u251  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier
Bioz Manufacturer Symbol Thermo Fisher manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Name:
    1405 F TEOM Continuous Ambhttp www thermofisher com order catalog product 75003424ient Air Monitor
    Description:
    Take continuous direct measurements of PM 10 PM 2 5 and PM 1 particulates using the Thermo Scientific 1405 F TEOM Continuous Ambient Air Monitor This monitor utilizes a tapered element oscillating microbalance TEOM and Filter Dynamics Measurement System FDMS to provide measurements with excellent short term precision and account for volatile and nonvolatile PM fractions
    Catalog Number:
    TEOM1405F
    Price:
    None
    Category:
    Instruments and Equipment
    Applications:
    Environmental|Industrial & Applied Science
    Buy from Supplier


    Structured Review

    Thermo Fisher u251
    Swainsonine affected glioma cell growth, migration and invasion by regulating miR-92a. MiR-92a mimic, inhibitor and the relevant negative control (NC) were transfected into <t>U251</t> cells, and ( a ) the transfection efficiency of miR-92a mimic and miR-92a inhibitor was appraised by quantitative real-time polymerase chain reaction (qRT-PCR); b BrdU positive cells was detected by Bromodeoxyuridine (BrdU) assay; c and d CyclinD1, Cyclin-dependent kinase 4 (CDK4), and p16 were appraised by western blot; e and f cell apoptosis and pro-Caspase-3, pro-Caspase-9, cleaved-Caspase-3 and cleaved-Caspase-9 were appraised by utilizing flow cytometry and western blot assays; g - l cell migration, invasion, the protein levels of matrix metalloproteinase (MMP)-2/− 9, Vimentin and E-cadherin were detected by Transwell and western blot assays. * p
    Take continuous direct measurements of PM 10 PM 2 5 and PM 1 particulates using the Thermo Scientific 1405 F TEOM Continuous Ambient Air Monitor This monitor utilizes a tapered element oscillating microbalance TEOM and Filter Dynamics Measurement System FDMS to provide measurements with excellent short term precision and account for volatile and nonvolatile PM fractions
    https://www.bioz.com/result/u251/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u251 - by Bioz Stars, 2021-05
    86/100 stars

    Images

    1) Product Images from "Swainsonine represses glioma cell proliferation, migration and invasion by reduction of miR-92a expression"

    Article Title: Swainsonine represses glioma cell proliferation, migration and invasion by reduction of miR-92a expression

    Journal: BMC Cancer

    doi: 10.1186/s12885-019-5425-7

    Swainsonine affected glioma cell growth, migration and invasion by regulating miR-92a. MiR-92a mimic, inhibitor and the relevant negative control (NC) were transfected into U251 cells, and ( a ) the transfection efficiency of miR-92a mimic and miR-92a inhibitor was appraised by quantitative real-time polymerase chain reaction (qRT-PCR); b BrdU positive cells was detected by Bromodeoxyuridine (BrdU) assay; c and d CyclinD1, Cyclin-dependent kinase 4 (CDK4), and p16 were appraised by western blot; e and f cell apoptosis and pro-Caspase-3, pro-Caspase-9, cleaved-Caspase-3 and cleaved-Caspase-9 were appraised by utilizing flow cytometry and western blot assays; g - l cell migration, invasion, the protein levels of matrix metalloproteinase (MMP)-2/− 9, Vimentin and E-cadherin were detected by Transwell and western blot assays. * p
    Figure Legend Snippet: Swainsonine affected glioma cell growth, migration and invasion by regulating miR-92a. MiR-92a mimic, inhibitor and the relevant negative control (NC) were transfected into U251 cells, and ( a ) the transfection efficiency of miR-92a mimic and miR-92a inhibitor was appraised by quantitative real-time polymerase chain reaction (qRT-PCR); b BrdU positive cells was detected by Bromodeoxyuridine (BrdU) assay; c and d CyclinD1, Cyclin-dependent kinase 4 (CDK4), and p16 were appraised by western blot; e and f cell apoptosis and pro-Caspase-3, pro-Caspase-9, cleaved-Caspase-3 and cleaved-Caspase-9 were appraised by utilizing flow cytometry and western blot assays; g - l cell migration, invasion, the protein levels of matrix metalloproteinase (MMP)-2/− 9, Vimentin and E-cadherin were detected by Transwell and western blot assays. * p

    Techniques Used: Migration, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, BrdU Staining, Western Blot, Flow Cytometry, Cytometry

    Swainsonine restrained U251 cells proliferation and evoked apoptosis. a The normal astrocyte NHA cells were disposed with diverse dosages of Swainsonine (0–40 μM) for 12 h, cell viability was appraised by utilizing Cell Counting Kit-8 (CCK-8); U251 cells were administrated with diverse dosages of Swainsonine (0–40 μM) for 12 h. b Cell viability, c cell proliferation, d cell cycle and ( e and f ) CyclinD1, Cyclin-dependent kinase 4 (CDK4), and p16 were appraised by exploiting CCK-8, Bromodeoxyuridine (BrdU), flow cytometry and western blot; g cell apoptosis and ( h ) pro-Caspase-3, pro-Caspase-9, cleaved-Caspase-3 and cleaved-Caspase-9 were assessed by flow cytometry and western blot. * p
    Figure Legend Snippet: Swainsonine restrained U251 cells proliferation and evoked apoptosis. a The normal astrocyte NHA cells were disposed with diverse dosages of Swainsonine (0–40 μM) for 12 h, cell viability was appraised by utilizing Cell Counting Kit-8 (CCK-8); U251 cells were administrated with diverse dosages of Swainsonine (0–40 μM) for 12 h. b Cell viability, c cell proliferation, d cell cycle and ( e and f ) CyclinD1, Cyclin-dependent kinase 4 (CDK4), and p16 were appraised by exploiting CCK-8, Bromodeoxyuridine (BrdU), flow cytometry and western blot; g cell apoptosis and ( h ) pro-Caspase-3, pro-Caspase-9, cleaved-Caspase-3 and cleaved-Caspase-9 were assessed by flow cytometry and western blot. * p

    Techniques Used: Cell Counting, CCK-8 Assay, Flow Cytometry, Cytometry, Western Blot

    Swainsonine repressed miR-92a expression in U251 cells. After disposing with 30 μM Swainsonine for 12 h, miR-92a expression was analyzed by utilizing quantitative real-time polymerase chain reaction (qRT-PCR) assay in U251 cells. ** p
    Figure Legend Snippet: Swainsonine repressed miR-92a expression in U251 cells. After disposing with 30 μM Swainsonine for 12 h, miR-92a expression was analyzed by utilizing quantitative real-time polymerase chain reaction (qRT-PCR) assay in U251 cells. ** p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Swainsonine restrained U251 cells migration and invasion. U251 cells were disposed by 30 μM Swainsonine for 12 h. a - c cell migration, as well as matrix metalloproteinase (MMP)-2 and MMP-9 were appraised by Transwell and western blot assays; d - f cell invasion as well as Vimentin and E-cadherin were detected by Transwell and western blot assays; g U251 cells were administrated with 10 μg/mL mitomycin C, and cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay; h the percentage of migration scratch coverage was examined by wound healing assay. * p
    Figure Legend Snippet: Swainsonine restrained U251 cells migration and invasion. U251 cells were disposed by 30 μM Swainsonine for 12 h. a - c cell migration, as well as matrix metalloproteinase (MMP)-2 and MMP-9 were appraised by Transwell and western blot assays; d - f cell invasion as well as Vimentin and E-cadherin were detected by Transwell and western blot assays; g U251 cells were administrated with 10 μg/mL mitomycin C, and cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay; h the percentage of migration scratch coverage was examined by wound healing assay. * p

    Techniques Used: Migration, Western Blot, Cell Counting, CCK-8 Assay, Wound Healing Assay

    2) Product Images from "ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells"

    Article Title: ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells

    Journal: Journal of neuro-oncology

    doi: 10.1007/s11060-012-0979-0

    KU55933 sensitizes TMZ responsive GBM cells to TMZ treatment. The effect of KU55933 on TMZ sensitivity of U251 (A) and U87 (B) cells was assessed in a clonogenic assay. Cell survival (mean ± SEM from three independent experiments) is plotted relative
    Figure Legend Snippet: KU55933 sensitizes TMZ responsive GBM cells to TMZ treatment. The effect of KU55933 on TMZ sensitivity of U251 (A) and U87 (B) cells was assessed in a clonogenic assay. Cell survival (mean ± SEM from three independent experiments) is plotted relative

    Techniques Used: Clonogenic Assay

    3) Product Images from "Constitutive UPRER activation sustains tumor cell differentiation"

    Article Title: Constitutive UPRER activation sustains tumor cell differentiation

    Journal: bioRxiv

    doi: 10.1101/594630

    Genetic perturbation of IRE1 effect on GBM cell reprogramming. A) Schematic representation of GBM cell working model of differentiated to stem cell phenotype culture. B) Phenotypic characterization of U251/ RADH85/ RADH87 parental and overexpressing WT, DN or Q* forms of IRE1 when grown in neurosphere media. C) Differentiated GBM cell lines U251, RADH85 and RADH87 were cultured in neurosphere medium and were passaged every 14 days. If the number of cells was under the initial number of cells seeded (10 6 ), the culture was stopped (n=2 to 4). D) Heat map representation of fold change of mRNA expression of genes involved in reprogramming, stemness and differentiation normalized to parental in U251, RADH85, RADH87 lines expressing WT, DN or Q* forms of IRE1 when grown in neurosphere media. E) Protein expression of reprogramming, stemness and differentiation markers in these lines compared to parental lines determined by flow cytometry. F) Clonogenicity of differentiated lines expressing WT, DN or Q* forms of IRE1 compared to parental lines when grown in neurosphere media. (ns): not significant; (*): p
    Figure Legend Snippet: Genetic perturbation of IRE1 effect on GBM cell reprogramming. A) Schematic representation of GBM cell working model of differentiated to stem cell phenotype culture. B) Phenotypic characterization of U251/ RADH85/ RADH87 parental and overexpressing WT, DN or Q* forms of IRE1 when grown in neurosphere media. C) Differentiated GBM cell lines U251, RADH85 and RADH87 were cultured in neurosphere medium and were passaged every 14 days. If the number of cells was under the initial number of cells seeded (10 6 ), the culture was stopped (n=2 to 4). D) Heat map representation of fold change of mRNA expression of genes involved in reprogramming, stemness and differentiation normalized to parental in U251, RADH85, RADH87 lines expressing WT, DN or Q* forms of IRE1 when grown in neurosphere media. E) Protein expression of reprogramming, stemness and differentiation markers in these lines compared to parental lines determined by flow cytometry. F) Clonogenicity of differentiated lines expressing WT, DN or Q* forms of IRE1 compared to parental lines when grown in neurosphere media. (ns): not significant; (*): p

    Techniques Used: Cell Culture, Expressing, Flow Cytometry

    XBP1s involvement in GBM cell reprogramming. A) Hierarchical clustering of genes involved in differentiation and stemness in GBM (TCGA cohort) based on high XBP1s or high RIDD activity (blue low levels, red high levels). B) mRNA expression of BMI1, CD133, nestin stem cell markers and SMA, vimentin, YKL40 differentiated cell markers based on microarray fluorescence intensity in high XBP1s and high RIDD activity tumors (TCGA cohort). C) Hierarchical clustering of genes involved in reprogramming in GBM (TCGA cohort) based on high XBP1s or high RIDD activity (blue low levels, red high levels). D) mRNA expression of SOX2, POU3F2, OLIG2, SALL2 reprogramming TFs based on microarray fluorescence intensity in high XBP1s and high RIDD activity tumors (TCGA cohort). E) mRNA levels of SOX2 upon XBP1s silencing in primary and classical adherent GBM lines (RADH85/87 and U251 respectively). (ns): not significant; (*): p
    Figure Legend Snippet: XBP1s involvement in GBM cell reprogramming. A) Hierarchical clustering of genes involved in differentiation and stemness in GBM (TCGA cohort) based on high XBP1s or high RIDD activity (blue low levels, red high levels). B) mRNA expression of BMI1, CD133, nestin stem cell markers and SMA, vimentin, YKL40 differentiated cell markers based on microarray fluorescence intensity in high XBP1s and high RIDD activity tumors (TCGA cohort). C) Hierarchical clustering of genes involved in reprogramming in GBM (TCGA cohort) based on high XBP1s or high RIDD activity (blue low levels, red high levels). D) mRNA expression of SOX2, POU3F2, OLIG2, SALL2 reprogramming TFs based on microarray fluorescence intensity in high XBP1s and high RIDD activity tumors (TCGA cohort). E) mRNA levels of SOX2 upon XBP1s silencing in primary and classical adherent GBM lines (RADH85/87 and U251 respectively). (ns): not significant; (*): p

    Techniques Used: Activity Assay, Expressing, Microarray, Fluorescence

    Pharmacological inhibition of IRE1 effect on GBM cell reprogramming. A) Differentiated GBM cell lines U251, RADH85 and RADH87 were cultured in neurosphere medium in the presence of MKC (5 µM), and were passaged every 14 days. If the number of cells was under the initial number of cells seeded (10 6 ), the culture is stopped (n=2 to 3). B) Phenotypic characterization of parental adherent (RADH85/87 and U251) lines through culture in neurosphere medium treated with MKC or DMSO. C) Heat map representation of fold change of mRNA expression of genes involved in reprogramming, stemness and differentiation normalized to parental in U251, RADH85, and RADH87 lines when grown in neurosphere media in the presence of MKC or DMSO. D) Protein expression of reprogramming, stemness and differentiation markers in these lines compared to parental lines determined by flow cytometry. E) Quantification of clonogenicity of single cell parental, WT or Q* IRE1 expressing RADH85/87 and U251 lines when seeded in serum-free medium in the presence of MKC or DMSO. (ns): not significant; (*): p
    Figure Legend Snippet: Pharmacological inhibition of IRE1 effect on GBM cell reprogramming. A) Differentiated GBM cell lines U251, RADH85 and RADH87 were cultured in neurosphere medium in the presence of MKC (5 µM), and were passaged every 14 days. If the number of cells was under the initial number of cells seeded (10 6 ), the culture is stopped (n=2 to 3). B) Phenotypic characterization of parental adherent (RADH85/87 and U251) lines through culture in neurosphere medium treated with MKC or DMSO. C) Heat map representation of fold change of mRNA expression of genes involved in reprogramming, stemness and differentiation normalized to parental in U251, RADH85, and RADH87 lines when grown in neurosphere media in the presence of MKC or DMSO. D) Protein expression of reprogramming, stemness and differentiation markers in these lines compared to parental lines determined by flow cytometry. E) Quantification of clonogenicity of single cell parental, WT or Q* IRE1 expressing RADH85/87 and U251 lines when seeded in serum-free medium in the presence of MKC or DMSO. (ns): not significant; (*): p

    Techniques Used: Inhibition, Cell Culture, Expressing, Flow Cytometry

    IRE1 activity is associated with cancer differentiated state in GBM specimens. A) Hierarchical clustering of GBM patients (TCGA cohort) based on high or low IRE1 activity confronted to differentiation and stem gene signatures derived from literature. B) mRNA expression of BMI1, CD133, nestin stem cell markers and SMA, vimentin, YKL40 differentiated cell markers based on microarray fluorescence intensity in high and low IRE1 activity tumors (TCGA cohort). C) Hierarchical clustering of GBM patients (TCGA cohort) based on high or low IRE1 activity confronted to a reprogramming TFs signature derived from literature. D) mRNA levels of reprogramming TFs OLIG2, POU3F2, SALL2, and SOX2 based on microarray fluorescence intensity in high and low IRE1 activity tumors (TCGA cohort). E) mRNA levels of reprogramming TFs in U251, RADH85 and RADH87 lines expressing WT, DN or Q* forms of IRE1 normalized to parental. (ns): not significant; (*): p
    Figure Legend Snippet: IRE1 activity is associated with cancer differentiated state in GBM specimens. A) Hierarchical clustering of GBM patients (TCGA cohort) based on high or low IRE1 activity confronted to differentiation and stem gene signatures derived from literature. B) mRNA expression of BMI1, CD133, nestin stem cell markers and SMA, vimentin, YKL40 differentiated cell markers based on microarray fluorescence intensity in high and low IRE1 activity tumors (TCGA cohort). C) Hierarchical clustering of GBM patients (TCGA cohort) based on high or low IRE1 activity confronted to a reprogramming TFs signature derived from literature. D) mRNA levels of reprogramming TFs OLIG2, POU3F2, SALL2, and SOX2 based on microarray fluorescence intensity in high and low IRE1 activity tumors (TCGA cohort). E) mRNA levels of reprogramming TFs in U251, RADH85 and RADH87 lines expressing WT, DN or Q* forms of IRE1 normalized to parental. (ns): not significant; (*): p

    Techniques Used: Activity Assay, Derivative Assay, Expressing, Microarray, Fluorescence

    XBP1s-dependent expression of miR148a prevents GBM cell reprogramming. A) Schematic representation of hypothesis of effect of IRE1 signaling on reprogramming TFs. B) Hierarchical clustering of miRNAs in GBM (TCGA cohort) confronted to high XBP1s or high RIDD activity (blue low levels, red high levels) with the best 5 candidates shown. C) mRNA expression of miR148a based on microarray fluorescence intensity in high XBP1s and high RIDD activity tumors (TCGA cohort). D) miR148a expression in adherent lines U251, RADH85/87 expressing DN or Q* forms of IRE1 normalized to parental. E) miR148a expression in adherent lines U251, RADH85/87 transiently deficient for XBP1s through siRNA transfection compared to control. F) SOX2 and miR148a expression levels in RADH85 IRE1 Q* expressing cells in the presence of miR148a mimic compared to control. G) XBP1s, SOX2 and miR148a expression levels in RADH85 IRE1 Q* expressing cells, over-expressing XBP1s compared to control. H) XBP1s, SOX2 and miR148a expression levels in RADH85 IRE1 Q* expressing cells, over-expressing XBP1s, in the presence of miR148a inhibitors compared to control. (ns): not significant; (*): p
    Figure Legend Snippet: XBP1s-dependent expression of miR148a prevents GBM cell reprogramming. A) Schematic representation of hypothesis of effect of IRE1 signaling on reprogramming TFs. B) Hierarchical clustering of miRNAs in GBM (TCGA cohort) confronted to high XBP1s or high RIDD activity (blue low levels, red high levels) with the best 5 candidates shown. C) mRNA expression of miR148a based on microarray fluorescence intensity in high XBP1s and high RIDD activity tumors (TCGA cohort). D) miR148a expression in adherent lines U251, RADH85/87 expressing DN or Q* forms of IRE1 normalized to parental. E) miR148a expression in adherent lines U251, RADH85/87 transiently deficient for XBP1s through siRNA transfection compared to control. F) SOX2 and miR148a expression levels in RADH85 IRE1 Q* expressing cells in the presence of miR148a mimic compared to control. G) XBP1s, SOX2 and miR148a expression levels in RADH85 IRE1 Q* expressing cells, over-expressing XBP1s compared to control. H) XBP1s, SOX2 and miR148a expression levels in RADH85 IRE1 Q* expressing cells, over-expressing XBP1s, in the presence of miR148a inhibitors compared to control. (ns): not significant; (*): p

    Techniques Used: Expressing, Activity Assay, Microarray, Fluorescence, Transfection

    4) Product Images from "ASPM promotes glioblastoma growth by regulating G1 restriction point progression and Wnt-β-catenin signaling"

    Article Title: ASPM promotes glioblastoma growth by regulating G1 restriction point progression and Wnt-β-catenin signaling

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102612

    ASPM enhances tumorigenicity of GBM cells in vitro and in vivo . ( A – C ) RT-qPCR analysis of ASPM expression in stable knockdown U87, U251 and U118 cells. GAPDH was used as an internal reference. sh-NC: shRNA control, sh-1/2: shRNA targeting ASPM. *p
    Figure Legend Snippet: ASPM enhances tumorigenicity of GBM cells in vitro and in vivo . ( A – C ) RT-qPCR analysis of ASPM expression in stable knockdown U87, U251 and U118 cells. GAPDH was used as an internal reference. sh-NC: shRNA control, sh-1/2: shRNA targeting ASPM. *p

    Techniques Used: In Vitro, In Vivo, Quantitative RT-PCR, Expressing, shRNA

    Downregulation of ASPM could arrest the cell cycle of GBM cells at the G0/G1 phase. ( A , B ) The enriched signalling pathways involved in ASPM-related genes by searching the LinkedOmics database. Cell cycle is the most enriched pathway. Volcano plot ( A ) and GSEA enrichment analysis ( B ) are shown. ( C ) Spearman’s correlation of the expression of cyclin E and ASPM in TCGA GBM, indicating a strong correlation. r = 0.53. ( D – F ) The cell cycle distribution of U87, U251 and U118 cells with stable ASPM knockdown was determined by PI staining and flow cytometry. Data shown are the mean ± SD (n = 3). G0/G1 phase distribution: *p
    Figure Legend Snippet: Downregulation of ASPM could arrest the cell cycle of GBM cells at the G0/G1 phase. ( A , B ) The enriched signalling pathways involved in ASPM-related genes by searching the LinkedOmics database. Cell cycle is the most enriched pathway. Volcano plot ( A ) and GSEA enrichment analysis ( B ) are shown. ( C ) Spearman’s correlation of the expression of cyclin E and ASPM in TCGA GBM, indicating a strong correlation. r = 0.53. ( D – F ) The cell cycle distribution of U87, U251 and U118 cells with stable ASPM knockdown was determined by PI staining and flow cytometry. Data shown are the mean ± SD (n = 3). G0/G1 phase distribution: *p

