean±S.D. from three different experiments. ★ P
    Figure Legend Snippet: AKT1 regulated Notch-1 and PTEN expression through NF κ B and had an anti-apoptotic role in gastric cancer cells. ( a ) Lysate from MKN-28 cells (parental), MKN-28 cells expressing lentiviral scramble shRNA (SCR shRNA), or lentiviral AKT1 shRNA were analyzed by anti-AKT1 blotting. The MKN-28 cells expressing lentiviral SCR shRNA or lentiviral AKT1 shRNA were treated with PS or 3 μ M doxorubicin for 12 h, respectively. The protein and mRNA levels of AKT1, Notch1 and PTEN were detected by immunoblotting and real-time RT-PCR. Immunoblotting results of β -actin were used to show equal loading. Values are the mean±S.D. from three different experiments. ★ P

    Techniques Used: Expressing, shRNA, Quantitative RT-PCR

    p65/NF κ B bound to the Notch1 promoter and regulated the transcription of Notch1 gene. ( a ) The human Notch1 promoter sequence. ( b ) The schematic diagram of constructs with different 5′ upstream deletions. ( c ) Luciferase activity of the deleted constructs in MKN-28 cells. After transfection of reporter gene, cells were treated by doxorubicin (3 μ M, 12 h) with or without pretreatment with PDTC or infection of Akt1 shRNA lentivirus and p65 siRNA. All transfection experiments were repeated three times, and luciferase activity was normalized to the Renilla minimal reporter. Mean data±S.D. were results from three different experiments. * P
    Figure Legend Snippet: p65/NF κ B bound to the Notch1 promoter and regulated the transcription of Notch1 gene. ( a ) The human Notch1 promoter sequence. ( b ) The schematic diagram of constructs with different 5′ upstream deletions. ( c ) Luciferase activity of the deleted constructs in MKN-28 cells. After transfection of reporter gene, cells were treated by doxorubicin (3 μ M, 12 h) with or without pretreatment with PDTC or infection of Akt1 shRNA lentivirus and p65 siRNA. All transfection experiments were repeated three times, and luciferase activity was normalized to the Renilla minimal reporter. Mean data±S.D. were results from three different experiments. * P

    Techniques Used: Sequencing, Construct, Luciferase, Activity Assay, Transfection, Infection, shRNA

    Doxorubicin-induced PTEN expression had a pro-apoptotic function in gastric cancer cells. ( a ) PTEN transactivation was mediated by NF κ B, AKT1, and Notch1. MKN-28 cells were transfected for 9 h with the PTEN-luc reporter construct. Some cells were transfected together with control or Notch1 siRNA. Twenty-four hours later, cells were either left or pretreated with 10 μ M PDTC for 1 h, left untreated or treated with 3 μ M doxorubicin for an additional 12-h period, after which the luciferase activity was determined. The MKN-28 cells expressing lentiviral SCR shRNA or lentiviral AKT1 shRNA were also transfected with PTEN-luc reporter construct followed by treatment with or without 3 μ M doxorubicin, after which the luciferase activity was determined. ( b ) Knockdown of PTEN by siRNA inhibited the basal and doxorubicin-induced cell apoptosis in gastric cancer cells. ( c ) Upregulation of PTEN expression by wild-type PTEN plasmid transfection promoted cell apoptosis in gastric cancer cells
    Figure Legend Snippet: Doxorubicin-induced PTEN expression had a pro-apoptotic function in gastric cancer cells. ( a ) PTEN transactivation was mediated by NF κ B, AKT1, and Notch1. MKN-28 cells were transfected for 9 h with the PTEN-luc reporter construct. Some cells were transfected together with control or Notch1 siRNA. Twenty-four hours later, cells were either left or pretreated with 10 μ M PDTC for 1 h, left untreated or treated with 3 μ M doxorubicin for an additional 12-h period, after which the luciferase activity was determined. The MKN-28 cells expressing lentiviral SCR shRNA or lentiviral AKT1 shRNA were also transfected with PTEN-luc reporter construct followed by treatment with or without 3 μ M doxorubicin, after which the luciferase activity was determined. ( b ) Knockdown of PTEN by siRNA inhibited the basal and doxorubicin-induced cell apoptosis in gastric cancer cells. ( c ) Upregulation of PTEN expression by wild-type PTEN plasmid transfection promoted cell apoptosis in gastric cancer cells

    Techniques Used: Expressing, Transfection, Construct, Luciferase, Activity Assay, shRNA, Plasmid Preparation

    5) Product Images from "Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells"

    Article Title: Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25961

    Knock-down of cofilin using siRNA disrupts F-actin organization and inhibits invasion of PC-3 cells PC-3 cells were seeded on 6-well plates or glass slides and transfected with 50, 100 and 200 nM cofilin 1 siRNA or with control siRNA as described in Materials and Methods. After 60 hours, the cells were lysed for Western blotting to detect the levels of total and p-cofilin (A) , fixed on glass slides and immunostained for cofilin (B) , or detached and incubated for 48 hours in an invasion assay (C) . After invasion assay, cells were fixed, stained and counted as described in Materials and Methods. * p
    Figure Legend Snippet: Knock-down of cofilin using siRNA disrupts F-actin organization and inhibits invasion of PC-3 cells PC-3 cells were seeded on 6-well plates or glass slides and transfected with 50, 100 and 200 nM cofilin 1 siRNA or with control siRNA as described in Materials and Methods. After 60 hours, the cells were lysed for Western blotting to detect the levels of total and p-cofilin (A) , fixed on glass slides and immunostained for cofilin (B) , or detached and incubated for 48 hours in an invasion assay (C) . After invasion assay, cells were fixed, stained and counted as described in Materials and Methods. * p

    Techniques Used: Transfection, Western Blot, Incubation, Invasion Assay, Staining

    6) Product Images from "RAPGEF5 regulates nuclear translocation of β-catenin"

    Article Title: RAPGEF5 regulates nuclear translocation of β-catenin

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2017.12.001

    Rapgef5 acts downstream of β-catenin cytoplasmic stabilization (A) The ability to induce secondary axes in development can be used as a read out of Wnt signaling activity. Uninjected embryos have a single axis (dotted green line) while embryos injected with β-catenin mRNA can have a second embryonic axis (two dotted red lines) that are readily detected at st 16–19 embryos. Injection of WT, stabilized (ST), or NLS tagged β-catenin can induce secondary axes. Rapgef5 depletion significantly decreases the percentage of secondary axes induced by WT and ST β-catenin. Injection of GSK3 mRNA reduces secondary axes induced by WT β-catenin but has no effect on ST β-catenin. The ability of NLS-β-catenin to induce secondary axes is unaffected by reduction of Rapgef5 levels. (B) Rapgef5 knockdown reduces luciferase activity in embryos injected with WT or ST β-catenin mRNA in a TOPFlash assay. Data are represented as mean ± SD. (C) Wnt signaling activity is reduced in ST β-catenin MEFs (Δexon3) following siRNA depletion of Rapgef5. The schematic depicts the Catnblox(ex3) mouse allele. Exon 3 (E3), containing the GSK3 phosphorylation sites that target β-catenin for cytoplasmic degradation, is flanked by loxp sites allowing for its conditional removal and production of a stabilized (ST) β-catenin allele. WT: WT cells; FOP: ST β-catenin cells transfected with FOPFlash negative control; TOP: ST β-catenin cells transfected with TOPFlash reporter plasmid; TOP + R5 siRNA: ST β-catenin cells transfected with Rapgef5 siRNA and TOPFlash reporter plasmid; Top + C. siRNA: ST β-catenin cells transfected with control siRNA and TOPFlash reporter plasmid. A Renilla luciferase transfection control was included in each treatment to allow normalization. Data are represented as mean ± SEM. (D) Pharmacological inhibition of GSK3 by the addition of BIO between stages 9 – 11 results in increased β-catenin signaling and loss of anterior development in Xenopus embryos. Depletion of Rapgef5 can counteract this effect and rescue development of the head demonstrating that Rapgef5 regulates Wnt signaling downstream of GSK3. A single asterisk indicates P
    Figure Legend Snippet: Rapgef5 acts downstream of β-catenin cytoplasmic stabilization (A) The ability to induce secondary axes in development can be used as a read out of Wnt signaling activity. Uninjected embryos have a single axis (dotted green line) while embryos injected with β-catenin mRNA can have a second embryonic axis (two dotted red lines) that are readily detected at st 16–19 embryos. Injection of WT, stabilized (ST), or NLS tagged β-catenin can induce secondary axes. Rapgef5 depletion significantly decreases the percentage of secondary axes induced by WT and ST β-catenin. Injection of GSK3 mRNA reduces secondary axes induced by WT β-catenin but has no effect on ST β-catenin. The ability of NLS-β-catenin to induce secondary axes is unaffected by reduction of Rapgef5 levels. (B) Rapgef5 knockdown reduces luciferase activity in embryos injected with WT or ST β-catenin mRNA in a TOPFlash assay. Data are represented as mean ± SD. (C) Wnt signaling activity is reduced in ST β-catenin MEFs (Δexon3) following siRNA depletion of Rapgef5. The schematic depicts the Catnblox(ex3) mouse allele. Exon 3 (E3), containing the GSK3 phosphorylation sites that target β-catenin for cytoplasmic degradation, is flanked by loxp sites allowing for its conditional removal and production of a stabilized (ST) β-catenin allele. WT: WT cells; FOP: ST β-catenin cells transfected with FOPFlash negative control; TOP: ST β-catenin cells transfected with TOPFlash reporter plasmid; TOP + R5 siRNA: ST β-catenin cells transfected with Rapgef5 siRNA and TOPFlash reporter plasmid; Top + C. siRNA: ST β-catenin cells transfected with control siRNA and TOPFlash reporter plasmid. A Renilla luciferase transfection control was included in each treatment to allow normalization. Data are represented as mean ± SEM. (D) Pharmacological inhibition of GSK3 by the addition of BIO between stages 9 – 11 results in increased β-catenin signaling and loss of anterior development in Xenopus embryos. Depletion of Rapgef5 can counteract this effect and rescue development of the head demonstrating that Rapgef5 regulates Wnt signaling downstream of GSK3. A single asterisk indicates P

    Techniques Used: Activity Assay, Injection, Luciferase, TOPFlash assay, Transfection, Negative Control, Plasmid Preparation, Inhibition

    7) Product Images from "Fucoxanthin Inhibits Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts via Modulation of Smad-Dependent and Smad-Independent Signaling Pathways"

    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

    Journal: Marine Drugs

    doi: 10.3390/md16090323

    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.
    Figure Legend 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.

