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    Name:
    PML siRNA
    Description:
    Gene Silencers generally consist of pools of three to five target specific 19 25 nucleotide sequences in length For independent verification of PML gene silencing results individual duplex components or plasmids are also available upon request Suitable control antibody PML Antibody E 11 sc 377390 is recommended as control antibody for monitoring of PML expression knockdown by Western blotting or immunofluorescence
    Catalog Number:
    SC-36284
    Price:
    None
    Category:
    Gene Editing siRNA shRNA Gene Silencers Transcription Regulator PML siRNA shRNA Plasmid and Lentiviral Particle Gene Silencers PML siRNA and shRNA Plasmids h
    Buy from Supplier


    Structured Review

    Santa Cruz Biotechnology sirnas
    Effect of <t>PML</t> silencing and overexpression on infectious particle production and antigen expression. A549 cells were non-transfected, transfected with <t>X-siRNAs,</t> PML-siRNAs or pcDNA-PMLIV and infected with DENV-2. Non-infected cells were also included as a control (A) At 24 h p.i. viral yields were determined by a standard plaque assay. The reported values are mean ± SD (n = 3). Asterisks indicate a significant difference (*** p
    Gene Silencers generally consist of pools of three to five target specific 19 25 nucleotide sequences in length For independent verification of PML gene silencing results individual duplex components or plasmids are also available upon request Suitable control antibody PML Antibody E 11 sc 377390 is recommended as control antibody for monitoring of PML expression knockdown by Western blotting or immunofluorescence
    https://www.bioz.com/result/sirnas/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sirnas - by Bioz Stars, 2021-09
    92/100 stars

    Images

    1) Product Images from "Cellular Promyelocytic Leukemia Protein Is an Important Dengue Virus Restriction Factor"

    Article Title: Cellular Promyelocytic Leukemia Protein Is an Important Dengue Virus Restriction Factor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125690

    Effect of PML silencing and overexpression on infectious particle production and antigen expression. A549 cells were non-transfected, transfected with X-siRNAs, PML-siRNAs or pcDNA-PMLIV and infected with DENV-2. Non-infected cells were also included as a control (A) At 24 h p.i. viral yields were determined by a standard plaque assay. The reported values are mean ± SD (n = 3). Asterisks indicate a significant difference (*** p
    Figure Legend Snippet: Effect of PML silencing and overexpression on infectious particle production and antigen expression. A549 cells were non-transfected, transfected with X-siRNAs, PML-siRNAs or pcDNA-PMLIV and infected with DENV-2. Non-infected cells were also included as a control (A) At 24 h p.i. viral yields were determined by a standard plaque assay. The reported values are mean ± SD (n = 3). Asterisks indicate a significant difference (*** p

    Techniques Used: Over Expression, Expressing, Transfection, Infection, Plaque Assay

    Assessment of PML silencing and overexpression in A549 cells. A549 cells were non-transfected, transfected with X-siRNAs, PML-siRNAs or pcDNA-PMLIV. (A) At 24 h post transfection, cells were fixed and PML protein was stained using anti-PML monoclonal antibody and TRITC-labeled anti-mouse IgG. Cells were visualized by fluorescence microscopy. Magnification: 400 X. (B) In parallel, cells were harvested for determination of PML-mRNA expression levels by real time PCR. PML-mRNA expression level is represented as fold difference relative to X-siRNAs-transfected cells and normalized to β-actin-mRNA. The reported values are mean ± SD (n = 3). Asterisks indicate a significant difference (*** p
    Figure Legend Snippet: Assessment of PML silencing and overexpression in A549 cells. A549 cells were non-transfected, transfected with X-siRNAs, PML-siRNAs or pcDNA-PMLIV. (A) At 24 h post transfection, cells were fixed and PML protein was stained using anti-PML monoclonal antibody and TRITC-labeled anti-mouse IgG. Cells were visualized by fluorescence microscopy. Magnification: 400 X. (B) In parallel, cells were harvested for determination of PML-mRNA expression levels by real time PCR. PML-mRNA expression level is represented as fold difference relative to X-siRNAs-transfected cells and normalized to β-actin-mRNA. The reported values are mean ± SD (n = 3). Asterisks indicate a significant difference (*** p

    Techniques Used: Over Expression, Transfection, Staining, Labeling, Fluorescence, Microscopy, Expressing, Real-time Polymerase Chain Reaction

    2) Product Images from "Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition"

    Article Title: Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition

    Journal: Scientific Reports

    doi: 10.1038/srep16077

    Role of FBXL5 in snail turnover and reversal of EMT. ( A , B ) Cells growing in chambered slides (3000 cells per well) overnight and exposed to SINE compounds at indicated concentrations for an additional 24 hrs. Immunofluorescence assay was performed as described in methods section. Immunofluorescence images (40×) of HMLE-snail and HMLER-snail cells showing nuclear retention of FBXL5 (Abcam, USA). ( C ) Photomicrographs of HMLE-snail cells under different treatment conditions. Note. Lack of EMT reversal in the presence of FBXL5 siRNA. ( D ) Histone DNA ELISA apoptosis and ( E ) MTT assay at 72 hrs post Selinexor treatment (150 nM) in the absence or presence of siRNAs for FBXL5 (see methods section for siRNA procedures). **P
    Figure Legend Snippet: Role of FBXL5 in snail turnover and reversal of EMT. ( A , B ) Cells growing in chambered slides (3000 cells per well) overnight and exposed to SINE compounds at indicated concentrations for an additional 24 hrs. Immunofluorescence assay was performed as described in methods section. Immunofluorescence images (40×) of HMLE-snail and HMLER-snail cells showing nuclear retention of FBXL5 (Abcam, USA). ( C ) Photomicrographs of HMLE-snail cells under different treatment conditions. Note. Lack of EMT reversal in the presence of FBXL5 siRNA. ( D ) Histone DNA ELISA apoptosis and ( E ) MTT assay at 72 hrs post Selinexor treatment (150 nM) in the absence or presence of siRNAs for FBXL5 (see methods section for siRNA procedures). **P

    Techniques Used: Immunofluorescence, Enzyme-linked Immunosorbent Assay, MTT Assay

    3) Product Images from "Adiponectin Receptor Signaling on Dendritic Cells Blunts Antitumor Immunity"

    Article Title: Adiponectin Receptor Signaling on Dendritic Cells Blunts Antitumor Immunity

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-13-1397

    Both APN receptors can independently modulate DC function. A, restoration of NF-κB activity in APN-conditioned mDC by siRNA knockdown of AdipoR1 and/or AdipoR2. Human DCs transfected with siRNAs specific for either AdipoR1 or AdipoR2 or both or
    Figure Legend Snippet: Both APN receptors can independently modulate DC function. A, restoration of NF-κB activity in APN-conditioned mDC by siRNA knockdown of AdipoR1 and/or AdipoR2. Human DCs transfected with siRNAs specific for either AdipoR1 or AdipoR2 or both or

    Techniques Used: Activity Assay, Transfection

    APN signals through AdipoR1 to induce IL10 production via activation of AMPK and MAPKp38. A, DCs were transfected with siRNAs as indicated, conditioned with or without APN, and then activated (mDCs). Supernatants were collected 24 hours after activation
    Figure Legend Snippet: APN signals through AdipoR1 to induce IL10 production via activation of AMPK and MAPKp38. A, DCs were transfected with siRNAs as indicated, conditioned with or without APN, and then activated (mDCs). Supernatants were collected 24 hours after activation

    Techniques Used: Activation Assay, Transfection

    4) Product Images from "Autocrine stimulation of clear-cell renal carcinoma cell migration in hypoxia via HIF-independent suppression of thrombospondin-1"

    Article Title: Autocrine stimulation of clear-cell renal carcinoma cell migration in hypoxia via HIF-independent suppression of thrombospondin-1

    Journal: Scientific Reports

    doi: 10.1038/srep00788

    TSP-1 regulation in RCC cell lines is independent of HIF. TSP-1 and HIF2α protein levels in 786-O cells transfected with a scrambled siRNA (scr), or siRNAs specific for HIF2α (siHIF2α) were analyzed after 24 h under normoxic or hypoxic conditions (left panel). TSP-1, HIF2α and HIF-1α protein levels in RCC4 cells transfected with scrambled (scr) or siRNA to HIF-1α (siHIF1α) or HIF-2α (siHIF2α) were analyzed after 24 h in normoxic or hypoxic conditions (right), with α-tubulin as a loading control. The results are representative of at least three experiments performed.
    Figure Legend Snippet: TSP-1 regulation in RCC cell lines is independent of HIF. TSP-1 and HIF2α protein levels in 786-O cells transfected with a scrambled siRNA (scr), or siRNAs specific for HIF2α (siHIF2α) were analyzed after 24 h under normoxic or hypoxic conditions (left panel). TSP-1, HIF2α and HIF-1α protein levels in RCC4 cells transfected with scrambled (scr) or siRNA to HIF-1α (siHIF1α) or HIF-2α (siHIF2α) were analyzed after 24 h in normoxic or hypoxic conditions (right), with α-tubulin as a loading control. The results are representative of at least three experiments performed.

    Techniques Used: Transfection

    5) Product Images from "Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis"

    Article Title: Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00551-2

    ADORA2A regulates HRMEC proliferation, sprouting and tube formation. a , b Bromodeoxyuridine (BrdU) staining of HRMECs transfected with siRNAs targeting human ADORA2A (siA 2A R) or with a non-targeting negative control (siCtrl) under hypoxia conditions (0.5% O 2 ). n = 6. * P
    Figure Legend Snippet: ADORA2A regulates HRMEC proliferation, sprouting and tube formation. a , b Bromodeoxyuridine (BrdU) staining of HRMECs transfected with siRNAs targeting human ADORA2A (siA 2A R) or with a non-targeting negative control (siCtrl) under hypoxia conditions (0.5% O 2 ). n = 6. * P

    Techniques Used: BrdU Staining, Transfection, Negative Control

    6) Product Images from "Vav1 Down-Modulates Akt2 Expression in Cells from Pancreatic Ductal Adenocarcinoma: Nuclear Vav1 as a Potential Regulator of Akt Related Malignancy in Pancreatic Cancer"

    Article Title: Vav1 Down-Modulates Akt2 Expression in Cells from Pancreatic Ductal Adenocarcinoma: Nuclear Vav1 as a Potential Regulator of Akt Related Malignancy in Pancreatic Cancer

    Journal: Biomedicines

    doi: 10.3390/biomedicines8100379

    Vav1-dependent modulation of Akt2 and motility in PDAC-derived cell lines. ( A ) Representative Western blot analysis with the indicated antibodies of total lysates from HPAF-2, HPAC, and PL-45 pancreatic cancer cells transfected with siRNAs specific for Vav1 (Vav1 siRNAs) or with a construct expressing the full-length human Vav1 (Over Vav1). Scramble siRNAs (Ctrl siRNAs) and an empty vector were controls. In ( B ), levels of pAkt1/2/3, Akt1, and Akt2 as deduced from the densitometry of immunochemical bands normalized with β-Tubulin. Levels are shown as fold changes relative to the respective controls (Ctrl), taken as 1. In ( C ), percentage of viable cells relative to controls (Ctrl). In ( D , E ), xCELLigence-driven dynamic monitoring of migration and invasion of cells under the same experimental conditions. Cell Index (CI) after 24 h was reported. Error bars indicate ± SD from three experiments in triplicate. * p
    Figure Legend Snippet: Vav1-dependent modulation of Akt2 and motility in PDAC-derived cell lines. ( A ) Representative Western blot analysis with the indicated antibodies of total lysates from HPAF-2, HPAC, and PL-45 pancreatic cancer cells transfected with siRNAs specific for Vav1 (Vav1 siRNAs) or with a construct expressing the full-length human Vav1 (Over Vav1). Scramble siRNAs (Ctrl siRNAs) and an empty vector were controls. In ( B ), levels of pAkt1/2/3, Akt1, and Akt2 as deduced from the densitometry of immunochemical bands normalized with β-Tubulin. Levels are shown as fold changes relative to the respective controls (Ctrl), taken as 1. In ( C ), percentage of viable cells relative to controls (Ctrl). In ( D , E ), xCELLigence-driven dynamic monitoring of migration and invasion of cells under the same experimental conditions. Cell Index (CI) after 24 h was reported. Error bars indicate ± SD from three experiments in triplicate. * p

    Techniques Used: Derivative Assay, Western Blot, Transfection, Construct, Expressing, Plasmid Preparation, Migration

    7) Product Images from "ROR2 receptor promotes the migration of osteosarcoma cells in response to Wnt5a"

    Article Title: ROR2 receptor promotes the migration of osteosarcoma cells in response to Wnt5a

    Journal: Cancer Cell International

    doi: 10.1186/s12935-017-0482-y

    ROR2 participates in Wnt5a-induced osteosarcoma cell migration. a Stable ROR2 knockdown MG-63 cells and osteosarcoma U2OS cells transfected with ROR2-siRNA or scrambled siRNA were subjected to Western blotting assays. The expression of ROR2 in MG-63 and U2OS cells was significantly knockdown by specific shRNAs or siRNAs targeting ROR2. GAPDH was used as an internal control. b and c Stable ROR2 knockdown MG-63 cells and osteosarcoma U2OS cells transfected with ROR2-siRNA or scrambled siRNA were subjected to wound healing assays. Cells incubated with 100 ng/mL Wnt5a were allowed to migrate for 12 h. Data were presented as mean ± SD of 5 determinations. The relative migration distance was normalized to the average value of scrambled group
    Figure Legend Snippet: ROR2 participates in Wnt5a-induced osteosarcoma cell migration. a Stable ROR2 knockdown MG-63 cells and osteosarcoma U2OS cells transfected with ROR2-siRNA or scrambled siRNA were subjected to Western blotting assays. The expression of ROR2 in MG-63 and U2OS cells was significantly knockdown by specific shRNAs or siRNAs targeting ROR2. GAPDH was used as an internal control. b and c Stable ROR2 knockdown MG-63 cells and osteosarcoma U2OS cells transfected with ROR2-siRNA or scrambled siRNA were subjected to wound healing assays. Cells incubated with 100 ng/mL Wnt5a were allowed to migrate for 12 h. Data were presented as mean ± SD of 5 determinations. The relative migration distance was normalized to the average value of scrambled group

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

    8) Product Images from "RACK1 depletion in the ribosome induces selective translation for non-canonical autophagy"

    Article Title: RACK1 depletion in the ribosome induces selective translation for non-canonical autophagy

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.204

    RACK1 depletion-induced autophagy is not a cell-line-specific phenomenon. ( a – d and g – i ) Control or RACK1 siRNAs were transfected (20 pmol) into HeLa ( a ), HDF ( b ), HepG2 and Hep3B ( c , d ), MCF7 and MDA-MB231 ( g ), MEF ( h ) and U2OS ( i ) cells. After 48 h, immunoblot analysis was performed using the indicated antibodies. ( e , f and j ) Indicated siRNAs (50 pmol) were transfected into EGFP-LC3 stably expressing HepG2, Hep3B and U2OS cells, followed by fluorescence microscopy ( f and j ) and immunoblot analyses using the indicated antibodies ( e ). ( k ) In each cell line, the intensities of LC3 proteins were normalized against that of tubulin protein, and the relative expression in RACK1 siRNA-treated cells compared with that in control cells was plotted. * P
    Figure Legend Snippet: RACK1 depletion-induced autophagy is not a cell-line-specific phenomenon. ( a – d and g – i ) Control or RACK1 siRNAs were transfected (20 pmol) into HeLa ( a ), HDF ( b ), HepG2 and Hep3B ( c , d ), MCF7 and MDA-MB231 ( g ), MEF ( h ) and U2OS ( i ) cells. After 48 h, immunoblot analysis was performed using the indicated antibodies. ( e , f and j ) Indicated siRNAs (50 pmol) were transfected into EGFP-LC3 stably expressing HepG2, Hep3B and U2OS cells, followed by fluorescence microscopy ( f and j ) and immunoblot analyses using the indicated antibodies ( e ). ( k ) In each cell line, the intensities of LC3 proteins were normalized against that of tubulin protein, and the relative expression in RACK1 siRNA-treated cells compared with that in control cells was plotted. * P