    Techniques Used: Expressing, Staining, Flow Cytometry

    5) Product Images from "Enhanced MGMT expression contributes to temozolomide resistance in glioma stem-like cells"

    Article Title: Enhanced MGMT expression contributes to temozolomide resistance in glioma stem-like cells

    Journal: Chinese Journal of Cancer

    doi: 10.5732/cjc.012.10236

    O 6 -methylguanine DNA methyltransferase (MGMT) and nuclear factor-κB (NF-κB) expression in the GSC lines and the parental glioma cell lines. A, reverse transcription-polymerase chain reaction (RT-PCR) shows that the glioma cell lines U251, SKMG-4, SF295, SKMG-1, U87, MGR1, and MGR2 are MGMT-negative, whereas the SF767 cell line is MGMT-positive. However, the GSC lines U251G, SKMG-4G, SF295G, SKMG-1G, and SF767G become MGMT-positive, whereas the U87G, MGR1G, and MGR2G lines remain MGMT-negative. Meanwhile, all GSC lines and their parental glioma cell lines displayed high NF-κB expression. B, the Western blotting data are consistent with the RT-PCR results. C, methylation-specific polymerase chain reaction (MSP) shows MGMT promoter methylation in all GSC lines and their parental glioma cell lines except for the SF767 and SF767G lines. In addition, unmethylated MGMT promoters exist in the glioma cell line SF767 and the GSC lines SF767G, U251G, SKMG-4G, SKMG-1G, and SF295G; H 2 O was used as the blank control.
    Figure Legend Snippet: O 6 -methylguanine DNA methyltransferase (MGMT) and nuclear factor-κB (NF-κB) expression in the GSC lines and the parental glioma cell lines. A, reverse transcription-polymerase chain reaction (RT-PCR) shows that the glioma cell lines U251, SKMG-4, SF295, SKMG-1, U87, MGR1, and MGR2 are MGMT-negative, whereas the SF767 cell line is MGMT-positive. However, the GSC lines U251G, SKMG-4G, SF295G, SKMG-1G, and SF767G become MGMT-positive, whereas the U87G, MGR1G, and MGR2G lines remain MGMT-negative. Meanwhile, all GSC lines and their parental glioma cell lines displayed high NF-κB expression. B, the Western blotting data are consistent with the RT-PCR results. C, methylation-specific polymerase chain reaction (MSP) shows MGMT promoter methylation in all GSC lines and their parental glioma cell lines except for the SF767 and SF767G lines. In addition, unmethylated MGMT promoters exist in the glioma cell line SF767 and the GSC lines SF767G, U251G, SKMG-4G, SKMG-1G, and SF295G; H 2 O was used as the blank control.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Methylation, Polymerase Chain Reaction

    6) Product Images from "Long Noncoding RNA MEG3 Suppresses Glioma Cell Proliferation, Migration, and Invasion by Acting as a Competing Endogenous RNA of miR-19a"

    Article Title: Long Noncoding RNA MEG3 Suppresses Glioma Cell Proliferation, Migration, and Invasion by Acting as a Competing Endogenous RNA of miR-19a

    Journal: Oncology Research

    doi: 10.3727/096504017X14886689179993

    lncRNA MEG3 and miR-19a mediated glioma migration and invasion. (A) Representative images of migration and invasion assays in glioma cell lines U87 and U251 are presented. (B) Quantification of glioma cells accompanying migration and invasion assays. (C) Colony formation assay was performed to determine the proliferation, and colonies were counted and captured. (D) Quantification of relative colony formation. Experiments were repeated three independent times. * p
    Figure Legend Snippet: lncRNA MEG3 and miR-19a mediated glioma migration and invasion. (A) Representative images of migration and invasion assays in glioma cell lines U87 and U251 are presented. (B) Quantification of glioma cells accompanying migration and invasion assays. (C) Colony formation assay was performed to determine the proliferation, and colonies were counted and captured. (D) Quantification of relative colony formation. Experiments were repeated three independent times. * p

    Techniques Used: Migration, Colony Assay

    lncRNA MEG3 and miR-19a mediated glioma proliferation, apoptosis, and cell cycle. (A) Cell counting kit-8 (CCK-8) assays showed the proliferation of U87 and U251 cells in various groups. (B) The apoptosis of U87 and U251 cells was assessed by flow cytometry analysis. (C) The apoptotic cells in each group are shown. (D) Flow cytometry analysis was applied to evaluate the effect on cell cycle in U87 and U251 cells. (E) Cell cycle distribution of each group. Experiments were repeated three independent times. * p
    Figure Legend Snippet: lncRNA MEG3 and miR-19a mediated glioma proliferation, apoptosis, and cell cycle. (A) Cell counting kit-8 (CCK-8) assays showed the proliferation of U87 and U251 cells in various groups. (B) The apoptosis of U87 and U251 cells was assessed by flow cytometry analysis. (C) The apoptotic cells in each group are shown. (D) Flow cytometry analysis was applied to evaluate the effect on cell cycle in U87 and U251 cells. (E) Cell cycle distribution of each group. Experiments were repeated three independent times. * p

    Techniques Used: Cell Counting, CCK-8 Assay, Flow Cytometry

    Expressions of long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) and miR-19a in glioma tissues and cell lines were detected by real-time quantitative PCR (RT-qPCR). (A) MEG3 relative expression was analyzed by RT-qPCR in glioma tissues from 40 patients (Cancer) compared to normal tissues (Control). (B) miR-19a relative expression was similarly analyzed by RT-qPCR. (C) Pearson’s correlation was performed to analyze the correlations between MEG3 and miR-19a expression in glioma tissues ( R 2 = 0.6159). (D) MEG3 relative expression in normal human astrocytes (NHAs) and glioma cell lines U87 and U251. (E) miR-19a relative expression in NHA, U87, and U251. All assays were performed in triplicate. ** p
    Figure Legend Snippet: Expressions of long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) and miR-19a in glioma tissues and cell lines were detected by real-time quantitative PCR (RT-qPCR). (A) MEG3 relative expression was analyzed by RT-qPCR in glioma tissues from 40 patients (Cancer) compared to normal tissues (Control). (B) miR-19a relative expression was similarly analyzed by RT-qPCR. (C) Pearson’s correlation was performed to analyze the correlations between MEG3 and miR-19a expression in glioma tissues ( R 2 = 0.6159). (D) MEG3 relative expression in normal human astrocytes (NHAs) and glioma cell lines U87 and U251. (E) miR-19a relative expression in NHA, U87, and U251. All assays were performed in triplicate. ** p

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    Alignment of complementary regions of lncRNA MEG3 and phosphatase and tensin homolog (PTEN) 3′-UTR between miR-19a. (A) A putative complementary binding target site between miR-19a and PTEN 3′-UTR was shown according to bioinformatics analyses (TargetScan, miRanda, and miRGen). (B, C) The luciferase results showed the probability of binding of miR-19a with the 3′-UTR of PTEN mRNA in the U87 and U251 cell lines. (D) A putative binding target site of miR-19a with MEG3 3′-UTR according to starBase V2.0. (E, F) The luciferase results of wild-type and mutant MEG3 3′-UTR with miR-19a. (G–I) RT-qPCR and Western blot analysis revealed the regulation of miR-19a and pcDNA-MEG3 on the expression of PTEN. Values represented the mean ± SD. Experiments were repeated three independent times. * p
    Figure Legend Snippet: Alignment of complementary regions of lncRNA MEG3 and phosphatase and tensin homolog (PTEN) 3′-UTR between miR-19a. (A) A putative complementary binding target site between miR-19a and PTEN 3′-UTR was shown according to bioinformatics analyses (TargetScan, miRanda, and miRGen). (B, C) The luciferase results showed the probability of binding of miR-19a with the 3′-UTR of PTEN mRNA in the U87 and U251 cell lines. (D) A putative binding target site of miR-19a with MEG3 3′-UTR according to starBase V2.0. (E, F) The luciferase results of wild-type and mutant MEG3 3′-UTR with miR-19a. (G–I) RT-qPCR and Western blot analysis revealed the regulation of miR-19a and pcDNA-MEG3 on the expression of PTEN. Values represented the mean ± SD. Experiments were repeated three independent times. * p

    Techniques Used: Binding Assay, Luciferase, Mutagenesis, Quantitative RT-PCR, Western Blot, Expressing

    7) Product Images from "Propofol Inhibits the Migration and Invasion of Glioma Cells by Blocking the PI3K/AKT Pathway Through miR-206/ROCK1 Axis"

    Article Title: Propofol Inhibits the Migration and Invasion of Glioma Cells by Blocking the PI3K/AKT Pathway Through miR-206/ROCK1 Axis

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S232601

    Propofol represses ROCK1 expression in glioma cells. ( A, B ) The expression of ROCK1 in glioma tissues, non-tumor tissues, glioma cell lines, and NHAs was detected using qRT-PCR. ( C, D ) The expression of ROCK1 in U251 and LN229 cells exposed to propofol was determined by qRT-PCR. * P
    Figure Legend Snippet: Propofol represses ROCK1 expression in glioma cells. ( A, B ) The expression of ROCK1 in glioma tissues, non-tumor tissues, glioma cell lines, and NHAs was detected using qRT-PCR. ( C, D ) The expression of ROCK1 in U251 and LN229 cells exposed to propofol was determined by qRT-PCR. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Propofol suppresses migration, invasion, and PI3K/AKT pathway activation of glioma cells. U251 and LN229 cells were treated with or without 5.1% sevoflurane. ( A, B ) Cell proliferation was detected using the MTT assay. ( C, D ) Transwell assay was used to measure cell migration and invasion. ( E ) Western blot was applied to examine the levels of p-PI3K, PI3K, p-AKT, and AKT. * P
    Figure Legend Snippet: Propofol suppresses migration, invasion, and PI3K/AKT pathway activation of glioma cells. U251 and LN229 cells were treated with or without 5.1% sevoflurane. ( A, B ) Cell proliferation was detected using the MTT assay. ( C, D ) Transwell assay was used to measure cell migration and invasion. ( E ) Western blot was applied to examine the levels of p-PI3K, PI3K, p-AKT, and AKT. * P

    Techniques Used: Migration, Activation Assay, MTT Assay, Transwell Assay, Western Blot

    ROCK1 is a target of miR-206. ( A ) A graphical representation of the miR-206 binding site within ROCK1 was presented. ( B, C ) The luciferase activity was examined in U251 and LN229 cells co-transfected with ROCK1-WT, or ROCK1-MUT and miR-206, or miR-NC. ( D, E ) The luciferase activity was analyzed in U251 and LN229 cells co-transfected with ROCK1-WT, or ROCK1-MUT and miR-206-I, or miR-NC-I. ( F ) The abundance of ROCK1 was detected in U251 and LN229 cells transfected with miR-NC or miR-206 after RIP. ( G, H ) The interaction between miR-206 and ROCK1 was confirmed by RNA pull-down assay. * P
    Figure Legend Snippet: ROCK1 is a target of miR-206. ( A ) A graphical representation of the miR-206 binding site within ROCK1 was presented. ( B, C ) The luciferase activity was examined in U251 and LN229 cells co-transfected with ROCK1-WT, or ROCK1-MUT and miR-206, or miR-NC. ( D, E ) The luciferase activity was analyzed in U251 and LN229 cells co-transfected with ROCK1-WT, or ROCK1-MUT and miR-206-I, or miR-NC-I. ( F ) The abundance of ROCK1 was detected in U251 and LN229 cells transfected with miR-NC or miR-206 after RIP. ( G, H ) The interaction between miR-206 and ROCK1 was confirmed by RNA pull-down assay. * P

    Techniques Used: Binding Assay, Luciferase, Activity Assay, Transfection, Pull Down Assay

    MiR-206 enhances propofol treatment mediated inhibition on the migration, invasion, and PI3K/AKT pathway activation of glioma cells. U251 and LN229 cells were transfected with miR-206 or miR-206-I prior to propofol exposure. ( A–F ) Cell migration and invasion abilities were detected using a transwell assay. ( G, H ) The levels of p-PI3K, PI3K, p-AKT, and AKT were determined by Western blot. * P
    Figure Legend Snippet: MiR-206 enhances propofol treatment mediated inhibition on the migration, invasion, and PI3K/AKT pathway activation of glioma cells. U251 and LN229 cells were transfected with miR-206 or miR-206-I prior to propofol exposure. ( A–F ) Cell migration and invasion abilities were detected using a transwell assay. ( G, H ) The levels of p-PI3K, PI3K, p-AKT, and AKT were determined by Western blot. * P

    Techniques Used: Inhibition, Migration, Activation Assay, Transfection, Transwell Assay, Western Blot

    Propofol promotes the expression of miR-206 in glioma cells. ( A, B ) The expression of miR-206 in glioma tissues, non-tumor tissues, glioma cell lines, and NHAs was measured using qRT-PCR. ( C, D ) The expression of miR-206 in U251 and LN229 cells treated with or without propofol was examined by qRT-PCR. * P
    Figure Legend Snippet: Propofol promotes the expression of miR-206 in glioma cells. ( A, B ) The expression of miR-206 in glioma tissues, non-tumor tissues, glioma cell lines, and NHAs was measured using qRT-PCR. ( C, D ) The expression of miR-206 in U251 and LN229 cells treated with or without propofol was examined by qRT-PCR. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Propofol inhibits the migration, invasion, and PI3K/AKT pathway activation of glioma cells by miR-206/ROCK1 axis. U251 and LN229 cells were transfected with miR-NC-I, miR-206-I, miR-206-I + si-NC, or miR-206-I + si-ROCK1 prior to propofol treatment. ( A–F ) Cell migration and invasion abilities were demonstrated using a transwell assay. ( G, H ) Western blot was utilized to examine the expression of p-PI3K, PI3K, p-AKT, and AKT. * P
    Figure Legend Snippet: Propofol inhibits the migration, invasion, and PI3K/AKT pathway activation of glioma cells by miR-206/ROCK1 axis. U251 and LN229 cells were transfected with miR-NC-I, miR-206-I, miR-206-I + si-NC, or miR-206-I + si-ROCK1 prior to propofol treatment. ( A–F ) Cell migration and invasion abilities were demonstrated using a transwell assay. ( G, H ) Western blot was utilized to examine the expression of p-PI3K, PI3K, p-AKT, and AKT. * P

    Techniques Used: Migration, Activation Assay, Transfection, Transwell Assay, Western Blot, Expressing

    8) Product Images from "Autophagy mediates glucose starvation-induced glioblastoma cell quiescence and chemoresistance through coordinating cell metabolism, cell cycle, and survival"

    Article Title: Autophagy mediates glucose starvation-induced glioblastoma cell quiescence and chemoresistance through coordinating cell metabolism, cell cycle, and survival

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0242-x

    Glucose starvation sensitizes glioblastoma cells to chemotherapies. a – b Glucose starvation (1.0 g/L) rendered both U87 ( a ) and U251 ( b ) cells more sensitive to chemotherapeutic drugs. By day 5, 40–50% cell death was induced by temozolomide (TMZ, 200 μM) or carboplatin (Carbo, 50 μM) treatment in U87 ( a ) and U251 cells ( b ) under normal glucose condition (4.5 g/L). The cell death rate was nearly doubled to 70–90% under glucose starvation condition. c Flow cytometry analysis by PI staining confirmed the synergetic cytotoxic effects between chemotherapies and glucose starvation. Dying cells were identified as the hypodiploids as denoted by the green circles. As depicted, the cell death rates in the lower panels were higher than that of the upper panels. In particular, when chemotherapeutic drugs were combined with glucose starvation as in lower panels (GS + TMZ, GS + Carbo), there were much more hypodiploid cells
    Figure Legend Snippet: Glucose starvation sensitizes glioblastoma cells to chemotherapies. a – b Glucose starvation (1.0 g/L) rendered both U87 ( a ) and U251 ( b ) cells more sensitive to chemotherapeutic drugs. By day 5, 40–50% cell death was induced by temozolomide (TMZ, 200 μM) or carboplatin (Carbo, 50 μM) treatment in U87 ( a ) and U251 cells ( b ) under normal glucose condition (4.5 g/L). The cell death rate was nearly doubled to 70–90% under glucose starvation condition. c Flow cytometry analysis by PI staining confirmed the synergetic cytotoxic effects between chemotherapies and glucose starvation. Dying cells were identified as the hypodiploids as denoted by the green circles. As depicted, the cell death rates in the lower panels were higher than that of the upper panels. In particular, when chemotherapeutic drugs were combined with glucose starvation as in lower panels (GS + TMZ, GS + Carbo), there were much more hypodiploid cells

    Techniques Used: Flow Cytometry, Cytometry, Staining

    9) Product Images from "Efficacy of a novel double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 against glioblastoma cells"

    Article Title: Efficacy of a novel double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 against glioblastoma cells

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-020-00485-1

    Tube formation capacity of HUVECs incubated in different U251 conditioned media. a Glioma cells U251 were respectively treated with Ad5-GFP, Ad5-Ki67/GFP, Ad5-Ki67/IL15 (MOI = 40) for 72 h, and at which time different U251 conditioned media (Ad5-GFP-CM, Ad5-Ki67/GFP-CM and Ad5-Ki67/IL15-CM) were collected. Angiogenic capacity of HUVECs cultured by CM, Ad5-GFP-CM, Ad5-Ki67/GFP-CM and Ad5-Ki67/IL15-CM (MOI = 40) for 6 h on Matrigel (×100, scale bars = 500 µm). CM represented the untreated U251 conditioned medium. b, c Total segments length and quantification of number of tubes generated by HUVECs incubated with different conditioned media. *P
    Figure Legend Snippet: Tube formation capacity of HUVECs incubated in different U251 conditioned media. a Glioma cells U251 were respectively treated with Ad5-GFP, Ad5-Ki67/GFP, Ad5-Ki67/IL15 (MOI = 40) for 72 h, and at which time different U251 conditioned media (Ad5-GFP-CM, Ad5-Ki67/GFP-CM and Ad5-Ki67/IL15-CM) were collected. Angiogenic capacity of HUVECs cultured by CM, Ad5-GFP-CM, Ad5-Ki67/GFP-CM and Ad5-Ki67/IL15-CM (MOI = 40) for 6 h on Matrigel (×100, scale bars = 500 µm). CM represented the untreated U251 conditioned medium. b, c Total segments length and quantification of number of tubes generated by HUVECs incubated with different conditioned media. *P

    Techniques Used: Incubation, Cell Culture, Generated

    Tube formation capacity of HUVECs incubated in different U251 conditioned media. a Angiogenic capacity of HUVECs cultured in Ad5-GFP-CM, Ad5-Ki67/GFP-CM, Ad5-Ki67/IL15-CM, Ad5-GFP-CM + VEGF, Ad5-Ki67/GFP-CM + VEGF and Ad5-Ki67/IL15-CM + VEGF (MOI = 40) for 6 h on Matrigel (×100, scale bars = 500 µm). b, c Total segments length and quantification of number of tubes generated by HUVECs incubated with different culture conditions. *P
    Figure Legend Snippet: Tube formation capacity of HUVECs incubated in different U251 conditioned media. a Angiogenic capacity of HUVECs cultured in Ad5-GFP-CM, Ad5-Ki67/GFP-CM, Ad5-Ki67/IL15-CM, Ad5-GFP-CM + VEGF, Ad5-Ki67/GFP-CM + VEGF and Ad5-Ki67/IL15-CM + VEGF (MOI = 40) for 6 h on Matrigel (×100, scale bars = 500 µm). b, c Total segments length and quantification of number of tubes generated by HUVECs incubated with different culture conditions. *P

    Techniques Used: Incubation, Cell Culture, Generated

    VEGF levels in different glioma conditioned media measured by ELISA. a VEGF levels were significantly lower in Ad5-GFP-CM, Ad5-Ki67/GFP-CM, and Ad5-Ki67/IL15-CM (MOI = 40) than in untreated conditioned medium in GL261 cells. b U251. c U87. d Primary cells BT-01. *P
    Figure Legend Snippet: VEGF levels in different glioma conditioned media measured by ELISA. a VEGF levels were significantly lower in Ad5-GFP-CM, Ad5-Ki67/GFP-CM, and Ad5-Ki67/IL15-CM (MOI = 40) than in untreated conditioned medium in GL261 cells. b U251. c U87. d Primary cells BT-01. *P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Expression of IL-15 in GBM cells treated with Ad5-Ki67/IL-15. a – d ELISA was used to measure IL-15 levels in conditioned media that different glioma cells were treated with Ad5-Ki67/IL-15 (MOI = 40) for 24, 48, 72, and 96 h. Untreated conditioned medium was used as a control. e – h IL-15 gene expression in GL261, U251, U87 and primary cells BT-01 treated with Ad5-Ki67/IL-15 for 72 h by qPCR. Untreated GBM cells were used as a control
    Figure Legend Snippet: Expression of IL-15 in GBM cells treated with Ad5-Ki67/IL-15. a – d ELISA was used to measure IL-15 levels in conditioned media that different glioma cells were treated with Ad5-Ki67/IL-15 (MOI = 40) for 24, 48, 72, and 96 h. Untreated conditioned medium was used as a control. e – h IL-15 gene expression in GL261, U251, U87 and primary cells BT-01 treated with Ad5-Ki67/IL-15 for 72 h by qPCR. Untreated GBM cells were used as a control