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

    8) Product Images from "D-AKAP2 Interacts with Rab4 and Rab11 through Its RGS Domains and Regulates Transferrin Receptor Recycling *"

    Article Title: D-AKAP2 Interacts with Rab4 and Rab11 through Its RGS Domains and Regulates Transferrin Receptor Recycling *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.022582

    Knockdown of D-AKAP2 increases the rate of transferrin recycling. A–F , after 2 days of treatment with control ( A–C ) or D-AKAP2 siRNA ( D–F ), HeLa cells were incubated for 20 min with fluorescently labeled transferrin and then chased with unlabeled transferrin for 0 ( A and D ), 15 ( B and E ), or 30 min ( C and F ) before being washed and fixed. Pulse-chased transferrin showed a more perinuclear localization in control cells and a more peripheral localization in D-AKAP2 knockdown cells, similar to the change seen with the Rab11 compartment. G , to quantify transferrin recycling, HeLa cells were pulse-chased in the same manner as described in A–F but were analyzed by flow cytometry. Fluorescence values ± S.E. are normalized to the value for the control cells at 0 min. p values were determined by a two-tailed t test. H , proposed model for the localization and function of D-AKAP2 in protein recycling with respect to Rab proteins as adapted from Sönnichsen et al. ( 13 ). Endocytosed cargo recycles to the plasma membrane either directly from the Rab5/Rab4 sorting endosome ( SE ) by the fast recycling pathway or via the Rab4/Rab11 ERC in the slow recycling pathway. D-AKAP2 is proposed to localize to Rab4/Rab11-positive membranes and to negatively regulate trafficking of Rab11 recycling vesicles to the surface.
    Figure Legend Snippet: Knockdown of D-AKAP2 increases the rate of transferrin recycling. A–F , after 2 days of treatment with control ( A–C ) or D-AKAP2 siRNA ( D–F ), HeLa cells were incubated for 20 min with fluorescently labeled transferrin and then chased with unlabeled transferrin for 0 ( A and D ), 15 ( B and E ), or 30 min ( C and F ) before being washed and fixed. Pulse-chased transferrin showed a more perinuclear localization in control cells and a more peripheral localization in D-AKAP2 knockdown cells, similar to the change seen with the Rab11 compartment. G , to quantify transferrin recycling, HeLa cells were pulse-chased in the same manner as described in A–F but were analyzed by flow cytometry. Fluorescence values ± S.E. are normalized to the value for the control cells at 0 min. p values were determined by a two-tailed t test. H , proposed model for the localization and function of D-AKAP2 in protein recycling with respect to Rab proteins as adapted from Sönnichsen et al. ( 13 ). Endocytosed cargo recycles to the plasma membrane either directly from the Rab5/Rab4 sorting endosome ( SE ) by the fast recycling pathway or via the Rab4/Rab11 ERC in the slow recycling pathway. D-AKAP2 is proposed to localize to Rab4/Rab11-positive membranes and to negatively regulate trafficking of Rab11 recycling vesicles to the surface.

    Techniques Used: Incubation, Labeling, Flow Cytometry, Cytometry, Fluorescence, Two Tailed Test

    Knockdown of D-AKAP2 by RNA interference causes the Rab11 compartment to become more peripheral. A , siRNA of D-AKAP2 effectively knocked down the levels of endogenous protein in HeLa cells after 2 days as judged by immunoblotting of whole-cell extracts. B–K , HeLa cells were treated with either control ( B , D , F , H , and J ) or D-AKAP2 siRNA ( C , E , G , I , and K ). B–G , cells treated with siRNA were transfected with GFP-Rab4 WT and mCh-Rab11 WT ( B and C ), mCh-Rab11 WT ( D and E ), or GFP-Rab4 WT ( F and G ). Fixed coverslips were stained for endogenous EEA1 to mark early endosomes. Rab11 and, to a lesser extent, Rab4 became more peripheral in D-AKAP2 knockdown cells, whereas EEA1 staining remained relatively unchanged. H–K , HeLa cells treated with siRNA were fixed and stained for endogenous Rab11 and transferrin receptor ( H and I ) or γ-adaptin and giantin ( J and K ). Endogenous Rab11 and TfnR displayed similar shifts to the periphery of cells treated with D-AKAP2 siRNA. Knocking down D-AKAP2 also caused fragmentation of the Golgi as visualized by staining the cis/medial-Golgi marker giantin, as well as γ-adaptin, which localizes to the trans-Golgi network and to endosomes. Scale bars , 10 μm.
    Figure Legend Snippet: Knockdown of D-AKAP2 by RNA interference causes the Rab11 compartment to become more peripheral. A , siRNA of D-AKAP2 effectively knocked down the levels of endogenous protein in HeLa cells after 2 days as judged by immunoblotting of whole-cell extracts. B–K , HeLa cells were treated with either control ( B , D , F , H , and J ) or D-AKAP2 siRNA ( C , E , G , I , and K ). B–G , cells treated with siRNA were transfected with GFP-Rab4 WT and mCh-Rab11 WT ( B and C ), mCh-Rab11 WT ( D and E ), or GFP-Rab4 WT ( F and G ). Fixed coverslips were stained for endogenous EEA1 to mark early endosomes. Rab11 and, to a lesser extent, Rab4 became more peripheral in D-AKAP2 knockdown cells, whereas EEA1 staining remained relatively unchanged. H–K , HeLa cells treated with siRNA were fixed and stained for endogenous Rab11 and transferrin receptor ( H and I ) or γ-adaptin and giantin ( J and K ). Endogenous Rab11 and TfnR displayed similar shifts to the periphery of cells treated with D-AKAP2 siRNA. Knocking down D-AKAP2 also caused fragmentation of the Golgi as visualized by staining the cis/medial-Golgi marker giantin, as well as γ-adaptin, which localizes to the trans-Golgi network and to endosomes. Scale bars , 10 μm.

    Techniques Used: Transfection, Staining, Marker

    9) Product Images from "Twist1 confers multidrug resistance in colon cancer through upregulation of ATP-binding cassette transporters"

    Article Title: Twist1 confers multidrug resistance in colon cancer through upregulation of ATP-binding cassette transporters

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17548

    siRNA-mediated downregulation of Twist1 reversed EMT (A) Healing rate was assayed at 6 and 24 h. The left panel shows representative images; the right panel shows quantitative analysis. (B) Number of cells invaded through basement member. (C) Twist1 silencing significantly decreased the number of actin microfilaments. (D) Immunofluorescence staining of E-cadherin and vimentin in the HCT-8/V cells following the downregulation of Twist1. Green corresponds to E-cadherin; red corresponds to vimentin. A blue signal represents nuclear DNA staining by DAPI. (E) Western blot analysis of E-cadherin and vimentin was performed on the HCT-8/V cells following the inhibition of Twist1. GAPDH was used as a loading control. The right panel shows the quantitative analysis of the relative protein levels. (F) IHC staining for E-cadherin and vimentin in the HCT-8 transplanted tumors. Representative images are shown in the left panel; the staining index is shown in the right panel. Scale bars represent 50 μm in D and F and 10 μm in C . Data from the three independent experiments are graphed as mean ± SD. *P
    Figure Legend Snippet: siRNA-mediated downregulation of Twist1 reversed EMT (A) Healing rate was assayed at 6 and 24 h. The left panel shows representative images; the right panel shows quantitative analysis. (B) Number of cells invaded through basement member. (C) Twist1 silencing significantly decreased the number of actin microfilaments. (D) Immunofluorescence staining of E-cadherin and vimentin in the HCT-8/V cells following the downregulation of Twist1. Green corresponds to E-cadherin; red corresponds to vimentin. A blue signal represents nuclear DNA staining by DAPI. (E) Western blot analysis of E-cadherin and vimentin was performed on the HCT-8/V cells following the inhibition of Twist1. GAPDH was used as a loading control. The right panel shows the quantitative analysis of the relative protein levels. (F) IHC staining for E-cadherin and vimentin in the HCT-8 transplanted tumors. Representative images are shown in the left panel; the staining index is shown in the right panel. Scale bars represent 50 μm in D and F and 10 μm in C . Data from the three independent experiments are graphed as mean ± SD. *P

    Techniques Used: Immunofluorescence, Staining, Western Blot, Inhibition, Immunohistochemistry

    Downregulation of Twist1 increased the chemosensitivity of HCT-8 transplanted tumor to vincristine in vivo (A) Effects on the tumor growth rate. The left panel shows the representative images of tumors harvested at the end of the experiments; the right panel shows the curve of the xenograft growth. (B) Metastases in lung tissues of the tumor-bearing model. The left panel shows the representative images of lung tissues stained with H E; the right panel shows the number of metastases in lung tissues. The scale bars represent 100 μm. (C) Overall survival analyses in mice treated with Twist1-siRNA or control siRNA followed by vincristine treatment ( P = 0.000). (D) Immunohistochemical staining for ABCB1 and ABCC1 in tumors. Representative images are shown in the left panel; the staining index is shown in the right panel. Scale bars = 50 μm. Data from three independent experiments are graphed as mean ± SD. * P
    Figure Legend Snippet: Downregulation of Twist1 increased the chemosensitivity of HCT-8 transplanted tumor to vincristine in vivo (A) Effects on the tumor growth rate. The left panel shows the representative images of tumors harvested at the end of the experiments; the right panel shows the curve of the xenograft growth. (B) Metastases in lung tissues of the tumor-bearing model. The left panel shows the representative images of lung tissues stained with H E; the right panel shows the number of metastases in lung tissues. The scale bars represent 100 μm. (C) Overall survival analyses in mice treated with Twist1-siRNA or control siRNA followed by vincristine treatment ( P = 0.000). (D) Immunohistochemical staining for ABCB1 and ABCC1 in tumors. Representative images are shown in the left panel; the staining index is shown in the right panel. Scale bars = 50 μm. Data from three independent experiments are graphed as mean ± SD. * P

    Techniques Used: In Vivo, Staining, Mouse Assay, Immunohistochemistry

    10) Product Images from "Altered CXCR3 isoform expression regulates prostate cancer cell migration and invasion"

    Article Title: Altered CXCR3 isoform expression regulates prostate cancer cell migration and invasion

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-11-3

    CXCR3 chemokine induced cell motility and invasion via PLCβ3 signaling pathway in prostate cancer cells and blocked cell motility by m-calpain activity inhibition in normal cells . (A) PLCβ3 was significantly knocked down in DU-145 cells by siRNA. Protein expression was quantified by ImageJ and normalized to GAPDH. Histogram represent mean values (+/-s.d.) of three separate experiments (**P
    Figure Legend Snippet: CXCR3 chemokine induced cell motility and invasion via PLCβ3 signaling pathway in prostate cancer cells and blocked cell motility by m-calpain activity inhibition in normal cells . (A) PLCβ3 was significantly knocked down in DU-145 cells by siRNA. Protein expression was quantified by ImageJ and normalized to GAPDH. Histogram represent mean values (+/-s.d.) of three separate experiments (**P

    Techniques Used: Activity Assay, Inhibition, Expressing

    11) Product Images from "Triptolide Modulates TREM-1 Signal Pathway to Inhibit the Inflammatory Response in Rheumatoid Arthritis"

    Article Title: Triptolide Modulates TREM-1 Signal Pathway to Inhibit the Inflammatory Response in Rheumatoid Arthritis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17040498

    Effects of TP on inflammatory cytokines expression after TREM-1 knockdown. PMA-induced U937 cells were treated with or without 1 μg/mL LPS, and then transfected with TREM-1 siRNA or control siRNA, respectively. After 48 h, cells were treated with or without TP for another 48 h. ( A ) TREM-1 mRNA protein levels were determined by Western blot; and ( B – D ) TNF-α, IL-1β and IL-6 production were determined by ELISA. ## p
    Figure Legend Snippet: Effects of TP on inflammatory cytokines expression after TREM-1 knockdown. PMA-induced U937 cells were treated with or without 1 μg/mL LPS, and then transfected with TREM-1 siRNA or control siRNA, respectively. After 48 h, cells were treated with or without TP for another 48 h. ( A ) TREM-1 mRNA protein levels were determined by Western blot; and ( B – D ) TNF-α, IL-1β and IL-6 production were determined by ELISA. ## p

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

    12) Product Images from "Proliferating Cell Nuclear Antigen is a novel inhibitory ligand for the natural cytotoxicity receptor NKp44"

    Article Title: Proliferating Cell Nuclear Antigen is a novel inhibitory ligand for the natural cytotoxicity receptor NKp44