    Techniques Used: Transfection, Multiple Displacement Amplification, Stable Transfection, Expressing, Fluorescence, Microscopy

    RACK1 depletion-induced autophagy is non-canonical. ( a ) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies (left panel). Intensities of Atg5/12 and Beclin1 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells were plotted (right panel). ( b ) HT1080 cells were transfected with siRNAs against control or RACK1 in combination with or without Beclin1 siRNA (100 pmol). After 48 h, these cells were analyzed by immunoblotting using the indicated antibodies. ( c ) HT1080 cells were transfected with control or Atg5 siRNA (100 pmol). After 24 h, the cells were re-transfected with control or RACK1 siRNA. After a further 48 h incubation, the cell extracts were subjected to immunoblot analysis using the indicated antibodies. ( d ) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies. ( e ) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 24 h. Extracts of siRNA-transfected cells were pretreated with rapamycin 1 μM for 24 h, followed by immunoblot analysis using the indicated antibodies. Intensities of LAMP1 and LAMP2 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells were plotted. * P
    Figure Legend Snippet: RACK1 depletion-induced autophagy is non-canonical. ( a ) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies (left panel). Intensities of Atg5/12 and Beclin1 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells were plotted (right panel). ( b ) HT1080 cells were transfected with siRNAs against control or RACK1 in combination with or without Beclin1 siRNA (100 pmol). After 48 h, these cells were analyzed by immunoblotting using the indicated antibodies. ( c ) HT1080 cells were transfected with control or Atg5 siRNA (100 pmol). After 24 h, the cells were re-transfected with control or RACK1 siRNA. After a further 48 h incubation, the cell extracts were subjected to immunoblot analysis using the indicated antibodies. ( d ) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies. ( e ) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 24 h. Extracts of siRNA-transfected cells were pretreated with rapamycin 1 μM for 24 h, followed by immunoblot analysis using the indicated antibodies. Intensities of LAMP1 and LAMP2 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells were plotted. * P

    Techniques Used: Transfection, Incubation

    Knockdown of RACK1 increases Bcl-xL protein level. ( a ) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies (left panel). Intensities of Bcl-xL protein was normalized against that of tubulin protein, and the relative expression in RACK1 siRNA-treated cells compared with that in control cells was plotted (right panel). ** P
    Figure Legend Snippet: Knockdown of RACK1 increases Bcl-xL protein level. ( a ) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies (left panel). Intensities of Bcl-xL protein was normalized against that of tubulin protein, and the relative expression in RACK1 siRNA-treated cells compared with that in control cells was plotted (right panel). ** P

    Techniques Used: Transfection, Incubation, Expressing

    ( a ) HT1080 cells were transfected with control or RACK1 siRNAs (50 pmol), and then incubated for 48 h. The cell lysates were subjected to immunoblot analysis using the indicated antibodies. LC3-II and p62/SQSTM1 are markers of autophagic flux (left panel). Intensities of LC3 and p62 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells was plotted (right panel). ** P
    Figure Legend Snippet: ( a ) HT1080 cells were transfected with control or RACK1 siRNAs (50 pmol), and then incubated for 48 h. The cell lysates were subjected to immunoblot analysis using the indicated antibodies. LC3-II and p62/SQSTM1 are markers of autophagic flux (left panel). Intensities of LC3 and p62 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells was plotted (right panel). ** P

    Techniques Used: Transfection, Incubation

    9) Product Images from "Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways"

    Article Title: Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2009.12.004

    Knockdown of JNK1/2 or MEK1/2 by corresponding siRNAs inhibits PMA-induced ANGPTL4 mRNA expression. HASM cells were transfected with siJNK1/2 (A) or siMEK1/2 (B), or control (scrambled; siCON) siRNAs for 48 h before they were treated with or without 30
    Figure Legend Snippet: Knockdown of JNK1/2 or MEK1/2 by corresponding siRNAs inhibits PMA-induced ANGPTL4 mRNA expression. HASM cells were transfected with siJNK1/2 (A) or siMEK1/2 (B), or control (scrambled; siCON) siRNAs for 48 h before they were treated with or without 30

    Techniques Used: Expressing, Transfection

    10) Product Images from "Baicalein protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis through inhibition of reactive oxygen species production and CHOP induction"

    Article Title: Baicalein protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis through inhibition of reactive oxygen species production and CHOP induction

    Journal: Experimental & Molecular Medicine

    doi: 10.3858/emm.2010.42.12.084

    Effects of inhibitors of MAPKs and siRNAs for CHOP and caspase 12 on ER stress-induced cell death and CHOP expression and phosphorylation in HT22 neuronal cells. (A) HT22 cells were pretreated with vehicle or inhibitors of MAPKs (10 µM SB203580
    Figure Legend Snippet: Effects of inhibitors of MAPKs and siRNAs for CHOP and caspase 12 on ER stress-induced cell death and CHOP expression and phosphorylation in HT22 neuronal cells. (A) HT22 cells were pretreated with vehicle or inhibitors of MAPKs (10 µM SB203580

    Techniques Used: Expressing

    11) Product Images from "Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells"

    Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

    Journal: Cancer Cell International

    doi: 10.1186/s12935-019-1033-5

    TGF-β induced OPNc expression enhanced the mobility of SK-MES-1 cells with a dependence to HDACs. a TGF-β treatments for 48 h increased the invasiveness of SK-MES-1 cells. b TGF-β induction increased OPNt and OPN-SIs levels with a preference to OPNc. c TGF-β promoted OPNc splicing with most significance in SK-MES-1 cells. d Knockdown of HDAC1 or HDAC2 significantly decreased the RUNX2-induced OPNc splicing. e The HDAC1 and HDAC2 expression were markedly reduced in the SK-MES-1 cells transfected with the targeted siRNAs. f Overexpression of OPN-SIs significantly promoted the migration of SK-MES-1 cells
    Figure Legend Snippet: TGF-β induced OPNc expression enhanced the mobility of SK-MES-1 cells with a dependence to HDACs. a TGF-β treatments for 48 h increased the invasiveness of SK-MES-1 cells. b TGF-β induction increased OPNt and OPN-SIs levels with a preference to OPNc. c TGF-β promoted OPNc splicing with most significance in SK-MES-1 cells. d Knockdown of HDAC1 or HDAC2 significantly decreased the RUNX2-induced OPNc splicing. e The HDAC1 and HDAC2 expression were markedly reduced in the SK-MES-1 cells transfected with the targeted siRNAs. f Overexpression of OPN-SIs significantly promoted the migration of SK-MES-1 cells

    Techniques Used: Expressing, Transfection, Over Expression, Migration

    RUNX2 dependent OPNc splicing required normal activities of HDAC1 or HDAC2. a Treatment of A549 cells with HDACs inhibitor TSA suppressed OPNc splicing induced by RUNX2 overexpression. b Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells following TSA treatment. c Inhibition of HDAC1 activity by NaB deprived RUNX2 induced OPNc splicing. d Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells treated with NaB. e Knockdown of HDAC1 or HDAC2, but not HDAC3, decreased RUNX2-induced OPNc splicing. f Western blots of HDAC1, HDAC2 and HDAC3 from A549 cells transfected with the targeted siRNAs
    Figure Legend Snippet: RUNX2 dependent OPNc splicing required normal activities of HDAC1 or HDAC2. a Treatment of A549 cells with HDACs inhibitor TSA suppressed OPNc splicing induced by RUNX2 overexpression. b Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells following TSA treatment. c Inhibition of HDAC1 activity by NaB deprived RUNX2 induced OPNc splicing. d Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells treated with NaB. e Knockdown of HDAC1 or HDAC2, but not HDAC3, decreased RUNX2-induced OPNc splicing. f Western blots of HDAC1, HDAC2 and HDAC3 from A549 cells transfected with the targeted siRNAs

    Techniques Used: Over Expression, Western Blot, Inhibition, Activity Assay, Transfection

    12) Product Images from "The long noncoding RNA lncNB1 promotes tumorigenesis by interacting with ribosomal protein RPL35"

    Article Title: The long noncoding RNA lncNB1 promotes tumorigenesis by interacting with ribosomal protein RPL35

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12971-3

    LncNB1 is required for DEPDC1B mRNA and protein and E2F1 protein expression. a Genome-wide differential gene expression was examined with Affymetrix microarray in BE(2)-C cells 40 h after transfection with control siRNA, lncNB1 siRNA-1, or siRNA-2. Gene set enrichment analysis generated histograms confirming down-regulation of E2F1 target genes by lncNB1 siRNAs. FDR indicated false discovery rate. b , c BE(2)-C, Kelly, and CHP134 cells were transfected with control siRNA, lncNB1 siRNA-1, or lncNB1 siRNA-2 for 48 h. LncNB1, DEPDC1B, and E2F1 RNA expression was analyzed by RT-PCR ( b ), and DEPDC1B, E2F1, and N-Myc protein expression was analyzed by immunoblot ( c ). Data were shown as the mean ± standard error of three independent experiments, and evaluated by one-way ANOVA. *, **, and *** indicate P
    Figure Legend Snippet: LncNB1 is required for DEPDC1B mRNA and protein and E2F1 protein expression. a Genome-wide differential gene expression was examined with Affymetrix microarray in BE(2)-C cells 40 h after transfection with control siRNA, lncNB1 siRNA-1, or siRNA-2. Gene set enrichment analysis generated histograms confirming down-regulation of E2F1 target genes by lncNB1 siRNAs. FDR indicated false discovery rate. b , c BE(2)-C, Kelly, and CHP134 cells were transfected with control siRNA, lncNB1 siRNA-1, or lncNB1 siRNA-2 for 48 h. LncNB1, DEPDC1B, and E2F1 RNA expression was analyzed by RT-PCR ( b ), and DEPDC1B, E2F1, and N-Myc protein expression was analyzed by immunoblot ( c ). Data were shown as the mean ± standard error of three independent experiments, and evaluated by one-way ANOVA. *, **, and *** indicate P

    Techniques Used: Expressing, Genome Wide, Microarray, Transfection, Generated, RNA Expression, Reverse Transcription Polymerase Chain Reaction

    13) Product Images from "Novel androgen receptor co-regulator GRHL2 exerts both oncogenic and anti-metastatic functions in prostate cancer"

    Article Title: Novel androgen receptor co-regulator GRHL2 exerts both oncogenic and anti-metastatic functions in prostate cancer

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-16-1616

    Regulation of prostate cancer cell growth and AR expression and signaling by GRHL2 (A) Prostate cancer cell lines were transfected with 3 distinct siRNAs and Trypan blue growth assays were performed. Error bars represent ± SD. (B) LNCaP cells were transfected with GRHL2 siRNA (siGRHL2) or a control (siNC) and gene expression was measured by RT-qPCR after 48 hours. Error bars represent ± SEM of 3 independent experiments. (C) Prostate cancer cell lines were transfected with GRHL2 siRNA (siGRHL2) or a control (siNC) and, 2 days later, protein expression was assessed by Western blotting. Tubulin or GAPDH are shown as loading controls. (D) LNCaP cells were transfected with a GRHL2 over-expression vector (GRHL2-OE) or a control (Empty) and protein expression was assessed by Western blotting after 48 hours. GAPDH is shown as a loading control. (E) AR expression is higher in metastatic CRPC (mCRPC) tumors with GRHL2 copy number gain or amplification compared to tumors with no change in GRHL2 copy number (diploid). Data is from the Grasso cohort and was obtained via cBioPortal. P value was determined using an unpaired t test.
    Figure Legend Snippet: Regulation of prostate cancer cell growth and AR expression and signaling by GRHL2 (A) Prostate cancer cell lines were transfected with 3 distinct siRNAs and Trypan blue growth assays were performed. Error bars represent ± SD. (B) LNCaP cells were transfected with GRHL2 siRNA (siGRHL2) or a control (siNC) and gene expression was measured by RT-qPCR after 48 hours. Error bars represent ± SEM of 3 independent experiments. (C) Prostate cancer cell lines were transfected with GRHL2 siRNA (siGRHL2) or a control (siNC) and, 2 days later, protein expression was assessed by Western blotting. Tubulin or GAPDH are shown as loading controls. (D) LNCaP cells were transfected with a GRHL2 over-expression vector (GRHL2-OE) or a control (Empty) and protein expression was assessed by Western blotting after 48 hours. GAPDH is shown as a loading control. (E) AR expression is higher in metastatic CRPC (mCRPC) tumors with GRHL2 copy number gain or amplification compared to tumors with no change in GRHL2 copy number (diploid). Data is from the Grasso cohort and was obtained via cBioPortal. P value was determined using an unpaired t test.

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

    14) Product Images from "The Protein Arginine Methyltransferases 1 and 5 affect Myc properties in glioblastoma stem cells"

    Article Title: The Protein Arginine Methyltransferases 1 and 5 affect Myc properties in glioblastoma stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52291-6

    Symmetrical and asymmetrical Myc dimethylation. ( a ) Top, left. Western blot showing the efficacy of a siPRMT5 in inhibiting the expression of PRMT5-6myc (PRMT5-myc in the figure). Top, right. In vitro methylation assay showing S-dimethylation of a recombinant Myc protein in the presence of an overexpressed PRMT5. Bottom, left. Western blot showing the efficacy of a siPRMT1 in inhibiting the expression of PRMT1-myc-Dkk (PRMT1-myc in the figure). Bottom, right. In vitro methylation assay showing AS-dimethylation of a recombinant Myc protein in the presence of an overexpressed PRMT1. Uncropped images are shown in Supplementary Fig. S2a . ( b ) Left. Western blot analysis showing PRMT1 and PRMT5 expression levels upon FlagMyc transfection in HEK293T cells. Middle. FlagMyc/HEK293T cells underwent immunoprecipitation with control IgG or ASYM24, SYM10 and anti-Flag antibodies. Western blot were performed by using anti-Flag antibody for ASYM24 and SYM10 immunoprecipitations and ASYM24 or SYM10 antibodies for anti-Flag immunoprecipitation. Right. Western blot showing the input of the immunoprecipitations. Uncropped images are shown in Supplementary Fig. S2b . ( c ) Left. FlagMyc/HEK293T cells were treated for 24 hrs with EPZ015666 or 9i. After additional 24 hrs, immunoprecipitations were performed by using either SYM10 or ASYM24 antibody. Uncropped images are shown in Supplementary Fig. S2c . ( d ) The model depicts the competition between PRMT1 and PRMT5 for Myc. Inhibiting PRMT5 activity with EPZ015666 restrained FlagMyc S-dimethylation, while enhancing AS-dimethylation (left). The opposite was obtained by inhibiting PRMT1 (right). ( e ) HEK293T cells were transfected with a pool of specific siRNAs against either PRMT5 or PRMT1. The day after, cells were transfected with pCBS-FlagMyc. 48 hrs later, cells underwent immunoprecipitation with SYM10 or ASYM24 antibodies. Uncropped images are shown in Supplementary Fig. S2d . Abbreviations: SYM = SYM10; ASYM = ASYM24.
    Figure Legend Snippet: Symmetrical and asymmetrical Myc dimethylation. ( a ) Top, left. Western blot showing the efficacy of a siPRMT5 in inhibiting the expression of PRMT5-6myc (PRMT5-myc in the figure). Top, right. In vitro methylation assay showing S-dimethylation of a recombinant Myc protein in the presence of an overexpressed PRMT5. Bottom, left. Western blot showing the efficacy of a siPRMT1 in inhibiting the expression of PRMT1-myc-Dkk (PRMT1-myc in the figure). Bottom, right. In vitro methylation assay showing AS-dimethylation of a recombinant Myc protein in the presence of an overexpressed PRMT1. Uncropped images are shown in Supplementary Fig. S2a . ( b ) Left. Western blot analysis showing PRMT1 and PRMT5 expression levels upon FlagMyc transfection in HEK293T cells. Middle. FlagMyc/HEK293T cells underwent immunoprecipitation with control IgG or ASYM24, SYM10 and anti-Flag antibodies. Western blot were performed by using anti-Flag antibody for ASYM24 and SYM10 immunoprecipitations and ASYM24 or SYM10 antibodies for anti-Flag immunoprecipitation. Right. Western blot showing the input of the immunoprecipitations. Uncropped images are shown in Supplementary Fig. S2b . ( c ) Left. FlagMyc/HEK293T cells were treated for 24 hrs with EPZ015666 or 9i. After additional 24 hrs, immunoprecipitations were performed by using either SYM10 or ASYM24 antibody. Uncropped images are shown in Supplementary Fig. S2c . ( d ) The model depicts the competition between PRMT1 and PRMT5 for Myc. Inhibiting PRMT5 activity with EPZ015666 restrained FlagMyc S-dimethylation, while enhancing AS-dimethylation (left). The opposite was obtained by inhibiting PRMT1 (right). ( e ) HEK293T cells were transfected with a pool of specific siRNAs against either PRMT5 or PRMT1. The day after, cells were transfected with pCBS-FlagMyc. 48 hrs later, cells underwent immunoprecipitation with SYM10 or ASYM24 antibodies. Uncropped images are shown in Supplementary Fig. S2d . Abbreviations: SYM = SYM10; ASYM = ASYM24.