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    Characteristics of novel constructed oncolytic adenovirus. a , b Recombinant oncolytic adenovirus (MOI = 40) does not infect microglial HMC3 and BV2 cells (×50, scale bars = 1000 µm). c The capacity of recombinant oncolytic adenovirus to infect GBM cells. Representative photomicrographs were obtained from GL261, U251, U87, and primary cells BT-01 that treated with Ad5-GFP and Ad5-Ki67/GFP at an MOI of 40 for 72 h (×50, scale bars = 1000 µm). d Quantification of the average intensity difference in GBM cells treated with Ad5-GFP and Ad5-Ki67/GFP for 72 h using Image Pro Plus. (n ≥ 3) *P
    Figure Legend Snippet: Characteristics of novel constructed oncolytic adenovirus. a , b Recombinant oncolytic adenovirus (MOI = 40) does not infect microglial HMC3 and BV2 cells (×50, scale bars = 1000 µm). c The capacity of recombinant oncolytic adenovirus to infect GBM cells. Representative photomicrographs were obtained from GL261, U251, U87, and primary cells BT-01 that treated with Ad5-GFP and Ad5-Ki67/GFP at an MOI of 40 for 72 h (×50, scale bars = 1000 µm). d Quantification of the average intensity difference in GBM cells treated with Ad5-GFP and Ad5-Ki67/GFP for 72 h using Image Pro Plus. (n ≥ 3) *P

    Techniques Used: Construct, Recombinant

    The OAd driven by the Ki67 core promoter and armed with IL-15 enhanced GBM eradication. a – d CCK-8 assay was performed to evaluate cell viability of GL261, U251, U87 and primary cells BT-01 that treated with Ad5-GFP, Ad5-Ki67/GFP, and Ad5-Ki67/IL15 (MOI = 40). The inhibition ability of Ad5-Ki67/GFP and Ad5-Ki67/IL15 was more potent than Ad5-GFP at different time points. e GL261 cell proliferation in response to the conditioned medium from virus-treated BV2 microglial cells (BV2-Ad5-GFP-CM, BV2-Ad5-Ki67/GFP-CM, and BV2-Ad5-Ki67/IL15-CM, MOI = 40) at different time points was determined by CCK8. f – h U251, U87 and primary cells BT-01 in response to the conditioned medium from virus-treated HMC3 microglial cells (HMC3-Ad5-GFP-CM, HMC3-Ad5-Ki67/GFP-CM, and HMC3-Ad5-Ki67/IL15-CM, MOI = 40) at different time points was determined by CCK8. All results are expressed as a percentage of untreated controls. Data are presented as mean ± SD of three independent experiments. *P
    Figure Legend Snippet: The OAd driven by the Ki67 core promoter and armed with IL-15 enhanced GBM eradication. a – d CCK-8 assay was performed to evaluate cell viability of GL261, U251, U87 and primary cells BT-01 that treated with Ad5-GFP, Ad5-Ki67/GFP, and Ad5-Ki67/IL15 (MOI = 40). The inhibition ability of Ad5-Ki67/GFP and Ad5-Ki67/IL15 was more potent than Ad5-GFP at different time points. e GL261 cell proliferation in response to the conditioned medium from virus-treated BV2 microglial cells (BV2-Ad5-GFP-CM, BV2-Ad5-Ki67/GFP-CM, and BV2-Ad5-Ki67/IL15-CM, MOI = 40) at different time points was determined by CCK8. f – h U251, U87 and primary cells BT-01 in response to the conditioned medium from virus-treated HMC3 microglial cells (HMC3-Ad5-GFP-CM, HMC3-Ad5-Ki67/GFP-CM, and HMC3-Ad5-Ki67/IL15-CM, MOI = 40) at different time points was determined by CCK8. All results are expressed as a percentage of untreated controls. Data are presented as mean ± SD of three independent experiments. *P

    Techniques Used: CCK-8 Assay, Inhibition

    The novel double-controlled OAd driven by the Ki67 core promoter selectively kills GBM cells. a , b CCK-8 assay was performed to evaluate cell viability of HMC3 and BV2 cells that treated with Ad5-GFP, Ad5-Ki67/GFP, and Ad5-Ki67/IL15 at different MOI. c – f CCK-8 assay demonstrated that recombinant oncolytic adenovirus inhibited proliferation of GL261, U251, U87, and primary cells BT-01 at different MOI. Inhibition levels were significantly higher in Ad5-Ki67/GFP and Ad5-Ki67/IL15 compared to Ad5. Results are expressed as a percentage of untreated controls
    Figure Legend Snippet: The novel double-controlled OAd driven by the Ki67 core promoter selectively kills GBM cells. a , b CCK-8 assay was performed to evaluate cell viability of HMC3 and BV2 cells that treated with Ad5-GFP, Ad5-Ki67/GFP, and Ad5-Ki67/IL15 at different MOI. c – f CCK-8 assay demonstrated that recombinant oncolytic adenovirus inhibited proliferation of GL261, U251, U87, and primary cells BT-01 at different MOI. Inhibition levels were significantly higher in Ad5-Ki67/GFP and Ad5-Ki67/IL15 compared to Ad5. Results are expressed as a percentage of untreated controls

    Techniques Used: CCK-8 Assay, Recombinant, Inhibition

    10) Product Images from "Xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating endoplasmic reticulum stress-dependent CHOP pathway"

    Article Title: Xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating endoplasmic reticulum stress-dependent CHOP pathway

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/s41401-019-0318-5

    Xanthatin inhibits glioma cell viability without affecting the cell cycle. The effect of xanthatin on cell viability in glioma cells. U251 and C6 cells were incubated with xanthatin at the indicated concentrations for 12 h ( a ) or 24 h ( b ). Cell viability was determined by MTT assay. c The effect of xanthatin on cell viability in nonmalignant glial cells. Primary cultured rat glial cells were incubated with xanthatin at various concentrations for 12 or 24 h, followed by the MTT assay. d Xanthatin treatment had no apparent effect on the cell cycle distribution of glioma cells. C6 cells were incubated with xanthatin at the indicated concentrations for 12 h. Propidium iodide staining and flow cytometry were used to determine the proportion of cells in various phases of the cell cycle. e Statistical analysis of the cell cycle distribution in d . Statistical values are expressed as the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: Xanthatin inhibits glioma cell viability without affecting the cell cycle. The effect of xanthatin on cell viability in glioma cells. U251 and C6 cells were incubated with xanthatin at the indicated concentrations for 12 h ( a ) or 24 h ( b ). Cell viability was determined by MTT assay. c The effect of xanthatin on cell viability in nonmalignant glial cells. Primary cultured rat glial cells were incubated with xanthatin at various concentrations for 12 or 24 h, followed by the MTT assay. d Xanthatin treatment had no apparent effect on the cell cycle distribution of glioma cells. C6 cells were incubated with xanthatin at the indicated concentrations for 12 h. Propidium iodide staining and flow cytometry were used to determine the proportion of cells in various phases of the cell cycle. e Statistical analysis of the cell cycle distribution in d . Statistical values are expressed as the mean ± SEM of three independent experiments. * P

    Techniques Used: Incubation, MTT Assay, Cell Culture, Staining, Flow Cytometry

    Xanthatin promotes the apoptosis of glioma cells. a The apoptosis rate of glioma cells was determined by flow cytometry with Annexin V/PI staining. C6 cells were incubated with 10, 15, and 20 µM xanthatin for 12 h. DMSO was used as a vehicle control. b Quantitative analysis of apoptotic cells in a . c TUNEL staining of C6 cells treated with xanthatin at the indicated concentrations for 12 h. d Quantitative analysis of TUNEL-positive C6 cells in c . e C6 cells were incubated with xanthatin at various concentrations for 12 h, and the levels of cleaved caspase-3, Bcl-2, Bax, and tubulin were detected by WB. f C6 cells were incubated with 15 µM xanthatin for 6, 12, and 24 h, and the levels of cleaved caspase-3, Bcl-2, Bax, and tubulin were determined by WB. g U251 cells were incubated with various concentrations of xanthatin for 12 h, and the levels of cleaved caspase-3, Bcl-2, Bax, and tubulin were detected by WB. h U251 cells were incubated with 15 µM xanthatin for 6, 12, and 24 h, and the expression levels of cleaved caspase-3, Bcl-2, Bax, and tubulin were determined by WB. Densitometric quantitative analysis of the Bcl-2/Bax ratio, which is expressed as the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: Xanthatin promotes the apoptosis of glioma cells. a The apoptosis rate of glioma cells was determined by flow cytometry with Annexin V/PI staining. C6 cells were incubated with 10, 15, and 20 µM xanthatin for 12 h. DMSO was used as a vehicle control. b Quantitative analysis of apoptotic cells in a . c TUNEL staining of C6 cells treated with xanthatin at the indicated concentrations for 12 h. d Quantitative analysis of TUNEL-positive C6 cells in c . e C6 cells were incubated with xanthatin at various concentrations for 12 h, and the levels of cleaved caspase-3, Bcl-2, Bax, and tubulin were detected by WB. f C6 cells were incubated with 15 µM xanthatin for 6, 12, and 24 h, and the levels of cleaved caspase-3, Bcl-2, Bax, and tubulin were determined by WB. g U251 cells were incubated with various concentrations of xanthatin for 12 h, and the levels of cleaved caspase-3, Bcl-2, Bax, and tubulin were detected by WB. h U251 cells were incubated with 15 µM xanthatin for 6, 12, and 24 h, and the expression levels of cleaved caspase-3, Bcl-2, Bax, and tubulin were determined by WB. Densitometric quantitative analysis of the Bcl-2/Bax ratio, which is expressed as the mean ± SEM of three independent experiments. * P

    Techniques Used: Flow Cytometry, Staining, Incubation, TUNEL Assay, Western Blot, Expressing

    Xanthatin upregulates the expression of ER stress-related genes and induces the nuclear translocation of CHOP in glioma cells. a U251 cells were treated with xanthatin (15 and 20 µM) for 12 h, and the mRNA levels of GRP78, XBP1s, ATF4, CHOP, PERK , and eIF2α were assessed by qRT-PCR. GAPDH was used as a control. Values are expressed as the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: Xanthatin upregulates the expression of ER stress-related genes and induces the nuclear translocation of CHOP in glioma cells. a U251 cells were treated with xanthatin (15 and 20 µM) for 12 h, and the mRNA levels of GRP78, XBP1s, ATF4, CHOP, PERK , and eIF2α were assessed by qRT-PCR. GAPDH was used as a control. Values are expressed as the mean ± SEM of three independent experiments. * P

    Techniques Used: Expressing, Translocation Assay, Quantitative RT-PCR

    Xanthatin induces ER stress in glioma cells. a Representative immunoblots against ER stress-related proteins from C6 cells treated with xanthatin (1, 5, 10, and 15 µM) for 12 h. b Representative immunoblots against ER stress-related proteins from C6 cells treated with 15 µM xanthatin for the indicated times. c Quantitative analysis of protein levels in a and b . d The levels of ER stress-related proteins in U251 cells treated with xanthatin at the indicated concentrations for 12 h. e The levels of ER stress-related proteins in U251 cells treated with xanthatin for 6, 12, and 24 h. f Quantitative analysis of protein levels in d and e . Values are expressed as the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: Xanthatin induces ER stress in glioma cells. a Representative immunoblots against ER stress-related proteins from C6 cells treated with xanthatin (1, 5, 10, and 15 µM) for 12 h. b Representative immunoblots against ER stress-related proteins from C6 cells treated with 15 µM xanthatin for the indicated times. c Quantitative analysis of protein levels in a and b . d The levels of ER stress-related proteins in U251 cells treated with xanthatin at the indicated concentrations for 12 h. e The levels of ER stress-related proteins in U251 cells treated with xanthatin for 6, 12, and 24 h. f Quantitative analysis of protein levels in d and e . Values are expressed as the mean ± SEM of three independent experiments. * P

    Techniques Used: Western Blot

    11) Product Images from "Disulfiram, a Ferroptosis Inducer, Triggers Lysosomal Membrane Permeabilization by Up-Regulating ROS in Glioblastoma"

    Article Title: Disulfiram, a Ferroptosis Inducer, Triggers Lysosomal Membrane Permeabilization by Up-Regulating ROS in Glioblastoma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S272312

    DSF inhibits cell growth of GBM cells ( A ) Growth curves generated with colorimetric data (O.D. 450 nm) from the CCK-8 assay. U251 and LN229 cells were treated with increasing concentrations of DSF for 24 h. The IC 50 for U251 and LN229 was 30 μM and 13.5 μM, respectively. ( B ) Fluorescence images for EdU incorporation at 5 μM DSF and DMSO (vehicle control) for 24 h in U251 and LN229 cells. ( C ) Quantification of EdU positive cells treated with DMSO (vehicle control) and 5 μM DSF in B. ( D ) Cell cycle analysis of U251 and LN229 cells treated with DMSO (vehicle control) and 5 μM DSF for 24 h. ( E ) Quantification of cell cycle parameters G0-G1, S, and G2-M obtained from flow cytometric analysis in D. * P
    Figure Legend Snippet: DSF inhibits cell growth of GBM cells ( A ) Growth curves generated with colorimetric data (O.D. 450 nm) from the CCK-8 assay. U251 and LN229 cells were treated with increasing concentrations of DSF for 24 h. The IC 50 for U251 and LN229 was 30 μM and 13.5 μM, respectively. ( B ) Fluorescence images for EdU incorporation at 5 μM DSF and DMSO (vehicle control) for 24 h in U251 and LN229 cells. ( C ) Quantification of EdU positive cells treated with DMSO (vehicle control) and 5 μM DSF in B. ( D ) Cell cycle analysis of U251 and LN229 cells treated with DMSO (vehicle control) and 5 μM DSF for 24 h. ( E ) Quantification of cell cycle parameters G0-G1, S, and G2-M obtained from flow cytometric analysis in D. * P

    Techniques Used: Generated, CCK-8 Assay, Fluorescence, Cell Cycle Assay

    DSF induces lysosomal membrane permeabilization in GBM cells. ( A ) Fluorescence images of acridine orange staining of DSF treated U251 and LN229 cells compared with controls (DMSO, vehicle control). Red fluorescence is associated with high concentrations of acridine orange, while green fluorescence is associated with reduced concentrations of acridine orange. ( B ) Fluorescence images of U251 and LN229 cells treated with DSF and transfected with EGFP-Gal3 construct (green fluorescence). Green puncta result from the collection of EGFP-Gal3 collection on compromised lysosomes. ( C ) Quantification of green puncta in ( B ). ( D ) Fluorescence images of LysoTracker Red staining of DSF treated U251 and LN229 cells. *** P
    Figure Legend Snippet: DSF induces lysosomal membrane permeabilization in GBM cells. ( A ) Fluorescence images of acridine orange staining of DSF treated U251 and LN229 cells compared with controls (DMSO, vehicle control). Red fluorescence is associated with high concentrations of acridine orange, while green fluorescence is associated with reduced concentrations of acridine orange. ( B ) Fluorescence images of U251 and LN229 cells treated with DSF and transfected with EGFP-Gal3 construct (green fluorescence). Green puncta result from the collection of EGFP-Gal3 collection on compromised lysosomes. ( C ) Quantification of green puncta in ( B ). ( D ) Fluorescence images of LysoTracker Red staining of DSF treated U251 and LN229 cells. *** P

    Techniques Used: Fluorescence, Staining, Transfection, Construct

    DSF induces ferroptosis in GBM cells. ( A ) Representative fluorescence images of BODIPY 581/591 C11 staining of U251 and LN229 cells treated with DMSO (vehicle control) and 5 μM DSF for 24 h. ( B ) Representative fluorescence images of CellRox Green staining of U251 and LN229 cells treated with DMSO (vehicle control) and 5 μM DSF. ( C ) Quantification of fluorescence in ( B ). ( D ) Levels of ferrous iron in U251 and LN229 cells treated with DMSO (vehicle control) and 5 μM DSF. ( E ) Western blot analysis of lysates prepared from U251 and LN229 cells treated with 0, 2.5, 5 and 10 μM DSF for 24 h to detect levels of protein inhibitors of ferroptosis, xCT and GPX-4. GAPDH was used as a loading control. The xCT/GAPDH and GPX-4/GAPDH ratios were calculated using ImageJ (National Institutes of Health, version 1.52r). ( F ) Transmission electron microscopy image showing the ultrastructure of U251 cells treated with DMSO (vehicle control) or 5 μM DSF. Normal mitochondria in control cells highlighted with the white arrows; shrunken mitochondria and the enhanced density of mitochondrial membrane highlighted with red arrows in DSF treated U251 cells. ( G ) BODIPY 581/591 C11 staining of U251 and LN229 cells treated with DMSO, 5 μM DSF, 50 μM iron chelator deferoxamine mesylate (DFO) + 5 μM DSF, 2 μM Ferrostatin-1 (Fer-1) + 5 μM DSF. ( H ) LDH release assay was used to test the cytotoxic effect of U251 and LN229 cells treated with DMSO, 10 μM DSF, 50 μM iron chelator deferoxamine mesylate (DFO) + 10 μM DSF, 2 μM Ferrostatin-1 (Fer-1) + 10 μM DSF. ** P
    Figure Legend Snippet: DSF induces ferroptosis in GBM cells. ( A ) Representative fluorescence images of BODIPY 581/591 C11 staining of U251 and LN229 cells treated with DMSO (vehicle control) and 5 μM DSF for 24 h. ( B ) Representative fluorescence images of CellRox Green staining of U251 and LN229 cells treated with DMSO (vehicle control) and 5 μM DSF. ( C ) Quantification of fluorescence in ( B ). ( D ) Levels of ferrous iron in U251 and LN229 cells treated with DMSO (vehicle control) and 5 μM DSF. ( E ) Western blot analysis of lysates prepared from U251 and LN229 cells treated with 0, 2.5, 5 and 10 μM DSF for 24 h to detect levels of protein inhibitors of ferroptosis, xCT and GPX-4. GAPDH was used as a loading control. The xCT/GAPDH and GPX-4/GAPDH ratios were calculated using ImageJ (National Institutes of Health, version 1.52r). ( F ) Transmission electron microscopy image showing the ultrastructure of U251 cells treated with DMSO (vehicle control) or 5 μM DSF. Normal mitochondria in control cells highlighted with the white arrows; shrunken mitochondria and the enhanced density of mitochondrial membrane highlighted with red arrows in DSF treated U251 cells. ( G ) BODIPY 581/591 C11 staining of U251 and LN229 cells treated with DMSO, 5 μM DSF, 50 μM iron chelator deferoxamine mesylate (DFO) + 5 μM DSF, 2 μM Ferrostatin-1 (Fer-1) + 5 μM DSF. ( H ) LDH release assay was used to test the cytotoxic effect of U251 and LN229 cells treated with DMSO, 10 μM DSF, 50 μM iron chelator deferoxamine mesylate (DFO) + 10 μM DSF, 2 μM Ferrostatin-1 (Fer-1) + 10 μM DSF. ** P

    Techniques Used: Fluorescence, Staining, Western Blot, Transmission Assay, Electron Microscopy, Lactate Dehydrogenase Assay

    DSF enhances the radiosensitivity of GBM cells. ( A ) Fluorescence images of the Live/Dead staining assay (green fluorescence, live; red fluorescence, dead) performed on U251 and LN229 cells treated with DSF (5 µM), radiation (4 GY, IR) or a combination of DSF and radiation. ( B ) Quantification of dead cells (red fluorescence) in ( A ). ( C ) Immunofluorescence staining of γ-H2AX (red fluorescence) to determine the double strand breaks (DSBs) caused by DSF, radiation (IR) or a combination of DSF and radiation. ( D ) Quantification of red puncta associated with γ-H2AX in ( C ). ( E ) Western blot analysis of lysates prepared from U251 and LN229 cells treated with DSF (5 µM), radiation (4 GY, IR) or a combination of DSF and radiation. GAPDH was used as a loading control. The γ-H2AX/GAPDH ratio was calculated using ImageJ. **P
    Figure Legend Snippet: DSF enhances the radiosensitivity of GBM cells. ( A ) Fluorescence images of the Live/Dead staining assay (green fluorescence, live; red fluorescence, dead) performed on U251 and LN229 cells treated with DSF (5 µM), radiation (4 GY, IR) or a combination of DSF and radiation. ( B ) Quantification of dead cells (red fluorescence) in ( A ). ( C ) Immunofluorescence staining of γ-H2AX (red fluorescence) to determine the double strand breaks (DSBs) caused by DSF, radiation (IR) or a combination of DSF and radiation. ( D ) Quantification of red puncta associated with γ-H2AX in ( C ). ( E ) Western blot analysis of lysates prepared from U251 and LN229 cells treated with DSF (5 µM), radiation (4 GY, IR) or a combination of DSF and radiation. GAPDH was used as a loading control. The γ-H2AX/GAPDH ratio was calculated using ImageJ. **P