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

    doi: 10.4049/jimmunol.1102267

    PCNA down-regulation in various tumor cells enhances lysis by NK cells (A) HeLa cells transfected either with control-siRNA, BAT3-siRNA or with PCNA-siRNA were labeled radioactively and incubated with NKp44-expressing primary NK cells. NC, untransfected HeLa cells were used as additional negative control. The lysis of the various targets by NK cells was assayed in a standard 5-h S 35 release assay (left panel). Effect of siRNA transfection on protein expression, including control β-actin, was measured by western blotting (right panel). (B) Target PANC-1 cells, transfected with either control-siRNA or with PCNA-siRNA, were incubated with various anti-NKp44-activated NK effectors for 18 hrs. Relative IFNγ levels in the culture supernatants are shown. (C–H) Various cancer cell lines were transfected either with control siRNA or with PCNA-siRNA and assayed for lysis by NK92-44 cells. Effect of siRNA transfection on protein expression, including control β-actin, was measured by western blotting (inset). As measured by ImageJ freeware, the silencing of PCNA in the target cell lines relative to β-actin in the lysis assays were: HeLa- 61%, PANC-1- 60%, MCF7- 37%, Du145- 67%, U251- 63% and A375- 8%. The results are from one representative experiment of 2 performed. Bars ± SD. * P-value
    Figure Legend Snippet: PCNA down-regulation in various tumor cells enhances lysis by NK cells (A) HeLa cells transfected either with control-siRNA, BAT3-siRNA or with PCNA-siRNA were labeled radioactively and incubated with NKp44-expressing primary NK cells. NC, untransfected HeLa cells were used as additional negative control. The lysis of the various targets by NK cells was assayed in a standard 5-h S 35 release assay (left panel). Effect of siRNA transfection on protein expression, including control β-actin, was measured by western blotting (right panel). (B) Target PANC-1 cells, transfected with either control-siRNA or with PCNA-siRNA, were incubated with various anti-NKp44-activated NK effectors for 18 hrs. Relative IFNγ levels in the culture supernatants are shown. (C–H) Various cancer cell lines were transfected either with control siRNA or with PCNA-siRNA and assayed for lysis by NK92-44 cells. Effect of siRNA transfection on protein expression, including control β-actin, was measured by western blotting (inset). As measured by ImageJ freeware, the silencing of PCNA in the target cell lines relative to β-actin in the lysis assays were: HeLa- 61%, PANC-1- 60%, MCF7- 37%, Du145- 67%, U251- 63% and A375- 8%. The results are from one representative experiment of 2 performed. Bars ± SD. * P-value

    Techniques Used: Lysis, Transfection, Labeling, Incubation, Expressing, Negative Control, Release Assay, Western Blot

    13) Product Images from "Heme Mediated STAT3 Activation in Severe Malaria"

    Article Title: Heme Mediated STAT3 Activation in Severe Malaria

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034280

    STAT3 activation is induced by Heme in CRL-2581 cells. Decreased activation of STAT3 by siRNA-STAT3 (siSTAT3) and AG490 down regulates HO-1 and CXCL10 protein. pSTAT3 was increased by Heme from 1 µM and peaked at 5 µM and then decreased (A). When pSTAT3 was reduced by siSTAT3 (B) or pharmacological inhibitor of Jak, AG490 (C), HO-1 protein expression was inhibited correspondingly. These data indicated that Heme induced production of HO-1 by way of STAT3 signaling pathway. Reduced activation of STAT3 caused by siSTAT3 ( Figure 8D ) and AG490 ( Figure 8E ) also caused reduced expression of CXCL10. We futher found when CXCL10 was blocked by anti-CXCL10 antibody, HO-1 induction by Heme was decreased to one half, indicating that CXCL10 directly induce HO-1 expression ( Figure 8F ). Reduced HO-1 expression by siHO-1 increased CXCL10 expression which implies that HO-1 also modulates CXCL10 expression ( Figure 8G ). Interestingly, pSTAT3 level was increased by CoPP and decreased by ZnPP ( Figure 8H ), which indicates that HO-1 also regulates STAT3 signaling.
    Figure Legend Snippet: STAT3 activation is induced by Heme in CRL-2581 cells. Decreased activation of STAT3 by siRNA-STAT3 (siSTAT3) and AG490 down regulates HO-1 and CXCL10 protein. pSTAT3 was increased by Heme from 1 µM and peaked at 5 µM and then decreased (A). When pSTAT3 was reduced by siSTAT3 (B) or pharmacological inhibitor of Jak, AG490 (C), HO-1 protein expression was inhibited correspondingly. These data indicated that Heme induced production of HO-1 by way of STAT3 signaling pathway. Reduced activation of STAT3 caused by siSTAT3 ( Figure 8D ) and AG490 ( Figure 8E ) also caused reduced expression of CXCL10. We futher found when CXCL10 was blocked by anti-CXCL10 antibody, HO-1 induction by Heme was decreased to one half, indicating that CXCL10 directly induce HO-1 expression ( Figure 8F ). Reduced HO-1 expression by siHO-1 increased CXCL10 expression which implies that HO-1 also modulates CXCL10 expression ( Figure 8G ). Interestingly, pSTAT3 level was increased by CoPP and decreased by ZnPP ( Figure 8H ), which indicates that HO-1 also regulates STAT3 signaling.

    Techniques Used: Activation Assay, Expressing

    14) Product Images from "High-mobility group box 1 from reactive astrocytes enhances the accumulation of endothelial progenitor cells in damaged white matter"

    Article Title: High-mobility group box 1 from reactive astrocytes enhances the accumulation of endothelial progenitor cells in damaged white matter

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.12120

    HMGB1-siRNA treatment reduced proliferated endothelial number after white matter injury
    Figure Legend Snippet: HMGB1-siRNA treatment reduced proliferated endothelial number after white matter injury

    Techniques Used:

    15) Product Images from "Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells"

    Article Title: Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells

    Journal: Virology Journal

    doi: 10.1186/s12985-017-0875-5

    Antiviral activity of IFITM1 induced by H9N2 virus infection in HUVECs and BEAS-2Bs. HUVECs and BEAS-2Bs were infected with H9N2 virus at MOI of 5 and incubated for 1 h, then cells were transfected with control siRNA or IFITM1 specific siRNA for 36 h. Virus titer in each group was detected using plaque assay at 36 h postinfection. The effect of IFIMT1 specific siRNA on IFITM1 expression was detected using western blot. a IFITM1 protein level after transfected with siRNA in HUVECs. b IFITM1 protein level after transfected with siRNA in BEAS-2Bs. c Virus titers in HUVECs transfected with control siRNA or IFITM1 specific siRNA. Compared to virus group (control siRNA), the virus titer in virus + siRNA group (IFITM1 specific siRNA) was increased by 13.1 ± 2.4% ( P > 0.05, t-test). d Virus titers in BEAS-2Bs transfected with control siRNA or IFITM1 specific siRNA. Compared to virus group (control siRNA), the virus titer in virus + siRNA group (specific siRNA) was increased by 9.7% ± 3.8% ( P > 0.05, t-test)
    Figure Legend Snippet: Antiviral activity of IFITM1 induced by H9N2 virus infection in HUVECs and BEAS-2Bs. HUVECs and BEAS-2Bs were infected with H9N2 virus at MOI of 5 and incubated for 1 h, then cells were transfected with control siRNA or IFITM1 specific siRNA for 36 h. Virus titer in each group was detected using plaque assay at 36 h postinfection. The effect of IFIMT1 specific siRNA on IFITM1 expression was detected using western blot. a IFITM1 protein level after transfected with siRNA in HUVECs. b IFITM1 protein level after transfected with siRNA in BEAS-2Bs. c Virus titers in HUVECs transfected with control siRNA or IFITM1 specific siRNA. Compared to virus group (control siRNA), the virus titer in virus + siRNA group (IFITM1 specific siRNA) was increased by 13.1 ± 2.4% ( P > 0.05, t-test). d Virus titers in BEAS-2Bs transfected with control siRNA or IFITM1 specific siRNA. Compared to virus group (control siRNA), the virus titer in virus + siRNA group (specific siRNA) was increased by 9.7% ± 3.8% ( P > 0.05, t-test)

    Techniques Used: Activity Assay, Infection, Incubation, Transfection, Plaque Assay, Expressing, Western Blot

    Antiviral activity of IFITM1 induced by viral particle inoculation in HUVECs and BEAS-2Bs. HUVECs and BEAS-2Bs were inoculated with viral particle at MOI of 5 and incubated for 1 h, then cells were transfected with control siRNA or IFITM1 specific siRNA for 36 h before infected with H9N2 virus at MOI of 5. Virus titer of each group was detected by plaque assay at 36 h postinfection. The effect of IFIMT1 specific siRNA on IFITM1 expression was detected using western blot. a IFITM1 protein level after transfected with siRNA in HUVECs. b IFITM1 protein level after transfected with siRNA in BEAS-2Bs. c Virus titers in HUVECs transfected with control siRNA or IFITM1 specific siRNA. Compared to particle group (control siRNA), the virus titer in particle + siRNA group (IFITM1 specific siRNA) was increased by 60.5 ± 10.7% ( P
    Figure Legend Snippet: Antiviral activity of IFITM1 induced by viral particle inoculation in HUVECs and BEAS-2Bs. HUVECs and BEAS-2Bs were inoculated with viral particle at MOI of 5 and incubated for 1 h, then cells were transfected with control siRNA or IFITM1 specific siRNA for 36 h before infected with H9N2 virus at MOI of 5. Virus titer of each group was detected by plaque assay at 36 h postinfection. The effect of IFIMT1 specific siRNA on IFITM1 expression was detected using western blot. a IFITM1 protein level after transfected with siRNA in HUVECs. b IFITM1 protein level after transfected with siRNA in BEAS-2Bs. c Virus titers in HUVECs transfected with control siRNA or IFITM1 specific siRNA. Compared to particle group (control siRNA), the virus titer in particle + siRNA group (IFITM1 specific siRNA) was increased by 60.5 ± 10.7% ( P

    Techniques Used: Activity Assay, Incubation, Transfection, Infection, Plaque Assay, Expressing, Western Blot

    16) Product Images from "Angiogenic growth factors augment K–Cl cotransporter expression in erythroid cells via hypoxia-inducible factor-1α"

    Article Title: Angiogenic growth factors augment K–Cl cotransporter expression in erythroid cells via hypoxia-inducible factor-1α

    Journal: American Journal of Hematology

    doi: 10.1002/ajh.23631

    VEGF-induced KCC4 and KCC3b mRNA expression in K562 cells: K562 cells were transfected with siRNA for p38 MAP kinase, p47 phox , JNK kinase, and scrambled scRNA control, prior to stimulation with VEGF for 8 hr. (A) Effect of VEGF on KCC4 mRNA levels. (B) Effect of VEGF on KCC3b mRNA levels. (C,D) Effect of knock-down and overexpression of HIF-1α on KCC4 and KCC3b mRNA levels. Data is represented as mean ± SD of three, independent experiments. Data are expressed as mean ± SD of three independent experiments. *** P
    Figure Legend Snippet: VEGF-induced KCC4 and KCC3b mRNA expression in K562 cells: K562 cells were transfected with siRNA for p38 MAP kinase, p47 phox , JNK kinase, and scrambled scRNA control, prior to stimulation with VEGF for 8 hr. (A) Effect of VEGF on KCC4 mRNA levels. (B) Effect of VEGF on KCC3b mRNA levels. (C,D) Effect of knock-down and overexpression of HIF-1α on KCC4 and KCC3b mRNA levels. Data is represented as mean ± SD of three, independent experiments. Data are expressed as mean ± SD of three independent experiments. *** P