    Techniques Used: Western Blot, Expressing, In Vitro, Methylation, Recombinant, Transfection, Immunoprecipitation, Activity Assay

    Myc/PRMT5/ PRMT1 complex. ( a ) Western blot. HEK293T cells were transfected with an empty or a FlagMyc expression vector. After 48 hrs, proteins were resolved onto a 12% polyacrylamide gel. β-actin was used as loading control. Uncropped images are shown in Supplementary Fig. S1a . ( b ) Western blot. Both HEK293T cells and GSCs were infected with a doxycycline inducible lentivirus carrying a shRNA against Myc (shMyc). After 48 hrs from doxycycline treatment, cells were lysed and proteins resolved onto a 12% polyacrilamide gel. Uncropped images are shown in Supplementary Fig. S1b . ( c,d ) Immunoprecipitations. FlagMyc/HEK293T cells and GSCs underwent reciprocal immunoprecipitation by using anti-Flag, anti-Myc, anti-PRMT1 and anti-PRMT5 antibodies (and control IgGs). Uncropped images are shown in Supplementary Fig. S1c,d . ( e ) Western blot. HEK293T cells were transfected with a scrambled siRNA or a pool of siRNAs against PRMT5 or PRMT1. Uncropped images are shown in Supplementary Fig. S1e . ( f ) Immunoprecipitation. HEK293T cells were transfected with a scrambled siRNA or a siRNAs pool against PRMT5. The day after, cells were transfected again with the FlagMyc expression vector. After further 48 hrs cells were immunoprecipitated with anti-PRMT5, anti-PRMT1 or anti-Flag antibodies (or control IgGs). Input is shown in the middle panel. The cartoon on the right panel outlines immunoprecipitation results. Uncropped images are shown in Supplementary Fig. S1f . ( g ) Immunoprecipitation experiments as in ( f ) in cells partially depleted of PRMT1 (see input, middle panel). The right panel outlines immunoprecipitation results. Uncropped images are shown in Supplementary Fig. S1g .
    Figure Legend Snippet: Myc/PRMT5/ PRMT1 complex. ( a ) Western blot. HEK293T cells were transfected with an empty or a FlagMyc expression vector. After 48 hrs, proteins were resolved onto a 12% polyacrylamide gel. β-actin was used as loading control. Uncropped images are shown in Supplementary Fig. S1a . ( b ) Western blot. Both HEK293T cells and GSCs were infected with a doxycycline inducible lentivirus carrying a shRNA against Myc (shMyc). After 48 hrs from doxycycline treatment, cells were lysed and proteins resolved onto a 12% polyacrilamide gel. Uncropped images are shown in Supplementary Fig. S1b . ( c,d ) Immunoprecipitations. FlagMyc/HEK293T cells and GSCs underwent reciprocal immunoprecipitation by using anti-Flag, anti-Myc, anti-PRMT1 and anti-PRMT5 antibodies (and control IgGs). Uncropped images are shown in Supplementary Fig. S1c,d . ( e ) Western blot. HEK293T cells were transfected with a scrambled siRNA or a pool of siRNAs against PRMT5 or PRMT1. Uncropped images are shown in Supplementary Fig. S1e . ( f ) Immunoprecipitation. HEK293T cells were transfected with a scrambled siRNA or a siRNAs pool against PRMT5. The day after, cells were transfected again with the FlagMyc expression vector. After further 48 hrs cells were immunoprecipitated with anti-PRMT5, anti-PRMT1 or anti-Flag antibodies (or control IgGs). Input is shown in the middle panel. The cartoon on the right panel outlines immunoprecipitation results. Uncropped images are shown in Supplementary Fig. S1f . ( g ) Immunoprecipitation experiments as in ( f ) in cells partially depleted of PRMT1 (see input, middle panel). The right panel outlines immunoprecipitation results. Uncropped images are shown in Supplementary Fig. S1g .

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

    15) Product Images from "Uterine NDRG2 expression is increased at implantation sites during early pregnancy in mice, and its down-regulation inhibits decidualization of mouse endometrial stromal cells"

    Article Title: Uterine NDRG2 expression is increased at implantation sites during early pregnancy in mice, and its down-regulation inhibits decidualization of mouse endometrial stromal cells

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/s12958-015-0047-7

    Down-regulation of NDRG2 expression in ESCs inhibits in vitro decidualization. Cultured mouse ESCs were transfected with NDRG2 -targeting siRNAs (100 nM) (a non-targeting siRNA was used as a control) at the time of plating. Quantitative PCR analyses of NDRG2 mRNA expression ( a ) and DTPRP mRNA expression ( b ) in ESCs transfected with NDRG2 -targeting siRNAs or non-targeting siRNAs at 24 h and 48 h. Western blot ( c ) and densitometric analyses ( d ) of NDRG2 protein levels in ESCs at 72 h after transfection. The relative fold induction of NDRG2 protein expression compared with its expression in non-siRNA-treated group is shown. The values represent the mean ± SEM, as determined from three separate experiments. *, significantly different ( P
    Figure Legend Snippet: Down-regulation of NDRG2 expression in ESCs inhibits in vitro decidualization. Cultured mouse ESCs were transfected with NDRG2 -targeting siRNAs (100 nM) (a non-targeting siRNA was used as a control) at the time of plating. Quantitative PCR analyses of NDRG2 mRNA expression ( a ) and DTPRP mRNA expression ( b ) in ESCs transfected with NDRG2 -targeting siRNAs or non-targeting siRNAs at 24 h and 48 h. Western blot ( c ) and densitometric analyses ( d ) of NDRG2 protein levels in ESCs at 72 h after transfection. The relative fold induction of NDRG2 protein expression compared with its expression in non-siRNA-treated group is shown. The values represent the mean ± SEM, as determined from three separate experiments. *, significantly different ( P

    Techniques Used: Expressing, In Vitro, Cell Culture, Transfection, Real-time Polymerase Chain Reaction, Western Blot

    16) Product Images from "BAD, a Proapoptotic Protein, Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells"

    Article Title: BAD, a Proapoptotic Protein, Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145780

    The figure shows the blotting of various proteins isolated from normal cells and those treated with various doses of either deguelin or transfected with siRNAs specific for silencing of ERK1/2. (A) Cells transfected with various doses of ERK-1 specific siRNA. (B) Cells transfected with various doses of ERK-2 specific siRNA. (C) BAD expression in cells transfected with either ERK-1 or ERK-2 siRNAs. (D) ERK-1 and ERK-2, (E) BAD, (F) Bcl-xl, (G) BAX and (H) Cytochrome-C levels in cells treated with various doses of deguelin. (I) The house-keeping control (β-actin).
    Figure Legend Snippet: The figure shows the blotting of various proteins isolated from normal cells and those treated with various doses of either deguelin or transfected with siRNAs specific for silencing of ERK1/2. (A) Cells transfected with various doses of ERK-1 specific siRNA. (B) Cells transfected with various doses of ERK-2 specific siRNA. (C) BAD expression in cells transfected with either ERK-1 or ERK-2 siRNAs. (D) ERK-1 and ERK-2, (E) BAD, (F) Bcl-xl, (G) BAX and (H) Cytochrome-C levels in cells treated with various doses of deguelin. (I) The house-keeping control (β-actin).

    Techniques Used: Isolation, Transfection, Expressing

    17) Product Images from "An Autocrine Cytokine/JAK/STAT-Signaling Induces Kynurenine Synthesis in Multidrug Resistant Human Cancer Cells"

    Article Title: An Autocrine Cytokine/JAK/STAT-Signaling Induces Kynurenine Synthesis in Multidrug Resistant Human Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126159

    The inhibition of the STAT1/STAT3 signaling reverses the kynurenine-dependent immunosuppression in multidrug resistant cells. A549/dx cells were grown for 48 h in fresh medium (CTRL), treated with a non-targeting scrambled siRNA (scr) or with a specific siRNAs pool targeting STAT1 or STAT3, respectively (si STAT1, si STAT3). Untreated chemosensitive A549 cells were used as control. A . The expression of STAT1, STAT3, IDO1, IDO2 and TDO was measured in whole cell lysates by Western blotting, 48 h after the transfection. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. B . The kynurenine levels in the cell culture supernatants were measured spectrophotometrically. Data are presented as means ± SD (n = 4). * p
    Figure Legend Snippet: The inhibition of the STAT1/STAT3 signaling reverses the kynurenine-dependent immunosuppression in multidrug resistant cells. A549/dx cells were grown for 48 h in fresh medium (CTRL), treated with a non-targeting scrambled siRNA (scr) or with a specific siRNAs pool targeting STAT1 or STAT3, respectively (si STAT1, si STAT3). Untreated chemosensitive A549 cells were used as control. A . The expression of STAT1, STAT3, IDO1, IDO2 and TDO was measured in whole cell lysates by Western blotting, 48 h after the transfection. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. B . The kynurenine levels in the cell culture supernatants were measured spectrophotometrically. Data are presented as means ± SD (n = 4). * p

    Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Cell Culture

    18) Product Images from "A Switch from Canonical to Noncanonical Wnt Signaling Mediates Drug Resistance in Colon Cancer Cells"

    Article Title: A Switch from Canonical to Noncanonical Wnt Signaling Mediates Drug Resistance in Colon Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027308

    HDACi-resistant HCT-R cells suppress Wnt/beta-catenin activity by overexpressing mediators of noncanonical Wnt signaling. (A) Representative western blot analyses of CC cells and their conditioned media after exposure to mock treatment (M) or 5 mM butyrate (B) for 19 hrs. (B and C). Silencing of WNT5A and ROR2 expression increases Wnt/catenin transcriptional activity of HCT-116 (B) and HCT-R (C) cells. Cells (50,000/well) were co-transfected with reporters for Wnt/catenin transcriptional activity (TopFlash or FopFlash, 50 ng/well), pRL-TK as a control for transfection efficiency, and control, ROR2 , or WNT5A siRNAs (10 pmol/well). Nucleic acids were introduced in the cells via a reverse transfection protocol with Lipofectamine 2000 in 96-well plates. At 31 hrs post-transfection, cells were exposed for 20 hrs to mock (m) or 5 mM butyrate (b) treatment. Wnt transcriptional activity was calculated as the ratio of the activity of TopFlash (with wild type Lef/Tcf binding sites) and FopFlash (with mutant Lef/Tcf binding sites). Each transfection was performed in duplicate wells; data represent the mean from results of three experiments. (D). Overexpression of WNT5A suppresses canonical Wnt transcriptional activity of HCT-116 cells. The co-transfection of HCT-116 cells (50,000/well) with the Wnt activity reporters TopFlash or FopFlash (50 ng per well), and pcDNANeo3.1 or WNT5A-expressing construct (150 ng per well) were carried out through a reverse transfection with Lipofectamine (0.7 µl/well) in 96-well plates. Post-transfection, cells were exposed to mock treatment (m) or 5 mM butyrate (b) for 20 hrs. Each transfection was performed in duplicate wells; data represent the mean from results of at least three experiments. (E) Suppression of ROR 2 expression increases the sensitivity of the HDACi-resistant HCT-R cells to the growth-suppressive effect of butyrate. HCT-R cells (10 6 ) were nucleofected with 175 pmol of ROR2 (Invitrogen) or control siRNA, and 500,000 cells were plated per well in a 6-well plate. At 24 hrs, cells were exposed to mock treatment (m) or 5 mM butyrate (b) for 17 hrs. At 48 hrs post-nucleofection, cells were plated at 100 or 200 per well in triplicates. Percent clonal growth was calculated as the number of colonies arising from a single cell suspension of 100 cells. The experiment was repeated three times and the results are the mean from the data of the three experiments. Statistically significant differences are noted by stars.
    Figure Legend Snippet: HDACi-resistant HCT-R cells suppress Wnt/beta-catenin activity by overexpressing mediators of noncanonical Wnt signaling. (A) Representative western blot analyses of CC cells and their conditioned media after exposure to mock treatment (M) or 5 mM butyrate (B) for 19 hrs. (B and C). Silencing of WNT5A and ROR2 expression increases Wnt/catenin transcriptional activity of HCT-116 (B) and HCT-R (C) cells. Cells (50,000/well) were co-transfected with reporters for Wnt/catenin transcriptional activity (TopFlash or FopFlash, 50 ng/well), pRL-TK as a control for transfection efficiency, and control, ROR2 , or WNT5A siRNAs (10 pmol/well). Nucleic acids were introduced in the cells via a reverse transfection protocol with Lipofectamine 2000 in 96-well plates. At 31 hrs post-transfection, cells were exposed for 20 hrs to mock (m) or 5 mM butyrate (b) treatment. Wnt transcriptional activity was calculated as the ratio of the activity of TopFlash (with wild type Lef/Tcf binding sites) and FopFlash (with mutant Lef/Tcf binding sites). Each transfection was performed in duplicate wells; data represent the mean from results of three experiments. (D). Overexpression of WNT5A suppresses canonical Wnt transcriptional activity of HCT-116 cells. The co-transfection of HCT-116 cells (50,000/well) with the Wnt activity reporters TopFlash or FopFlash (50 ng per well), and pcDNANeo3.1 or WNT5A-expressing construct (150 ng per well) were carried out through a reverse transfection with Lipofectamine (0.7 µl/well) in 96-well plates. Post-transfection, cells were exposed to mock treatment (m) or 5 mM butyrate (b) for 20 hrs. Each transfection was performed in duplicate wells; data represent the mean from results of at least three experiments. (E) Suppression of ROR 2 expression increases the sensitivity of the HDACi-resistant HCT-R cells to the growth-suppressive effect of butyrate. HCT-R cells (10 6 ) were nucleofected with 175 pmol of ROR2 (Invitrogen) or control siRNA, and 500,000 cells were plated per well in a 6-well plate. At 24 hrs, cells were exposed to mock treatment (m) or 5 mM butyrate (b) for 17 hrs. At 48 hrs post-nucleofection, cells were plated at 100 or 200 per well in triplicates. Percent clonal growth was calculated as the number of colonies arising from a single cell suspension of 100 cells. The experiment was repeated three times and the results are the mean from the data of the three experiments. Statistically significant differences are noted by stars.