    Techniques Used: Fluorescence, Staining, Immunofluorescence, Western Blot

    DSF induces LMP in a ROS-dependent manner. ( A ) Fluorescence images of cells transfected with EGFP-Gal3. U251 and LN229 cells were treated with DSF and inhibitors of ferroptosis, 5 mM GSH or 2 µM ferrostatin-1 (Fer-1), and compared with DSF treatment alone. ( B ) Quantification of green punta in ( A ). ** P
    Figure Legend Snippet: DSF induces LMP in a ROS-dependent manner. ( A ) Fluorescence images of cells transfected with EGFP-Gal3. U251 and LN229 cells were treated with DSF and inhibitors of ferroptosis, 5 mM GSH or 2 µM ferrostatin-1 (Fer-1), and compared with DSF treatment alone. ( B ) Quantification of green punta in ( A ). ** P

    Techniques Used: Fluorescence, Transfection

    12) Product Images from "Sevoflurane inhibits cell proliferation and migration of glioma by targeting the miR-27b/VEGF axis"

    Article Title: Sevoflurane inhibits cell proliferation and migration of glioma by targeting the miR-27b/VEGF axis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2021.12047

    VEGF is a target of miR-27b. (A) A conserved binding site for miR-27b in the 3′UTR region of the VEGF gene was identified. miR-27b targeted VEGF 3′UTR in (B) U251 and (C) U87 cells. **P
    Figure Legend Snippet: VEGF is a target of miR-27b. (A) A conserved binding site for miR-27b in the 3′UTR region of the VEGF gene was identified. miR-27b targeted VEGF 3′UTR in (B) U251 and (C) U87 cells. **P

    Techniques Used: Binding Assay

    Sevoflurane-induced inhibition of glioma cell migration is mediated by the miR-27b/VEGF axis. The untreated control group acted as a negative control group for the experiments. (A) Images and (B) analysis of the inhibitory effects of sevoflurane on U251 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. (C) Images and (D) analysis of the inhibitory effects of sevoflurane on U87 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. Magnification, ×100. *P
    Figure Legend Snippet: Sevoflurane-induced inhibition of glioma cell migration is mediated by the miR-27b/VEGF axis. The untreated control group acted as a negative control group for the experiments. (A) Images and (B) analysis of the inhibitory effects of sevoflurane on U251 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. (C) Images and (D) analysis of the inhibitory effects of sevoflurane on U87 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. Magnification, ×100. *P

    Techniques Used: Inhibition, Migration, Negative Control

    Sevoflurane-induced inhibition of glioma cell proliferation is mediated by the miR-27b/VEGF axis. Compared with the siRNA-NC group, siRNA-1 VEGF and siRNA-2 VEGF significantly decreased the expression of VEGF in (A) U251 and (B) U87 cells. The untreated control group acted as a negative control group for the experiments. The inhibitory effects of sevoflurane on (C) U251 and (D) U87 cell proliferation were abolished by pre-treatment with miRNA-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. P
    Figure Legend Snippet: Sevoflurane-induced inhibition of glioma cell proliferation is mediated by the miR-27b/VEGF axis. Compared with the siRNA-NC group, siRNA-1 VEGF and siRNA-2 VEGF significantly decreased the expression of VEGF in (A) U251 and (B) U87 cells. The untreated control group acted as a negative control group for the experiments. The inhibitory effects of sevoflurane on (C) U251 and (D) U87 cell proliferation were abolished by pre-treatment with miRNA-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. P

    Techniques Used: Inhibition, Expressing, Negative Control

    Sevoflurane induces the expression of miR-27b in glioma cells. Decreased miR-27b lower expression was identified in (A) U251 cells compared with HEB cells, which was significantly increased by (B) sevoflurane. Similar miR-27b expression levels were found in (C) U87 cells and (D) after sevoflurane treatment. **P
    Figure Legend Snippet: Sevoflurane induces the expression of miR-27b in glioma cells. Decreased miR-27b lower expression was identified in (A) U251 cells compared with HEB cells, which was significantly increased by (B) sevoflurane. Similar miR-27b expression levels were found in (C) U87 cells and (D) after sevoflurane treatment. **P

    Techniques Used: Expressing

    Sevoflurane reduces the expression of VEGF in glioma cells by targeting miR-27b. (A) Increased expression of VEGF in U251 cells compared with HEB cells. The untreated control group acted as a negative control group for the experiments. (B) Compared with control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by miR-27b inhibitor. (C) Increased expression of VEGF in U87 cells compared with HEB cells. (D) Compared with the control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by the miR-27b inhibitor. P
    Figure Legend Snippet: Sevoflurane reduces the expression of VEGF in glioma cells by targeting miR-27b. (A) Increased expression of VEGF in U251 cells compared with HEB cells. The untreated control group acted as a negative control group for the experiments. (B) Compared with control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by miR-27b inhibitor. (C) Increased expression of VEGF in U87 cells compared with HEB cells. (D) Compared with the control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by the miR-27b inhibitor. P

    Techniques Used: Expressing, Negative Control

    Sevoflurane inhibits the expression levels of MMP-2 and MMP-9. (A) Decreased in protein expression levels of MMP-2 and MMP-9 in U251 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (B) Quantification of western blotting results. (C) Decreased in protein expression levels of MMP-2 and MMP-9 in U87 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (D) Quantification of western blotting results. **P
    Figure Legend Snippet: Sevoflurane inhibits the expression levels of MMP-2 and MMP-9. (A) Decreased in protein expression levels of MMP-2 and MMP-9 in U251 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (B) Quantification of western blotting results. (C) Decreased in protein expression levels of MMP-2 and MMP-9 in U87 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (D) Quantification of western blotting results. **P

    Techniques Used: Expressing, Western Blot

    miR-27b mimic and inhibitor affect miR-27b expression levels. In (A) U251 cells, miR-27b was significantly increased after miR-27b mimics transfection, while significantly decreased after (B) miR-27b inhibitor transfection. Similar results were obtained in U87 cells after (C) miR-27b mimic and (D) inhibitor transfection. **P
    Figure Legend Snippet: miR-27b mimic and inhibitor affect miR-27b expression levels. In (A) U251 cells, miR-27b was significantly increased after miR-27b mimics transfection, while significantly decreased after (B) miR-27b inhibitor transfection. Similar results were obtained in U87 cells after (C) miR-27b mimic and (D) inhibitor transfection. **P

    Techniques Used: Expressing, Transfection

    13) Product Images from "Gamma-Secretase Represents a Therapeutic Target for the Treatment of Invasive Glioma Mediated by the p75 Neurotrophin Receptor"

    Article Title: Gamma-Secretase Represents a Therapeutic Target for the Treatment of Invasive Glioma Mediated by the p75 Neurotrophin Receptor

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0060289

    Cleavage-Resistant Chimeric p75 NTR Proteins Do Not Induce Migration and Invasion In Vitro or In Vivo (A and B) The invasive ability of U87 (A) and U251 (B) stably transfected with p75 NTR or the p75 NTR cleavage-resistant chimeric constructs ( p75FasS and p75FasTM ) were assessed using 3D-invasion assays. Expression of p75 NTR significantly increased invasion in the genetically distinct glioma cell lines U87 and U251, whereas neither p75FasS nor p75FasTM stimulated glioma invasion as compared to the p75 NTR -negative control cells (pcDNA). Values shown are the mean ± s.e.m. from three independent experiments; (the less than symbol [
    Figure Legend Snippet: Cleavage-Resistant Chimeric p75 NTR Proteins Do Not Induce Migration and Invasion In Vitro or In Vivo (A and B) The invasive ability of U87 (A) and U251 (B) stably transfected with p75 NTR or the p75 NTR cleavage-resistant chimeric constructs ( p75FasS and p75FasTM ) were assessed using 3D-invasion assays. Expression of p75 NTR significantly increased invasion in the genetically distinct glioma cell lines U87 and U251, whereas neither p75FasS nor p75FasTM stimulated glioma invasion as compared to the p75 NTR -negative control cells (pcDNA). Values shown are the mean ± s.e.m. from three independent experiments; (the less than symbol [

    Techniques Used: Migration, In Vitro, In Vivo, Stable Transfection, Transfection, Construct, Expressing, Negative Control

    14) Product Images from "MicroRNA-6071 Suppresses Glioblastoma Progression Through the Inhibition of PI3K/AKT/mTOR Pathway by Binding to ULBP2"

    Article Title: MicroRNA-6071 Suppresses Glioblastoma Progression Through the Inhibition of PI3K/AKT/mTOR Pathway by Binding to ULBP2

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S265791

    miR-6071 inhibited the activation of PI3K/AKT/mTOR pathway in U251 and A172 cells. ( A ) The expressions of p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR in U251 cells were measured by Western blot. ( B ) The expressions of p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR in A172 cells were measured by Western blot. **P
    Figure Legend Snippet: miR-6071 inhibited the activation of PI3K/AKT/mTOR pathway in U251 and A172 cells. ( A ) The expressions of p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR in U251 cells were measured by Western blot. ( B ) The expressions of p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR in A172 cells were measured by Western blot. **P

    Techniques Used: Activation Assay, Western Blot

    miR-6071 inhibited the ability of migration and invasion in U251 and A172 cells. ( A ) The migration of U251 and A172 cells was tested by wound-healing assay. ( B ) The migration of U251 and A172 cells was tested by transwell assay. ( C ) The invasion of U251 and A172 cells was tested by transwell assay. ( D ) The expressions of MMP-2 and MMP-9 in U251 and A172 cells were measured by Western blot. **P
    Figure Legend Snippet: miR-6071 inhibited the ability of migration and invasion in U251 and A172 cells. ( A ) The migration of U251 and A172 cells was tested by wound-healing assay. ( B ) The migration of U251 and A172 cells was tested by transwell assay. ( C ) The invasion of U251 and A172 cells was tested by transwell assay. ( D ) The expressions of MMP-2 and MMP-9 in U251 and A172 cells were measured by Western blot. **P

    Techniques Used: Migration, Wound Healing Assay, Transwell Assay, Western Blot

    miR-6071 promoted the apoptosis ability of U251 and A172 cells. ( A ) The apoptosis of U251 and A172 cells was detected by flow cytometry. ( B ) The expressions of Bax, caspase-3 and Bcl-2 in U251 and A172 cells were measured by Western blot. **P
    Figure Legend Snippet: miR-6071 promoted the apoptosis ability of U251 and A172 cells. ( A ) The apoptosis of U251 and A172 cells was detected by flow cytometry. ( B ) The expressions of Bax, caspase-3 and Bcl-2 in U251 and A172 cells were measured by Western blot. **P

    Techniques Used: Flow Cytometry, Western Blot

    miR-6071 inhibited the proliferation ability of U251 and A172 cells. ( A ) The expression of miR-6071 in transfected U251 and A172 cells was detected by qRT-PCR. ( B ) The colonies number of U251 and A172 cells was detected by colony formation assay. ( C ) The proliferation of U251 and A172 cells was tested by EdU assay. **P
    Figure Legend Snippet: miR-6071 inhibited the proliferation ability of U251 and A172 cells. ( A ) The expression of miR-6071 in transfected U251 and A172 cells was detected by qRT-PCR. ( B ) The colonies number of U251 and A172 cells was detected by colony formation assay. ( C ) The proliferation of U251 and A172 cells was tested by EdU assay. **P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Colony Assay, EdU Assay

    15) Product Images from "p300- and Myc-mediated regulation of glioblastoma multiforme cell differentiation"

    Article Title: p300- and Myc-mediated regulation of glioblastoma multiforme cell differentiation

    Journal: Oncotarget

    doi:

    Myc overrides p300-mediated transcriptional regulation of GFAP and Nestin. U251 (A B) and U87 (C D) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A C) or 2 µg Nestin-Luc (B D) reporter construct along with 1 µg of p300 and 1 µg of Myc expression constructs, either separately or in combination. Luciferase activity was determined at 72 h posttransfection and normalized percent luciferase activities were plotted as mean ± SE (n=3) (* and ** indicate p
    Figure Legend Snippet: Myc overrides p300-mediated transcriptional regulation of GFAP and Nestin. U251 (A B) and U87 (C D) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A C) or 2 µg Nestin-Luc (B D) reporter construct along with 1 µg of p300 and 1 µg of Myc expression constructs, either separately or in combination. Luciferase activity was determined at 72 h posttransfection and normalized percent luciferase activities were plotted as mean ± SE (n=3) (* and ** indicate p

    Techniques Used: Transfection, Construct, Expressing, Luciferase, Activity Assay

    RNAi-mediated knockdown of p300 enhances invasion and migration of U251 cells in vitro. 2.5×10 4 parental U251 cells, U251 stable clones expressing two different p300-shRNA constructs (Sh#1-clone 10 and Sh#3-clone 10) or empty vector were used for modified Boyden chamber invasion assay in serum-free medium. After 16 h, cells invaded to the underside of the membrane were fixed with methanol, stained with hematoxylin blue and (A) visualized using phase contrast microscopy. The number of invaded cells in (A) were counted and plotted as mean ± SE (n=3) (B). The experiment was repeated two times, and similar results were obtained. (C) U251 cells as used in (A) (parental, vector, Sh#1-clone 10, Sh#3-clone 10) were used for wound healing assay in serum-free medium and phase-contrast images were obtained at 0, 24 and 48 h after scraping. (D) Cell proliferation/viability was determined using MTT assay after 0, 24 and 48 h of serum starvation. The values are plotted as mean ± SE (n=3). ** indicates p
    Figure Legend Snippet: RNAi-mediated knockdown of p300 enhances invasion and migration of U251 cells in vitro. 2.5×10 4 parental U251 cells, U251 stable clones expressing two different p300-shRNA constructs (Sh#1-clone 10 and Sh#3-clone 10) or empty vector were used for modified Boyden chamber invasion assay in serum-free medium. After 16 h, cells invaded to the underside of the membrane were fixed with methanol, stained with hematoxylin blue and (A) visualized using phase contrast microscopy. The number of invaded cells in (A) were counted and plotted as mean ± SE (n=3) (B). The experiment was repeated two times, and similar results were obtained. (C) U251 cells as used in (A) (parental, vector, Sh#1-clone 10, Sh#3-clone 10) were used for wound healing assay in serum-free medium and phase-contrast images were obtained at 0, 24 and 48 h after scraping. (D) Cell proliferation/viability was determined using MTT assay after 0, 24 and 48 h of serum starvation. The values are plotted as mean ± SE (n=3). ** indicates p

    Techniques Used: Migration, In Vitro, Clone Assay, Expressing, shRNA, Construct, Plasmid Preparation, Modification, Invasion Assay, Staining, Microscopy, Wound Healing Assay, MTT Assay

    Tumorigenicity of GBM cells correlates with expression of Nestin and GFAP (A) Rat right frontal lobes were implanted with 5×10 5 U251 or U87 cells in 5 µl PBS. After 3 weeks, tumor volumes were determined by pixel imaging analysis. (B) The right flanks of nude mice were injected (s.c.) with 2.5×10 6 U251 or U87 cells in 100 µl PBS (mixed with 100 µl matrigel). After 5 weeks, tumor volumes were calculated using the formula: volume = width 2 × length × 0.4. U251 or U87 cells grown on coverslips were fixed, permeabilized and stained for (C) GFAP (green) and (D) Nestin (red) using respective primary antibodies and secondary antibodies conjugated with Alexafluor488 and Alexafluor568 respectively. (E) Schematic of luciferase (Luc) reporter constructs: GFAP-Luc constitutes a 2.21 kb human GFAP promoter containing a GAS element (−1558 to −1547) in pGL3-Basic vector (upper panel) and Nestin-Luc constitutes a 714 bp enhancer fragment form the second intron of the human Nestin gene cloned upstream of an 81 bp minimal thymidine kinase (TK) promoter driving the luciferase gene in the vector, TP222 (lower panel). Activities of the GFAP promoter (F) and the Nestin enhancer (G) were determined at 72 h post transfection of U251 and U87 cells (1×10 6 ) with indicated reporter plasmids (or empty vectors) by luciferase assay. For (A) (B), each value represents mean ± SE of 5 individual animals of each group. Normalized percent luciferase values for (F G) are plotted as mean ± SE (n = 3). ** indicates p
    Figure Legend Snippet: Tumorigenicity of GBM cells correlates with expression of Nestin and GFAP (A) Rat right frontal lobes were implanted with 5×10 5 U251 or U87 cells in 5 µl PBS. After 3 weeks, tumor volumes were determined by pixel imaging analysis. (B) The right flanks of nude mice were injected (s.c.) with 2.5×10 6 U251 or U87 cells in 100 µl PBS (mixed with 100 µl matrigel). After 5 weeks, tumor volumes were calculated using the formula: volume = width 2 × length × 0.4. U251 or U87 cells grown on coverslips were fixed, permeabilized and stained for (C) GFAP (green) and (D) Nestin (red) using respective primary antibodies and secondary antibodies conjugated with Alexafluor488 and Alexafluor568 respectively. (E) Schematic of luciferase (Luc) reporter constructs: GFAP-Luc constitutes a 2.21 kb human GFAP promoter containing a GAS element (−1558 to −1547) in pGL3-Basic vector (upper panel) and Nestin-Luc constitutes a 714 bp enhancer fragment form the second intron of the human Nestin gene cloned upstream of an 81 bp minimal thymidine kinase (TK) promoter driving the luciferase gene in the vector, TP222 (lower panel). Activities of the GFAP promoter (F) and the Nestin enhancer (G) were determined at 72 h post transfection of U251 and U87 cells (1×10 6 ) with indicated reporter plasmids (or empty vectors) by luciferase assay. For (A) (B), each value represents mean ± SE of 5 individual animals of each group. Normalized percent luciferase values for (F G) are plotted as mean ± SE (n = 3). ** indicates p

    Techniques Used: Expressing, Imaging, Mouse Assay, Injection, Staining, Luciferase, Construct, Plasmid Preparation, Clone Assay, Transfection

    Effect of RNAi-mediated knockdown of p300 on GFAP and Nestin expression. (A B) Five p300-shRNA stable clones (Sh#1-clones: 10, 20 and 27; Sh#3-clones: 10 and 12), one vector control and parental U251 cells (1×10 6 ) were transfected with 2 µg of GFAP-Luc (A), and 2 µg of Nestin-Luc (B). Luciferase activity was measured at 72 h posttransfection and normalized percent luciferase activities were plotted as mean ± SE (n=3) (* and ** indicate p
    Figure Legend Snippet: Effect of RNAi-mediated knockdown of p300 on GFAP and Nestin expression. (A B) Five p300-shRNA stable clones (Sh#1-clones: 10, 20 and 27; Sh#3-clones: 10 and 12), one vector control and parental U251 cells (1×10 6 ) were transfected with 2 µg of GFAP-Luc (A), and 2 µg of Nestin-Luc (B). Luciferase activity was measured at 72 h posttransfection and normalized percent luciferase activities were plotted as mean ± SE (n=3) (* and ** indicate p

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

    Myc differentially regulates the transcription of GFAP and Nestin genes in GBM cells. U251 (A, B D) and U87 (C, E F) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A, D E) or 2 µg Nestin-Luc (B, C, F) reporter construct along with indicated amounts of Myc expression plasmid (or empty vector) (A, B C) or two different Myc-specific shRNAs (Sh #2 and #3) (D, E, F). GFAP promoter (A, D E) and Nestin enhancer (B, C F) activities were determined at 72 h post transfection by luciferase assay and normalized percent luciferase activities are plotted as mean ± SE (n=3). * and ** indicate p
    Figure Legend Snippet: Myc differentially regulates the transcription of GFAP and Nestin genes in GBM cells. U251 (A, B D) and U87 (C, E F) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A, D E) or 2 µg Nestin-Luc (B, C, F) reporter construct along with indicated amounts of Myc expression plasmid (or empty vector) (A, B C) or two different Myc-specific shRNAs (Sh #2 and #3) (D, E, F). GFAP promoter (A, D E) and Nestin enhancer (B, C F) activities were determined at 72 h post transfection by luciferase assay and normalized percent luciferase activities are plotted as mean ± SE (n=3). * and ** indicate p

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

    p300 differentially regulates transcription of GFAP and Nestin genes U251 (A) and U87 (B C) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A B) or 2 µg Nestin-Luc (C) reporter constructs along with indicated amounts of p300 or empty control vector (A, B C). Luciferase activity was measured at 72 h after transfection. (D) 2×10 6 cells treated with 20 ng/ml of IL-6 or 100 ng/ml of EGF for 30 min and subjected to ChIP using anti-p300 or matched IgG antibodies. p300 occupancy to the GFAP promoter was determined by PCR using radio-labeled primers and product densities plotted as ‘percent of input’. U251 (E F) and U87 (G) cells (1×10 6 ) were transfected with two different p300-shRNAs (Sh#1 and Sh#3) or 2 µg empty vector along with 2µg GFAP-Luc (E) or Nestin-Luc (F G) reporter constructs. The promoter/enhancer activity was determined at 72 h posttransfection by luciferase assay. Normalized percent luciferase values are plotted as mean ± SE (n = 3). * and ** indicate p
    Figure Legend Snippet: p300 differentially regulates transcription of GFAP and Nestin genes U251 (A) and U87 (B C) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A B) or 2 µg Nestin-Luc (C) reporter constructs along with indicated amounts of p300 or empty control vector (A, B C). Luciferase activity was measured at 72 h after transfection. (D) 2×10 6 cells treated with 20 ng/ml of IL-6 or 100 ng/ml of EGF for 30 min and subjected to ChIP using anti-p300 or matched IgG antibodies. p300 occupancy to the GFAP promoter was determined by PCR using radio-labeled primers and product densities plotted as ‘percent of input’. U251 (E F) and U87 (G) cells (1×10 6 ) were transfected with two different p300-shRNAs (Sh#1 and Sh#3) or 2 µg empty vector along with 2µg GFAP-Luc (E) or Nestin-Luc (F G) reporter constructs. The promoter/enhancer activity was determined at 72 h posttransfection by luciferase assay. Normalized percent luciferase values are plotted as mean ± SE (n = 3). * and ** indicate p