    Techniques Used: Expressing, Transfection, Over Expression

    17) Product Images from "MiR-193b regulates breast cancer cell migration and vasculogenic mimicry by targeting dimethylarginine dimethylaminohydrolase 1"

    Article Title: MiR-193b regulates breast cancer cell migration and vasculogenic mimicry by targeting dimethylarginine dimethylaminohydrolase 1

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14454-1

    Effect of DDAH1 inhibition on MDA-MB-231 cell proliferation and migration. ( A ) MDA-MB-231 cells were transfected with 30 nM DDAH1-targeting siRNA, miR-193b mimic or appropriate controls and proliferation assessed by staining with crystal violet at 6, 24 and 48 hr post transfection. Absorbance was measured at 570 nm after extensive washing. ( B ) MDA-MB-231 cells were transfected with 30 nM DDAH1-targeting siRNA, miR-193b mimic or appropriate controls and proliferation was assessed using the IncuCyte™ imaging system. Confluence was measured every 2 hr for a 48 hr period. ( C ) Scratch wound migration assays were performed with MDA-MB-231 cells using the IncuCyte™ imaging system following generation of a wound in a confluent cell monolayer. Relative wound density was determined across 3 sections per well over a 12 hr period. Data are represented as the mean of two independent experiments performed in triplicate. Error bars represent SEM. ***p
    Figure Legend Snippet: Effect of DDAH1 inhibition on MDA-MB-231 cell proliferation and migration. ( A ) MDA-MB-231 cells were transfected with 30 nM DDAH1-targeting siRNA, miR-193b mimic or appropriate controls and proliferation assessed by staining with crystal violet at 6, 24 and 48 hr post transfection. Absorbance was measured at 570 nm after extensive washing. ( B ) MDA-MB-231 cells were transfected with 30 nM DDAH1-targeting siRNA, miR-193b mimic or appropriate controls and proliferation was assessed using the IncuCyte™ imaging system. Confluence was measured every 2 hr for a 48 hr period. ( C ) Scratch wound migration assays were performed with MDA-MB-231 cells using the IncuCyte™ imaging system following generation of a wound in a confluent cell monolayer. Relative wound density was determined across 3 sections per well over a 12 hr period. Data are represented as the mean of two independent experiments performed in triplicate. Error bars represent SEM. ***p

    Techniques Used: Inhibition, Multiple Displacement Amplification, Migration, Transfection, Staining, Imaging

    DDAH1 regulates the formation of tube-like structures by MDA-MB-231 cells on Matrigel. ( A , B ) Validation of DDAH1 gene knockdown following transfection with 30 nM DDAH1-targeting or control siRNA for 48 hr. DDAH1-V1 mRNA expression was normalised to 18S expression and is shown relative to control siRNA transfection, set to 100% ( A ). DDAH1 protein expression was assessed by western blotting ( B ). ( C ) MDA-MB-231 cells were transfected with 30 nM DDAH1-targeting siRNA or NC and the number of tubes and branches formed per well were quantified 24 hr after seeding on Matrigel. ( D ) Representative phase contrast images. ( E ) MDA-MB-231 cells were transfected with 30 nM miR-193b mimic or NC and the number of tubes and branches formed per well were quantified 24 hr after seeding on Matrigel. ( F ) Representative phase contrast images. Data shown are mean of three independent experiments performed in triplicate. ( G ) Expression of VEGF-A mRNA in MDA-MB-231 cells following transfection of 30 nM miR-193b mimic, DDAH1-targeting siRNA or appropriate controls. Expression was assessed 48 hr post-transfection and normalised to expression of 18S. Data are average of at least two independent experiments. Error bars represent SEM. ***p
    Figure Legend Snippet: DDAH1 regulates the formation of tube-like structures by MDA-MB-231 cells on Matrigel. ( A , B ) Validation of DDAH1 gene knockdown following transfection with 30 nM DDAH1-targeting or control siRNA for 48 hr. DDAH1-V1 mRNA expression was normalised to 18S expression and is shown relative to control siRNA transfection, set to 100% ( A ). DDAH1 protein expression was assessed by western blotting ( B ). ( C ) MDA-MB-231 cells were transfected with 30 nM DDAH1-targeting siRNA or NC and the number of tubes and branches formed per well were quantified 24 hr after seeding on Matrigel. ( D ) Representative phase contrast images. ( E ) MDA-MB-231 cells were transfected with 30 nM miR-193b mimic or NC and the number of tubes and branches formed per well were quantified 24 hr after seeding on Matrigel. ( F ) Representative phase contrast images. Data shown are mean of three independent experiments performed in triplicate. ( G ) Expression of VEGF-A mRNA in MDA-MB-231 cells following transfection of 30 nM miR-193b mimic, DDAH1-targeting siRNA or appropriate controls. Expression was assessed 48 hr post-transfection and normalised to expression of 18S. Data are average of at least two independent experiments. Error bars represent SEM. ***p

    Techniques Used: Multiple Displacement Amplification, Transfection, Expressing, Western Blot

    18) Product Images from "Downregulation of Hes1 expression in experimental biliary atresia and its effects on bile duct structure"

    Article Title: Downregulation of Hes1 expression in experimental biliary atresia and its effects on bile duct structure

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v24.i29.3260

    Hes1 siRNA in 3D biliary epithelial cell culture. Biliary epithelial cells (BECs) were cultured in either normal growth medium (N. medium) or in matrix + collagen mixed gel (detailed components are given in the Materials and Methods section). The cells were cultured without a transfection reagent (Cont.), with Lipofectamine (Lipo) only, with irrelevant control siRNA (Cont. siRNA) or with Hes1 siRNA for seven days, then fixed and stained with anti-CK19, γ-GT and AFP antibodies, and finally counter-stained with Alexa Fluor ® 488. Photographs of the cells were taken with a Leica DMi8 inverted fluorescence microscope, and the groups were then compared. The experiment was duplicated, and one set of representative results is shown here.
    Figure Legend Snippet: Hes1 siRNA in 3D biliary epithelial cell culture. Biliary epithelial cells (BECs) were cultured in either normal growth medium (N. medium) or in matrix + collagen mixed gel (detailed components are given in the Materials and Methods section). The cells were cultured without a transfection reagent (Cont.), with Lipofectamine (Lipo) only, with irrelevant control siRNA (Cont. siRNA) or with Hes1 siRNA for seven days, then fixed and stained with anti-CK19, γ-GT and AFP antibodies, and finally counter-stained with Alexa Fluor ® 488. Photographs of the cells were taken with a Leica DMi8 inverted fluorescence microscope, and the groups were then compared. The experiment was duplicated, and one set of representative results is shown here.

    Techniques Used: Cell Culture, Transfection, Staining, Fluorescence, Microscopy

    19) Product Images from "MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes"

    Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

    Journal: bioRxiv

    doi: 10.1101/2020.04.10.036103

    Oncogenic Ras regulates ABCB1 expression via RSKs. (A) Representative western blot showing RSK phosphorylation in RasV12 cells with or without MEK inhibition. (B) (Top) Representative gel image showing RSK1, RSK2, and RSK3 cDNA levels in RasV12 cells treated with or without various RSK siRNAs. (Bottom) Representative western blot showing RSK2 expression in RasV12 cells treated with or without various RSK siRNAs. (D, E) Fold change in PpIX accumulation in RasV12 cells (D) transfected with siRNA against RSKs and (E) treated with different concentrations of SL0101, a pan-RSK inhibitor. *p
    Figure Legend Snippet: Oncogenic Ras regulates ABCB1 expression via RSKs. (A) Representative western blot showing RSK phosphorylation in RasV12 cells with or without MEK inhibition. (B) (Top) Representative gel image showing RSK1, RSK2, and RSK3 cDNA levels in RasV12 cells treated with or without various RSK siRNAs. (Bottom) Representative western blot showing RSK2 expression in RasV12 cells treated with or without various RSK siRNAs. (D, E) Fold change in PpIX accumulation in RasV12 cells (D) transfected with siRNA against RSKs and (E) treated with different concentrations of SL0101, a pan-RSK inhibitor. *p

    Techniques Used: Expressing, Western Blot, Inhibition, Transfection

    20) Product Images from "RNase L Triggers Autophagy in Response to Viral Infections"

    Article Title: RNase L Triggers Autophagy in Response to Viral Infections

    Journal: Journal of Virology

    doi: 10.1128/JVI.00270-12

    Depletion of ATG5 and beclin-1 by siRNA treatments impairs the antiviral activity of RNase L. (A and D) Effects of siRNA against ATG5 mRNA and beclin-1 (bec) mRNA, respectively, in WT and Rnasel −/− MEF as determined in immunoblots in comparison
    Figure Legend Snippet: Depletion of ATG5 and beclin-1 by siRNA treatments impairs the antiviral activity of RNase L. (A and D) Effects of siRNA against ATG5 mRNA and beclin-1 (bec) mRNA, respectively, in WT and Rnasel −/− MEF as determined in immunoblots in comparison

    Techniques Used: Activity Assay, Western Blot

    21) Product Images from "Teng-Long-Bu-Zhong-Tang induces p21-dependent cell senescence in colorectal carcinoma LS174T cells via histone acetylation"

    Article Title: Teng-Long-Bu-Zhong-Tang induces p21-dependent cell senescence in colorectal carcinoma LS174T cells via histone acetylation

    Journal: Journal of Experimental Pharmacology

    doi: 10.2147/JEP.S129272

    Role of p53 in TLBZT-induced p21 expression. Notes: After 24 h of transfection with p53 siRNA or control siRNA, LS174T cells were treated with 200 μg/mL of TLBZT for 5 days and subjected to Western blot probing p53 and p21; β-actin was used as a loading control. Abbreviations: siRNA, small interfering RNA; TLBZT, Teng-Long-Bu-Zhong-Tang.
    Figure Legend Snippet: Role of p53 in TLBZT-induced p21 expression. Notes: After 24 h of transfection with p53 siRNA or control siRNA, LS174T cells were treated with 200 μg/mL of TLBZT for 5 days and subjected to Western blot probing p53 and p21; β-actin was used as a loading control. Abbreviations: siRNA, small interfering RNA; TLBZT, Teng-Long-Bu-Zhong-Tang.