    Techniques Used: Activity Assay, Western Blot, Expressing, Transfection, Binding Assay, Mutagenesis, Over Expression, Cotransfection, Construct

    HDACi-resistant HCT-R cells exhibit high expression levels of pAKT, a downstream effector of WNT5A and ROR2. (A) HDACi-resistant HCT-R cells express higher levels of pAKT than HDACi-sensitive HCT-116 cells. Cells were plated in 6-well plates at 450,000 cells per well, and at 24 hrs were exposed to mock (M) or 5 mM butyrate (B) treatment for 17 hrs. Total protein lysates were analyzed by western blot analyses, and levels of Ser473-phosphorylated AKT (pAKT), total AKT(AKT), and ACTIN were detected as described in Materials and Methods . (B) HCT-R cells are more resistant to 5-fluorouracil (5-FU) induced apoptosis than parental HDACi-sensitive HCT-116 cells. Cells were exposed to 1.5 mM 5-FU for 24 hrs, and apoptotic assays were performed as described in Materials and Methods . Percentage of live cells was calculated by dividing the number of live cells by the total number of analyzed cells. (C) Silencing of ROR2 expression in HCT-R cells decreases the levels of Ser473-phosphorylated AKT. One-million cells were nucleofected with control or ROR 2 siRNAs (175 pmol, Invitrogen), and plated at 500,000 per well in 6-well plates. At 24 hrs post-transfection, the cells were exposed to mock (M) or 5 mM butyrate (B) for 19 hrs. Total cell lysates were analyzed by western blot; a representative western blot is shown. The bar graph under the western blot image represents the quantification of pAKT levels in three independent experiments by densitometry, the difference between butyrate-treated cells are statistically significant (P
    Figure Legend Snippet: HDACi-resistant HCT-R cells exhibit high expression levels of pAKT, a downstream effector of WNT5A and ROR2. (A) HDACi-resistant HCT-R cells express higher levels of pAKT than HDACi-sensitive HCT-116 cells. Cells were plated in 6-well plates at 450,000 cells per well, and at 24 hrs were exposed to mock (M) or 5 mM butyrate (B) treatment for 17 hrs. Total protein lysates were analyzed by western blot analyses, and levels of Ser473-phosphorylated AKT (pAKT), total AKT(AKT), and ACTIN were detected as described in Materials and Methods . (B) HCT-R cells are more resistant to 5-fluorouracil (5-FU) induced apoptosis than parental HDACi-sensitive HCT-116 cells. Cells were exposed to 1.5 mM 5-FU for 24 hrs, and apoptotic assays were performed as described in Materials and Methods . Percentage of live cells was calculated by dividing the number of live cells by the total number of analyzed cells. (C) Silencing of ROR2 expression in HCT-R cells decreases the levels of Ser473-phosphorylated AKT. One-million cells were nucleofected with control or ROR 2 siRNAs (175 pmol, Invitrogen), and plated at 500,000 per well in 6-well plates. At 24 hrs post-transfection, the cells were exposed to mock (M) or 5 mM butyrate (B) for 19 hrs. Total cell lysates were analyzed by western blot; a representative western blot is shown. The bar graph under the western blot image represents the quantification of pAKT levels in three independent experiments by densitometry, the difference between butyrate-treated cells are statistically significant (P

    Techniques Used: Expressing, Western Blot, Transfection

    19) Product Images from "The Effect of ROCK on TNF-α-Induced CXCL8 Secretion by Intestinal Epithelial Cell Lines is Mediated Through MKK4 and JNK Signaling"

    Article Title: The Effect of ROCK on TNF-α-Induced CXCL8 Secretion by Intestinal Epithelial Cell Lines is Mediated Through MKK4 and JNK Signaling

    Journal: Cellular immunology

    doi: 10.1016/j.cellimm.2014.12.011

    RNAi knockdown of ROCK2, but not ROCK1, significantly inhibits TNF-induced CXCL8 secretion by Caco-2 cells. Caco-2 cells were cultured in FN-coated wells for 24 h before adding Control or specific siRNAs in to the cells. After 24 hours, some cells were
    Figure Legend Snippet: RNAi knockdown of ROCK2, but not ROCK1, significantly inhibits TNF-induced CXCL8 secretion by Caco-2 cells. Caco-2 cells were cultured in FN-coated wells for 24 h before adding Control or specific siRNAs in to the cells. After 24 hours, some cells were

    Techniques Used: Cell Culture

    20) Product Images from "P2X7 receptor mediates NLRP3-dependent IL-1β secretion and parasite proliferation in Toxoplasma gondii-infected human small intestinal epithelial cells"

    Article Title: P2X7 receptor mediates NLRP3-dependent IL-1β secretion and parasite proliferation in Toxoplasma gondii-infected human small intestinal epithelial cells

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-017-2573-y

    T. gondii -induced NLRP3 inflammasome activation inhibits intracellular parasite proliferation. a FHs 74 Int cells were transfected with NLRP3, Casp-1, ASC or control siRNAs and then infected with T. gondii at an MOI of 10 for 2 h. Cells were fixed and stained with Texas Red-X phalloidin to label F-actin (red), and the nuclei were stained with DAPI (blue). The number of T. gondii -infected cells and the total number of cells were counted, and the T. gondii infection rates were calculated. b siRNA-transfected FHs 74 Int cells were infected with T. gondii at an MOI of 10 for 8 h. Cells were fixed and stained with Texas Red-X phalloidin and DAPI. Intracellular parasites were revealed by fluorescence microscopy. All data shown are representative of three independent experiments. Scale-bars : 20 μm
    Figure Legend Snippet: T. gondii -induced NLRP3 inflammasome activation inhibits intracellular parasite proliferation. a FHs 74 Int cells were transfected with NLRP3, Casp-1, ASC or control siRNAs and then infected with T. gondii at an MOI of 10 for 2 h. Cells were fixed and stained with Texas Red-X phalloidin to label F-actin (red), and the nuclei were stained with DAPI (blue). The number of T. gondii -infected cells and the total number of cells were counted, and the T. gondii infection rates were calculated. b siRNA-transfected FHs 74 Int cells were infected with T. gondii at an MOI of 10 for 8 h. Cells were fixed and stained with Texas Red-X phalloidin and DAPI. Intracellular parasites were revealed by fluorescence microscopy. All data shown are representative of three independent experiments. Scale-bars : 20 μm

    Techniques Used: Activation Assay, Transfection, Infection, Staining, Fluorescence, Microscopy

    21) Product Images from "An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer"

    Article Title: An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer

    Journal: Nature medicine

    doi: 10.1038/nm.4369

    BRAF amp is sufficient to confer a selective advantage in the presence of ERKi treatment ( a ) Immunoblot analysis of cell lines derived from parental (1D) or ERK inhibitor-resistant (1D-EiR) PDX. ( b ) Immunoblot analysis of signaling intermediates in 1D and 1D-EiR cells treated for 1h with SCH984. ( c ) Cell viability at 72h after treatment. ( d ) Cell viability of 1D-EiR cells transfected with BRAF specific or control siRNAs followed by drug treatment as in c. ( e f ) A375 cells, engineered to express BRAF V600E under a doxycycline (dox)-induced promoter, were treated as shown (dox, 2μg/mL; SCH984, 500 nM) to determine the effect on signaling by immunoblotting (e) or viability (f). Withdrawal of dox after a 6-week stimulation restored sensitivity to the ERKi. A representative of at least two independent experiments is shown for the immunoblots in this figure. In viability experiments, n = 3, mean ± s.e.m.
    Figure Legend Snippet: BRAF amp is sufficient to confer a selective advantage in the presence of ERKi treatment ( a ) Immunoblot analysis of cell lines derived from parental (1D) or ERK inhibitor-resistant (1D-EiR) PDX. ( b ) Immunoblot analysis of signaling intermediates in 1D and 1D-EiR cells treated for 1h with SCH984. ( c ) Cell viability at 72h after treatment. ( d ) Cell viability of 1D-EiR cells transfected with BRAF specific or control siRNAs followed by drug treatment as in c. ( e f ) A375 cells, engineered to express BRAF V600E under a doxycycline (dox)-induced promoter, were treated as shown (dox, 2μg/mL; SCH984, 500 nM) to determine the effect on signaling by immunoblotting (e) or viability (f). Withdrawal of dox after a 6-week stimulation restored sensitivity to the ERKi. A representative of at least two independent experiments is shown for the immunoblots in this figure. In viability experiments, n = 3, mean ± s.e.m.

    Techniques Used: Derivative Assay, Transfection, Western Blot

    22) Product Images from "Epigenetic repression of miR-17 contributed to di(2-ethylhexyl) phthalate-triggered insulin resistance by targeting Keap1-Nrf2/miR-200a axis in skeletal muscle"

    Article Title: Epigenetic repression of miR-17 contributed to di(2-ethylhexyl) phthalate-triggered insulin resistance by targeting Keap1-Nrf2/miR-200a axis in skeletal muscle

    Journal: Theranostics

    doi: 10.7150/thno.45253

    Dnmt3a and lncRNA Malat1 cooperatively suppressed miR-17 in SkM. A-C . The mRNA (A) and protein (B-C) levels of DNA methyltransferase in SkM of DEHP-exposed mice (n = 4 mice per group qRT-PCR analysis and n = 3 mice per group for western blot). Gapdh was used as the loading control. D . The mRNA expression of Dnmt3a in DEHP-exposed C2C12 myotubes co-treated with 5-Aza (n = 3 independent experiments). Gapdh was used as the loading control. E . The expression of miR-17 in DEHP-exposed C2C12 myotubes co-treated with 5-Aza (n = 3 independent experiments). U6 was used to normalized miR-17 expression. F . The insulin-stimulated 2-DG uptake in DEHP-exposed C2C12 myotubes co-treated with 5-Aza (n = 3 independent experiments). G-H . The expression of lncRNA Malat1 in SkM of DEHP-exposed mice (G, n = 4 mice per group) and DEHP-treated C2C12 myotubes (H, n = 3 independent experiments). Gapdh was used as the loading control. I . The expression of lncRNA Malat1 in C2C12 myotubes transfected with lncRNA Malat1 siRNAs and treated with 25 µM DEHP (n = 3 independent experiments). Gapdh was used as the loading control. J-O . C2C12 myotubes were co-treated with 25 µM DEHP, 5-Aza, Txnip siRNAs or corresponding control (n = 3 independent experiments). J . The expression of miR-17 normalized by U6. K . The GSH content normalized to protein content in C2C12 myotubes. L . The GSSG content normalized to protein content in C2C12 myotubes. M . The calculated GSH/GSSG ratio. N . The H 2 O 2 content. O . The insulin-stimulated 2-DG uptake. P . The expression of miR-200a normalized by U6. Q . The proposed signaling pathway involved in DEHP-induced IR. All data were presented as the mean ± SEM. * P
    Figure Legend Snippet: Dnmt3a and lncRNA Malat1 cooperatively suppressed miR-17 in SkM. A-C . The mRNA (A) and protein (B-C) levels of DNA methyltransferase in SkM of DEHP-exposed mice (n = 4 mice per group qRT-PCR analysis and n = 3 mice per group for western blot). Gapdh was used as the loading control. D . The mRNA expression of Dnmt3a in DEHP-exposed C2C12 myotubes co-treated with 5-Aza (n = 3 independent experiments). Gapdh was used as the loading control. E . The expression of miR-17 in DEHP-exposed C2C12 myotubes co-treated with 5-Aza (n = 3 independent experiments). U6 was used to normalized miR-17 expression. F . The insulin-stimulated 2-DG uptake in DEHP-exposed C2C12 myotubes co-treated with 5-Aza (n = 3 independent experiments). G-H . The expression of lncRNA Malat1 in SkM of DEHP-exposed mice (G, n = 4 mice per group) and DEHP-treated C2C12 myotubes (H, n = 3 independent experiments). Gapdh was used as the loading control. I . The expression of lncRNA Malat1 in C2C12 myotubes transfected with lncRNA Malat1 siRNAs and treated with 25 µM DEHP (n = 3 independent experiments). Gapdh was used as the loading control. J-O . C2C12 myotubes were co-treated with 25 µM DEHP, 5-Aza, Txnip siRNAs or corresponding control (n = 3 independent experiments). J . The expression of miR-17 normalized by U6. K . The GSH content normalized to protein content in C2C12 myotubes. L . The GSSG content normalized to protein content in C2C12 myotubes. M . The calculated GSH/GSSG ratio. N . The H 2 O 2 content. O . The insulin-stimulated 2-DG uptake. P . The expression of miR-200a normalized by U6. Q . The proposed signaling pathway involved in DEHP-induced IR. All data were presented as the mean ± SEM. * P

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

    23) Product Images from "Interferon-induced transmembrane protein 1 (IFITM1) overexpression enhances the aggressive phenotype of SUM149 inflammatory breast cancer cells in a signal transducer and activator of transcription 2 (STAT2)-dependent manner"

    Article Title: Interferon-induced transmembrane protein 1 (IFITM1) overexpression enhances the aggressive phenotype of SUM149 inflammatory breast cancer cells in a signal transducer and activator of transcription 2 (STAT2)-dependent manner

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-016-0683-7

    Effects of small interfering RNA (siRNA) knockdown of interferon-induced transmembrane protein 1 (IFITM1) on proliferation and tumorigenic potential of SUM149 cells. a Western blot analysis of SUM149 cells showing the protein levels of IFITM1. The IFITM1 gene was knocked down using three separate siRNAs (siRNA 1, siRNA2, and siRNA 3), and the control samples were transfected with a negative control siRNA (siCon) for 72 h. b Cell proliferation after 72 h of IFITM1 knockdown with three separate siRNAs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess cell proliferation. Bars represent mean ± standard deviation (SD). ** P
    Figure Legend Snippet: Effects of small interfering RNA (siRNA) knockdown of interferon-induced transmembrane protein 1 (IFITM1) on proliferation and tumorigenic potential of SUM149 cells. a Western blot analysis of SUM149 cells showing the protein levels of IFITM1. The IFITM1 gene was knocked down using three separate siRNAs (siRNA 1, siRNA2, and siRNA 3), and the control samples were transfected with a negative control siRNA (siCon) for 72 h. b Cell proliferation after 72 h of IFITM1 knockdown with three separate siRNAs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess cell proliferation. Bars represent mean ± standard deviation (SD). ** P

    Techniques Used: Small Interfering RNA, Western Blot, Transfection, Negative Control, Standard Deviation

    24) Product Images from "Coxsackievirus-Induced miR-21 Disrupts Cardiomyocyte Interactions via the Downregulation of Intercalated Disk Components"

    Article Title: Coxsackievirus-Induced miR-21 Disrupts Cardiomyocyte Interactions via the Downregulation of Intercalated Disk Components

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004070

    K108 and K406 are essential sites for desmin ubiquitination and degradation. HL-1 cells were co-transfected with miRNA mimics, siRNAs and desmin plasmids (wild type or mutants) or infected with CVB3 as indicated. Cellular proteins were collected for detection of desmin expression level using WB. The intensities of the bands were measured by using ImageJ and the signal ratios were listed below ( A ). The proteins were also pulled down by an anti-desmin antibody and subjected to WB detection of K48-linked polyubiquitin chains ( B ).
    Figure Legend Snippet: K108 and K406 are essential sites for desmin ubiquitination and degradation. HL-1 cells were co-transfected with miRNA mimics, siRNAs and desmin plasmids (wild type or mutants) or infected with CVB3 as indicated. Cellular proteins were collected for detection of desmin expression level using WB. The intensities of the bands were measured by using ImageJ and the signal ratios were listed below ( A ). The proteins were also pulled down by an anti-desmin antibody and subjected to WB detection of K48-linked polyubiquitin chains ( B ).

    Techniques Used: Transfection, Infection, Expressing, Western Blot

    YOD1 regulates desmin degradation during CVB3 infection. ( A ) Silencing YOD1 expression downregulates desmin. HL-1 cells were transfected and infected as indicated. YOD1 and desmin proteins were detected by WB. ( B ) YOD1 siRNA enhances desmin ubiquitination. HL-1 cells were transfected and infected as indicated. Desmin was immunoprecipitated and analyzed by WB detection of ubiquitin. ( C ) Proteasome inhibitor blocks the YOD1 siRNA-mediated desmin degradation. HL-1 cells were transfected and treated with DMSO or MG132 as indicated. Proteins were extracted to detect the desmin levels. ( D ) Overexpression of YOD1 inhibits desmin degradation. HL-1 cells were divided into two groups: one group was transfected with empty vector or YOD1-expressing vector and then infected with CVB3; the other group was co-transfected with miRNA mimics and YOD1-expressing plasmid or empty vector. Desmin and YOD1 expression levels were evaluated by WB. ( E ) YOD1 siRNA disrupts desmosome structure. HL-1 cells were transfected with scrambled siRNAs or YOD1 siRNAs and the desmosome structures were analyzed by EM. Three representative views were listed for each sample and desmosomes were indicated with black arrows. For each sample, 100 cells were analyzed and the numbers of desmosomes observed were indicated. Magnification: 37000×. Bar: 0.5 µm.
    Figure Legend Snippet: YOD1 regulates desmin degradation during CVB3 infection. ( A ) Silencing YOD1 expression downregulates desmin. HL-1 cells were transfected and infected as indicated. YOD1 and desmin proteins were detected by WB. ( B ) YOD1 siRNA enhances desmin ubiquitination. HL-1 cells were transfected and infected as indicated. Desmin was immunoprecipitated and analyzed by WB detection of ubiquitin. ( C ) Proteasome inhibitor blocks the YOD1 siRNA-mediated desmin degradation. HL-1 cells were transfected and treated with DMSO or MG132 as indicated. Proteins were extracted to detect the desmin levels. ( D ) Overexpression of YOD1 inhibits desmin degradation. HL-1 cells were divided into two groups: one group was transfected with empty vector or YOD1-expressing vector and then infected with CVB3; the other group was co-transfected with miRNA mimics and YOD1-expressing plasmid or empty vector. Desmin and YOD1 expression levels were evaluated by WB. ( E ) YOD1 siRNA disrupts desmosome structure. HL-1 cells were transfected with scrambled siRNAs or YOD1 siRNAs and the desmosome structures were analyzed by EM. Three representative views were listed for each sample and desmosomes were indicated with black arrows. For each sample, 100 cells were analyzed and the numbers of desmosomes observed were indicated. Magnification: 37000×. Bar: 0.5 µm.