    Techniques Used: Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Labeling

    16) Product Images from "Sevoflurane inhibits cell proliferation and migration of glioma by targeting the miR-27b/VEGF axis"

    Article Title: Sevoflurane inhibits cell proliferation and migration of glioma by targeting the miR-27b/VEGF axis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2021.12047

    VEGF is a target of miR-27b. (A) A conserved binding site for miR-27b in the 3′UTR region of the VEGF gene was identified. miR-27b targeted VEGF 3′UTR in (B) U251 and (C) U87 cells. **P
    Figure Legend Snippet: VEGF is a target of miR-27b. (A) A conserved binding site for miR-27b in the 3′UTR region of the VEGF gene was identified. miR-27b targeted VEGF 3′UTR in (B) U251 and (C) U87 cells. **P

    Techniques Used: Binding Assay

    Sevoflurane-induced inhibition of glioma cell migration is mediated by the miR-27b/VEGF axis. The untreated control group acted as a negative control group for the experiments. (A) Images and (B) analysis of the inhibitory effects of sevoflurane on U251 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. (C) Images and (D) analysis of the inhibitory effects of sevoflurane on U87 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. Magnification, ×100. *P
    Figure Legend Snippet: Sevoflurane-induced inhibition of glioma cell migration is mediated by the miR-27b/VEGF axis. The untreated control group acted as a negative control group for the experiments. (A) Images and (B) analysis of the inhibitory effects of sevoflurane on U251 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. (C) Images and (D) analysis of the inhibitory effects of sevoflurane on U87 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. Magnification, ×100. *P

    Techniques Used: Inhibition, Migration, Negative Control

    Sevoflurane-induced inhibition of glioma cell proliferation is mediated by the miR-27b/VEGF axis. Compared with the siRNA-NC group, siRNA-1 VEGF and siRNA-2 VEGF significantly decreased the expression of VEGF in (A) U251 and (B) U87 cells. The untreated control group acted as a negative control group for the experiments. The inhibitory effects of sevoflurane on (C) U251 and (D) U87 cell proliferation were abolished by pre-treatment with miRNA-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. P
    Figure Legend Snippet: Sevoflurane-induced inhibition of glioma cell proliferation is mediated by the miR-27b/VEGF axis. Compared with the siRNA-NC group, siRNA-1 VEGF and siRNA-2 VEGF significantly decreased the expression of VEGF in (A) U251 and (B) U87 cells. The untreated control group acted as a negative control group for the experiments. The inhibitory effects of sevoflurane on (C) U251 and (D) U87 cell proliferation were abolished by pre-treatment with miRNA-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. P

    Techniques Used: Inhibition, Expressing, Negative Control

    Sevoflurane induces the expression of miR-27b in glioma cells. Decreased miR-27b lower expression was identified in (A) U251 cells compared with HEB cells, which was significantly increased by (B) sevoflurane. Similar miR-27b expression levels were found in (C) U87 cells and (D) after sevoflurane treatment. **P
    Figure Legend Snippet: Sevoflurane induces the expression of miR-27b in glioma cells. Decreased miR-27b lower expression was identified in (A) U251 cells compared with HEB cells, which was significantly increased by (B) sevoflurane. Similar miR-27b expression levels were found in (C) U87 cells and (D) after sevoflurane treatment. **P

    Techniques Used: Expressing

    Sevoflurane reduces the expression of VEGF in glioma cells by targeting miR-27b. (A) Increased expression of VEGF in U251 cells compared with HEB cells. The untreated control group acted as a negative control group for the experiments. (B) Compared with control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by miR-27b inhibitor. (C) Increased expression of VEGF in U87 cells compared with HEB cells. (D) Compared with the control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by the miR-27b inhibitor. P
    Figure Legend Snippet: Sevoflurane reduces the expression of VEGF in glioma cells by targeting miR-27b. (A) Increased expression of VEGF in U251 cells compared with HEB cells. The untreated control group acted as a negative control group for the experiments. (B) Compared with control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by miR-27b inhibitor. (C) Increased expression of VEGF in U87 cells compared with HEB cells. (D) Compared with the control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by the miR-27b inhibitor. P

    Techniques Used: Expressing, Negative Control

    Sevoflurane inhibits the expression levels of MMP-2 and MMP-9. (A) Decreased in protein expression levels of MMP-2 and MMP-9 in U251 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (B) Quantification of western blotting results. (C) Decreased in protein expression levels of MMP-2 and MMP-9 in U87 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (D) Quantification of western blotting results. **P
    Figure Legend Snippet: Sevoflurane inhibits the expression levels of MMP-2 and MMP-9. (A) Decreased in protein expression levels of MMP-2 and MMP-9 in U251 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (B) Quantification of western blotting results. (C) Decreased in protein expression levels of MMP-2 and MMP-9 in U87 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (D) Quantification of western blotting results. **P

    Techniques Used: Expressing, Western Blot

    miR-27b mimic and inhibitor affect miR-27b expression levels. In (A) U251 cells, miR-27b was significantly increased after miR-27b mimics transfection, while significantly decreased after (B) miR-27b inhibitor transfection. Similar results were obtained in U87 cells after (C) miR-27b mimic and (D) inhibitor transfection. **P
    Figure Legend Snippet: miR-27b mimic and inhibitor affect miR-27b expression levels. In (A) U251 cells, miR-27b was significantly increased after miR-27b mimics transfection, while significantly decreased after (B) miR-27b inhibitor transfection. Similar results were obtained in U87 cells after (C) miR-27b mimic and (D) inhibitor transfection. **P

    Techniques Used: Expressing, Transfection

    17) Product Images from "D-2-Hydroxyglutarate does not mimic all the IDH mutation effects, in particular the reduced etoposide-triggered apoptosis mediated by an alteration in mitochondrial NADH"

    Article Title: D-2-Hydroxyglutarate does not mimic all the IDH mutation effects, in particular the reduced etoposide-triggered apoptosis mediated by an alteration in mitochondrial NADH

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.13

    ( a ) Expression of proteins involved in apoptosis in U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms. Whole lysates of cells were isolated 24 h after vehicule (V) or etoposide (ETO) treatment and analyzed (40 μ g) by immunoblotting with the indicated antibodies (left panel). Actin was used as a loading control. ( b ) ROS production was measured using the DCFDA probe. U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms were seeded at 2.5 × 10 4 cells then incubated with the DCFDA probe. Fluorescence was measured at 538 nm every 3 min for 75 min and the slope corresponding to ROS production was calculated. ( c ) Oxygen consumption rate (OCR) of stable cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured over time. Cells (4 × 10 4 ) were plated and OCR was measured 24 h later by a XF24 Analyzer (Seahorse Bioscience). Mitochondrial inhibitors (oligomycin(a), CCCP(b), and rotenone and antimycin A(c)) were added as indicated with arrows. ( d ) Basal oxygen consumption was determined by measuring OCR as in c removing the non-mitochondrial oxygen consumption (OCR upon rotenone and antimycin A treatment). ( e ) The respiratory reserve of U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured by a XF24 Analyzer (Seahorse Bioscience). The respiratory reserve was determined as the difference between maximal OCR and basal OCR. ( f ) NADH production of cells expressing wild-type or mutant IDH1 isoforms. Cells (1 × 10 6 ) were plated, lysed the next day and subsequently assayed for their ability to produce NADH. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate
    Figure Legend Snippet: ( a ) Expression of proteins involved in apoptosis in U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms. Whole lysates of cells were isolated 24 h after vehicule (V) or etoposide (ETO) treatment and analyzed (40 μ g) by immunoblotting with the indicated antibodies (left panel). Actin was used as a loading control. ( b ) ROS production was measured using the DCFDA probe. U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms were seeded at 2.5 × 10 4 cells then incubated with the DCFDA probe. Fluorescence was measured at 538 nm every 3 min for 75 min and the slope corresponding to ROS production was calculated. ( c ) Oxygen consumption rate (OCR) of stable cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured over time. Cells (4 × 10 4 ) were plated and OCR was measured 24 h later by a XF24 Analyzer (Seahorse Bioscience). Mitochondrial inhibitors (oligomycin(a), CCCP(b), and rotenone and antimycin A(c)) were added as indicated with arrows. ( d ) Basal oxygen consumption was determined by measuring OCR as in c removing the non-mitochondrial oxygen consumption (OCR upon rotenone and antimycin A treatment). ( e ) The respiratory reserve of U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured by a XF24 Analyzer (Seahorse Bioscience). The respiratory reserve was determined as the difference between maximal OCR and basal OCR. ( f ) NADH production of cells expressing wild-type or mutant IDH1 isoforms. Cells (1 × 10 6 ) were plated, lysed the next day and subsequently assayed for their ability to produce NADH. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate

    Techniques Used: Expressing, Plasmid Preparation, Mutagenesis, Isolation, Incubation, Fluorescence

    ( a ) Caspase 3 activation was determined with DEVDase activity assay in stable transfected U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms after induction of apoptosis. Cells were plated at 5 × 10 5 cells and treated the next day with different inducers of cell apoptosis. Cellular extracts were prepared from untreated cells (CTR), 5 h after treatment with TRAIL (50 ng/ml), 24 h after ETO (50 μ g/ml), FASL (60ng/ml) or cisplatin (CIS) (15 μ g/ml) and 72 h after γ -irradiation (IRR; 5 Gy). ( b ) The number of dead cells 24 h after ETO (50 μ g/ml) treatment was determined by FACS. Cells were incubated 5 min with propidium iodide (1 μ g/ml) and analyzed by FACS. ( c ) The mitochondrial membrane potential was determined by FACS after ETO (50 μ g/ml) treatment at different time points. Cells were incubated 15 min with JC-1 probe and analyzed by FACS. ( d and e ) Caspase 3 activation after 24 h ETO (50 μ g/ml) exposure was determined with DEVDase activity assay, respectively, in wild-type and mutant IDH1-overexpressing LN18 and T98 cells. Results are expressed relative to wild-type IDH1-expressing cells. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH1, wild-type IDH1-expressing cells; R132, IDH1 R132 -expressing cells transfected. * P
    Figure Legend Snippet: ( a ) Caspase 3 activation was determined with DEVDase activity assay in stable transfected U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms after induction of apoptosis. Cells were plated at 5 × 10 5 cells and treated the next day with different inducers of cell apoptosis. Cellular extracts were prepared from untreated cells (CTR), 5 h after treatment with TRAIL (50 ng/ml), 24 h after ETO (50 μ g/ml), FASL (60ng/ml) or cisplatin (CIS) (15 μ g/ml) and 72 h after γ -irradiation (IRR; 5 Gy). ( b ) The number of dead cells 24 h after ETO (50 μ g/ml) treatment was determined by FACS. Cells were incubated 5 min with propidium iodide (1 μ g/ml) and analyzed by FACS. ( c ) The mitochondrial membrane potential was determined by FACS after ETO (50 μ g/ml) treatment at different time points. Cells were incubated 15 min with JC-1 probe and analyzed by FACS. ( d and e ) Caspase 3 activation after 24 h ETO (50 μ g/ml) exposure was determined with DEVDase activity assay, respectively, in wild-type and mutant IDH1-overexpressing LN18 and T98 cells. Results are expressed relative to wild-type IDH1-expressing cells. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH1, wild-type IDH1-expressing cells; R132, IDH1 R132 -expressing cells transfected. * P

    Techniques Used: Activation Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Irradiation, FACS, Incubation

    Metabolic alterations driven by IDH mutation in U251 cells. In cancer cells, glycolysis upregulation generates the production of reducing equivalents NADH, which is then shuttles from the cytosol to mitochondria with the MAS. Glutamine is converted to glutamate, which is further converted to α KG by GDH. IDH also produces α KG from isocitrate. In cells expressing IDH1 R132 , IDH1 R132 converts α KG into D-2HG. To limit cellular α KG depletion, IDH1 R132 -overexpressing cells diverts AAT from MAS function to produce α KG. As a result, glycolytic NADH is no longer fully imported into mitochondria. AAT, aspartate aminotransferase; ETC, electron transport chain; GDH, glutamate dehydrogenase; Gln, Glutamine; Glu, Glutamate; IDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; MAS, malate–aspartate shuttle; Pyr, pyruvate; TCA, tricarboxylic acid cycle
    Figure Legend Snippet: Metabolic alterations driven by IDH mutation in U251 cells. In cancer cells, glycolysis upregulation generates the production of reducing equivalents NADH, which is then shuttles from the cytosol to mitochondria with the MAS. Glutamine is converted to glutamate, which is further converted to α KG by GDH. IDH also produces α KG from isocitrate. In cells expressing IDH1 R132 , IDH1 R132 converts α KG into D-2HG. To limit cellular α KG depletion, IDH1 R132 -overexpressing cells diverts AAT from MAS function to produce α KG. As a result, glycolytic NADH is no longer fully imported into mitochondria. AAT, aspartate aminotransferase; ETC, electron transport chain; GDH, glutamate dehydrogenase; Gln, Glutamine; Glu, Glutamate; IDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; MAS, malate–aspartate shuttle; Pyr, pyruvate; TCA, tricarboxylic acid cycle

    Techniques Used: Mutagenesis, Expressing

    ( a ) Expression of wild-type (IDH2) or mutant IDH2 (R140 and R172) isoforms in stable overexpressing U251 cells was confirmed by immunoblotting using whole lysates (40 μg) or purified mitochondria. Actin and porin were used as a loading control, respectively, for whole lysates and mitochondria. ( b ) IDH activity in cells overexpressing wild-type or mutant IDH2 isoforms. Cells (1 × 10 6 ) were plated, lysed the next day and subsequently assayed for their ability to generate NADPH. ( c ) Caspase 3 activation was determined with DEVDase activity assay in stable U251 cells expressing wild-type and mutant IDH2 isoforms after 24 h ETO (50 μ g/ml) exposure. Results are expressed relative to wild-type IDH2-expressing cells. ( d ) Caspase 3 activation was determined with DEVDase activity assay after 24 h ETO (50 μ g/ml) exposure in U251 cells treated with D-2HG (3 mM) for 6 days. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH2, wild-type IDH2-expressing cells; R140, IDH1 R140 -expressing cells transfected; R172, IDH1 R172 -expressing cells transfected. * P
    Figure Legend Snippet: ( a ) Expression of wild-type (IDH2) or mutant IDH2 (R140 and R172) isoforms in stable overexpressing U251 cells was confirmed by immunoblotting using whole lysates (40 μg) or purified mitochondria. Actin and porin were used as a loading control, respectively, for whole lysates and mitochondria. ( b ) IDH activity in cells overexpressing wild-type or mutant IDH2 isoforms. Cells (1 × 10 6 ) were plated, lysed the next day and subsequently assayed for their ability to generate NADPH. ( c ) Caspase 3 activation was determined with DEVDase activity assay in stable U251 cells expressing wild-type and mutant IDH2 isoforms after 24 h ETO (50 μ g/ml) exposure. Results are expressed relative to wild-type IDH2-expressing cells. ( d ) Caspase 3 activation was determined with DEVDase activity assay after 24 h ETO (50 μ g/ml) exposure in U251 cells treated with D-2HG (3 mM) for 6 days. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH2, wild-type IDH2-expressing cells; R140, IDH1 R140 -expressing cells transfected; R172, IDH1 R172 -expressing cells transfected. * P

    Techniques Used: Expressing, Mutagenesis, Purification, Activity Assay, Activation Assay, Plasmid Preparation, Transfection

    Lactate deshydrogenase activity ( a ) and lactate production ( b ) of U251 cells expressing IDH1 isoforms. Cells were treated or not with 3 mM oxamate (OX) for 48 h. ( c ) Basal oxygen consumption was determined by measuring OCR as in Figure 4c in cells expressing IDH1 isoforms treated or not with 3 mM oxamate for 7 days. ( d ) Mitochondrial respiratory reserve was determined as in Figure 4e in cells treated or not with 3 mM oxamate for 7 days. ( e ) Caspase 3 activation was determined using a DEVDase activity assay 24 h after ETO-induced apoptosis in U251 cells treated or not for 7 days with 3 mM oxamate. ( f ) The number of dead cells was measured 24 h after a concomitant treatment with CCCP (1 μ M) or malate (3 mM) and ETO (50 μ g/ml). The number of dead cells was determined by FACS after propidium iodide (1 μ g/ml) staining. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate
    Figure Legend Snippet: Lactate deshydrogenase activity ( a ) and lactate production ( b ) of U251 cells expressing IDH1 isoforms. Cells were treated or not with 3 mM oxamate (OX) for 48 h. ( c ) Basal oxygen consumption was determined by measuring OCR as in Figure 4c in cells expressing IDH1 isoforms treated or not with 3 mM oxamate for 7 days. ( d ) Mitochondrial respiratory reserve was determined as in Figure 4e in cells treated or not with 3 mM oxamate for 7 days. ( e ) Caspase 3 activation was determined using a DEVDase activity assay 24 h after ETO-induced apoptosis in U251 cells treated or not for 7 days with 3 mM oxamate. ( f ) The number of dead cells was measured 24 h after a concomitant treatment with CCCP (1 μ M) or malate (3 mM) and ETO (50 μ g/ml). The number of dead cells was determined by FACS after propidium iodide (1 μ g/ml) staining. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate

    Techniques Used: Activity Assay, Expressing, Activation Assay, FACS, Staining

    18) Product Images from "Long Noncoding RNA H19 Promotes Proliferation and Invasion in Human Glioma Cells by Downregulating miR-152"

    Article Title: Long Noncoding RNA H19 Promotes Proliferation and Invasion in Human Glioma Cells by Downregulating miR-152

    Journal: Oncology Research

    doi: 10.3727/096504018X15178768577951

    The expression of H19 was inversely associated with miR-152 expression. (A) The potential binding sites for miR-152 on H19. (B, C) Luciferase activity was detected after U251 and U87 cells were cotransfected with miR-152 mimic or miR-control and WT-H19 (wild type) or MUT-H19 (mutated) reporter. (D, E) The expression of H19 in U251 and U87 cells transfected with miR-control, miR-152 mimics, or anti-miR-152 was quantified by RT-qPCR. (F, G) The expression of miR-152 in U251 and U87 cells transfected with si-control or si-H19, and either with pcDNA-vector or pcDNA-H19 was examined by RT-qPCR. ** p
    Figure Legend Snippet: The expression of H19 was inversely associated with miR-152 expression. (A) The potential binding sites for miR-152 on H19. (B, C) Luciferase activity was detected after U251 and U87 cells were cotransfected with miR-152 mimic or miR-control and WT-H19 (wild type) or MUT-H19 (mutated) reporter. (D, E) The expression of H19 in U251 and U87 cells transfected with miR-control, miR-152 mimics, or anti-miR-152 was quantified by RT-qPCR. (F, G) The expression of miR-152 in U251 and U87 cells transfected with si-control or si-H19, and either with pcDNA-vector or pcDNA-H19 was examined by RT-qPCR. ** p

    Techniques Used: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Plasmid Preparation

    H19 promoted cell proliferation and invasion by downregulating miR-152. (A, B) Cell proliferation was tested by MTT assay after U251 and U87 cells were transfected with si-control, si-H19, si-H19 + anti-miR-control, or si-H19 + anti-miR-152. (C, D) Cell invasion was examined by Transwell invasion assay after U251 and U87 cells were transfected with si-control, si-H19, si-H19 + anti-miR-control, or si-H19 + anti-miR-152. ** p
    Figure Legend Snippet: H19 promoted cell proliferation and invasion by downregulating miR-152. (A, B) Cell proliferation was tested by MTT assay after U251 and U87 cells were transfected with si-control, si-H19, si-H19 + anti-miR-control, or si-H19 + anti-miR-152. (C, D) Cell invasion was examined by Transwell invasion assay after U251 and U87 cells were transfected with si-control, si-H19, si-H19 + anti-miR-control, or si-H19 + anti-miR-152. ** p

    Techniques Used: MTT Assay, Transfection, Transwell Invasion Assay

    Downregulation of H19 suppressed cell proliferation and invasion in glioma cell lines. H19 expression in U251 (A) and U87 (B) cells transfected with siRNA against H19 (si-H19) or siRNA control (si-control) was determined by RT-qPCR. MTT assays were performed to assess the proliferation of U251 (C) and U87 (D) cells transfected with si-H19 or si-control. Transwell invasion assays were used to evaluate the invasive ability of U251 (E) and U87 (F) cells transfected with si-H19 or si-control. * p
    Figure Legend Snippet: Downregulation of H19 suppressed cell proliferation and invasion in glioma cell lines. H19 expression in U251 (A) and U87 (B) cells transfected with siRNA against H19 (si-H19) or siRNA control (si-control) was determined by RT-qPCR. MTT assays were performed to assess the proliferation of U251 (C) and U87 (D) cells transfected with si-H19 or si-control. Transwell invasion assays were used to evaluate the invasive ability of U251 (E) and U87 (F) cells transfected with si-H19 or si-control. * p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, MTT Assay