    Techniques Used: Expressing, Transfection, Western Blot, Small Interfering RNA

    Role of p21 in TLBZT-induced cell senescence. Notes: After 24 h of transfection with p21 siRNA or control siRNA, LS174T cells were treated with 200 μg/mL of TLBZT for 5 days and subjected to Western blot analyses ( A ) and cell senescence detection ( B ). Data are representative of three independent experiments. ** P
    Figure Legend Snippet: Role of p21 in TLBZT-induced cell senescence. Notes: After 24 h of transfection with p21 siRNA or control siRNA, LS174T cells were treated with 200 μg/mL of TLBZT for 5 days and subjected to Western blot analyses ( A ) and cell senescence detection ( B ). Data are representative of three independent experiments. ** P

    Techniques Used: Transfection, Western Blot

    22) Product Images from "Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes"

    Article Title: Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar4254

    Knockdown of LKB1 in articular chondrocytes attenuates AMPK activity and promotes matrix catabolic responses . Human primary normal knee articular chondrocytes were transfected with liver kinase B1 (LKB1) and control siRNA. Two days after transfection, cells were either (A) used directly for western blot analysis of LKB1 expression or (B) treated with IL-1β (10 ng/ml) and TNFα (10 ng/ml) for 18 hours followed by western blot analysis for phosphorylation of AMP-activated protein kinase alpha (AMPKα) and total AMPKα. (C) Nitric oxide (NO) release, (D) matrix metalloproteinase (MMP)-3 release and (E) MMP-13 release were then analyzed from the conditioned media by Griess reaction and ELISA, respectively. Data representative of three individual experiments. * P
    Figure Legend Snippet: Knockdown of LKB1 in articular chondrocytes attenuates AMPK activity and promotes matrix catabolic responses . Human primary normal knee articular chondrocytes were transfected with liver kinase B1 (LKB1) and control siRNA. Two days after transfection, cells were either (A) used directly for western blot analysis of LKB1 expression or (B) treated with IL-1β (10 ng/ml) and TNFα (10 ng/ml) for 18 hours followed by western blot analysis for phosphorylation of AMP-activated protein kinase alpha (AMPKα) and total AMPKα. (C) Nitric oxide (NO) release, (D) matrix metalloproteinase (MMP)-3 release and (E) MMP-13 release were then analyzed from the conditioned media by Griess reaction and ELISA, respectively. Data representative of three individual experiments. * P

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

    23) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.343707

    Effects of GH and FGF21 on total [ 3 H]thymidine incorporation and collagen X expression in FGFR1-siRNA- and ERK 1 siRNA-transfected chondrocytes. After being previously cultured without or with graded concentrations of FGF21, siRNA-transfected chondrocytes were cultured for 24 h in the absence or presence of rmGH (10 ng/ml). A , at the end of culture period, 2.5 μCi/well of [ 3 H]thymidine (Amersham Biosciences) was added to the culture medium for an additional 3 h. Incorporation of [ 3 H]thymidine was measured by liquid scintillation counting. Results are expressed as % of untreated and represent mean values obtained from three independent experiments. B , collagen X mRNA expression was determined by real time PCR. Total RNA was extracted from transfected chondrocytes and then processed as described under “Materials and Methods.” The relative expression levels of mRNA were normalized by β-actin in the same samples. Results were expressed as -fold change compared with control chondrocytes (mean ± S.E.).
    Figure Legend Snippet: Effects of GH and FGF21 on total [ 3 H]thymidine incorporation and collagen X expression in FGFR1-siRNA- and ERK 1 siRNA-transfected chondrocytes. After being previously cultured without or with graded concentrations of FGF21, siRNA-transfected chondrocytes were cultured for 24 h in the absence or presence of rmGH (10 ng/ml). A , at the end of culture period, 2.5 μCi/well of [ 3 H]thymidine (Amersham Biosciences) was added to the culture medium for an additional 3 h. Incorporation of [ 3 H]thymidine was measured by liquid scintillation counting. Results are expressed as % of untreated and represent mean values obtained from three independent experiments. B , collagen X mRNA expression was determined by real time PCR. Total RNA was extracted from transfected chondrocytes and then processed as described under “Materials and Methods.” The relative expression levels of mRNA were normalized by β-actin in the same samples. Results were expressed as -fold change compared with control chondrocytes (mean ± S.E.).

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

    24) Product Images from "High expression of ENPP1 in high-grade serous ovarian carcinoma predicts poor prognosis and as a molecular therapy target"

    Article Title: High expression of ENPP1 in high-grade serous ovarian carcinoma predicts poor prognosis and as a molecular therapy target

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0245733

    ENPP1 mRNA and protein expression of A2780 after 48h of PC-1 siRNA transfection. A : Western Blot electrophoresis; B : Analysis of protein relative expression. C : qRT-PCR. Blank: A2780 cells was interfered by cell culture medium without any siRNA; Control siRNA: A2780 cells was interfered by Control siRNA; PC-1 siRNA: A2780 cells was interfered by PC-1 siRNA. *P
    Figure Legend Snippet: ENPP1 mRNA and protein expression of A2780 after 48h of PC-1 siRNA transfection. A : Western Blot electrophoresis; B : Analysis of protein relative expression. C : qRT-PCR. Blank: A2780 cells was interfered by cell culture medium without any siRNA; Control siRNA: A2780 cells was interfered by Control siRNA; PC-1 siRNA: A2780 cells was interfered by PC-1 siRNA. *P

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

    25) Product Images from "Cigarette smoke supports stemness and epithelial-mesenchymal transition in bladder cancer stem cells through SHH signaling"

    Article Title: Cigarette smoke supports stemness and epithelial-mesenchymal transition in bladder cancer stem cells through SHH signaling

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    SHH pathway regulates the promotive effects of CSE on bladder CSCs. The tumor spheres were treated with CSE and Vismodegib or Gli1-siRNA. A. SHH pathway related-proteins were detected by western blotting. B. Images of tumorspheres. C. Expression of bladder CSCs markers was measured by western blotting. D. Expression of cell proliferation related-proteins was determined by western blotting. E. The invasive ability was determined by Transwell assay. F. Expression of EMT related-proteins was determined by western blotting. G. Expression of bladder CSC markers after Gli1 siRNA treatment was measured by western blotting. Data are expressed as mean ± SD. * P
    Figure Legend Snippet: SHH pathway regulates the promotive effects of CSE on bladder CSCs. The tumor spheres were treated with CSE and Vismodegib or Gli1-siRNA. A. SHH pathway related-proteins were detected by western blotting. B. Images of tumorspheres. C. Expression of bladder CSCs markers was measured by western blotting. D. Expression of cell proliferation related-proteins was determined by western blotting. E. The invasive ability was determined by Transwell assay. F. Expression of EMT related-proteins was determined by western blotting. G. Expression of bladder CSC markers after Gli1 siRNA treatment was measured by western blotting. Data are expressed as mean ± SD. * P

    Techniques Used: Western Blot, Expressing, Transwell Assay

    26) Product Images from "Vertebrate Lonesome Kinase Regulated Extracellular Matrix Protein Phosphorylation, Cell Shape and Adhesion in Trabecular Meshwork Cells"

    Article Title: Vertebrate Lonesome Kinase Regulated Extracellular Matrix Protein Phosphorylation, Cell Shape and Adhesion in Trabecular Meshwork Cells

    Journal: Journal of cellular physiology

    doi: 10.1002/jcp.25582

    VLK deficiency in human TM cells induces changes in cell shape and decreases in actin stress fibers and focal adhesions. TM cells (lanes 1–3 represent 3 individual samples) treated with siRNA specific to VLK for 72 h showed a significant decrease in VLK protein levels in both cell lysates (A C) and media (B D) compared to the control cells treated with scrambled siRNA, based on immunoblotting and densitometric analyses. Values are mean ± SEM of 12 independent analyses. *P
    Figure Legend Snippet: VLK deficiency in human TM cells induces changes in cell shape and decreases in actin stress fibers and focal adhesions. TM cells (lanes 1–3 represent 3 individual samples) treated with siRNA specific to VLK for 72 h showed a significant decrease in VLK protein levels in both cell lysates (A C) and media (B D) compared to the control cells treated with scrambled siRNA, based on immunoblotting and densitometric analyses. Values are mean ± SEM of 12 independent analyses. *P

    Techniques Used:

    27) 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

    28) Product Images from "MUC18 Regulates Lung Rhinovirus Infection and Inflammation"

    Article Title: MUC18 Regulates Lung Rhinovirus Infection and Inflammation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0163927

    Reduced viral load and IL-8 induction in human airway epithelial cells with MUC18 knockdown (KD). Tracheobronchial epithelial cells from three donors (n = 3 replicates per donor) were transfected with control or MUC18 siRNA and then treated with culture medium (–) or HRV for 2 hours. Cells were harvested 24 hours post infection. Viral load was significantly lower in MUC18 KD cells compared to control (A). Expression of the antiviral gene MX1 was similar between KD and control cells (B). There was less induction of the pro-inflammatory cytokine, IL-8, in MUC18 KD samples compared to control (C) , but was not statistically significant. Successful knockdown of MUC18 was confirmed via Western Blot and densitometry (D) in cells after 24 hours of MUC18 or control siRNA transfection. Data are presented as means ± SEMs. A single star (*) indicates p
    Figure Legend Snippet: Reduced viral load and IL-8 induction in human airway epithelial cells with MUC18 knockdown (KD). Tracheobronchial epithelial cells from three donors (n = 3 replicates per donor) were transfected with control or MUC18 siRNA and then treated with culture medium (–) or HRV for 2 hours. Cells were harvested 24 hours post infection. Viral load was significantly lower in MUC18 KD cells compared to control (A). Expression of the antiviral gene MX1 was similar between KD and control cells (B). There was less induction of the pro-inflammatory cytokine, IL-8, in MUC18 KD samples compared to control (C) , but was not statistically significant. Successful knockdown of MUC18 was confirmed via Western Blot and densitometry (D) in cells after 24 hours of MUC18 or control siRNA transfection. Data are presented as means ± SEMs. A single star (*) indicates p

    Techniques Used: Transfection, Infection, Expressing, Western Blot

    Fluorescent Green Dye to indicate endosome acidity in human tracheobronchial epithelial cells infected with rhinovirus 1B. Tracheobronchial epithelial cells from donors were seeded onto cover slips and transfected with control or MUC18 siRNA and infected with culture medium (–) or HRV-1B for 2 hours. 30 minutes prior to the end of infection, LysoSensor Dye was added to the cultures. Cover slips were removed and stained with DAPI to indicate the nucleus and mounted onto microscope slides. Control siRNA cells were more green, indicating a lower pH (A) and cells transfected with MUC18 siRNA were less green, indicating a higher pH (B) .
    Figure Legend Snippet: Fluorescent Green Dye to indicate endosome acidity in human tracheobronchial epithelial cells infected with rhinovirus 1B. Tracheobronchial epithelial cells from donors were seeded onto cover slips and transfected with control or MUC18 siRNA and infected with culture medium (–) or HRV-1B for 2 hours. 30 minutes prior to the end of infection, LysoSensor Dye was added to the cultures. Cover slips were removed and stained with DAPI to indicate the nucleus and mounted onto microscope slides. Control siRNA cells were more green, indicating a lower pH (A) and cells transfected with MUC18 siRNA were less green, indicating a higher pH (B) .