    Techniques Used: Infection, Expressing, Transfection, Western Blot, Immunoprecipitation, Over Expression, Plasmid Preparation

    25) Product Images from "Novel miR-5088-5p promotes malignancy of breast cancer by inhibiting DBC2"

    Article Title: Novel miR-5088-5p promotes malignancy of breast cancer by inhibiting DBC2

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2021.05.004

    Fyn promotes biogenesis of miR-5088-5p by inhibiting its methylation (A) Levels of miR-5088-5p expression were measured in MCF-7 and MDA-MB-231 cells, which were transfected with siRNAs against Lyn, Fyn, and EGFR (10 nM) and treated with inhibitors of PI3K (LY294002), p38 (SB203580), and JNK (SP600125) (20 nM) by qRT-PCR. (B) Fyn expression was confirmed in public microarray data of breast cancer patients (normal, n = 14; breast cancer patients, n = 11; RePORTER: Hs.169370.1.A2_3p_a_at; https://www.oncomine.org/resource/login.html ). (C) The pattern of Fyn expression was confirmed in four types of breast cancer patients using TCGA dataset (luminal A, 619 cases; luminal B, 603 cases; Her2 + , 188 cases; triple-negative type, 290 cases). (D and E) Primary, precursor, and mature forms of miR-5088-5p were identified in MCF-7 and MDA-MB-231 after knockdown (D) or overexpression (E) of Fyn using siRNA or overexpressing vector, respectively. (F) After transfection with miR-5088-5p inhibitor and/or Fyn-overexpressing vectors in MCF-7 and MDA-MB-231 cells, the expression of EMT- and stemness-related proteins was measured by western blot analysis. The data are presented as the mean ± SD. ∗p
    Figure Legend Snippet: Fyn promotes biogenesis of miR-5088-5p by inhibiting its methylation (A) Levels of miR-5088-5p expression were measured in MCF-7 and MDA-MB-231 cells, which were transfected with siRNAs against Lyn, Fyn, and EGFR (10 nM) and treated with inhibitors of PI3K (LY294002), p38 (SB203580), and JNK (SP600125) (20 nM) by qRT-PCR. (B) Fyn expression was confirmed in public microarray data of breast cancer patients (normal, n = 14; breast cancer patients, n = 11; RePORTER: Hs.169370.1.A2_3p_a_at; https://www.oncomine.org/resource/login.html ). (C) The pattern of Fyn expression was confirmed in four types of breast cancer patients using TCGA dataset (luminal A, 619 cases; luminal B, 603 cases; Her2 + , 188 cases; triple-negative type, 290 cases). (D and E) Primary, precursor, and mature forms of miR-5088-5p were identified in MCF-7 and MDA-MB-231 after knockdown (D) or overexpression (E) of Fyn using siRNA or overexpressing vector, respectively. (F) After transfection with miR-5088-5p inhibitor and/or Fyn-overexpressing vectors in MCF-7 and MDA-MB-231 cells, the expression of EMT- and stemness-related proteins was measured by western blot analysis. The data are presented as the mean ± SD. ∗p

    Techniques Used: Methylation, Expressing, Multiple Displacement Amplification, Transfection, Quantitative RT-PCR, Microarray, Over Expression, Plasmid Preparation, Western Blot

    26) Product Images from "Sphingosine-1-Phosphate Promotes Extravillous Trophoblast Cell Invasion by Activating MEK/ERK/MMP-2 Signaling Pathways via S1P/S1PR1 Axis Activation"

    Article Title: Sphingosine-1-Phosphate Promotes Extravillous Trophoblast Cell Invasion by Activating MEK/ERK/MMP-2 Signaling Pathways via S1P/S1PR1 Axis Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106725

    S1P-induced MMP-2 upregulation and signaling pathways is mediated through S1PR1 receptor. (A) HTR8/SVneo cells were pretreated for 30 minutes with the S1PR1 and S1PR3 receptor antagonist VPC23019 (1 µM) before stimulation with S1P (10 nM). (B) HTR8/SVneo were pretreated for 30 minutes with the selective S1PR3 receptor antagonist CAY10444 (1 µM) before stimulation with S1P. (C) HTR8/SVneo were pretreated for 30 minutes with VPC23019 (1 µM) before stimulation with the selective S1PR1 receptor agonist SEW2871 (1 µM). (D) Cells were transfected with control siRNA or siRNAs targeting S1PR1 (50 pmol). Knockdown of S1P1 was confirmed by Real-time PCR. (E) Induction of MMP-2 by S1P was disrupted in siRNA transfected cells. (F) The levels of activated MEK1/2, ERK1/2 were determined by western blot using phospho-specific antibodies (p-MEK1/2, p-ERK1/2, respectively). N = 3 performed in triplicate and values were expressed as mean ±SEM with p
    Figure Legend Snippet: S1P-induced MMP-2 upregulation and signaling pathways is mediated through S1PR1 receptor. (A) HTR8/SVneo cells were pretreated for 30 minutes with the S1PR1 and S1PR3 receptor antagonist VPC23019 (1 µM) before stimulation with S1P (10 nM). (B) HTR8/SVneo were pretreated for 30 minutes with the selective S1PR3 receptor antagonist CAY10444 (1 µM) before stimulation with S1P. (C) HTR8/SVneo were pretreated for 30 minutes with VPC23019 (1 µM) before stimulation with the selective S1PR1 receptor agonist SEW2871 (1 µM). (D) Cells were transfected with control siRNA or siRNAs targeting S1PR1 (50 pmol). Knockdown of S1P1 was confirmed by Real-time PCR. (E) Induction of MMP-2 by S1P was disrupted in siRNA transfected cells. (F) The levels of activated MEK1/2, ERK1/2 were determined by western blot using phospho-specific antibodies (p-MEK1/2, p-ERK1/2, respectively). N = 3 performed in triplicate and values were expressed as mean ±SEM with p

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

    27) Product Images from "Nucleolin Interacts with the Feline Calicivirus 3? Untranslated Region and the Protease-Polymerase NS6 and NS7 Proteins, Playing a Role in Virus Replication ▿"

    Article Title: Nucleolin Interacts with the Feline Calicivirus 3? Untranslated Region and the Protease-Polymerase NS6 and NS7 Proteins, Playing a Role in Virus Replication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01878-10

    Effect of nucleolin silencing on the expression of FCV NS6/7. (A) CrFK cells were either left untreated (lane 1), treated with siPort reagent alone (lane 2), or transfected with a nontargeting siRNA (lane 3) or with two siRNAs specific for feline nucleolin (lane 4). The levels of nucleolin expression were evaluated by Western blotting. (B) CrFK cells were either not treated (lanes 1 and 2) or transfected with a nontargeting siRNA (lanes 3 and 4) or with two siRNAs specific for feline nucleolin (lanes 5 and 6) for 48 h. Cells were then either mock infected or infected with FCV (MOI 10) for 5 h, and the expression levels of nucleolin, FCV NS6/7, NS3 or p39, and hnRNP A1 (A1), used as the loading control, were evaluated by Western blotting in FCV-infected (lanes 2, 4, and 6) and uninfected (lanes 1, 3, and 5) cells. (C) Nucleolin and NS6/7 band intensities were quantified by densitometric analysis using Quantity-One software (Bio-Rad) and are expressed as the percentage in relation to the hnRNP A1 expression level. Error bars represent the standard deviation from three independent experiments. (D) Forty-eight hours after siRNA transfection, cell viability of untreated and nucleolin siRNA-treated cells was measured as described in Materials and Methods. Error bars show standard deviations.
    Figure Legend Snippet: Effect of nucleolin silencing on the expression of FCV NS6/7. (A) CrFK cells were either left untreated (lane 1), treated with siPort reagent alone (lane 2), or transfected with a nontargeting siRNA (lane 3) or with two siRNAs specific for feline nucleolin (lane 4). The levels of nucleolin expression were evaluated by Western blotting. (B) CrFK cells were either not treated (lanes 1 and 2) or transfected with a nontargeting siRNA (lanes 3 and 4) or with two siRNAs specific for feline nucleolin (lanes 5 and 6) for 48 h. Cells were then either mock infected or infected with FCV (MOI 10) for 5 h, and the expression levels of nucleolin, FCV NS6/7, NS3 or p39, and hnRNP A1 (A1), used as the loading control, were evaluated by Western blotting in FCV-infected (lanes 2, 4, and 6) and uninfected (lanes 1, 3, and 5) cells. (C) Nucleolin and NS6/7 band intensities were quantified by densitometric analysis using Quantity-One software (Bio-Rad) and are expressed as the percentage in relation to the hnRNP A1 expression level. Error bars represent the standard deviation from three independent experiments. (D) Forty-eight hours after siRNA transfection, cell viability of untreated and nucleolin siRNA-treated cells was measured as described in Materials and Methods. Error bars show standard deviations.

    Techniques Used: Expressing, Transfection, Western Blot, Infection, Software, Standard Deviation

    28) Product Images from "Activation of Peroxisome Proliferator-activated Receptor ? (PPAR?) Suppresses Hypoxia-inducible Factor-1? (HIF-1?) Signaling in Cancer Cells *"

    Article Title: Activation of Peroxisome Proliferator-activated Receptor ? (PPAR?) Suppresses Hypoxia-inducible Factor-1? (HIF-1?) Signaling in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.367367

    Knockdown of pVHL attenuates clofibrate-induced suppression of HIF-1α signaling. A , left panel , MCF-7 cells were transfected with a pool of pVHL siRNAs (50–100 pmol/ml; siRNA-a, siRNA-b, and siRNA-c) for 48 h. Right panel , MCF-7 cells
    Figure Legend Snippet: Knockdown of pVHL attenuates clofibrate-induced suppression of HIF-1α signaling. A , left panel , MCF-7 cells were transfected with a pool of pVHL siRNAs (50–100 pmol/ml; siRNA-a, siRNA-b, and siRNA-c) for 48 h. Right panel , MCF-7 cells

    Techniques Used: Transfection

    29) Product Images from "Interferon-induced transmembrane protein 1 (IFITM1) overexpression enhances the aggressive phenotype of SUM149 inflammatory breast cancer cells in a signal transducer and activator of transcription 2 (STAT2)-dependent manner"

    Article Title: Interferon-induced transmembrane protein 1 (IFITM1) overexpression enhances the aggressive phenotype of SUM149 inflammatory breast cancer cells in a signal transducer and activator of transcription 2 (STAT2)-dependent manner

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-016-0683-7

    Effects of small interfering RNA (siRNA) knockdown of interferon-induced transmembrane protein 1 (IFITM1) on proliferation and tumorigenic potential of SUM149 cells. a Western blot analysis of SUM149 cells showing the protein levels of IFITM1. The IFITM1 gene was knocked down using three separate siRNAs (siRNA 1, siRNA2, and siRNA 3), and the control samples were transfected with a negative control siRNA (siCon) for 72 h. b Cell proliferation after 72 h of IFITM1 knockdown with three separate siRNAs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess cell proliferation. Bars represent mean ± standard deviation (SD). ** P
    Figure Legend Snippet: Effects of small interfering RNA (siRNA) knockdown of interferon-induced transmembrane protein 1 (IFITM1) on proliferation and tumorigenic potential of SUM149 cells. a Western blot analysis of SUM149 cells showing the protein levels of IFITM1. The IFITM1 gene was knocked down using three separate siRNAs (siRNA 1, siRNA2, and siRNA 3), and the control samples were transfected with a negative control siRNA (siCon) for 72 h. b Cell proliferation after 72 h of IFITM1 knockdown with three separate siRNAs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess cell proliferation. Bars represent mean ± standard deviation (SD). ** P

    Techniques Used: Small Interfering RNA, Western Blot, Transfection, Negative Control, Standard Deviation

    30) Product Images from "Identification of a wide spectrum of ciliary gene mutations in nonsyndromic biliary atresia patients implicates ciliary dysfunction as a novel disease mechanism"

    Article Title: Identification of a wide spectrum of ciliary gene mutations in nonsyndromic biliary atresia patients implicates ciliary dysfunction as a novel disease mechanism

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2021.103530

    Knockdown of PCNT, KIF 3B and TTC17 resulted in reduced number of cilia in cells. (a) Examination of the knockdown efficiency by siRNAs in human fibroblasts by real-time quantitative PCR assay using GAPDH as internal controls. Three independent experiments were performed and data represented as means ± standard deviation (SD). *** p
    Figure Legend Snippet: Knockdown of PCNT, KIF 3B and TTC17 resulted in reduced number of cilia in cells. (a) Examination of the knockdown efficiency by siRNAs in human fibroblasts by real-time quantitative PCR assay using GAPDH as internal controls. Three independent experiments were performed and data represented as means ± standard deviation (SD). *** p

    Techniques Used: Real-time Polymerase Chain Reaction, Standard Deviation

    31) Product Images from "IFITM Proteins Inhibit Entry Driven by the MERS-Coronavirus Spike Protein: Evidence for Cholesterol-Independent Mechanisms"

    Article Title: IFITM Proteins Inhibit Entry Driven by the MERS-Coronavirus Spike Protein: Evidence for Cholesterol-Independent Mechanisms

    Journal: Viruses

    doi: 10.3390/v6093683

    Inhibition of S protein-driven cell entry by IFITM proteins. ( A ) 293T cells, transfected to express the viral receptors and transduced to express IFITM1, 2, 3, or cat as control, were transduced with infectivity-normalized retroviral vectors bearing the S proteins of the globally circulating human coronaviruses NL63 and 229E as well as the S proteins of the emerging SARS- and MERS-CoV. Transduction efficiency was determined at 72 h post inoculation by measuring luciferase activity in cell lysates. Transduction of control cells was set as 100%. The average of three independent experiments carried out with triplicate samples is shown, error bars indicate SEM. Statistical significance was calculated using one tailed, paired t -test. * p ≤ 0.05; ** p ≤ 0.01; ( B ) A549 wild type cells (control) and A549 cells transduced to stably express IFITM3 were transfected with siRNA directed against IFITM3. Scrambled siRNA were used as a control. Knockdown of IFITM3 expression was analyzed by Western blot. Detection of β-actin served as a loading control; ( C ) A549 control cells or A549-IFITM3 cells were transfected with siRNA directed against IFITM3 or scrambled siRNA as control. Cells were then transduced with the retroviral vectors described in (A). Transduction efficiency was analyzed at 72 h post transduction. Transduction of cells transfected with the scrambled siRNA was set as 1. The average of three independent experiments performed with triplicate samples is shown; error bars indicate SEM. The Welch-Test for independent samples was used to determine whether the effects of the siRNAs on transduction of A549 control and A549-IFITM3 cells were significantly different. * p ≤ 0.05; ** p ≤ 0.01.
    Figure Legend Snippet: Inhibition of S protein-driven cell entry by IFITM proteins. ( A ) 293T cells, transfected to express the viral receptors and transduced to express IFITM1, 2, 3, or cat as control, were transduced with infectivity-normalized retroviral vectors bearing the S proteins of the globally circulating human coronaviruses NL63 and 229E as well as the S proteins of the emerging SARS- and MERS-CoV. Transduction efficiency was determined at 72 h post inoculation by measuring luciferase activity in cell lysates. Transduction of control cells was set as 100%. The average of three independent experiments carried out with triplicate samples is shown, error bars indicate SEM. Statistical significance was calculated using one tailed, paired t -test. * p ≤ 0.05; ** p ≤ 0.01; ( B ) A549 wild type cells (control) and A549 cells transduced to stably express IFITM3 were transfected with siRNA directed against IFITM3. Scrambled siRNA were used as a control. Knockdown of IFITM3 expression was analyzed by Western blot. Detection of β-actin served as a loading control; ( C ) A549 control cells or A549-IFITM3 cells were transfected with siRNA directed against IFITM3 or scrambled siRNA as control. Cells were then transduced with the retroviral vectors described in (A). Transduction efficiency was analyzed at 72 h post transduction. Transduction of cells transfected with the scrambled siRNA was set as 1. The average of three independent experiments performed with triplicate samples is shown; error bars indicate SEM. The Welch-Test for independent samples was used to determine whether the effects of the siRNAs on transduction of A549 control and A549-IFITM3 cells were significantly different. * p ≤ 0.05; ** p ≤ 0.01.