    Overexpression of miR-152 suppressed cell proliferation and invasion in glioma cell lines. Expression of miR-152 in U251 (A) and U87 (B) cells transfected with miR-152 mimic or miR-control was quantified by RT-qPCR. MTT assays were applied to observe the proliferation of U251 (C) and U87 (D) cells transfected with miR-152 mimic or miR-control. Transwell invasion assays were performed to assess the invasive ability of U251 (E) and U87 (F) cells transfected with miR-152 mimic or miR-control. * p
    Figure Legend Snippet: Overexpression of miR-152 suppressed cell proliferation and invasion in glioma cell lines. Expression of miR-152 in U251 (A) and U87 (B) cells transfected with miR-152 mimic or miR-control was quantified by RT-qPCR. MTT assays were applied to observe the proliferation of U251 (C) and U87 (D) cells transfected with miR-152 mimic or miR-control. Transwell invasion assays were performed to assess the invasive ability of U251 (E) and U87 (F) cells transfected with miR-152 mimic or miR-control. * p

    Techniques Used: Over Expression, Expressing, Transfection, Quantitative RT-PCR, MTT Assay

    H19 was upregulated and microRNA-152 (miR-152) was downregulated in glioma cell lines in comparison with that in normal human astrocytes (NHAs). (A) H19 expression in glioma cell lines (A172, U251, U87, U373, and U563) and NHAs was assessed by quantitative real-time PCR (RT-qPCR). (B) miR-152 expression in glioma cell lines (A172, U251, U87, U373, and U563) and NHAs was determined by RT-qPCR. * p
    Figure Legend Snippet: H19 was upregulated and microRNA-152 (miR-152) was downregulated in glioma cell lines in comparison with that in normal human astrocytes (NHAs). (A) H19 expression in glioma cell lines (A172, U251, U87, U373, and U563) and NHAs was assessed by quantitative real-time PCR (RT-qPCR). (B) miR-152 expression in glioma cell lines (A172, U251, U87, U373, and U563) and NHAs was determined by RT-qPCR. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Downregulation of H19 inhibited glioma growth in vivo. U251 cells stably transfected with short hairpin RNA (shRNA) targeting H19 (sh-H19) or shRNA control (sh-con) were subcutaneously inoculated into the right side of the groin of nude mice. (A) Seven days later, the volume of the tumor xenografts was measured every 6 days using a caliper. (B) After 25 days of injection, the tumor-bearing mice were sacrificed, and visible tumors were excised and weighted. * p
    Figure Legend Snippet: Downregulation of H19 inhibited glioma growth in vivo. U251 cells stably transfected with short hairpin RNA (shRNA) targeting H19 (sh-H19) or shRNA control (sh-con) were subcutaneously inoculated into the right side of the groin of nude mice. (A) Seven days later, the volume of the tumor xenografts was measured every 6 days using a caliper. (B) After 25 days of injection, the tumor-bearing mice were sacrificed, and visible tumors were excised and weighted. * p

    Techniques Used: In Vivo, Stable Transfection, Transfection, shRNA, Mouse Assay, Injection

    19) Product Images from "PDGFA/PDGFRα-regulated GOLM1 promotes human glioma progression through activation of AKT"

    Article Title: PDGFA/PDGFRα-regulated GOLM1 promotes human glioma progression through activation of AKT

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0665-3

    GOLM1 may mediate PDGFA/PDGFRα signaling in A172 cells in vitro . a Western blot analysis of p-PDGFRα in lysates prepared from PDGFA treated (20 ng/mL) and untreated U251 and A172 cells. PBS was used as the vehicle control. b IF staining of GOLM1 (red) in A172 cells treated with 0 ng/mL, 20 ng/mL and 50 ng/mL PDGFA for 24 h. Nuclei were labeled with DAPI (blue). Scale bar = 20 μm. c qRT-PCR (upper) and Western blot analysis (lower) of GOLM1 in lysates prepared from A172 cells treated with 0, 20, and 50 ng/mL PDGFA for 48 h. d qRT-PCR (upper) and Western blot analysis (lower) of GOLM1 in untreated cells or cells treated with increasing amounts of PDGFA in the presence of DMSO (vehicle control) or an inhibitor of PDGFRα AG1296 (5 μM) for 48 h. A172-NC and -sh-GOLM1 cells were treated with PBS (0 ng/mL PDGFA) as negative control or PDGFA (20 ng/mL) for 48 h. e EdU assays to evaluate cell proliferation under indicated treatment. Scale bar = 100 μm. f Representative images of Transwell migration and invasion assays performed in cells with indicated treatment. g Graphic representation of ratios of EdU positive cells under different treatments. Data are presented as the mean ± SEM. h Quantification of invaded and migrated cells in Transwell assays after incubation for 24 h. Data is presented as the mean ± SEM. Scale bar = 50 μm. i Western blot analysis of p-AKT (S473), AKT, p-ERK1/2 and ERK in A172-NC and -sh-GOLM1 cells after treatment with PDGFA (20 μg/mL) for 0, 10, 20, and 30 min. (NS, not significant; * P
    Figure Legend Snippet: GOLM1 may mediate PDGFA/PDGFRα signaling in A172 cells in vitro . a Western blot analysis of p-PDGFRα in lysates prepared from PDGFA treated (20 ng/mL) and untreated U251 and A172 cells. PBS was used as the vehicle control. b IF staining of GOLM1 (red) in A172 cells treated with 0 ng/mL, 20 ng/mL and 50 ng/mL PDGFA for 24 h. Nuclei were labeled with DAPI (blue). Scale bar = 20 μm. c qRT-PCR (upper) and Western blot analysis (lower) of GOLM1 in lysates prepared from A172 cells treated with 0, 20, and 50 ng/mL PDGFA for 48 h. d qRT-PCR (upper) and Western blot analysis (lower) of GOLM1 in untreated cells or cells treated with increasing amounts of PDGFA in the presence of DMSO (vehicle control) or an inhibitor of PDGFRα AG1296 (5 μM) for 48 h. A172-NC and -sh-GOLM1 cells were treated with PBS (0 ng/mL PDGFA) as negative control or PDGFA (20 ng/mL) for 48 h. e EdU assays to evaluate cell proliferation under indicated treatment. Scale bar = 100 μm. f Representative images of Transwell migration and invasion assays performed in cells with indicated treatment. g Graphic representation of ratios of EdU positive cells under different treatments. Data are presented as the mean ± SEM. h Quantification of invaded and migrated cells in Transwell assays after incubation for 24 h. Data is presented as the mean ± SEM. Scale bar = 50 μm. i Western blot analysis of p-AKT (S473), AKT, p-ERK1/2 and ERK in A172-NC and -sh-GOLM1 cells after treatment with PDGFA (20 μg/mL) for 0, 10, 20, and 30 min. (NS, not significant; * P

    Techniques Used: In Vitro, Western Blot, Staining, Labeling, Quantitative RT-PCR, Negative Control, Migration, Incubation

    GOLM1 promotes human glioma progression through activation of AKT. a Image of phospho-kinase array performed with lysates prepared from U251-NC and -sh-GOLM1 cells. Spots with significant decreases in phosphorylation are numbered and quantification is shown in ( b ). c Western blot analysis of p-AKT (S473), AKT, p-ERK1/2, ERK1/2 in indicated cells. d Kinases and genes downstream of AKT in U251, A172 and U87MG cells were analyzed by western blot. U87MG-Lenti-NC and -Lenti-GOLM1 cells were treated with the AKT inhibitor MK-2206 (2 μM) or DMSO (vehicle control) and evaluated for e cell viability in the CCK8 assay and f cell proliferation in EdU (red) assays. Scale bar = 100 μm. g Graphic representation of ratios of EdU positive cells. Data are presented as the mean ± SEM. h Transwell migration and invasion assays were performed on U87MG-Lenti-NC and -Lenti-GOLM1cells with indicated treatment. i Quantification of invaded and migrated cells in Transwell assays after incubation for 24 h. Scale bar = 50 μm. (* P
    Figure Legend Snippet: GOLM1 promotes human glioma progression through activation of AKT. a Image of phospho-kinase array performed with lysates prepared from U251-NC and -sh-GOLM1 cells. Spots with significant decreases in phosphorylation are numbered and quantification is shown in ( b ). c Western blot analysis of p-AKT (S473), AKT, p-ERK1/2, ERK1/2 in indicated cells. d Kinases and genes downstream of AKT in U251, A172 and U87MG cells were analyzed by western blot. U87MG-Lenti-NC and -Lenti-GOLM1 cells were treated with the AKT inhibitor MK-2206 (2 μM) or DMSO (vehicle control) and evaluated for e cell viability in the CCK8 assay and f cell proliferation in EdU (red) assays. Scale bar = 100 μm. g Graphic representation of ratios of EdU positive cells. Data are presented as the mean ± SEM. h Transwell migration and invasion assays were performed on U87MG-Lenti-NC and -Lenti-GOLM1cells with indicated treatment. i Quantification of invaded and migrated cells in Transwell assays after incubation for 24 h. Scale bar = 50 μm. (* P

    Techniques Used: Activation Assay, Western Blot, CCK-8 Assay, Migration, Incubation

    GOLM1 expression is elevated in human gliomas. a qRT-PCR to measure relative expression levels of GOLM1 in different grade glioma and non-neoplastic tissue samples. b Representative images of IHC staining for GOLM1 in human glioma and non-neoplastic brain tissue samples. Scale bar = 200 μm; c Graphic representation of scoring performed on IHC staining for GOLM1 in glioma and non-neoplastic tissue samples. Bar graphs show the mean ± the standard error of the mean (SEM) for each group. d qRT-PCR (upper) and Western blot analysis (lower) of GOLM1 levels in NHA, U87MG, A172 and U251 cells. e Representative images of immunofluorescence staining performed for GOLM1 (red) in U87MG and U251 cells and visualized under fluorescence microscopy. F-actin was stained with phalloidin (green). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (** P
    Figure Legend Snippet: GOLM1 expression is elevated in human gliomas. a qRT-PCR to measure relative expression levels of GOLM1 in different grade glioma and non-neoplastic tissue samples. b Representative images of IHC staining for GOLM1 in human glioma and non-neoplastic brain tissue samples. Scale bar = 200 μm; c Graphic representation of scoring performed on IHC staining for GOLM1 in glioma and non-neoplastic tissue samples. Bar graphs show the mean ± the standard error of the mean (SEM) for each group. d qRT-PCR (upper) and Western blot analysis (lower) of GOLM1 levels in NHA, U87MG, A172 and U251 cells. e Representative images of immunofluorescence staining performed for GOLM1 (red) in U87MG and U251 cells and visualized under fluorescence microscopy. F-actin was stained with phalloidin (green). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (** P

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Western Blot, Immunofluorescence, Fluorescence, Microscopy

    GOLM1 knockdown inhibits glioma progression in U251 and A172 cells in vitro . Knockdown efficiency of lentiviral shRNA constructs, sh-GOLM1–1 and sh-GOLM1–2 in U251 and A172 cells assessed with a qRT-PCR and b western blot analysis. U251- and A172-NC and -sh-GOLM1 cell lines evaluated in c CCK8 assay for cell viability. d EdU (red) assays for proliferation rate. Nuclei are stained with DAPI (blue). Scale bar = 100 μm. e Graphic representation of ratios of EdU positive cells in U251- and A172-NC and sh-GOLM1 cells. Data are presented as the mean ± SEM. f Representative images of the morphology of U251- and A172-NC and sh-GOLM1 cells under bright field microscopy. Scale bar = 100 μm. g Images of Transwell assays performed with U251- and A172-NC and sh-GOLM1 expressing cells. Scale bar = 50 μm. h Quantification of invaded and migrated cells in Transwell assays after incubation for 24 h. Data are presented as the mean ± SEM. (** P
    Figure Legend Snippet: GOLM1 knockdown inhibits glioma progression in U251 and A172 cells in vitro . Knockdown efficiency of lentiviral shRNA constructs, sh-GOLM1–1 and sh-GOLM1–2 in U251 and A172 cells assessed with a qRT-PCR and b western blot analysis. U251- and A172-NC and -sh-GOLM1 cell lines evaluated in c CCK8 assay for cell viability. d EdU (red) assays for proliferation rate. Nuclei are stained with DAPI (blue). Scale bar = 100 μm. e Graphic representation of ratios of EdU positive cells in U251- and A172-NC and sh-GOLM1 cells. Data are presented as the mean ± SEM. f Representative images of the morphology of U251- and A172-NC and sh-GOLM1 cells under bright field microscopy. Scale bar = 100 μm. g Images of Transwell assays performed with U251- and A172-NC and sh-GOLM1 expressing cells. Scale bar = 50 μm. h Quantification of invaded and migrated cells in Transwell assays after incubation for 24 h. Data are presented as the mean ± SEM. (** P

    Techniques Used: In Vitro, shRNA, Construct, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Staining, Microscopy, Expressing, Incubation

    GOLM1 knockdown inhibits glioma progression in P3#GBM cells in vitro and in vivo . Expression of GOLM1 in U251, A172 and P3#GBM cells was analyzed by a qRT-PCR and b western blot. Overexpression of GOLM1 in P3#GBM cells was confirmed by c qRT-PCR and d western blot analysis. e CCK8 assay for cell viability. f Representative images of invaded spheroids in 3D invasion assay for P3#GBM-NC and -sh-GOLM1 cells. Scale bar = 200 μm. g The area covered by invading cells was quantitated after 96 h of incubation. h Kaplan-Meier survival analysis of mice implanted with P3#GBM -NC ( n = 8) and -sh-GOLM1 ( n = 8) cells. The log-rank test was used to calculate P -values, which were
    Figure Legend Snippet: GOLM1 knockdown inhibits glioma progression in P3#GBM cells in vitro and in vivo . Expression of GOLM1 in U251, A172 and P3#GBM cells was analyzed by a qRT-PCR and b western blot. Overexpression of GOLM1 in P3#GBM cells was confirmed by c qRT-PCR and d western blot analysis. e CCK8 assay for cell viability. f Representative images of invaded spheroids in 3D invasion assay for P3#GBM-NC and -sh-GOLM1 cells. Scale bar = 200 μm. g The area covered by invading cells was quantitated after 96 h of incubation. h Kaplan-Meier survival analysis of mice implanted with P3#GBM -NC ( n = 8) and -sh-GOLM1 ( n = 8) cells. The log-rank test was used to calculate P -values, which were

    Techniques Used: In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, CCK-8 Assay, Invasion Assay, Incubation, Mouse Assay

    Silencing of GOLM1 inhibits invasive growth of U251 cells in vivo . a Kaplan-Meier survival analysis of mice implanted with U251-NC ( n = 8) and -sh-GOLM1 ( n = 8) cells. The log-rank test was used to calculate P -values, which were
    Figure Legend Snippet: Silencing of GOLM1 inhibits invasive growth of U251 cells in vivo . a Kaplan-Meier survival analysis of mice implanted with U251-NC ( n = 8) and -sh-GOLM1 ( n = 8) cells. The log-rank test was used to calculate P -values, which were

    Techniques Used: In Vivo, Mouse Assay

    20) Product Images from "p73 promotes glioblastoma cell invasion by directly activating POSTN (periostin) expression"

    Article Title: p73 promotes glioblastoma cell invasion by directly activating POSTN (periostin) expression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7600

    POSTN overexpression rescues invasion ability of glioblastoma cells with p73 knock down A. Invasion into matrigel of U251 cells that were transfected with siRNA targeting p73, POSTN, both or a nontargeting control is shown. B . As in A, but cells were transfected with siRNA (scr or p73) and plasmids encoding POSTN or empty vector as indicated.
    Figure Legend Snippet: POSTN overexpression rescues invasion ability of glioblastoma cells with p73 knock down A. Invasion into matrigel of U251 cells that were transfected with siRNA targeting p73, POSTN, both or a nontargeting control is shown. B . As in A, but cells were transfected with siRNA (scr or p73) and plasmids encoding POSTN or empty vector as indicated.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation

    P73 knock down reduces migration and invasion of glioblastoma cells A. Whole protein extract of U251 cells 72 h post-transfection with scr or p73 siRNA was analysed by immunoblotting with antibodies against p73, SNAIL, E-cadherin and GAPDH. Line indicates where 2 lanes that are not next to each other on the gel were moved side by side (see Figure S3A for full scan). B. The migration ability of U251 cells after siRNA transfection (scr or p73) was determined using the xCelligence. C. Invasion into matrigel of U251 cells that were transfected with siRNAs targeting p73 or a non-targeting control is shown. D. As in C but cells were transiently transfected with plasmids encoding TAp73α, ΔNp73α or empty vector control. P -values shown are for EV compared to TAp73 and EV compared ΔNp73. * p
    Figure Legend Snippet: P73 knock down reduces migration and invasion of glioblastoma cells A. Whole protein extract of U251 cells 72 h post-transfection with scr or p73 siRNA was analysed by immunoblotting with antibodies against p73, SNAIL, E-cadherin and GAPDH. Line indicates where 2 lanes that are not next to each other on the gel were moved side by side (see Figure S3A for full scan). B. The migration ability of U251 cells after siRNA transfection (scr or p73) was determined using the xCelligence. C. Invasion into matrigel of U251 cells that were transfected with siRNAs targeting p73 or a non-targeting control is shown. D. As in C but cells were transiently transfected with plasmids encoding TAp73α, ΔNp73α or empty vector control. P -values shown are for EV compared to TAp73 and EV compared ΔNp73. * p

    Techniques Used: Migration, Transfection, Plasmid Preparation

    Analysis of mRNA expression after p73 knock down suggests a role for p73 in cell migration and invasion A. Results of the microarray revealed 632 genes that were differentially expressed in control cells compared to knock down of endogenous p73 (Schematic illustration of gene array results). Pathway analysis of microarray results comparing U251 transfected with scr siRNA and p73 siRNA indicated changes in the pathways shown. Odds ratios for pathways are Integrin binding = 4.4, Fibronectin binding 9.05 and Cell adhesion = 2.04. p- values refer to the significance of enrichment of genes of the pathways shown. Heat maps of B. “Integrin and Fibronectin binding” (combined), 10 genes for integrin binding and 5 genes for fibronectin binding were identified, while CTGF is involved in both pathways. C. “Cell Adhesion” genes identified by GO term analysis, as differentially expressed between si scr and si p73.
    Figure Legend Snippet: Analysis of mRNA expression after p73 knock down suggests a role for p73 in cell migration and invasion A. Results of the microarray revealed 632 genes that were differentially expressed in control cells compared to knock down of endogenous p73 (Schematic illustration of gene array results). Pathway analysis of microarray results comparing U251 transfected with scr siRNA and p73 siRNA indicated changes in the pathways shown. Odds ratios for pathways are Integrin binding = 4.4, Fibronectin binding 9.05 and Cell adhesion = 2.04. p- values refer to the significance of enrichment of genes of the pathways shown. Heat maps of B. “Integrin and Fibronectin binding” (combined), 10 genes for integrin binding and 5 genes for fibronectin binding were identified, while CTGF is involved in both pathways. C. “Cell Adhesion” genes identified by GO term analysis, as differentially expressed between si scr and si p73.