    Techniques Used: Infection, Transfection, Staining, Microscopy

    Reduced viral load and IL-8 induction in human airway epithelial cells with MUC18 knockdown (KD). Tracheobronchial epithelial cells from three donors (n = 3 replicates per donor) were transfected with control or MUC18 siRNA and then treated with culture medium (–) or HRV for 2 hours. Cells were harvested 24 hours post infection. Viral load was significantly lower in MUC18 KD cells compared to control (A). Expression of the antiviral gene MX1 was similar between KD and control cells (B). There was less induction of the pro-inflammatory cytokine, IL-8, in MUC18 KD samples compared to control (C) , but was not statistically significant. Successful knockdown of MUC18 was confirmed via Western Blot and densitometry (D) in cells after 24 hours of MUC18 or control siRNA transfection. Data are presented as means ± SEMs. A single star (*) indicates p
    Figure Legend Snippet: Reduced viral load and IL-8 induction in human airway epithelial cells with MUC18 knockdown (KD). Tracheobronchial epithelial cells from three donors (n = 3 replicates per donor) were transfected with control or MUC18 siRNA and then treated with culture medium (–) or HRV for 2 hours. Cells were harvested 24 hours post infection. Viral load was significantly lower in MUC18 KD cells compared to control (A). Expression of the antiviral gene MX1 was similar between KD and control cells (B). There was less induction of the pro-inflammatory cytokine, IL-8, in MUC18 KD samples compared to control (C) , but was not statistically significant. Successful knockdown of MUC18 was confirmed via Western Blot and densitometry (D) in cells after 24 hours of MUC18 or control siRNA transfection. Data are presented as means ± SEMs. A single star (*) indicates p

    Techniques Used: Transfection, Infection, Expressing, Western Blot

    Fluorescent Green Dye to indicate endosome acidity in human tracheobronchial epithelial cells infected with rhinovirus 1B. Tracheobronchial epithelial cells from donors were seeded onto cover slips and transfected with control or MUC18 siRNA and infected with culture medium (–) or HRV-1B for 2 hours. 30 minutes prior to the end of infection, LysoSensor Dye was added to the cultures. Cover slips were removed and stained with DAPI to indicate the nucleus and mounted onto microscope slides. Control siRNA cells were more green, indicating a lower pH (A) and cells transfected with MUC18 siRNA were less green, indicating a higher pH (B) .
    Figure Legend Snippet: Fluorescent Green Dye to indicate endosome acidity in human tracheobronchial epithelial cells infected with rhinovirus 1B. Tracheobronchial epithelial cells from donors were seeded onto cover slips and transfected with control or MUC18 siRNA and infected with culture medium (–) or HRV-1B for 2 hours. 30 minutes prior to the end of infection, LysoSensor Dye was added to the cultures. Cover slips were removed and stained with DAPI to indicate the nucleus and mounted onto microscope slides. Control siRNA cells were more green, indicating a lower pH (A) and cells transfected with MUC18 siRNA were less green, indicating a higher pH (B) .

    Techniques Used: Infection, Transfection, Staining, Microscopy

    29) Product Images from "Ectodomain Shedding of Preadipocyte Factor 1 (Pref-1) by Tumor Necrosis Factor Alpha Converting Enzyme (TACE) and Inhibition of Adipocyte Differentiation"

    Article Title: Ectodomain Shedding of Preadipocyte Factor 1 (Pref-1) by Tumor Necrosis Factor Alpha Converting Enzyme (TACE) and Inhibition of Adipocyte Differentiation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.02437-05

    Effect of TACE-mediated Pref-1 release on 3T3-L1 adipocyte differentiation. (A) 3T3-L1 cells were infected with control lentivirus (a and d) and Lenti-TACE virus (b and e) overnight, and the cells were subjected to differentiation in the absence (a, b, d, and e) or presence (c and f) of GM6001 as described in Materials and Methods. Cells under a microscope (a to c) and Oil red O staining for lipid accumulation (d to f and right panel) are shown. cDNAs to adipocyte markers, PPARγ, and C/EBPα were 32 P labeled and used for Northern blot analysis. The 28S and 18S rRNAs are shown as a loading control (lower left panel). We use 1 μg of total RNA for RT-PCR analysis in determining the mRNA levels of adipocyte markers, ADSF/resistin, adiponectin, FAS, and leptin. β-Actin was used as a control (lower right panel). Representative results from at least three separate experiments are shown. (B) siRNA for TACE inhibits TACE expression and regulates Pref-1 shedding. COS cells stably expressing Pref-1A were transfected with control siRNA or TACE siRNA. After 30 h of transfection, cells were incubated in serum-free media for indicated time periods. The 50-kDa Pref-1 ectodomain and Pref-1 membrane forms were detected in media and lysates by Western blotting with anti-HA antibody, and TACE was detected in lysates by using the anti-TACE antibody. Molecular weight markers are shown on the right. (C) siRNA for TACE inhibits Pref-1 shedding and enhances adipocyte differentiation. 3T3-L1 control cells were transfected with control siRNA and infected with control lentivirus. Compared to the control cells, the infection of Lenti-TACE virus inhibited adipocyte differentiation; in contrast, transfection of TACE siRNA enhanced adipocyte differentiation. Oil red O staining for lipid accumulation is shown (upper panel). cDNAs to PPARγ and C/EBPα were 32 P labeled and used for Northern blot analysis. The 28S and 18S rRNAs are shown as a loading control (lower left panel). We use 1 μg of total RNA for RT-PCR analysis to determine the mRNA levels of the adipocyte markers, ADSF/resistin, adiponectin, FAS, and leptin. GAPDH was used as a control (lower right panel). Representative results from two independent experiments are shown.
    Figure Legend Snippet: Effect of TACE-mediated Pref-1 release on 3T3-L1 adipocyte differentiation. (A) 3T3-L1 cells were infected with control lentivirus (a and d) and Lenti-TACE virus (b and e) overnight, and the cells were subjected to differentiation in the absence (a, b, d, and e) or presence (c and f) of GM6001 as described in Materials and Methods. Cells under a microscope (a to c) and Oil red O staining for lipid accumulation (d to f and right panel) are shown. cDNAs to adipocyte markers, PPARγ, and C/EBPα were 32 P labeled and used for Northern blot analysis. The 28S and 18S rRNAs are shown as a loading control (lower left panel). We use 1 μg of total RNA for RT-PCR analysis in determining the mRNA levels of adipocyte markers, ADSF/resistin, adiponectin, FAS, and leptin. β-Actin was used as a control (lower right panel). Representative results from at least three separate experiments are shown. (B) siRNA for TACE inhibits TACE expression and regulates Pref-1 shedding. COS cells stably expressing Pref-1A were transfected with control siRNA or TACE siRNA. After 30 h of transfection, cells were incubated in serum-free media for indicated time periods. The 50-kDa Pref-1 ectodomain and Pref-1 membrane forms were detected in media and lysates by Western blotting with anti-HA antibody, and TACE was detected in lysates by using the anti-TACE antibody. Molecular weight markers are shown on the right. (C) siRNA for TACE inhibits Pref-1 shedding and enhances adipocyte differentiation. 3T3-L1 control cells were transfected with control siRNA and infected with control lentivirus. Compared to the control cells, the infection of Lenti-TACE virus inhibited adipocyte differentiation; in contrast, transfection of TACE siRNA enhanced adipocyte differentiation. Oil red O staining for lipid accumulation is shown (upper panel). cDNAs to PPARγ and C/EBPα were 32 P labeled and used for Northern blot analysis. The 28S and 18S rRNAs are shown as a loading control (lower left panel). We use 1 μg of total RNA for RT-PCR analysis to determine the mRNA levels of the adipocyte markers, ADSF/resistin, adiponectin, FAS, and leptin. GAPDH was used as a control (lower right panel). Representative results from two independent experiments are shown.

    Techniques Used: Infection, Microscopy, Staining, Labeling, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Stable Transfection, Transfection, Incubation, Western Blot, Molecular Weight

    Effect of TACE on adipocyte differentiation of MEFs. (A) Total RNA extracted from MEFs isolated from Pref-1 KO and wild-type (WT) embryos were used for Northern blot analysis to detect the expression levels of Pref-1 and TACE (left panel). Pref-1 KO and WT MEFs were transfected with either control siRNA or TACE siRNA and then subjected to treatment with differentiation-inducing agents. Total RNA was extracted from the cells after differentiation and used for Northern blot analysis. cDNAs to Pref-1, TACE, PPARγ, and C/EBPα were 32 P labeled and used for hybridization (right panel). The 28S and 18S rRNAs were used as a loading control. (B) Pref-1 KO MEFs were infected with control lentivirus, Lenti-TACE virus alone, or Lenti-TACE and Lenti-Pref-1 virus together. Oil red O for lipid accumulation is shown (left panel). Northern blot analysis for adipocyte markers, PPARγ, C/EBPα, and 28S and 18S rRNAs as a loading control are shown (right panel). Representative results from two separate experiments are shown.
    Figure Legend Snippet: Effect of TACE on adipocyte differentiation of MEFs. (A) Total RNA extracted from MEFs isolated from Pref-1 KO and wild-type (WT) embryos were used for Northern blot analysis to detect the expression levels of Pref-1 and TACE (left panel). Pref-1 KO and WT MEFs were transfected with either control siRNA or TACE siRNA and then subjected to treatment with differentiation-inducing agents. Total RNA was extracted from the cells after differentiation and used for Northern blot analysis. cDNAs to Pref-1, TACE, PPARγ, and C/EBPα were 32 P labeled and used for hybridization (right panel). The 28S and 18S rRNAs were used as a loading control. (B) Pref-1 KO MEFs were infected with control lentivirus, Lenti-TACE virus alone, or Lenti-TACE and Lenti-Pref-1 virus together. Oil red O for lipid accumulation is shown (left panel). Northern blot analysis for adipocyte markers, PPARγ, C/EBPα, and 28S and 18S rRNAs as a loading control are shown (right panel). Representative results from two separate experiments are shown.

    Techniques Used: Isolation, Northern Blot, Expressing, Transfection, Labeling, Hybridization, Infection

    Effect of TACE on adipocyte differentiation of MEFs is predominantly through Pref-1. (A) KO MEFs were infected with control lentivirus (a and e), Lenti-Pref-1A virus (b to d and f to h), or Lenti-TACE virus (c and g) expressing vector or transfected with control siRNA (a to c and e to g) or TACE siRNA (d and h). Cells under a microscope (a to d) and Oil red O for lipid accumulation (e to h and B, upper panel) are shown. cDNAs to PPARγ and C/EBPα were 32 P labeled and used for Northern blot analysis. The 28S and 18S rRNAs are shown as a loading control (B, lower left panel). We used 1 μg of total RNA for RT-PCR analysis to determine the mRNA levels of adipocyte markers, ADSF/resistin, adiponectin, FAS, and leptin. β-Actin was used as a control (B, lower right panel). Representative results from at least three separate experiments are shown.
    Figure Legend Snippet: Effect of TACE on adipocyte differentiation of MEFs is predominantly through Pref-1. (A) KO MEFs were infected with control lentivirus (a and e), Lenti-Pref-1A virus (b to d and f to h), or Lenti-TACE virus (c and g) expressing vector or transfected with control siRNA (a to c and e to g) or TACE siRNA (d and h). Cells under a microscope (a to d) and Oil red O for lipid accumulation (e to h and B, upper panel) are shown. cDNAs to PPARγ and C/EBPα were 32 P labeled and used for Northern blot analysis. The 28S and 18S rRNAs are shown as a loading control (B, lower left panel). We used 1 μg of total RNA for RT-PCR analysis to determine the mRNA levels of adipocyte markers, ADSF/resistin, adiponectin, FAS, and leptin. β-Actin was used as a control (B, lower right panel). Representative results from at least three separate experiments are shown.