    Techniques Used: Inhibition, Transfection, Transduction, Infection, Luciferase, Activity Assay, One-tailed Test, Stable Transfection, Expressing, Western Blot

    32) Product Images from "A cooperative activation loop among SWI/SNF, ?-H2AX and H3 acetylation for DNA double-strand break repair"

    Article Title: A cooperative activation loop among SWI/SNF, ?-H2AX and H3 acetylation for DNA double-strand break repair

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2010.27

    S139ph is required for the acetylation of H3 on γ-H2AX nucleosomes. ( A ; ac, acetylation; me, monomethylation; me2, dimethylation; ph, phosphorylation. ( B ) Vector and f-H2AX cells were transfected with either the siRNAs specific for BRG1 or hBrm (B/h), or non-specific control siRNA. Cells were collected at 1 h after 10-Gy IR for the analysis of BRG1 and hBrm knockdown by immunoblot with specific antibodies; α-tubulin was also analysed for internal control. ( C ) Flag-tagged nucleosomes were immunoprecipitated from the aliquots of the cells prepared in ( B ) were analysed for the indicated modifications of H3 by immunoblot using specific antibodies. The fold reduction of H3 acetylation on precipitated f-H2AX nucleosomes by SWI/SNF knockdown was calculated as per in ( A ) and shown at the right side of the corresponding gel. ( D ) Cells were transfected with either control or BRG1/hBrm-specific siRNAs, and at 1 h after 10-Gy IR, whole cell lysates were prepared for the analysis of BRG1 and hBrm expression, and acid-extracted histones for the analysis of H3 acetylation as indicated.
    Figure Legend Snippet: S139ph is required for the acetylation of H3 on γ-H2AX nucleosomes. ( A ; ac, acetylation; me, monomethylation; me2, dimethylation; ph, phosphorylation. ( B ) Vector and f-H2AX cells were transfected with either the siRNAs specific for BRG1 or hBrm (B/h), or non-specific control siRNA. Cells were collected at 1 h after 10-Gy IR for the analysis of BRG1 and hBrm knockdown by immunoblot with specific antibodies; α-tubulin was also analysed for internal control. ( C ) Flag-tagged nucleosomes were immunoprecipitated from the aliquots of the cells prepared in ( B ) were analysed for the indicated modifications of H3 by immunoblot using specific antibodies. The fold reduction of H3 acetylation on precipitated f-H2AX nucleosomes by SWI/SNF knockdown was calculated as per in ( A ) and shown at the right side of the corresponding gel. ( D ) Cells were transfected with either control or BRG1/hBrm-specific siRNAs, and at 1 h after 10-Gy IR, whole cell lysates were prepared for the analysis of BRG1 and hBrm expression, and acid-extracted histones for the analysis of H3 acetylation as indicated.

    Techniques Used: Plasmid Preparation, Transfection, Immunoprecipitation, Expressing

    BRG1 binding to γ-H2AX nucleosomes is important for S139ph and DSB repair. ( A ) Cells were cotransfected with non-specific (lane 1) or BRG1-specific siRNAs (lanes 2 and 3), plus either empty vectors (lanes 1 and 2) or the expression vectors for HA-tagged siRNA-resistant BRG1 (BRG1 R ) (lane 3). Whole cell lysates were analysed for the expression of BRG1 and BRG1 R by immunoblot. ( B ) Cells were cotransfected with non-specific (lanes 1 and 2) or BRG1-specific siRNAs (lanes 3–5), plus either empty vectors (lanes 1–3) or the expression vectors for BRG1 R (lane 4) or BRD-deleted BRG1 R (BRG1 R ΔBRG1, lane 5). At 1 h after untreated (lane 1) or 10-Gy IR (lanes 2–5), cells were collected to prepare whole cell lysates and acid-extracted histones for immunoblots with anti-BRG1 or anti-γ-H2AX antibodies, respectively; α-tubulin and H2A were also analysed for loading control. ( C ) Cells were transfected as described in lanes 2–5 of ( B ) and exposed to 2-Gy IR. After 1 h, cells were fixed and dually stained with anti-BRG1 or anti-γ-H2AX antibodies before confocal images were captured. Average number of γ-H2AX foci per cell was depicted as graph by counting at least 50 cells. The error bar indicates mean±s.d. of three independent experiments. ( D ) Representative confocal images from the experiments in ( C ) are shown. ( E ) Cells transfected as per in ( D ) were untreated (0 Gy) or exposed to 1–5 Gy IR before the viability was determined by colony formation assays with triplicates per sample. The graph shows average number of colonies with mean±s.d. of four independent experiments. ( F ) Cells were transfected with empty or Myc-BRD expression vectors. At 1 h after 10-Gy IR, cells were collected to prepare whole cell lysates and acid-extracted histones for immunoblots with anti-Myc or anti-γ-H2AX antibodies, respectively; α-tubulin and H2A were also analysed for loading control. ( G ) Cells transfected with empty, Myc-BRD or Myc-BRG1(588–748) vectors were irradiated by 2 Gy, and after 1 h, cells were fixed for dual staining with the antibodies against Myc or γ-H2AX. The Myc-BRG1(588–748) vector expresses the sequences of 588–748 amino acids of BRG1, outside the BRD, and was used as a control. Average number of γ-H2AX foci per cell was depicted as graph by counting at least 50 each of untransfected and transfected cells. The error bar indicates mean±s.d. of three independent experiments. ( H ) Representative confocal images from the experiments in ( G ) are shown. ( I ) Cells were transfected with indicated vectors, and after irradiation, cells were subjected to colony formation assays as described in ( E ). The graph shows average number of colonies with mean±s.d. of three independent experiments.
    Figure Legend Snippet: BRG1 binding to γ-H2AX nucleosomes is important for S139ph and DSB repair. ( A ) Cells were cotransfected with non-specific (lane 1) or BRG1-specific siRNAs (lanes 2 and 3), plus either empty vectors (lanes 1 and 2) or the expression vectors for HA-tagged siRNA-resistant BRG1 (BRG1 R ) (lane 3). Whole cell lysates were analysed for the expression of BRG1 and BRG1 R by immunoblot. ( B ) Cells were cotransfected with non-specific (lanes 1 and 2) or BRG1-specific siRNAs (lanes 3–5), plus either empty vectors (lanes 1–3) or the expression vectors for BRG1 R (lane 4) or BRD-deleted BRG1 R (BRG1 R ΔBRG1, lane 5). At 1 h after untreated (lane 1) or 10-Gy IR (lanes 2–5), cells were collected to prepare whole cell lysates and acid-extracted histones for immunoblots with anti-BRG1 or anti-γ-H2AX antibodies, respectively; α-tubulin and H2A were also analysed for loading control. ( C ) Cells were transfected as described in lanes 2–5 of ( B ) and exposed to 2-Gy IR. After 1 h, cells were fixed and dually stained with anti-BRG1 or anti-γ-H2AX antibodies before confocal images were captured. Average number of γ-H2AX foci per cell was depicted as graph by counting at least 50 cells. The error bar indicates mean±s.d. of three independent experiments. ( D ) Representative confocal images from the experiments in ( C ) are shown. ( E ) Cells transfected as per in ( D ) were untreated (0 Gy) or exposed to 1–5 Gy IR before the viability was determined by colony formation assays with triplicates per sample. The graph shows average number of colonies with mean±s.d. of four independent experiments. ( F ) Cells were transfected with empty or Myc-BRD expression vectors. At 1 h after 10-Gy IR, cells were collected to prepare whole cell lysates and acid-extracted histones for immunoblots with anti-Myc or anti-γ-H2AX antibodies, respectively; α-tubulin and H2A were also analysed for loading control. ( G ) Cells transfected with empty, Myc-BRD or Myc-BRG1(588–748) vectors were irradiated by 2 Gy, and after 1 h, cells were fixed for dual staining with the antibodies against Myc or γ-H2AX. The Myc-BRG1(588–748) vector expresses the sequences of 588–748 amino acids of BRG1, outside the BRD, and was used as a control. Average number of γ-H2AX foci per cell was depicted as graph by counting at least 50 each of untransfected and transfected cells. The error bar indicates mean±s.d. of three independent experiments. ( H ) Representative confocal images from the experiments in ( G ) are shown. ( I ) Cells were transfected with indicated vectors, and after irradiation, cells were subjected to colony formation assays as described in ( E ). The graph shows average number of colonies with mean±s.d. of three independent experiments.

    Techniques Used: Binding Assay, Expressing, Western Blot, Transfection, Staining, Irradiation, Plasmid Preparation

    33) Product Images from "Analysis of Ebola Virus Entry Into Macrophages"

    Article Title: Analysis of Ebola Virus Entry Into Macrophages

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiv140

    Niemann-Pick C1 (NPC1), but not Axl, T cell immunoglobulin and mucin domain-1 (TIM-1), or mannose-specific lectins, promotes Ebola virus (EBOV) glycoprotein (GP)–mediated transduction of macrophages. A , Total RNA isolated from the indicated cell lines and human monocyte-derived macrophages (MDMs) was treated with deoxyribonuclease (DNase) and analyzed by quantitative reverse-transcriptase polymerase chain reaction for the presence of Axl, TIM-1, and NPC1 transcripts. β-Glucuronidase (GUSB) served as a housekeeping reference. Results represent the mean of 3 independent analyses; error bars indicate standard deviations. B , Human MDMs were transfected with the respective siRNAs and transduced with the indicated pseudotypes. Luciferase activities in cell lysates were determined 72 hours after transduction. Comparable results were obtained in 8 independent experiments using different pseudotype preparations. C , MDMs were incubated with the indicated concentrations of U18666A, transduced with the indicated pseudotypes and luciferase activity analyzed 72 hours after transduction. Results of a single representative experiment performed with triplicate samples are shown; 2 independent experiments yielded similar results. D , 293T cells ( left ) transfected with empty plasmid (pcDNA) or dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin (DC-SIGN) encoding plasmid as well as MDMs ( right ) were incubated with 200 µg/mL mannan or solvent (control) and then transduced with the indicated pseudotypes. Transduction efficiency was analyzed by quantification of luciferase activities in cell lysates. Results of a representative experiment performed with triplicate samples are shown; an independent experiment yielded comparable results. Statistical significance was calculated using the 2-tailed Student t test. * P
    Figure Legend Snippet: Niemann-Pick C1 (NPC1), but not Axl, T cell immunoglobulin and mucin domain-1 (TIM-1), or mannose-specific lectins, promotes Ebola virus (EBOV) glycoprotein (GP)–mediated transduction of macrophages. A , Total RNA isolated from the indicated cell lines and human monocyte-derived macrophages (MDMs) was treated with deoxyribonuclease (DNase) and analyzed by quantitative reverse-transcriptase polymerase chain reaction for the presence of Axl, TIM-1, and NPC1 transcripts. β-Glucuronidase (GUSB) served as a housekeeping reference. Results represent the mean of 3 independent analyses; error bars indicate standard deviations. B , Human MDMs were transfected with the respective siRNAs and transduced with the indicated pseudotypes. Luciferase activities in cell lysates were determined 72 hours after transduction. Comparable results were obtained in 8 independent experiments using different pseudotype preparations. C , MDMs were incubated with the indicated concentrations of U18666A, transduced with the indicated pseudotypes and luciferase activity analyzed 72 hours after transduction. Results of a single representative experiment performed with triplicate samples are shown; 2 independent experiments yielded similar results. D , 293T cells ( left ) transfected with empty plasmid (pcDNA) or dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin (DC-SIGN) encoding plasmid as well as MDMs ( right ) were incubated with 200 µg/mL mannan or solvent (control) and then transduced with the indicated pseudotypes. Transduction efficiency was analyzed by quantification of luciferase activities in cell lysates. Results of a representative experiment performed with triplicate samples are shown; an independent experiment yielded comparable results. Statistical significance was calculated using the 2-tailed Student t test. * P

    Techniques Used: Transduction, Isolation, Derivative Assay, Polymerase Chain Reaction, Transfection, Luciferase, Incubation, Activity Assay, Plasmid Preparation

    Transfection of macrophages with small interfering RNAs (siRNA) against Niemann-Pick C1 (NPC1), Mer, and scavenger receptor A (SR-A) reduces Ebola virus (EBOV) infection. Monocyte-derived macrophages (MDMs) were transfected with the indicated siRNAs and infected with EBOV strain Mayinga at a multiplicity of infection of 10. At 16 hours after infection, cells were extensively washed, fixed with 4% paraformaldehyde, and stained against EBOV nucleoprotein (NP) and nuclei (using 4',6-diamidino-2-phenylindole [DAPI]). Staining was analyzed with microscopy, and infection efficiency was calculated by determining the ratio of NP-positive to DAPI-positive cells. Results represent the mean of 4 separate experiments performed with MDMs from different donors is shown. Error bars indicate standard errors of the mean. Infection of cells transfected with control siRNA was set at 100%. Statistical significance was calculated using the 2-tailed Student t test. * P
    Figure Legend Snippet: Transfection of macrophages with small interfering RNAs (siRNA) against Niemann-Pick C1 (NPC1), Mer, and scavenger receptor A (SR-A) reduces Ebola virus (EBOV) infection. Monocyte-derived macrophages (MDMs) were transfected with the indicated siRNAs and infected with EBOV strain Mayinga at a multiplicity of infection of 10. At 16 hours after infection, cells were extensively washed, fixed with 4% paraformaldehyde, and stained against EBOV nucleoprotein (NP) and nuclei (using 4',6-diamidino-2-phenylindole [DAPI]). Staining was analyzed with microscopy, and infection efficiency was calculated by determining the ratio of NP-positive to DAPI-positive cells. Results represent the mean of 4 separate experiments performed with MDMs from different donors is shown. Error bars indicate standard errors of the mean. Infection of cells transfected with control siRNA was set at 100%. Statistical significance was calculated using the 2-tailed Student t test. * P