    Techniques Used: Expressing, Migration, Microarray, Transfection, Binding Assay

    POSTN is a direct target of p73 A. High POSTN mRNA expression is strongly correlated with reduced survival of glioblastoma patients. Data is from the REMBRANT database/NIH ( https://caintegrator.nci.nih.gov/rembrandt/ ). p -value corresponds to comparison between all three conditions with each other. B. Expression of POSTN mRNA 72 h after transfection with p73 or scr siRNA was determined using RT- qPCR in U251 (left panel) and U87 (right panel) cells. C. U251 protein extracts of cells transfected with siRNA for p73 or a scrambled control and analysed by immunoblotting using p73, POSTN and β-tubulin antibodies. Asterisk indicates an unspecific band of the p73 antibody. D. Expression of POSTN mRNA 24 h after transfection with plasmids encoding TAp73α, ΔNp73α or an empty vector control was determined using RT-qPCR in U251 (left panel) and U87 (right panel) cells. E. Analysis of the POSTN promoter revealed one potential binding site for transcription factors of the p53 family 600 bp upstream of the transcriptional start site. F. ChIP assay demonstrating binding of TAp73α to the POSTN promoter in U251 cells. G. U251 (left panel) or U87 (right panel) cells were transfected with p73 constructs (TAp73α or ΔNp73α) plus POSTN-Luc wt or mutant and control Renilla-Luc. Post transfection (24 h), cells were harvested and dual luciferase reporter assays performed. Results were normalised by expressing firefly/renilla luciferase activity in relative light units (RLU) as the mean +/− S.D.H. H. U251 (left panel) or U87 (right panel) cells were transfected with siRNA (p73 or scrambled control), 48 h post transfection cells were treated with 5 ng/ml TGFβ for another 24 h. Cells were harvested and mRNA expression of p73 and POSTN was analysed using RT-qPCR. * p
    Figure Legend Snippet: POSTN is a direct target of p73 A. High POSTN mRNA expression is strongly correlated with reduced survival of glioblastoma patients. Data is from the REMBRANT database/NIH ( https://caintegrator.nci.nih.gov/rembrandt/ ). p -value corresponds to comparison between all three conditions with each other. B. Expression of POSTN mRNA 72 h after transfection with p73 or scr siRNA was determined using RT- qPCR in U251 (left panel) and U87 (right panel) cells. C. U251 protein extracts of cells transfected with siRNA for p73 or a scrambled control and analysed by immunoblotting using p73, POSTN and β-tubulin antibodies. Asterisk indicates an unspecific band of the p73 antibody. D. Expression of POSTN mRNA 24 h after transfection with plasmids encoding TAp73α, ΔNp73α or an empty vector control was determined using RT-qPCR in U251 (left panel) and U87 (right panel) cells. E. Analysis of the POSTN promoter revealed one potential binding site for transcription factors of the p53 family 600 bp upstream of the transcriptional start site. F. ChIP assay demonstrating binding of TAp73α to the POSTN promoter in U251 cells. G. U251 (left panel) or U87 (right panel) cells were transfected with p73 constructs (TAp73α or ΔNp73α) plus POSTN-Luc wt or mutant and control Renilla-Luc. Post transfection (24 h), cells were harvested and dual luciferase reporter assays performed. Results were normalised by expressing firefly/renilla luciferase activity in relative light units (RLU) as the mean +/− S.D.H. H. U251 (left panel) or U87 (right panel) cells were transfected with siRNA (p73 or scrambled control), 48 h post transfection cells were treated with 5 ng/ml TGFβ for another 24 h. Cells were harvested and mRNA expression of p73 and POSTN was analysed using RT-qPCR. * p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Construct, Mutagenesis, Luciferase, Activity Assay

    Glioblastoma are more chemo-sensitive after p73 knock down A. U251 cells were treated with 50 μM Temozolomide for 48 h. Early and late apoptosis was detected using Annexin V/PI-double staining followed by flow cytometry analysis. B. As in A, but with additional time points of Temozolomide treatment of 24 and 72 h, total apoptosis was quantified and is shown. C. Colony formation assay after p73 knock down. * p = 0.05, ** p
    Figure Legend Snippet: Glioblastoma are more chemo-sensitive after p73 knock down A. U251 cells were treated with 50 μM Temozolomide for 48 h. Early and late apoptosis was detected using Annexin V/PI-double staining followed by flow cytometry analysis. B. As in A, but with additional time points of Temozolomide treatment of 24 and 72 h, total apoptosis was quantified and is shown. C. Colony formation assay after p73 knock down. * p = 0.05, ** p

    Techniques Used: Double Staining, Flow Cytometry, Cytometry, Colony Assay

    P73 knock down induces morphological transformation of glioblastoma cells A. p73 gene deletion is correlated with prolonged survival of glioblastoma patients. Data is from the REMBRANT database/NIH ( https://caintegrator.nci.nih.gov/rembrandt/ ). The p -value corresponds to the gene deletion group in comparison to all other patients. B. Morphological changes of U251 cells after 72 h of p73 knock down using siRNA transfection. C. Cells as in B but fixed and stained with an anti-GFAP antibody. Pictures were taken using the Cellomics, GFAP signal was quantified and is expressed as arbitrary units (right panel). D. Cells were grown in media that was serum rich (10%), serum free or media complemented with growth factors (GF) (20 ng/ml EGF and FGF) and p27 supplement. Total protein was extracted and blotted with antibodies against p73 and GAPDH. E. Cells were incubated in serum free medium or full medium for 72 h and visualised and quantified (F) using a GFAP antibody as in C. Scale Bars in B, C and E represent 100 μm. * p
    Figure Legend Snippet: P73 knock down induces morphological transformation of glioblastoma cells A. p73 gene deletion is correlated with prolonged survival of glioblastoma patients. Data is from the REMBRANT database/NIH ( https://caintegrator.nci.nih.gov/rembrandt/ ). The p -value corresponds to the gene deletion group in comparison to all other patients. B. Morphological changes of U251 cells after 72 h of p73 knock down using siRNA transfection. C. Cells as in B but fixed and stained with an anti-GFAP antibody. Pictures were taken using the Cellomics, GFAP signal was quantified and is expressed as arbitrary units (right panel). D. Cells were grown in media that was serum rich (10%), serum free or media complemented with growth factors (GF) (20 ng/ml EGF and FGF) and p27 supplement. Total protein was extracted and blotted with antibodies against p73 and GAPDH. E. Cells were incubated in serum free medium or full medium for 72 h and visualised and quantified (F) using a GFAP antibody as in C. Scale Bars in B, C and E represent 100 μm. * p

    Techniques Used: Transformation Assay, Transfection, Staining, Incubation

    21) Product Images from "Transferrin Receptor 2 Is Frequently and Highly Expressed in Glioblastomas"

    Article Title: Transferrin Receptor 2 Is Frequently and Highly Expressed in Glioblastomas

    Journal: Translational Oncology

    doi:

    Inhibition of TfR2 expression by siRNA treatment reduces GBM cell growth. (A) Western blot analysis of TfR1 and TfR2 expression in U251 cells transfected with TfR2 siRNA or nontargeting control siRNA (CsiRNA), compared with untreated (C) cells. (B) Flow cytometry analysis of TfR2 expression in U251 and TB10 cells transfected with TfR2 siRNA or CsiRNA. Data represent the mean values ± SEM observed in three independent experiments (* P
    Figure Legend Snippet: Inhibition of TfR2 expression by siRNA treatment reduces GBM cell growth. (A) Western blot analysis of TfR1 and TfR2 expression in U251 cells transfected with TfR2 siRNA or nontargeting control siRNA (CsiRNA), compared with untreated (C) cells. (B) Flow cytometry analysis of TfR2 expression in U251 and TB10 cells transfected with TfR2 siRNA or CsiRNA. Data represent the mean values ± SEM observed in three independent experiments (* P

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

    Association between TfR2 and caveolin-1 in lipid rafts as shown by immunofluorescence experiments. TB10 and U251 cells were plated on glass coverslips and left to adhere overnight. TfR-2 was visualized by incubating cells with anti-TfR_2 mAb and then with Texas Red-conjugated antimouse IgG (red fluorescence). Cells were then fixed, permeabilized, and then labeled with caveolin-1 antibody followed by Alexa 488-conjugated antirabbit antibody (green fluorescence). An image of a representative cells is shown reporting the red (TfR-2), green (Cav-1), merged fluorescence (at two different magnifications), and phase contrast.
    Figure Legend Snippet: Association between TfR2 and caveolin-1 in lipid rafts as shown by immunofluorescence experiments. TB10 and U251 cells were plated on glass coverslips and left to adhere overnight. TfR-2 was visualized by incubating cells with anti-TfR_2 mAb and then with Texas Red-conjugated antimouse IgG (red fluorescence). Cells were then fixed, permeabilized, and then labeled with caveolin-1 antibody followed by Alexa 488-conjugated antirabbit antibody (green fluorescence). An image of a representative cells is shown reporting the red (TfR-2), green (Cav-1), merged fluorescence (at two different magnifications), and phase contrast.

    Techniques Used: Immunofluorescence, Fluorescence, Labeling

    TfR2 stimulation activates ERK1/ERK2 MAPK. (A) Activation of ERK1/ERK2 MAPK. TB10 (left panel) and U87MG (middle panel) cells were serum-starved, treated with 30 µ M human holo-Tf for the indicated times and subjected to immunoblot analysis with anti-phospho-ERK1/ERK2. Blots were stripped and reprobed for total ERK1/ERK2 and for TfR2 to ensure equivalent loading and transfer. U251 cells (right panel) were serum-starved, treated with the stimulation medium in the absence ( NT indicates not treated) or in the presence of 30 µ M human holo-Tf and 30 µ M human apo-Tf for 30 minutes at 37°C and subjected to immunoblot analysis with anti-phospho-ERK1/ERK2. (B) Effect of ERK1/ERK2 phosphorylation inhibitor on the growth of TB10 cells. TB10 cells have been grown either in the absence or in the presence of the MEK-1 inhibitor PD184352, added either at 1 or 2 µ M.
    Figure Legend Snippet: TfR2 stimulation activates ERK1/ERK2 MAPK. (A) Activation of ERK1/ERK2 MAPK. TB10 (left panel) and U87MG (middle panel) cells were serum-starved, treated with 30 µ M human holo-Tf for the indicated times and subjected to immunoblot analysis with anti-phospho-ERK1/ERK2. Blots were stripped and reprobed for total ERK1/ERK2 and for TfR2 to ensure equivalent loading and transfer. U251 cells (right panel) were serum-starved, treated with the stimulation medium in the absence ( NT indicates not treated) or in the presence of 30 µ M human holo-Tf and 30 µ M human apo-Tf for 30 minutes at 37°C and subjected to immunoblot analysis with anti-phospho-ERK1/ERK2. (B) Effect of ERK1/ERK2 phosphorylation inhibitor on the growth of TB10 cells. TB10 cells have been grown either in the absence or in the presence of the MEK-1 inhibitor PD184352, added either at 1 or 2 µ M.

    Techniques Used: Activation Assay

    Effect of hypoxia on TfR2 expression. (A) Flow cytometry analysis of TfR2 expression in TB10 cells grown for 40 hours either in the absence (C) or in the presence of 100 µ M CoCl 2 . (B) Western blot analysis of HIF-1α expression in U251 cells grown for either 24 or 48 hours either in the absence (C) or in the presence of 100 µ M CoCl 2 and in TB10 cells grown for 48 hours under normal (20% O 2 ) and reduced (5% and 1% O 2 ) oxygen tension. (C) Analysis of the level of fluorescence (mean fluorescence intensity values normalized with respect to their negative control) of TfR2 labeling observed in TB10 and U251 cells grown for either 24 or 48 hours either in the absence (C) or in the presence of 100 µ M CoCl 2 or under controlled (20%, 5%, and 1% O 2 ) oxygen tension. Data represent the mean values ± SEM observed in three independent experiments. * P
    Figure Legend Snippet: Effect of hypoxia on TfR2 expression. (A) Flow cytometry analysis of TfR2 expression in TB10 cells grown for 40 hours either in the absence (C) or in the presence of 100 µ M CoCl 2 . (B) Western blot analysis of HIF-1α expression in U251 cells grown for either 24 or 48 hours either in the absence (C) or in the presence of 100 µ M CoCl 2 and in TB10 cells grown for 48 hours under normal (20% O 2 ) and reduced (5% and 1% O 2 ) oxygen tension. (C) Analysis of the level of fluorescence (mean fluorescence intensity values normalized with respect to their negative control) of TfR2 labeling observed in TB10 and U251 cells grown for either 24 or 48 hours either in the absence (C) or in the presence of 100 µ M CoCl 2 or under controlled (20%, 5%, and 1% O 2 ) oxygen tension. Data represent the mean values ± SEM observed in three independent experiments. * P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Western Blot, Fluorescence, Negative Control, Labeling

    Effect of temozolomide on cell proliferation and cell cycle. (A) Cell lines TB10, U251, T98G, and U87MG were treated with control medium (C) or temozolomide (TMZ) at the indicated concentrations and then trypsinized and counted after 72 hours of incubation at 37°C (left panel). The cells were subjected to cell cycle analysis in flow cytometry. Data shown are percentages of the cells in the G 2 /M phase (right panel). (B) U251 cells were transfected with control siRNA (40 nM) or TfR2 siRNA (60 nM), treated for 72 hours with temozolomide 300 µ M, and then analyzed for the number of living cells (left panel) and the percentage of the cells in G 2 /M phase (right panel).
    Figure Legend Snippet: Effect of temozolomide on cell proliferation and cell cycle. (A) Cell lines TB10, U251, T98G, and U87MG were treated with control medium (C) or temozolomide (TMZ) at the indicated concentrations and then trypsinized and counted after 72 hours of incubation at 37°C (left panel). The cells were subjected to cell cycle analysis in flow cytometry. Data shown are percentages of the cells in the G 2 /M phase (right panel). (B) U251 cells were transfected with control siRNA (40 nM) or TfR2 siRNA (60 nM), treated for 72 hours with temozolomide 300 µ M, and then analyzed for the number of living cells (left panel) and the percentage of the cells in G 2 /M phase (right panel).

    Techniques Used: Incubation, Cell Cycle Assay, Flow Cytometry, Cytometry, Transfection

    TfR2 expression in GBM cell lines. (A) Flow cytometry analysis of TfR1 and TfR2 expression in TB10, U87MG, T89G, and U251 GBMcell lines and in BTSC1 and BTSC83 GBM stem cell lines. Cells have been labeled with anti-TfR1 and anti-TfR2 mAbs and then analyzed by flow cytometry. (B) Western blot analysis of TfR1 and TfR2 expression in TB10, U87GM, T98G, and U251 GBM cell lines and in BTSC1 and BTSC83 GBM stem cell lines. Blots were stripped and reprobed for actin to ensure equivalent loading and transfer. (C and D) Association of TfR2 with lipid rafts/caveolae by density gradient centrifugation. TB10 (C) and U251 (D) cells were lysed in Triton X-100 and subjected to sucrose gradient centrifugation. Aliquots of fractions collected from the top of the gradient were analyzed by Western blot analysis with the use of antibodies against TfR1, TfR2, and caveolin-1 (Cav-1). Equal protein amounts for each fraction were loaded on SDS-PAGE.
    Figure Legend Snippet: TfR2 expression in GBM cell lines. (A) Flow cytometry analysis of TfR1 and TfR2 expression in TB10, U87MG, T89G, and U251 GBMcell lines and in BTSC1 and BTSC83 GBM stem cell lines. Cells have been labeled with anti-TfR1 and anti-TfR2 mAbs and then analyzed by flow cytometry. (B) Western blot analysis of TfR1 and TfR2 expression in TB10, U87GM, T98G, and U251 GBM cell lines and in BTSC1 and BTSC83 GBM stem cell lines. Blots were stripped and reprobed for actin to ensure equivalent loading and transfer. (C and D) Association of TfR2 with lipid rafts/caveolae by density gradient centrifugation. TB10 (C) and U251 (D) cells were lysed in Triton X-100 and subjected to sucrose gradient centrifugation. Aliquots of fractions collected from the top of the gradient were analyzed by Western blot analysis with the use of antibodies against TfR1, TfR2, and caveolin-1 (Cav-1). Equal protein amounts for each fraction were loaded on SDS-PAGE.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Labeling, Western Blot, Gradient Centrifugation, SDS Page

    22) Product Images from "Canonical NFκB signaling in myeloid cells is required for the glioblastoma growth"

    Article Title: Canonical NFκB signaling in myeloid cells is required for the glioblastoma growth

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14079-4

    p65 KO chimera failed to inhibit human GBM growth in immune-deficient host. ( A and B ) The p65 KO and control recipient mice bone marrow was isolated and transplanted into irradiated donor nude mice. Once mice achieved 70% engraftment (chimera) in 2 weeks, U251 or PDX GBM811 cells were orthotopically implanted and followed-up for MRI at the 3-week or 8-week protocol, respectively. MRI data showing p65KO chimera failed to decrease the U251 or PDX GBM811 human tumor growth. Shown is one of the two experiments performed. Quantitative data is expressed in mean ± SD.
    Figure Legend Snippet: p65 KO chimera failed to inhibit human GBM growth in immune-deficient host. ( A and B ) The p65 KO and control recipient mice bone marrow was isolated and transplanted into irradiated donor nude mice. Once mice achieved 70% engraftment (chimera) in 2 weeks, U251 or PDX GBM811 cells were orthotopically implanted and followed-up for MRI at the 3-week or 8-week protocol, respectively. MRI data showing p65KO chimera failed to decrease the U251 or PDX GBM811 human tumor growth. Shown is one of the two experiments performed. Quantitative data is expressed in mean ± SD.

    Techniques Used: Mouse Assay, Isolation, Irradiation, Magnetic Resonance Imaging

    23) Product Images from "Association between sister chromatid exchange and double minute chromosomes in human tumor cells"

    Article Title: Association between sister chromatid exchange and double minute chromosomes in human tumor cells

    Journal: Molecular Cytogenetics

    doi: 10.1186/s13039-015-0192-x

    Location of SCE on the chromosome of each cell line. a – d . The numbers of SCEs on the p arm or the q arm of the chromosome are shown for the UACC-1598, HO-8910, SK-PN-DW, and U251 cells. Data are show as mean ± standard deviation (SD). *** indicate P
    Figure Legend Snippet: Location of SCE on the chromosome of each cell line. a – d . The numbers of SCEs on the p arm or the q arm of the chromosome are shown for the UACC-1598, HO-8910, SK-PN-DW, and U251 cells. Data are show as mean ± standard deviation (SD). *** indicate P

    Techniques Used: Standard Deviation

    The SCE phenomenon in each cell line. a . SCEs were found in both groups of cell lines and are indicated by the straight arrows. The dotted arrows indicate DMs in the DM-positive cells. b – d The numbers of SCEs are shown for the UACC-1598, HO-8910, SK-PN-DW, U251, NCI-N87 and MGC-803 cells. Data are shown as mean ± standard deviation (SD). *** indicate P
    Figure Legend Snippet: The SCE phenomenon in each cell line. a . SCEs were found in both groups of cell lines and are indicated by the straight arrows. The dotted arrows indicate DMs in the DM-positive cells. b – d The numbers of SCEs are shown for the UACC-1598, HO-8910, SK-PN-DW, U251, NCI-N87 and MGC-803 cells. Data are shown as mean ± standard deviation (SD). *** indicate P

    Techniques Used: Standard Deviation

    24) Product Images from "GFAP hyperpalmitoylation exacerbates astrogliosis and neurodegenerative pathology in PPT1-deficient mice"

    Article Title: GFAP hyperpalmitoylation exacerbates astrogliosis and neurodegenerative pathology in PPT1-deficient mice

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

    doi: 10.1073/pnas.2022261118

    Palmitoylated GFAP is involved in controlling astrocyte proliferation. ( A ) Constructed GFAP-KO cell was examined by Western blot to confirm its loss of GFAP in U251. ( B ) The removal of GFAP does not cause apparent morphological alteration in U251 cell. ( C ) The CCK8 was used to quantify cell proliferation in U251 cells for different genotypes (WT and GFAP-KO). ( D and E ) CCK8 was used to quantify cell proliferation in U251 ( D ) or U87 cells ( E ) expressing either empty vector (Flag), GFAP-Flag or GFAP-C291A-Flag construct. ( F and G ) CCK8 assay was conducted to quantify cell proliferation in U251 ( F ) or U87 ( G ) as treated with either DMSO, 2-BP, or none (WT). Data are mean ± SEM; P values calculated using paired two-sided t test. n = 3. ** P ≤ 0.01, *** P ≤ 0.001.
    Figure Legend Snippet: Palmitoylated GFAP is involved in controlling astrocyte proliferation. ( A ) Constructed GFAP-KO cell was examined by Western blot to confirm its loss of GFAP in U251. ( B ) The removal of GFAP does not cause apparent morphological alteration in U251 cell. ( C ) The CCK8 was used to quantify cell proliferation in U251 cells for different genotypes (WT and GFAP-KO). ( D and E ) CCK8 was used to quantify cell proliferation in U251 ( D ) or U87 cells ( E ) expressing either empty vector (Flag), GFAP-Flag or GFAP-C291A-Flag construct. ( F and G ) CCK8 assay was conducted to quantify cell proliferation in U251 ( F ) or U87 ( G ) as treated with either DMSO, 2-BP, or none (WT). Data are mean ± SEM; P values calculated using paired two-sided t test. n = 3. ** P ≤ 0.01, *** P ≤ 0.001.

    Techniques Used: Construct, Western Blot, Expressing, Plasmid Preparation, CCK-8 Assay

    GFAP is palmitoylated at C291. ( A ) GFAP expressed in HEK-293T cells or from mice brain was analyzed for protein palmitoylation by Acyl-RAC assay. HA + , with HA, HA − , without HA. ( B ) Astroglioma cell line U251 was incubated with 2-BP for different period of time, and then were subjected for evaluating the level of GFAP palmitoylation by Acyl-RAC assay. ( C ) Protein sequence of GFAP from various species were aligned together for analyzing cysteine conservation. ( D ) Purified GFAP was probed by mass-spectrometry, a mass-shift of 238 Da linked to cysteine is a hallmark for palmitoylation. ( E ) WT or point-mutated (C291A, cysteine to alanine mutation) GFAP were expressed in HEK-293T cells and subjected for protein palmitoylation by Acyl-RAC assay.
    Figure Legend Snippet: GFAP is palmitoylated at C291. ( A ) GFAP expressed in HEK-293T cells or from mice brain was analyzed for protein palmitoylation by Acyl-RAC assay. HA + , with HA, HA − , without HA. ( B ) Astroglioma cell line U251 was incubated with 2-BP for different period of time, and then were subjected for evaluating the level of GFAP palmitoylation by Acyl-RAC assay. ( C ) Protein sequence of GFAP from various species were aligned together for analyzing cysteine conservation. ( D ) Purified GFAP was probed by mass-spectrometry, a mass-shift of 238 Da linked to cysteine is a hallmark for palmitoylation. ( E ) WT or point-mutated (C291A, cysteine to alanine mutation) GFAP were expressed in HEK-293T cells and subjected for protein palmitoylation by Acyl-RAC assay.