    Techniques Used: Infection, Expressing, Plasmid Preparation, Transfection, Microscopy, Labeling, Northern Blot, Reverse Transcription Polymerase Chain Reaction

    30) Product Images from "Impact of PKCε downregulation on autophagy in glioblastoma cells"

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

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4095-1

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

    Techniques Used: Immunofluorescence, Transfection, Imaging

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

    Techniques Used: Transfection

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

    Techniques Used: Expressing, Transfection

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

    Techniques Used: Activation Assay, Transfection, Construct

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

    Techniques Used: Transfection

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

    Techniques Used: Transfection

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

    Techniques Used: Expressing, Transfection

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

    Techniques Used: Transfection, Microscopy, Expressing

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

    Techniques Used: Transfection

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

    Techniques Used: Transfection, Expressing

    31) Product Images from "Hypochlorous acid via peroxynitrite activates protein kinase Cθ and insulin resistance in adipocytes"

    Article Title: Hypochlorous acid via peroxynitrite activates protein kinase Cθ and insulin resistance in adipocytes

    Journal: Journal of Molecular Endocrinology

    doi: 10.1530/JME-14-0213

    Inhibition of JNK prevents HOCl-induced phosphorylation of IRS1 at Ser307 and insulin resistance. (A) 3T3-L1 adipocytes were pretreated with 30 μmol/l SP600125 for 90 min and incubated with 200 μmol/l HOCl for 1 h and then stimulated with 100 nmol/l insulin or left unstimulated for 15 min. (B) 3T3-L1 adipocytes were transfected with JNK2 siRNA or control siRNA for 48 h and treated with 200 μmol/l HOCl for 1 h, and then stimulated with 100 nmol/l insulin or left unstimulated for 15 min. Western analysis of protein expression and phosphorylation of JNK, IRS1, Akt, and GSK3β was performed. Blots are representative of the results from five independent experiments.
    Figure Legend Snippet: Inhibition of JNK prevents HOCl-induced phosphorylation of IRS1 at Ser307 and insulin resistance. (A) 3T3-L1 adipocytes were pretreated with 30 μmol/l SP600125 for 90 min and incubated with 200 μmol/l HOCl for 1 h and then stimulated with 100 nmol/l insulin or left unstimulated for 15 min. (B) 3T3-L1 adipocytes were transfected with JNK2 siRNA or control siRNA for 48 h and treated with 200 μmol/l HOCl for 1 h, and then stimulated with 100 nmol/l insulin or left unstimulated for 15 min. Western analysis of protein expression and phosphorylation of JNK, IRS1, Akt, and GSK3β was performed. Blots are representative of the results from five independent experiments.

    Techniques Used: Inhibition, Incubation, Transfection, Western Blot, Expressing

    PKCθ mediates phosphorylation of IKK, JNK, and IRS1 and insulin resistance. 3T3-L1 adipocytes were transfected with PKCθ siRNA or control siRNA for 48 h and treated with 200 μmol/l HOCl for 1 h and subsequently stimulated with 100 nmol/l insulin for 15 min or left unstimulated. Western blot analysis of protein expression and phosphorylation of PKCθ, IKK, JNK, and IRS1 (A and B). Akt kinase activity in cell lysates was measured (C). The membrane fraction was isolated and proteins were subjected to SDS–PAGE and immunoblotted with GTLU4 antibody (D). The blots are representative of results obtained from five independent experiments.
    Figure Legend Snippet: PKCθ mediates phosphorylation of IKK, JNK, and IRS1 and insulin resistance. 3T3-L1 adipocytes were transfected with PKCθ siRNA or control siRNA for 48 h and treated with 200 μmol/l HOCl for 1 h and subsequently stimulated with 100 nmol/l insulin for 15 min or left unstimulated. Western blot analysis of protein expression and phosphorylation of PKCθ, IKK, JNK, and IRS1 (A and B). Akt kinase activity in cell lysates was measured (C). The membrane fraction was isolated and proteins were subjected to SDS–PAGE and immunoblotted with GTLU4 antibody (D). The blots are representative of results obtained from five independent experiments.

    Techniques Used: Transfection, Western Blot, Expressing, Activity Assay, Isolation, SDS Page

    Inhibition of IKK blocks HOCl-induced serine phosphorylation of IRS1 and insulin resistance. (A) 3T3-L1 adipocytes were pretreated with 10 μmol/l PS1145 for 90 min and incubated with 200 μmol/l HOCl for 1 h and then stimulated with 100 nmol/l insulin or left unstimulated for 15 min. (B) 3T3-L1 adipocytes were transfected with IKKβ siRNA or control siRNA for 48 h and treated with 200 μmol/l HOCl for 1 h and then stimulated with or without 100 nmol/l insulin for 15 min. Western analysis of protein expression and phosphorylation of IKK, IRS1, Akt, and GSK3β was performed. Blots are representative of the results from five independent experiments.
    Figure Legend Snippet: Inhibition of IKK blocks HOCl-induced serine phosphorylation of IRS1 and insulin resistance. (A) 3T3-L1 adipocytes were pretreated with 10 μmol/l PS1145 for 90 min and incubated with 200 μmol/l HOCl for 1 h and then stimulated with 100 nmol/l insulin or left unstimulated for 15 min. (B) 3T3-L1 adipocytes were transfected with IKKβ siRNA or control siRNA for 48 h and treated with 200 μmol/l HOCl for 1 h and then stimulated with or without 100 nmol/l insulin for 15 min. Western analysis of protein expression and phosphorylation of IKK, IRS1, Akt, and GSK3β was performed. Blots are representative of the results from five independent experiments.

    Techniques Used: Inhibition, Incubation, Transfection, Western Blot, Expressing

    32) Product Images from "DHX32 Promotes Angiogenesis in Colorectal Cancer Through Augmenting β-catenin Signaling to Induce Expression of VEGFA"

    Article Title: DHX32 Promotes Angiogenesis in Colorectal Cancer Through Augmenting β-catenin Signaling to Induce Expression of VEGFA

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2017.03.012

    DHX32 promotes transcription of VEGFA in CRC cells. (a) SW480 stable cells were transfected with β-catenin/TCF luciferase reporter gene pTOPFLASH or mutated pFOPFLASH, pRL-TK as internal control. The mean ± SD of a representative result of three independent experiments is shown. (b) Real-time RT-PCR analyses of VEGFA expression in SW480 cells with DHX32 depletion or overexpression. (c) Endogenous VEGFA protein levels in SW480 cells with DHX32 overexpression or depletion were detected by immunoblotting. (d) SW480 stable cells with depletion DHX32 were transfected with β-catenin and DHX32-overexpressed stable cells were transfected with control siRNA or siRNA against β-catenin. VEGFA protein levels in condition medium were quantified by ELISA analysis. (e) Representative PCR gel of ChIP assays showing binding of β-catenin to the VEGF promoter over the IgG control. Immunoprecipitate was carried out using an antibody to β-catenin. Nonimmunoprecipitated chromatin was used as an “input” control, and an IgG antibody control was performed on all occasions. The PCR primers were amplified in the − 262 to − 101 region of the VEGF promoter. ** P
    Figure Legend Snippet: DHX32 promotes transcription of VEGFA in CRC cells. (a) SW480 stable cells were transfected with β-catenin/TCF luciferase reporter gene pTOPFLASH or mutated pFOPFLASH, pRL-TK as internal control. The mean ± SD of a representative result of three independent experiments is shown. (b) Real-time RT-PCR analyses of VEGFA expression in SW480 cells with DHX32 depletion or overexpression. (c) Endogenous VEGFA protein levels in SW480 cells with DHX32 overexpression or depletion were detected by immunoblotting. (d) SW480 stable cells with depletion DHX32 were transfected with β-catenin and DHX32-overexpressed stable cells were transfected with control siRNA or siRNA against β-catenin. VEGFA protein levels in condition medium were quantified by ELISA analysis. (e) Representative PCR gel of ChIP assays showing binding of β-catenin to the VEGF promoter over the IgG control. Immunoprecipitate was carried out using an antibody to β-catenin. Nonimmunoprecipitated chromatin was used as an “input” control, and an IgG antibody control was performed on all occasions. The PCR primers were amplified in the − 262 to − 101 region of the VEGF promoter. ** P

    Techniques Used: Transfection, Luciferase, Quantitative RT-PCR, Expressing, Over Expression, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Amplification

    33) Product Images from "The Rate of NF-?B Nuclear Translocation Is Regulated by PKA and A Kinase Interacting Protein 1"

    Article Title: The Rate of NF-?B Nuclear Translocation Is Regulated by PKA and A Kinase Interacting Protein 1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018713

    Knock down of endogenous PKAc accelerates nuclear accumulation of endogenous p65. HeLa cells were transfected with mock siRNA (lanes 1–3) or PKAc siRNA (lanes 4–6) for 36 hours, serum starved for 12 hours, and then stimulated with TNFα for 0, 15, or 45 min. Samples were analyzed on 10% SDS-polyacrylamide gels and Western blotted with antibodies to p65, PKAc, and lamin B.
    Figure Legend Snippet: Knock down of endogenous PKAc accelerates nuclear accumulation of endogenous p65. HeLa cells were transfected with mock siRNA (lanes 1–3) or PKAc siRNA (lanes 4–6) for 36 hours, serum starved for 12 hours, and then stimulated with TNFα for 0, 15, or 45 min. Samples were analyzed on 10% SDS-polyacrylamide gels and Western blotted with antibodies to p65, PKAc, and lamin B.

    Techniques Used: Transfection, Western Blot

    34) Product Images from "The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2"

    Article Title: The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005934

    ORC2 knockdown enhances PV replication. (A) Replication luciferase assays were completed with pFLORI31 and ORC2 depletion. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. ORC2 shRNA decreased ORC2 protein expression in C33A cells. (B) ORC2 shRNA decreased EBNA-1 based replication using the pREP4 plasmid (EBNA-1 based replication) in C33A cells. (C) ORC2 shRNA decreased ORC2 protein expression in HPV-BP and SiHa cells. (D) ORC2 knockdown increased HPV replication in cells containing HPV-16 episomes (HPV-BP) but not in cells with integrated HPV-16 (SiHa). HPV-BP and SiHa cells were transfected with 5.5 μg of shRNA hairpins. 7d later, cells were harvested and HPV-16 copy number (HPV-16 LCR) as normalized to GM-CSF ori #2. This experiment was performed three times with a representative image presented here.
    Figure Legend Snippet: ORC2 knockdown enhances PV replication. (A) Replication luciferase assays were completed with pFLORI31 and ORC2 depletion. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. ORC2 shRNA decreased ORC2 protein expression in C33A cells. (B) ORC2 shRNA decreased EBNA-1 based replication using the pREP4 plasmid (EBNA-1 based replication) in C33A cells. (C) ORC2 shRNA decreased ORC2 protein expression in HPV-BP and SiHa cells. (D) ORC2 knockdown increased HPV replication in cells containing HPV-16 episomes (HPV-BP) but not in cells with integrated HPV-16 (SiHa). HPV-BP and SiHa cells were transfected with 5.5 μg of shRNA hairpins. 7d later, cells were harvested and HPV-16 copy number (HPV-16 LCR) as normalized to GM-CSF ori #2. This experiment was performed three times with a representative image presented here.

    Techniques Used: Luciferase, shRNA, Expressing, Plasmid Preparation, Transfection

    ORC2 depletion increases E1 and E2 occupancy at the viral origin in CIN612-9E cells. CIN612-9E cells were transfected with 30 nM of control or ORC2 siRNAs for 48 h. ChIP experiments were performed using (A) rabbit anti-HPV-31 E2 antibodies or (B) rat E1 antibodies. Real-time PCR was performed with primers listed in Materials and Methods located in HPV-31 LCR previously found to be enriched for E2 binding. E2 binding sites are located between the LCR2 and LCR4 primer sets. Ct values are normalized to input and values are expressed as mean +/- SEM. * p-value ≤ 0.05 compared to control siRNA.
    Figure Legend Snippet: ORC2 depletion increases E1 and E2 occupancy at the viral origin in CIN612-9E cells. CIN612-9E cells were transfected with 30 nM of control or ORC2 siRNAs for 48 h. ChIP experiments were performed using (A) rabbit anti-HPV-31 E2 antibodies or (B) rat E1 antibodies. Real-time PCR was performed with primers listed in Materials and Methods located in HPV-31 LCR previously found to be enriched for E2 binding. E2 binding sites are located between the LCR2 and LCR4 primer sets. Ct values are normalized to input and values are expressed as mean +/- SEM. * p-value ≤ 0.05 compared to control siRNA.