    Techniques Used: Transfection, Infection, Derivative Assay, Staining, Microscopy

    Transfection of macrophages with small interfering RNAs (siRNAs) against Niemann-Pick C1 (NPC1), Mer, and scavenger receptor A (SR-A) reduces Ebola virus (EBOV) glycoprotein (GP)–mediated entry. A , Monocyte-derived macrophages (MDMs) were transfected with the indicated single siRNAs ( left ) or combinations of siRNAs ( right ) and transduced with the indicated pseudotypes. Luciferase activities in cell lysates were determined 72 hours after transduction. Transduction of cells transfected with control siRNA (control) was set at 100%. Results on the left represent the mean of 3 (Mer), 6 (SR-A), and up to 15 experiments (NPC1, CD14, CD36, and integrin αV); results on the right, the mean of 3 independent experiments; error bars indicate standard errors of the mean. B , THP-1 cells induced with phorbol-12-myristate-13-acetate were transfected with the indicated siRNAs, and expression of Mer ( white bars ) was analyzed with flow cytometry. MDMs were transfected with the indicated siRNAs, and SR-A expression ( black bars ) was analyzed by means of Western blotting. The signals measured were quantified with ImageJ software. Expression of Mer and SR-A in cells transfected with control siRNA was set at 100%. Results represent the mean of 4 (Mer expression) to 6 (SR-A expression) independent experiments. C , HeLa cells, which do not express endogenous Mer or SR-A, were transfected with the respective siRNAs and transduced with the indicated pseudotypes. Luciferase activities in cell lysates were analyzed 72 hours after transduction. Results represent the mean of 4 independent experiment performed with triplicate samples. The transduction of cells transfected with control siRNA was set at 100%. D , MDMs were incubated with the indicated concentrations of tannic acid transduced with the indicated pseudotypes, and transduction efficiency was determined as described above. Transduction of cells incubated with solvent (water) was set at 100%. Results represent the mean of 3 separate experiments performed with triplicate samples. Statistical significance was calculated using the 2-tailed Student t test. * P
    Figure Legend Snippet: Transfection of macrophages with small interfering RNAs (siRNAs) against Niemann-Pick C1 (NPC1), Mer, and scavenger receptor A (SR-A) reduces Ebola virus (EBOV) glycoprotein (GP)–mediated entry. A , Monocyte-derived macrophages (MDMs) were transfected with the indicated single siRNAs ( left ) or combinations of siRNAs ( right ) and transduced with the indicated pseudotypes. Luciferase activities in cell lysates were determined 72 hours after transduction. Transduction of cells transfected with control siRNA (control) was set at 100%. Results on the left represent the mean of 3 (Mer), 6 (SR-A), and up to 15 experiments (NPC1, CD14, CD36, and integrin αV); results on the right, the mean of 3 independent experiments; error bars indicate standard errors of the mean. B , THP-1 cells induced with phorbol-12-myristate-13-acetate were transfected with the indicated siRNAs, and expression of Mer ( white bars ) was analyzed with flow cytometry. MDMs were transfected with the indicated siRNAs, and SR-A expression ( black bars ) was analyzed by means of Western blotting. The signals measured were quantified with ImageJ software. Expression of Mer and SR-A in cells transfected with control siRNA was set at 100%. Results represent the mean of 4 (Mer expression) to 6 (SR-A expression) independent experiments. C , HeLa cells, which do not express endogenous Mer or SR-A, were transfected with the respective siRNAs and transduced with the indicated pseudotypes. Luciferase activities in cell lysates were analyzed 72 hours after transduction. Results represent the mean of 4 independent experiment performed with triplicate samples. The transduction of cells transfected with control siRNA was set at 100%. D , MDMs were incubated with the indicated concentrations of tannic acid transduced with the indicated pseudotypes, and transduction efficiency was determined as described above. Transduction of cells incubated with solvent (water) was set at 100%. Results represent the mean of 3 separate experiments performed with triplicate samples. Statistical significance was calculated using the 2-tailed Student t test. * P

    Techniques Used: Transfection, Derivative Assay, Transduction, Luciferase, Expressing, Flow Cytometry, Cytometry, Western Blot, Software, Incubation

    34) Product Images from "Sprouty Proteins Are Negative Regulators of Interferon (IFN) Signaling and IFN-inducible Biological Responses *"

    Article Title: Sprouty Proteins Are Negative Regulators of Interferon (IFN) Signaling and IFN-inducible Biological Responses *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.400721

    Negative regulatory effects of Spry proteins in induction of IFN-dependent antiviral and antileukemic effects. A , Spry1,2,4 flox/flox MEFs and Spry1,2,4 −/− MEFs were incubated with the indicated doses of mouse IFNα. The cells were subsequently challenged with encephalomyocarditis virus ( EMCV ), and the viral cytopathic effects ( CPE ) were quantified 4 days later. Data are expressed as the percentage of protection from viral cytopathic effects of encephalomyocarditis virus. B and C , U937 cells were transfected with the indicated siRNAs and plated in a methylcellulose assay system in the absence or presence of human IFNα and IFNβ. Data are expressed as a percentage of the control ( Ctrl ) siRNA-transfected cell-derived colony formation and represent means ± S.E. of four independent experiments. For panel B , paired t test analysis showed p = 0.001 for the combination of control siRNA and IFNα versus the combination of Spry1 siRNA and IFNα; p = 0.00009 for the combination of control siRNA and IFNα versus the combination of Spry2 siRNA and IFNα; p = 0.019 for the combination of control siRNA and IFNβ versus the combination of Spry1 siRNA and IFNβ for CFU-L; and p = 0.035 for the combination of control siRNA and IFNβ versus the combination of Spry2 siRNA and IFNβ for CFU-L colonies. For panel C , paired t test analysis showed p = 0.02 for the combination of control siRNA and IFNα versus the combination of Spry4 siRNA and IFNα; and p = 0.008 for the combination of control siRNA and IFNβ versus the combination of Spry4 siRNA and IFNβ for CFU-L. D , mononuclear cells derived from peripheral blood of patients with polycythemia vera were transfected with the indicated siRNAs and were then plated in a methylcellulose assay system, in the absence or presence of human IFNα. BFU-E progenitor colonies were scored after 14 days in culture. Data are expressed as the percentage of control colony formation of the control untreated siRNA-transfected cells and represent means ± S.E. of five independent experiments. For panel D , paired t test analysis showed p = 0.0008 for the combination of control siRNA and IFNα versus the combination of Spry1 siRNA and IFNα; p = 0.0017 for the combination of control siRNA and IFNα versus the combination of Spry2 siRNA and IFNα; and p = 0.0074 for the combination of control siRNA and IFNα versus the combination of Spry4 siRNA and IFNα.
    Figure Legend Snippet: Negative regulatory effects of Spry proteins in induction of IFN-dependent antiviral and antileukemic effects. A , Spry1,2,4 flox/flox MEFs and Spry1,2,4 −/− MEFs were incubated with the indicated doses of mouse IFNα. The cells were subsequently challenged with encephalomyocarditis virus ( EMCV ), and the viral cytopathic effects ( CPE ) were quantified 4 days later. Data are expressed as the percentage of protection from viral cytopathic effects of encephalomyocarditis virus. B and C , U937 cells were transfected with the indicated siRNAs and plated in a methylcellulose assay system in the absence or presence of human IFNα and IFNβ. Data are expressed as a percentage of the control ( Ctrl ) siRNA-transfected cell-derived colony formation and represent means ± S.E. of four independent experiments. For panel B , paired t test analysis showed p = 0.001 for the combination of control siRNA and IFNα versus the combination of Spry1 siRNA and IFNα; p = 0.00009 for the combination of control siRNA and IFNα versus the combination of Spry2 siRNA and IFNα; p = 0.019 for the combination of control siRNA and IFNβ versus the combination of Spry1 siRNA and IFNβ for CFU-L; and p = 0.035 for the combination of control siRNA and IFNβ versus the combination of Spry2 siRNA and IFNβ for CFU-L colonies. For panel C , paired t test analysis showed p = 0.02 for the combination of control siRNA and IFNα versus the combination of Spry4 siRNA and IFNα; and p = 0.008 for the combination of control siRNA and IFNβ versus the combination of Spry4 siRNA and IFNβ for CFU-L. D , mononuclear cells derived from peripheral blood of patients with polycythemia vera were transfected with the indicated siRNAs and were then plated in a methylcellulose assay system, in the absence or presence of human IFNα. BFU-E progenitor colonies were scored after 14 days in culture. Data are expressed as the percentage of control colony formation of the control untreated siRNA-transfected cells and represent means ± S.E. of five independent experiments. For panel D , paired t test analysis showed p = 0.0008 for the combination of control siRNA and IFNα versus the combination of Spry1 siRNA and IFNα; p = 0.0017 for the combination of control siRNA and IFNα versus the combination of Spry2 siRNA and IFNα; and p = 0.0074 for the combination of control siRNA and IFNα versus the combination of Spry4 siRNA and IFNα.

    Techniques Used: Incubation, Transfection, Methylcellulose Assay, Derivative Assay

    35) Product Images from "Calmodulin antagonist enhances DR5-mediated apoptotic signaling in TRA-8 resistant triple negative breast cancer cells"

    Article Title: Calmodulin antagonist enhances DR5-mediated apoptotic signaling in TRA-8 resistant triple negative breast cancer cells

    Journal: Journal of cellular biochemistry

    doi: 10.1002/jcb.26848

    CaM expression knock-down increased DR5-mediated DISC formation and caspase cleavage in TRA-8 resistant TNBC cells (A) Co-IP of CaM, DR5, FADD and caspase-8 from HCC1143 cells with control siRNA or CaM 1, 2, and 3 siRNAs pretreatment, then followed with or without 500 ng/mL TRA-8 treatment for 3 hours. Cell Lysate lane: cell lysates before IP. IP: DISC lane: Co-IP of CaM with DR5-mediated DISC from cell lysates using anti-DR5 (D-6) antibody with protein A/G Plus-Agarose beads. (B) ]. (C) Western blot analysis of lysates of HCC1143 cells with control siRNA or CaM 1, 2, and 3 siRNAs pretreatment, then followed with or without 500 ng/mL TRA-8 treatment for 6 hours, for caspase-8, caspase-3, and PARP-1 and their cleavages. Western blot of lysate results are from two independent experiments. Co-IP results are from three independent experiments and error bars show the standard error of the mean. * p
    Figure Legend Snippet: CaM expression knock-down increased DR5-mediated DISC formation and caspase cleavage in TRA-8 resistant TNBC cells (A) Co-IP of CaM, DR5, FADD and caspase-8 from HCC1143 cells with control siRNA or CaM 1, 2, and 3 siRNAs pretreatment, then followed with or without 500 ng/mL TRA-8 treatment for 3 hours. Cell Lysate lane: cell lysates before IP. IP: DISC lane: Co-IP of CaM with DR5-mediated DISC from cell lysates using anti-DR5 (D-6) antibody with protein A/G Plus-Agarose beads. (B) ]. (C) Western blot analysis of lysates of HCC1143 cells with control siRNA or CaM 1, 2, and 3 siRNAs pretreatment, then followed with or without 500 ng/mL TRA-8 treatment for 6 hours, for caspase-8, caspase-3, and PARP-1 and their cleavages. Western blot of lysate results are from two independent experiments. Co-IP results are from three independent experiments and error bars show the standard error of the mean. * p

    Techniques Used: Chick Chorioallantoic Membrane Assay, Expressing, Co-Immunoprecipitation Assay, Western Blot

    36) Product Images from "14-3-3 Proteins recognize a histone code at histone H3 and are required for transcriptional activation"

    Article Title: 14-3-3 Proteins recognize a histone code at histone H3 and are required for transcriptional activation

    Journal:

    doi: 10.1038/sj.emboj.7601954

    14-3-3ζ Is required for transcriptional activation of the HDAC1 gene by anisomycin and TSA. ( A ) Isoform-specific depletion of 14-3-3 proteins by RNA interference. HeLa cells were transfected with siRNAs against 14-3-3ɛ or ζ and
    Figure Legend Snippet: 14-3-3ζ Is required for transcriptional activation of the HDAC1 gene by anisomycin and TSA. ( A ) Isoform-specific depletion of 14-3-3 proteins by RNA interference. HeLa cells were transfected with siRNAs against 14-3-3ɛ or ζ and

    Techniques Used: Activation Assay, Transfection

    37) Product Images from "Nicotinic acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote stemness in an ARRB1/ β-arrestin-1 dependent manner in NSCLC"

    Article Title: Nicotinic acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote stemness in an ARRB1/ β-arrestin-1 dependent manner in NSCLC

    Journal: Oncotarget

    doi:

    Differential contributions of Src, EGFR, PI3K and MEK signaling in regulating SCF expression (A) Real time PCR showing the inhibition of nicotine-mediated induction of SCF by inhibitors of Src (PP2), EGFR (Gefitinib), PI3 kinase (LY294002) and MEK (PD98509). (B-D) siRNAs to Src and EGFR prevented nicotine-mediated induction of SCF , as seen by RT-PCR. (E-F) ELISA assay shows increased secretion of SCF in the medium of A549 and H1650 cells treated with nicotine and EGF; levels were significantly reduced in conditioned media from cells transfected with siRNAs to β-arrestin-1 or E2F1.
    Figure Legend Snippet: Differential contributions of Src, EGFR, PI3K and MEK signaling in regulating SCF expression (A) Real time PCR showing the inhibition of nicotine-mediated induction of SCF by inhibitors of Src (PP2), EGFR (Gefitinib), PI3 kinase (LY294002) and MEK (PD98509). (B-D) siRNAs to Src and EGFR prevented nicotine-mediated induction of SCF , as seen by RT-PCR. (E-F) ELISA assay shows increased secretion of SCF in the medium of A549 and H1650 cells treated with nicotine and EGF; levels were significantly reduced in conditioned media from cells transfected with siRNAs to β-arrestin-1 or E2F1.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Inhibition, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection

    38) Product Images from "LRP6 Mediates cAMP Generation by G Protein-Coupled Receptors Through Regulating the Membrane Targeting of G?s"

    Article Title: LRP6 Mediates cAMP Generation by G Protein-Coupled Receptors Through Regulating the Membrane Targeting of G?s

    Journal: Science signaling

    doi: 10.1126/scisignal.2001464

    LRP6 is required for PTH-induced production of cAMP. ( A ) LRP6-specific siRNA inhibited PTH-induced cAMP accumulation in UMR-106 cells. Cells were transfected with siRNAs specific for GFP, LRP5, or LRP6 or with siRNAs against LRP5 and LRP6. Cells were
    Figure Legend Snippet: LRP6 is required for PTH-induced production of cAMP. ( A ) LRP6-specific siRNA inhibited PTH-induced cAMP accumulation in UMR-106 cells. Cells were transfected with siRNAs specific for GFP, LRP5, or LRP6 or with siRNAs against LRP5 and LRP6. Cells were

    Techniques Used: Transfection

    LRP6 is required for receptor-induced, Gα s -coupled cAMP production. ( A ) LRP6-specific siRNA abolished isoproterenol-induced cAMP accumulation in C2C12 cells. Cells were trans fected with siRNAs against GFP, LRP5, or LRP6 or with siRNAs specific
    Figure Legend Snippet: LRP6 is required for receptor-induced, Gα s -coupled cAMP production. ( A ) LRP6-specific siRNA abolished isoproterenol-induced cAMP accumulation in C2C12 cells. Cells were trans fected with siRNAs against GFP, LRP5, or LRP6 or with siRNAs specific

    Techniques Used:

    PTH induces LRP6 aggregation and Gα s βγ accumulation in UMR-106 cells, as analyzed by sucrose gradient sedimentation. UMR-106 cells were transfected with siRNAs specific for GFP or LRP6, and were treated with PTH(1–34) (50
    Figure Legend Snippet: PTH induces LRP6 aggregation and Gα s βγ accumulation in UMR-106 cells, as analyzed by sucrose gradient sedimentation. UMR-106 cells were transfected with siRNAs specific for GFP or LRP6, and were treated with PTH(1–34) (50

    Techniques Used: Sedimentation, Transfection

    39) Product Images from "Virion Background and Efficiency of Virion Incorporation Determine Susceptibility of Simian Immunodeficiency Virus Env-Driven Viral Entry to Inhibition by IFITM Proteins"

    Article Title: Virion Background and Efficiency of Virion Incorporation Determine Susceptibility of Simian Immunodeficiency Virus Env-Driven Viral Entry to Inhibition by IFITM Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.01488-16

    The efficiency of SIV-Env incorporation into virions determines IFITM sensitivity. (A) MLV vectors encoding luciferase and bearing escalating amounts of SIV-Env or MLV-Env were produced by transient transfection of 293T cells with equal amounts of vector plasmid and the indicated amounts of Env-encoding plasmids. Equal volumes of the vector preparations were then inoculated onto 293T cells previously transduced to express IFITMs, and luciferase expression in cell lysates was analyzed at 72 h postransduction. The results of a single representative experiment conducted with triplicate samples are shown. Error bars indicate SD. The results were confirmed in three separate experiments. Numbers above bars indicate the averages from four independent experiments for which transduction of control cells was set as 100%. Statistical analysis was carried out for normalized data. (B) sMAGI cells were transfected with the indicated siRNAs, and IFITM3 expression was analyzed by an immunoblot assay employing an IFITM3-specific antibody. Similar results were obtained in two separate experiments. (C) sMAGI cells transfected as described for panel B were transduced with MLV vectors bearing escalating amounts of SIV-Env, and transduction efficiency was determined as described for panel A. In addition, the cells were infected with FLUAV encoding Gaussia luciferase, and luciferase expression in culture supernatants was analyzed at 48 h postinfection. The average from three independent experiments (two for FLUAV) is shown. Error bars indicate SEM.
    Figure Legend Snippet: The efficiency of SIV-Env incorporation into virions determines IFITM sensitivity. (A) MLV vectors encoding luciferase and bearing escalating amounts of SIV-Env or MLV-Env were produced by transient transfection of 293T cells with equal amounts of vector plasmid and the indicated amounts of Env-encoding plasmids. Equal volumes of the vector preparations were then inoculated onto 293T cells previously transduced to express IFITMs, and luciferase expression in cell lysates was analyzed at 72 h postransduction. The results of a single representative experiment conducted with triplicate samples are shown. Error bars indicate SD. The results were confirmed in three separate experiments. Numbers above bars indicate the averages from four independent experiments for which transduction of control cells was set as 100%. Statistical analysis was carried out for normalized data. (B) sMAGI cells were transfected with the indicated siRNAs, and IFITM3 expression was analyzed by an immunoblot assay employing an IFITM3-specific antibody. Similar results were obtained in two separate experiments. (C) sMAGI cells transfected as described for panel B were transduced with MLV vectors bearing escalating amounts of SIV-Env, and transduction efficiency was determined as described for panel A. In addition, the cells were infected with FLUAV encoding Gaussia luciferase, and luciferase expression in culture supernatants was analyzed at 48 h postinfection. The average from three independent experiments (two for FLUAV) is shown. Error bars indicate SEM.

    Techniques Used: Luciferase, Produced, Transfection, Plasmid Preparation, Expressing, Transduction, Infection

    40) Product Images from "Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression"

    Article Title: Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003077

    Fhit activates TK1 expression. (A) Western blot analysis of TK1 and TYMS expression in siRNA transfected HEK293 cells. Western blots were performed on 5 independent experiments, and a representative blot is shown. (B) Western blot analysis of TK1 expression in siRNA transfected HEK293 cells with or with exogenous Fhit overexpression. pRcCMV expression plasmid carrying FHIT cDNA was co-transfected with FHIT siRNAs to achieve exogenous Fhit overexpression. A representative blot is shown. (C) Western blot analysis of TK1 expression in Fhit +/+ and Fhit −/− mouse kidney epithelial cells. A representative blot is shown. (D) Western blot analysis of TK1 expression in A549 cells with Fhit stably knocked down for 7–9 weeks. A representative blot is shown.
    Figure Legend Snippet: Fhit activates TK1 expression. (A) Western blot analysis of TK1 and TYMS expression in siRNA transfected HEK293 cells. Western blots were performed on 5 independent experiments, and a representative blot is shown. (B) Western blot analysis of TK1 expression in siRNA transfected HEK293 cells with or with exogenous Fhit overexpression. pRcCMV expression plasmid carrying FHIT cDNA was co-transfected with FHIT siRNAs to achieve exogenous Fhit overexpression. A representative blot is shown. (C) Western blot analysis of TK1 expression in Fhit +/+ and Fhit −/− mouse kidney epithelial cells. A representative blot is shown. (D) Western blot analysis of TK1 expression in A549 cells with Fhit stably knocked down for 7–9 weeks. A representative blot is shown.

    Techniques Used: Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Stable Transfection

    Fhit-deficient cells exhibit spontaneous DNA breaks. (A) Neutral comet assays of HEK293 cells 2 days after transfections with siRNAs and pRcCMV- FHIT -flag or pRcCMV-empty-flag plasmids. Representative nuclei are shown; bars, 20 µm. (B) Box plots of Tail moments include data (siCtrl, n = 183; si FHIT , n = 142; si FHIT +CMV-ev, n = 135; si FHIT +CMV-Fhit, n = 132) from 3 separate experiments. Statistical significance was determined using the Kruskal-Wallis rank sum test. (C) Indirect immunofluorescence of γH2AX and 53BP1, 2 days after Fhit knockdown. Representative nuclei are shown; bars, 10 µm. (D and E) Quantification of γH2AX-positive cells (D) and 53BP1-positive cells (E). Bar graphs indicate the means, and error bars represent the standard deviations. Data were collected from 3 independent experiments. Statistical significance was assessed using a 2-sided Student's T-test. (F) Neutral comet assays of Fhit +/+ or Fhit −/− mouse kidney cells 48 h after transfection with pRcCMV- FHIT -flag or pRcCMV-empty-flag plasmids. Box plots of Tail moments are shown. Statistical significance was determined using the Mann-Whitney rank sum test. (G) Neutral comet assays of Fhit-deficient H1299 lung carcinomas cells, with or without induction of Fhit expression. Comet assays were performed 48 h after ponasterone A-induction of Fhit expression. Box plots of Tail moments are shown. Statistical significance was determined using the Mann-Whitney rank sum test.
    Figure Legend Snippet: Fhit-deficient cells exhibit spontaneous DNA breaks. (A) Neutral comet assays of HEK293 cells 2 days after transfections with siRNAs and pRcCMV- FHIT -flag or pRcCMV-empty-flag plasmids. Representative nuclei are shown; bars, 20 µm. (B) Box plots of Tail moments include data (siCtrl, n = 183; si FHIT , n = 142; si FHIT +CMV-ev, n = 135; si FHIT +CMV-Fhit, n = 132) from 3 separate experiments. Statistical significance was determined using the Kruskal-Wallis rank sum test. (C) Indirect immunofluorescence of γH2AX and 53BP1, 2 days after Fhit knockdown. Representative nuclei are shown; bars, 10 µm. (D and E) Quantification of γH2AX-positive cells (D) and 53BP1-positive cells (E). Bar graphs indicate the means, and error bars represent the standard deviations. Data were collected from 3 independent experiments. Statistical significance was assessed using a 2-sided Student's T-test. (F) Neutral comet assays of Fhit +/+ or Fhit −/− mouse kidney cells 48 h after transfection with pRcCMV- FHIT -flag or pRcCMV-empty-flag plasmids. Box plots of Tail moments are shown. Statistical significance was determined using the Mann-Whitney rank sum test. (G) Neutral comet assays of Fhit-deficient H1299 lung carcinomas cells, with or without induction of Fhit expression. Comet assays were performed 48 h after ponasterone A-induction of Fhit expression. Box plots of Tail moments are shown. Statistical significance was determined using the Mann-Whitney rank sum test.

    Techniques Used: Transfection, Immunofluorescence, MANN-WHITNEY, Expressing

    Loss of Fhit causes replication stress-induced chromosomal instability. (A) Cyclin A and 53BP1 immunofluorescence after Fhit knockdown in HEK293 cells. Representative images are shown; bars, 20 µm. (B and C) Histograms of 53BP1 nuclear bodies/G1 phase cell 3 days following siRNA transfection (B) or 14 days after siRNA transfection with fresh siRNAs transfected every 4 days (C). G1 phase cells were defined as cells negative for Cyclin A staining. Mann-Whitney rank sum test was used to determine P-values. (D) Representative images of DAPI-stained nuclei in siCtrl or siFHIT cells. Arrow marks a micronucleus; bars, 5 µm. (E) Quantification of micronucleated cells 3 days after siRNA transfections. Bar graphs represent the means, and error bars mark the standard deviations. P-value determined using a 2-sided T-test. (F) Percentage of aneuploid or tetraploid kidney cells established from Fhit +/+ or Fhit −/− mouse kidney epithelial cells. Cells were sub-cultured 8 times and metaphase chromosomes were prepared and counted (n = 37 for Fhit +/+ ; n = 40 for Fhit −/− metaphases). (G) Quantification of the number of breaks/metaphase for the mouse kidney cells described in (F).
    Figure Legend Snippet: Loss of Fhit causes replication stress-induced chromosomal instability. (A) Cyclin A and 53BP1 immunofluorescence after Fhit knockdown in HEK293 cells. Representative images are shown; bars, 20 µm. (B and C) Histograms of 53BP1 nuclear bodies/G1 phase cell 3 days following siRNA transfection (B) or 14 days after siRNA transfection with fresh siRNAs transfected every 4 days (C). G1 phase cells were defined as cells negative for Cyclin A staining. Mann-Whitney rank sum test was used to determine P-values. (D) Representative images of DAPI-stained nuclei in siCtrl or siFHIT cells. Arrow marks a micronucleus; bars, 5 µm. (E) Quantification of micronucleated cells 3 days after siRNA transfections. Bar graphs represent the means, and error bars mark the standard deviations. P-value determined using a 2-sided T-test. (F) Percentage of aneuploid or tetraploid kidney cells established from Fhit +/+ or Fhit −/− mouse kidney epithelial cells. Cells were sub-cultured 8 times and metaphase chromosomes were prepared and counted (n = 37 for Fhit +/+ ; n = 40 for Fhit −/− metaphases). (G) Quantification of the number of breaks/metaphase for the mouse kidney cells described in (F).

    Techniques Used: Immunofluorescence, Transfection, Staining, MANN-WHITNEY, Cell Culture

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    Article Snippet: .. 2. siRNA and plasmid transfections PML silencing was accomplished by using commercial siRNAs (sc-36283; Santa Cruz Biotechnology). ..

    Article Title: Death Domain-associated Protein DAXX Promotes Ovarian Cancer Development and Chemoresistance *
    Article Snippet: .. The 21-nucleotide RNA oligonucleotides of Daxx (sc-35178) and Pml (sc-36284) were purchased from Santa Cruz Biotechnology and were transfected into cells using Lipofectamine 2000 (Invitrogen). ..

    Article Title: Dengue Non-structural Protein 5 Polymerase Complexes With Promyelocytic Leukemia Protein (PML) Isoforms III and IV to Disrupt PML-Nuclear Bodies in Infected Cells
    Article Snippet: .. siRNA, Plasmid Constructs and Transfections For siRNA transfection, A549 cells were seeded into 24-well microplates at a density of 1* 105 cells per well prior to transfection using Lipofectamine 2000 (Thermo Fisher Scientific) the following day with 50 nM siRNA (sc-36283, Santa Cruz Biotechnology) following the manufacturer's specifications. ..

    Small Interfering RNA:

    Article Title: Promyelocytic Leukemia Restricts Enterovirus 71 Replication by Inhibiting Autophagy
    Article Snippet: .. PML small interfering RNA (siRNA) (sc-36284) was purchased from Santa Cruz (Santa Cruz, CA, USA). ..

    Construct:

    Article Title: Dengue Non-structural Protein 5 Polymerase Complexes With Promyelocytic Leukemia Protein (PML) Isoforms III and IV to Disrupt PML-Nuclear Bodies in Infected Cells
    Article Snippet: .. siRNA, Plasmid Constructs and Transfections For siRNA transfection, A549 cells were seeded into 24-well microplates at a density of 1* 105 cells per well prior to transfection using Lipofectamine 2000 (Thermo Fisher Scientific) the following day with 50 nM siRNA (sc-36283, Santa Cruz Biotechnology) following the manufacturer's specifications. ..

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    Santa Cruz Biotechnology smad2 sirna
    Effects of <t>Smad2</t> on Col I and Col III expression in hHSFs. hHSFs cells were transfected with Smad2-plasmid or <t>Smad2-siRNA.</t> Smad2, Col I and Col III expression were determined by (A) western blotting and (B-D) reverse transcription-quantitative polymerase chain reaction at 48 h post-transfection. Data are presented as the mean ± standard deviation. *P
    Smad2 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology sirna against mouse ahr
    I3C inhibits the development of intestinal organoids from crypts or Lgr5 + stem cells <t>AhR-dependently</t> Crypts, isolated from the small intestine, were resuspended with Matrigel and applied into a 24-well plate and then cultured in complete culture medium for 4 days. I3C (0.1–1 μM), α-naphthoflavone (1 μM), and DMSO (0.1% v/v) were added to the culture medium 1.5 h before the addition of growth and differentiation factors. Then, organoids were observed (A) and counted (B) under the inverted microscope, and assayed for RNA analysis (C), cell proliferation (G), or cell viability assay (H). For <t>siRNA</t> nucleofection, 1 x 10 3 crypt cells were suspended in 0.1 ml of Nucleofector solution and 5 μl of siRNA against mouse AhR or control siRNA and electroporated. Cells were cultured and harvested 24 h later and assayed for AhR expression (D). Crypt cells containing Lgr5 + cells, which were nucleofected with siRNA, were mixed with Matrigel (200 cells/25 μl) with 1 μM Jagged-1 peptide, subjected to organoid development, and observed and counted under the inverted microscope (E, F). Results are presented as mean ± SD of 5 (B, G, and H) or 3 (F) independent experiments. Statistical significance was analyzed using the paired Student’s t -test. * p
    Sirna Against Mouse Ahr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology control sirna
    Effects of fucoxanthin (Fx) on Smad2/Smad3 activation in NPDFs. ( A ) The NPDFs were pretreated with Fx (5–30 µM) for 30 min, followed by stimulation with TGF-β1 (1 ng/mL) for 30 min. ( B ) The <t>Smad</t> 2/3 nuclear translocation was determined via western blot analysis. ( C ) Smad 2/3 silencing inhibits the expression of α-SMA and Col-1 proteins in TGF-β1-stimulated NPDFs. The NPDFs were transfected with Smad2/3 <t>siRNA</t> (40 and 80 nM) for 24 h, followed by stimulation with TGF-β1 (1 ng/mL) for 24 h. ( D ) The NPDFs were transfected with either the control siRNA (80 nM) or the Smad 2/3 siRNA (80 nM) for 24 h, followed by stimulation with TGF-β1 (1 ng/mL) for 30 min. Each bar represents the mean ± S.E.M. from three independent experiments. Untreated cells were used as a control (CON). p-Smad 2/3, phosphor-Smad 2/3; t-Smad 2/3, total-Smad 2/3; siCon, control siRNA; siSmad, Smad 2/3 siRNA.
    Control Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology control non silencing sirna
    KLF4 depletion increases endothelial barrier permeability A, Timeline of TER assay. B, HLMVECs plated on gold microelectrodes were left untreated (control) or transfected with a non-silencing <t>siRNA</t> or KLF4 -silencing siRNA, followed by TER assay. Note that KLF4 silencing inhibited the thrombin response relative to untreated group (no transfection) or negative control group (non-silencing siRNA; n = 5 per group). Mean value (± s.e.m.) of maximal TER responses to thrombin (50 nM) stimulation (n = 7). Thrombin-induced decrease in TER was significantly attenuated in HLMVECs transfected by KLF4-depletion compared with untreated control or negative control group transfected with a non-silencing siRNA. C-E, KLF4 knockdown increases transendothelial permeability of fluorescein isothiocyanate (FITC)-conjugated albumin by decreasing <t>VE-cadherin</t> expression and AJ integrity. C , Timeline of experiments. D , Confluent HLMVEC monolayers were grown on microporous filters for 36 h, either left alone (control) or treated with control siRNA or with KLF4 -siRNA for 12 hours. At 18 hr post transfection, transendothelial FITC-albumin permeability was measured. Control HLMVECs showed basal transendothelial FITC-albumin permeability values, while KLF4 knockdown increased transendothelial FITC-albumin permeability. Re-expression of VE-cadherin into KLF4-depleted ECs partially restored the effect of loss of KLF4. Values are mean ± s.e.m. (n= 10). *p
    Control Non Silencing Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of Smad2 on Col I and Col III expression in hHSFs. hHSFs cells were transfected with Smad2-plasmid or Smad2-siRNA. Smad2, Col I and Col III expression were determined by (A) western blotting and (B-D) reverse transcription-quantitative polymerase chain reaction at 48 h post-transfection. Data are presented as the mean ± standard deviation. *P

    Journal: Experimental and Therapeutic Medicine

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

    doi: 10.3892/etm.2018.5984

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

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

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

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

    Journal: Molecules and Cells

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

    doi: 10.14348/molcells.2018.2167

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

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

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

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

    Journal: Marine Drugs

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

    doi: 10.3390/md16090323

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

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

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

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

    Journal: Circulation research

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

    doi: 10.1161/CIRCRESAHA.110.219592

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

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

    Techniques: Permeability, Transfection, Negative Control, Expressing

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

    Journal: Circulation research

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

    doi: 10.1161/CIRCRESAHA.110.219592

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

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

    Techniques: Mouse Assay