    Techniques Used: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    ORC2 silencing enhanced PV replication in proliferating and differentiating CIN612-9E cells. (A) ORC2 siRNA duplexes decreased ORC2 protein levels at a concentration of 15 or 5 nM after 48 h. (B) CIN612-9E cells were transfected with RLuc and pFLORI31. 48h later cells were lysed and luciferase activity measured. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. (C) CIN612-9E cells were transfected with 15 nM ORC2 and differentiated in 10% FBS DMEM + 2 mM CaCl 2 for 48h. Involucrin and β-actin levels were analyzed. (D) CIN612-9E cells were transfected with RLuc and pFLORI31. Five hours later, the transfection was removed and cells were placed in either E-medium or 10% FBS DMEM + 2 mM CaCl 2 . 48h later cells were lysed and luciferase activity measured. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. (E) CIN612-9E cells were transfected with 15 nM control or ORC2 siRNAs, RLuc and pFLORI31. Five hours later, the transfection was removed and cells were placed in 10% FBS DMEM + 2 mM CaCl 2 . 48h later cells were lysed and luciferase activity measured. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. (F) CIN612-9E cells were transfected with 15 nM control or ORC2 siRNAs and placed in 10% FBS DMEM + 2 mM CaCl 2 for 72 h. DNA was isolated and HPV-31 DNA content was measured by RT-PCR and normalized to β-actin levels. The ORC2 siRNA group was normalized to the control siRNA group (control siRNA = 1). Values are expressed mean +/-. * p-value ≤ 0.05.
    Figure Legend Snippet: ORC2 silencing enhanced PV replication in proliferating and differentiating CIN612-9E cells. (A) ORC2 siRNA duplexes decreased ORC2 protein levels at a concentration of 15 or 5 nM after 48 h. (B) CIN612-9E cells were transfected with RLuc and pFLORI31. 48h later cells were lysed and luciferase activity measured. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. (C) CIN612-9E cells were transfected with 15 nM ORC2 and differentiated in 10% FBS DMEM + 2 mM CaCl 2 for 48h. Involucrin and β-actin levels were analyzed. (D) CIN612-9E cells were transfected with RLuc and pFLORI31. Five hours later, the transfection was removed and cells were placed in either E-medium or 10% FBS DMEM + 2 mM CaCl 2 . 48h later cells were lysed and luciferase activity measured. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. (E) CIN612-9E cells were transfected with 15 nM control or ORC2 siRNAs, RLuc and pFLORI31. Five hours later, the transfection was removed and cells were placed in 10% FBS DMEM + 2 mM CaCl 2 . 48h later cells were lysed and luciferase activity measured. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. (F) CIN612-9E cells were transfected with 15 nM control or ORC2 siRNAs and placed in 10% FBS DMEM + 2 mM CaCl 2 for 72 h. DNA was isolated and HPV-31 DNA content was measured by RT-PCR and normalized to β-actin levels. The ORC2 siRNA group was normalized to the control siRNA group (control siRNA = 1). Values are expressed mean +/-. * p-value ≤ 0.05.

    Techniques Used: Concentration Assay, Transfection, Luciferase, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "HDAC and Ku70 axis- an effective target for apoptosis induction by a new 2-cyano-3-oxo-1,9-dien glycyrrhetinic acid analogue"

    Article Title: HDAC and Ku70 axis- an effective target for apoptosis induction by a new 2-cyano-3-oxo-1,9-dien glycyrrhetinic acid analogue

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0602-1

    Bak and Bax are activated in 10e-treated cells and contribute to the apoptosis induction. A The activated Bak or Bax proteins in THP-1, HL-60, and I9.2 cells treated with 2 μM or 4 μM 10e for the given times were immunoprecipitated with the anti-Bak(Ab-1) or anti-Bax (6A7) antibody (detecting the active forms), respectively, followed by the western blotting using poly anti-Bak or anti-Bax antibody. B THP-1 cells were transfected with Bak siRNA or a negative control siRNA for 30 h, then treated with 4 μM 10e for additional 6 h. The levels of PARP and Bak were determined by western blotting. The apoptotic cells were measured by FACS after staining with Annexin V-FITC. C The THP-1 and HL-60 cell lysates treated with 4 μM or 2 μM 10e for 6 h were immunoprecipitated with anti-Bax antibody and immunoblotted with an anti-Ku70, Bax, or Bcl-2 antibody. D THP-1 cells were transfected with Bax siRNA or a negative control siRNA for 30 h, then treated with 4 μM 10e for additional 6 h. The levels of PARP and Bax were determined by western blotting. The active form of Bax was detected with IP. The apoptotic cells were measured by FACS after staining with Annexin V-FITC
    Figure Legend Snippet: Bak and Bax are activated in 10e-treated cells and contribute to the apoptosis induction. A The activated Bak or Bax proteins in THP-1, HL-60, and I9.2 cells treated with 2 μM or 4 μM 10e for the given times were immunoprecipitated with the anti-Bak(Ab-1) or anti-Bax (6A7) antibody (detecting the active forms), respectively, followed by the western blotting using poly anti-Bak or anti-Bax antibody. B THP-1 cells were transfected with Bak siRNA or a negative control siRNA for 30 h, then treated with 4 μM 10e for additional 6 h. The levels of PARP and Bak were determined by western blotting. The apoptotic cells were measured by FACS after staining with Annexin V-FITC. C The THP-1 and HL-60 cell lysates treated with 4 μM or 2 μM 10e for 6 h were immunoprecipitated with anti-Bax antibody and immunoblotted with an anti-Ku70, Bax, or Bcl-2 antibody. D THP-1 cells were transfected with Bax siRNA or a negative control siRNA for 30 h, then treated with 4 μM 10e for additional 6 h. The levels of PARP and Bax were determined by western blotting. The active form of Bax was detected with IP. The apoptotic cells were measured by FACS after staining with Annexin V-FITC

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, Negative Control, FACS, Staining

    36) Product Images from "THE ROLE OF IGF-1 SIGNALING PATHWAY IN CISPLATIN-RESISTANT LUNG CANCER CELLS"

    Article Title: THE ROLE OF IGF-1 SIGNALING PATHWAY IN CISPLATIN-RESISTANT LUNG CANCER CELLS

    Journal: International Journal of Radiation Oncology, Biology, Physics

    doi: 10.1016/j.ijrobp.2011.06.1999

    IGF-1R inhibition sensitizes CDDP-R cells to cisplatin and radiation A. Parental and CDDP-R cells were transfected with control siRNA or IGF-1R siRNA. After 48h, cells were lysed and probed for IGF-1R by Western blot. Actin was used as control. B. Transfected cells were treated with 0, 3, or 20µM cisplatin. After 72h, MTS assay was performed according to the manufacture’s instruction and absorbance at 490nm was recorded. Shown are bar graphs representing mean survival fraction relative to control and standard deviations. *: p
    Figure Legend Snippet: IGF-1R inhibition sensitizes CDDP-R cells to cisplatin and radiation A. Parental and CDDP-R cells were transfected with control siRNA or IGF-1R siRNA. After 48h, cells were lysed and probed for IGF-1R by Western blot. Actin was used as control. B. Transfected cells were treated with 0, 3, or 20µM cisplatin. After 72h, MTS assay was performed according to the manufacture’s instruction and absorbance at 490nm was recorded. Shown are bar graphs representing mean survival fraction relative to control and standard deviations. *: p

    Techniques Used: Inhibition, Transfection, Western Blot, MTS Assay

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    Article Title: Twist1 confers multidrug resistance in colon cancer through upregulation of ATP-binding cassette transporters
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    Small Interfering RNA:

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    Negative Control:

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    other:

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    Incubation:

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    Santa Cruz Biotechnology control sirna
    Effects of WTD on production of TNF-α and RANTES in MIP-1β-induced U937 cells after <t>CCR5</t> knockdown. U937 cells were transfected with or without CCR5 <t>siRNA,</t> then treated with or without WTD for 2 h, and then exposed to MIP-1β (25 nM) for 24 h. Total CCR5 and its phosphorylation form (A) were detected by western blotting. The levels of TNF-α (B) and RANTES (C) in cell supernatants were detected by ELISA. ∗∗ P
    Control Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology sirna
    Impacts of <t>PRDX3</t> 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 <t>siRNA</t> (+). ( 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.
    Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of WTD on production of TNF-α and RANTES in MIP-1β-induced U937 cells after CCR5 knockdown. U937 cells were transfected with or without CCR5 siRNA, then treated with or without WTD for 2 h, and then exposed to MIP-1β (25 nM) for 24 h. Total CCR5 and its phosphorylation form (A) were detected by western blotting. The levels of TNF-α (B) and RANTES (C) in cell supernatants were detected by ELISA. ∗∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Wu-Tou Decoction in Rheumatoid Arthritis: Integrating Network Pharmacology and In Vivo Pharmacological Evaluation

    doi: 10.3389/fphar.2017.00230

    Figure Lengend Snippet: Effects of WTD on production of TNF-α and RANTES in MIP-1β-induced U937 cells after CCR5 knockdown. U937 cells were transfected with or without CCR5 siRNA, then treated with or without WTD for 2 h, and then exposed to MIP-1β (25 nM) for 24 h. Total CCR5 and its phosphorylation form (A) were detected by western blotting. The levels of TNF-α (B) and RANTES (C) in cell supernatants were detected by ELISA. ∗∗ P

    Article Snippet: SiRNA Transfection CCR5 siRNA was obtained from Dharmacon (Lafayette, CO, USA), and control siRNA was purchased from Santa Cruz Biotechnology Inc. (Delaware, CA, USA).

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

    Knockdown of importin β1 increases DR5-mediated sensitivity of HeLa cells to rTRAIL. A , importin β1 siRNA or control siRNA was transfected into HeLa cells (5 × 10 3 ) in a 96-well plate. At 24 h post-transfection, cells were treated with or without 25 ng/ml rTRAIL for 24 h and observed using a phase-contrast microscope. Scale bars indicate 50 μm. B , at 24 h after transfection with importin β1 siRNA or control siRNA, the indicated concentrations of rTRAIL were added. Cell death was quantified by WST assay as described under “Experimental Procedures.” Data are the mean ± S.D. of triplicate samples. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Importin ?1 Protein-mediated Nuclear Localization of Death Receptor 5 (DR5) Limits DR5/Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Cell Death of Human Tumor Cells *

    doi: 10.1074/jbc.M111.309377

    Figure Lengend Snippet: Knockdown of importin β1 increases DR5-mediated sensitivity of HeLa cells to rTRAIL. A , importin β1 siRNA or control siRNA was transfected into HeLa cells (5 × 10 3 ) in a 96-well plate. At 24 h post-transfection, cells were treated with or without 25 ng/ml rTRAIL for 24 h and observed using a phase-contrast microscope. Scale bars indicate 50 μm. B , at 24 h after transfection with importin β1 siRNA or control siRNA, the indicated concentrations of rTRAIL were added. Cell death was quantified by WST assay as described under “Experimental Procedures.” Data are the mean ± S.D. of triplicate samples. **, p

    Article Snippet: Mouse anti-tubulin mAb was from Sigma-Aldrich; rabbit anti-human DR5 pAb (Ab-1) was from Calbiochem; and rabbit anti-human YY1 pAb (C-20), rabbit anti-human importin β1 pAb (H-300), goat anti-human importin β1 pAb (C-19), rabbit anti-STAT6 pAb (M-200), importin β1 siRNA, and control siRNA were obtained from Santa Cruz Biotechnology.

    Techniques: Transfection, Microscopy, WST Assay

    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.

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.17927

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.17927

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.17927

    Figure Lengend 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.

    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.

    Techniques: Expressing, Standard Deviation