    Techniques Used: Mouse Assay, Incubation, Sequencing, Purification, Mass Spectrometry, Mutagenesis

    25) Product Images from "A New Patient-Derived Metastatic Glioblastoma Cell Line: Characterisation and Response to Sodium Selenite Anticancer Agent"

    Article Title: A New Patient-Derived Metastatic Glioblastoma Cell Line: Characterisation and Response to Sodium Selenite Anticancer Agent

    Journal: Cancers

    doi: 10.3390/cancers11010012

    SS’s potential way of action was studied by evaluating ( a ) oxidative stress by thiol group levels in R2J and U251 cell lines after being treated for 24h with SS or TMZ. Cell lysates were obtained after five thaw-freeze cycles. Thiol groups (µM) were normalized by the protein concentration in each sample. Results, expressed in percentage vs. control (not treated cells), are the mean ± SD of three independent experiments with * p
    Figure Legend Snippet: SS’s potential way of action was studied by evaluating ( a ) oxidative stress by thiol group levels in R2J and U251 cell lines after being treated for 24h with SS or TMZ. Cell lysates were obtained after five thaw-freeze cycles. Thiol groups (µM) were normalized by the protein concentration in each sample. Results, expressed in percentage vs. control (not treated cells), are the mean ± SD of three independent experiments with * p

    Techniques Used: Protein Concentration

    Sodium selenite (SS) cytotoxicity and cell death triggered. SS is more cytotoxic than TMZ in R2J (( a ) and ( b )) and in U251 (( c ) and ( d )) cells: Cell survival was evaluated by an MTT assay performed on cell growing without (control = 100%) or with variable doses of sodium selenite for 24 h and 72 h ( a , c ) or sodium selenite or TMZ for 72 h, followed by 72 h of wash-out ( b , d ). Results, expressed in percentage of cell survival vs. the control, are the mean ± SD of three independent experiments with * p
    Figure Legend Snippet: Sodium selenite (SS) cytotoxicity and cell death triggered. SS is more cytotoxic than TMZ in R2J (( a ) and ( b )) and in U251 (( c ) and ( d )) cells: Cell survival was evaluated by an MTT assay performed on cell growing without (control = 100%) or with variable doses of sodium selenite for 24 h and 72 h ( a , c ) or sodium selenite or TMZ for 72 h, followed by 72 h of wash-out ( b , d ). Results, expressed in percentage of cell survival vs. the control, are the mean ± SD of three independent experiments with * p

    Techniques Used: MTT Assay

    26) Product Images from "Long non-coding RNA ATB promotes glioma malignancy by negatively regulating miR-200a"

    Article Title: Long non-coding RNA ATB promotes glioma malignancy by negatively regulating miR-200a

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-016-0367-2

    Knockdown of ATB inhibited U251 cell growth in vivo. a sh-control or sh-ATB was transfected into U251 cells, which were injected in nude mice, respectively. b Tumor volumes were calculated every 5 days after injection. Bars indicate SD. c Tumor weights are represented as means of tumor weights ± SD. d Immunohistochemical (IHC) staining expression of Ki-67 in subcutaneous tumors of mice injected with sh-control or sh-ATB cells. Data are presented as mean ± s.d. from three independent experiments. ** P
    Figure Legend Snippet: Knockdown of ATB inhibited U251 cell growth in vivo. a sh-control or sh-ATB was transfected into U251 cells, which were injected in nude mice, respectively. b Tumor volumes were calculated every 5 days after injection. Bars indicate SD. c Tumor weights are represented as means of tumor weights ± SD. d Immunohistochemical (IHC) staining expression of Ki-67 in subcutaneous tumors of mice injected with sh-control or sh-ATB cells. Data are presented as mean ± s.d. from three independent experiments. ** P

    Techniques Used: In Vivo, Transfection, Injection, Mouse Assay, Immunohistochemistry, Staining, Expressing

    miR-200a directly targeted the 3′UTR of oncogene TGF-β2 and inhibits its expression in glioma cells. a The predicted of miR-200a binding sites in the 3′-UTR region of TGF-β2 (TGF-β2-3′-UTR-WT) and the corresponding mutant sequence (TGF-β2-3′UTR-MUT) was shown. b - c Relative expression of TGF-β2 mRNA and protein levels in U251 and A172 cells after transfected with miR-200a mimics, miR-200a inhibitors, and miR-200a NC. * P
    Figure Legend Snippet: miR-200a directly targeted the 3′UTR of oncogene TGF-β2 and inhibits its expression in glioma cells. a The predicted of miR-200a binding sites in the 3′-UTR region of TGF-β2 (TGF-β2-3′-UTR-WT) and the corresponding mutant sequence (TGF-β2-3′UTR-MUT) was shown. b - c Relative expression of TGF-β2 mRNA and protein levels in U251 and A172 cells after transfected with miR-200a mimics, miR-200a inhibitors, and miR-200a NC. * P

    Techniques Used: Expressing, Binding Assay, Mutagenesis, Sequencing, Transfection

    Knockdown of ATB inhibited the proliferation, migration, and invasion of glioma cells in vitro. a CCK-8 assay was performed to determine the proliferation effect of sh-ATB and sh-control transfected U251 and A172 cells. * P
    Figure Legend Snippet: Knockdown of ATB inhibited the proliferation, migration, and invasion of glioma cells in vitro. a CCK-8 assay was performed to determine the proliferation effect of sh-ATB and sh-control transfected U251 and A172 cells. * P

    Techniques Used: Migration, In Vitro, CCK-8 Assay, Transfection

    ATB directly targeted miR-200a in glioma. a The predicted miR-200a binding sites on ATB. b - c Luciferase activity in U251 and A172 glioma cells co-transfected with miR-200a mimics and luciferase reporters containing ATB-WT or ATB-MUT transcript. ** P
    Figure Legend Snippet: ATB directly targeted miR-200a in glioma. a The predicted miR-200a binding sites on ATB. b - c Luciferase activity in U251 and A172 glioma cells co-transfected with miR-200a mimics and luciferase reporters containing ATB-WT or ATB-MUT transcript. ** P

    Techniques Used: Binding Assay, Luciferase, Activity Assay, Transfection

    27) Product Images from "Identification of miRNA-7 by genome-wide analysis as a critical sensitizer for TRAIL-induced apoptosis in glioblastoma cells"

    Article Title: Identification of miRNA-7 by genome-wide analysis as a critical sensitizer for TRAIL-induced apoptosis in glioblastoma cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx317

    Further validation of miR-7-XIAP axis. ( A ) XIAP knock-down by siRNA (siXIAP) in four cancer cell lines. Western blot assays 48 h after transfection. β-actin was utilized as a loading reference. ( B ) Percent cell apoptosis of cancer cells for the aforementioned sets determined by FACS assay after 48 h treatment of TRAIL. t -test, mean ± SD, n = 3. ( C ) Western blot analysis determining the changes of XIAP expression level in control, miR-7 with/without XIAP-treated sets. ( D ) Percent cell apoptosis of cancer cells for the aforementioned sets. One-way ANOVA, mean ± SD, n = 3. ( E ) The relationship between miR-7 and XIAP in GBM cell lines (U87, U251, A172, T98G) was evaluated using qRT-PCR assay. ( F ) The relationship in four hepatocellular carcinoma cell lines (Hep G2, SMMC-7721, HuH-7 and MHCC-97H) and five prostatic carcinoma cell lines (PC-3, PC-3M, DU 145, 22Rv1 and LNCaP). ( G ) The relationship in GBM clinical samples. * P
    Figure Legend Snippet: Further validation of miR-7-XIAP axis. ( A ) XIAP knock-down by siRNA (siXIAP) in four cancer cell lines. Western blot assays 48 h after transfection. β-actin was utilized as a loading reference. ( B ) Percent cell apoptosis of cancer cells for the aforementioned sets determined by FACS assay after 48 h treatment of TRAIL. t -test, mean ± SD, n = 3. ( C ) Western blot analysis determining the changes of XIAP expression level in control, miR-7 with/without XIAP-treated sets. ( D ) Percent cell apoptosis of cancer cells for the aforementioned sets. One-way ANOVA, mean ± SD, n = 3. ( E ) The relationship between miR-7 and XIAP in GBM cell lines (U87, U251, A172, T98G) was evaluated using qRT-PCR assay. ( F ) The relationship in four hepatocellular carcinoma cell lines (Hep G2, SMMC-7721, HuH-7 and MHCC-97H) and five prostatic carcinoma cell lines (PC-3, PC-3M, DU 145, 22Rv1 and LNCaP). ( G ) The relationship in GBM clinical samples. * P

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

    28) Product Images from "uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells"

    Article Title: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2012.1496

    Effect of pUC and radiation treatment on pre-established intracranial tumors. U251 non-GICs and GICs were implanted intracranially into nude mice and treated with 450 μ g of pUC seven days after implantation. Radiation treatment was given in two doses (5 Gy at days 8 and 10). When chronic symptoms were observed, the mice were sacrificed, and their brains were collected and embedded in paraffin. Paraffin-embedded sections were deparaffinized, antigen retrieved, and co-localization studies were carried out. The brain sections were incubated overnight with primary antibodies in 10% goat serum at 4°C in a humidified chamber, counterstained with Alexa Fluor-conjugated secondary antibodies, and incubated with DAPI for nuclear staining before mounting. A, Co-localization of integrin β1 (green), pPKC θ/δ (red) and DAPI (blue) in the paraffin sections of the nude mice established with U251 non-GICs and GICs and treated with shRNA and radiation alone or in combination. B, Interaction between integrin β1 (green) and PKC ζ (red) in the various in vivo combination treatments. DAPI (blue) was used for nuclear staining. C, In vivo brain sections of immunocompromised mice implanted with U251 non-GICs and GICs were labeled with FAK (green) and α-actinin (red) and processed for immunofluorescence. The sections were incubated with DAPI for a brief period of time for nuclear staining. D, Mock, pUC-treated, irradiated, and pUC + irradiated brain sections of the nude mice pre-established with U251 non-GICs and GICs were immunoprocessed and labeled with FAK (green), vinculin (red) and DAPI (blue) in order to observe the co-localization of FAK and vinculin in in vivo samples.
    Figure Legend Snippet: Effect of pUC and radiation treatment on pre-established intracranial tumors. U251 non-GICs and GICs were implanted intracranially into nude mice and treated with 450 μ g of pUC seven days after implantation. Radiation treatment was given in two doses (5 Gy at days 8 and 10). When chronic symptoms were observed, the mice were sacrificed, and their brains were collected and embedded in paraffin. Paraffin-embedded sections were deparaffinized, antigen retrieved, and co-localization studies were carried out. The brain sections were incubated overnight with primary antibodies in 10% goat serum at 4°C in a humidified chamber, counterstained with Alexa Fluor-conjugated secondary antibodies, and incubated with DAPI for nuclear staining before mounting. A, Co-localization of integrin β1 (green), pPKC θ/δ (red) and DAPI (blue) in the paraffin sections of the nude mice established with U251 non-GICs and GICs and treated with shRNA and radiation alone or in combination. B, Interaction between integrin β1 (green) and PKC ζ (red) in the various in vivo combination treatments. DAPI (blue) was used for nuclear staining. C, In vivo brain sections of immunocompromised mice implanted with U251 non-GICs and GICs were labeled with FAK (green) and α-actinin (red) and processed for immunofluorescence. The sections were incubated with DAPI for a brief period of time for nuclear staining. D, Mock, pUC-treated, irradiated, and pUC + irradiated brain sections of the nude mice pre-established with U251 non-GICs and GICs were immunoprocessed and labeled with FAK (green), vinculin (red) and DAPI (blue) in order to observe the co-localization of FAK and vinculin in in vivo samples.

    Techniques Used: Mouse Assay, Incubation, Staining, shRNA, In Vivo, Labeling, Immunofluorescence, Irradiation

    Depletion of uPAR and cathepsin B inhibits PKC/integrin signaling to FAK and the cytoskeletal molecules. A, Cell lysates from U251 and 5310 non-GICs and GICs were extracted using RIPA buffer after treatment with pUC and radiation alone or in combination, and western blot analysis was performed to determine the expression levels of FAK, pFAK (Tyr 397 ), pFAK (Tyr 925 ), vinculin, and α-actinin using their specific antibodies. GAPDH was used as a loading control. B, The total cell lysates of U251 and 5310 non-GICs and GICs as described above were immunoprecipitated with FAK antibody (2 μ g). The protein precipitates were washed with lysis buffer and incubated with 1X loading dye at 90°C for 10 min. SDS-PAGE was conducted and western blotting was performed with vinculin and α-actinin antibodies as described in Materials and methods.
    Figure Legend Snippet: Depletion of uPAR and cathepsin B inhibits PKC/integrin signaling to FAK and the cytoskeletal molecules. A, Cell lysates from U251 and 5310 non-GICs and GICs were extracted using RIPA buffer after treatment with pUC and radiation alone or in combination, and western blot analysis was performed to determine the expression levels of FAK, pFAK (Tyr 397 ), pFAK (Tyr 925 ), vinculin, and α-actinin using their specific antibodies. GAPDH was used as a loading control. B, The total cell lysates of U251 and 5310 non-GICs and GICs as described above were immunoprecipitated with FAK antibody (2 μ g). The protein precipitates were washed with lysis buffer and incubated with 1X loading dye at 90°C for 10 min. SDS-PAGE was conducted and western blotting was performed with vinculin and α-actinin antibodies as described in Materials and methods.

    Techniques Used: Western Blot, Expressing, Immunoprecipitation, Lysis, Incubation, SDS Page

    pUC inhibits the FAK migratory signaling pathway and inhibition of ERK can further influence the FAK signal. A, Cell lysates of control, pSV-transfected, and pUC-transfected U251 and 5310 non-GICs and GICs with and without irradiation were collected. Western blot analysis was performed for FAK migratory signaling molecules Rac-1, Cdc42, pCdc42/Rac-1, Ras, ERK, and pERK using their specific antibodies. GAPDH served as a loading control. B, The total protein lysates from U251 and 5310 non-GICs and GICs were collected from U0126 (10 μ M) and DMSO treatments. SDS-PAGE was conducted as described in Materials and methods. Western blotting was performed to determine the protein expression levels of ERK, pERK, FAK, pFAK (Tyr 397 ), pFAK (Tyr 925 ), vinculin, α-actinin, Rac-1, Cdc42, pCdc42/Rac-1, and Ras using their specific antibodies.
    Figure Legend Snippet: pUC inhibits the FAK migratory signaling pathway and inhibition of ERK can further influence the FAK signal. A, Cell lysates of control, pSV-transfected, and pUC-transfected U251 and 5310 non-GICs and GICs with and without irradiation were collected. Western blot analysis was performed for FAK migratory signaling molecules Rac-1, Cdc42, pCdc42/Rac-1, Ras, ERK, and pERK using their specific antibodies. GAPDH served as a loading control. B, The total protein lysates from U251 and 5310 non-GICs and GICs were collected from U0126 (10 μ M) and DMSO treatments. SDS-PAGE was conducted as described in Materials and methods. Western blotting was performed to determine the protein expression levels of ERK, pERK, FAK, pFAK (Tyr 397 ), pFAK (Tyr 925 ), vinculin, α-actinin, Rac-1, Cdc42, pCdc42/Rac-1, and Ras using their specific antibodies.

    Techniques Used: Inhibition, Transfection, Irradiation, Western Blot, SDS Page, Expressing

    Effect of radiation and pUC on migration and cytoskeleton of non-GICs and GICs. A, U251 and 5310 non-GICs and GICs were irradiated with 5 and 10 Gy at 24 and 48 h. Cell lysates were extracted and analyzed by SDS-PAGE to determine the expression levels of uPAR and cathepsin B with and without radiation. GAPDH served as a loading control. All the results are representative of three individual experiments. B, U251 and 5310 non-GICs and GICs were transfected with pSV and pUC with and without irradiation for 24 and 48 h. Cell lysates were prepared and analyzed for uPAR and cathepsin B expression levels using western blotting. C, The protein expression levels of uPAR and cathepsin B in U251 and 5310 cells treated with pUC and radiation alone and in combination were analyzed by densitometric analysis and are depicted in the graph as relative protein expression (control of each set as 100%). D, Wound healing assay was performed with pSV and pUC and/or radiation treatments in U251 and 5310 non-GICs and GICs as described in Materials and methods. The cells were then fixed, stained with DAPI, and imaged using a fluorescence microscope (Olympus IX71, USA). Scale bar, 500 μ m. E, The migration and wound healing capacities of U251 and 5310 non-GICs and GICs treated under the previously described conditions were measured using a microscope, analyzed, and are graphically represented as a relative percentage of wound healing (migration of pSV-treated samples as 100%). F, U251 and 5310 non-GICs and GICs treated with pSV and pUC with and without radiation were grown on fibronectin-coated 4-well chamber slides. The cells were then fixed with 4% buffered formalin, stained with vinculin antibody, and incubated with DAPI for a brief period of time before mounting. Scale bar, 200 μ m. Values are mean ± SD of three different experiments ( * p
    Figure Legend Snippet: Effect of radiation and pUC on migration and cytoskeleton of non-GICs and GICs. A, U251 and 5310 non-GICs and GICs were irradiated with 5 and 10 Gy at 24 and 48 h. Cell lysates were extracted and analyzed by SDS-PAGE to determine the expression levels of uPAR and cathepsin B with and without radiation. GAPDH served as a loading control. All the results are representative of three individual experiments. B, U251 and 5310 non-GICs and GICs were transfected with pSV and pUC with and without irradiation for 24 and 48 h. Cell lysates were prepared and analyzed for uPAR and cathepsin B expression levels using western blotting. C, The protein expression levels of uPAR and cathepsin B in U251 and 5310 cells treated with pUC and radiation alone and in combination were analyzed by densitometric analysis and are depicted in the graph as relative protein expression (control of each set as 100%). D, Wound healing assay was performed with pSV and pUC and/or radiation treatments in U251 and 5310 non-GICs and GICs as described in Materials and methods. The cells were then fixed, stained with DAPI, and imaged using a fluorescence microscope (Olympus IX71, USA). Scale bar, 500 μ m. E, The migration and wound healing capacities of U251 and 5310 non-GICs and GICs treated under the previously described conditions were measured using a microscope, analyzed, and are graphically represented as a relative percentage of wound healing (migration of pSV-treated samples as 100%). F, U251 and 5310 non-GICs and GICs treated with pSV and pUC with and without radiation were grown on fibronectin-coated 4-well chamber slides. The cells were then fixed with 4% buffered formalin, stained with vinculin antibody, and incubated with DAPI for a brief period of time before mounting. Scale bar, 200 μ m. Values are mean ± SD of three different experiments ( * p

    Techniques Used: Migration, Irradiation, SDS Page, Expressing, Transfection, Western Blot, Wound Healing Assay, Staining, Fluorescence, Microscopy, Incubation

    Isolation and characterization of GICs in U251 glioblastoma cells and 5310 xenograft cells. A, U251 and 5310 cells were enriched with knockout DMEM supplemented with growth factors as described in Materials and methods. After a considerable amount of sphere formation was observed, cells were collected, dissociated, and labeled with CD133 for isolation of positive cells using IgG-stained cells as a negative control. The CD133 + population is indicated as a black line over the dotted line, representing the total population in the histogram. B, Western blot analysis of non-GICs and GICs showing the expression of stem cell markers CD133, CD44, Nestin, Sox-2, proliferation marker Ki67, and the lineage markers GFAP and Tuj-1. C, Densitometric analysis indicating the increase in the protein expression levels of CD133, CD44, Nestin, and Sox-2 markers in GICs as compared to that of their non-GIC counterparts. D, Total RNA was extracted from the non-GICs and GICs and the mRNA expression levels of the stem cell, proliferation, and lineage markers were determined by RT-PCR analysis. E, GICs were further characterized by immunofluorescence analysis. GICs were immunoprocessed with the respective antibodies and then conjugated with species-specific Alexa Fluor 594-conjugated secondary antibodies and visualized under a microscope. Scale bar, 200 μ m.
    Figure Legend Snippet: Isolation and characterization of GICs in U251 glioblastoma cells and 5310 xenograft cells. A, U251 and 5310 cells were enriched with knockout DMEM supplemented with growth factors as described in Materials and methods. After a considerable amount of sphere formation was observed, cells were collected, dissociated, and labeled with CD133 for isolation of positive cells using IgG-stained cells as a negative control. The CD133 + population is indicated as a black line over the dotted line, representing the total population in the histogram. B, Western blot analysis of non-GICs and GICs showing the expression of stem cell markers CD133, CD44, Nestin, Sox-2, proliferation marker Ki67, and the lineage markers GFAP and Tuj-1. C, Densitometric analysis indicating the increase in the protein expression levels of CD133, CD44, Nestin, and Sox-2 markers in GICs as compared to that of their non-GIC counterparts. D, Total RNA was extracted from the non-GICs and GICs and the mRNA expression levels of the stem cell, proliferation, and lineage markers were determined by RT-PCR analysis. E, GICs were further characterized by immunofluorescence analysis. GICs were immunoprocessed with the respective antibodies and then conjugated with species-specific Alexa Fluor 594-conjugated secondary antibodies and visualized under a microscope. Scale bar, 200 μ m.

    Techniques Used: Isolation, Knock-Out, Labeling, Staining, Negative Control, Western Blot, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Microscopy

    PKCs regulate the ECM-integrin interaction signal and vice versa in U251 and 5310 non-GICs and GICs. A, SDS-PAGE was conducted with the cell lysates of control, DMSO-treated, rottlerin (200 μ M) and integrin β1 siRNA-treated U251 and 5310 non-GICs and GICs grown in non-coated and collagen-coated culture dishes. The cell lysates were immunoblotted with PKC θ, PKC δ, pPKC θ/δ, PKC ζ, integrin β1 and integrin α2. B, SDS-PAGE was conducted with the cell lysates of control, DMSO-treated, rottlerin (200 μ M) and integrin β1 siRNA-treated U251 and 5310 non-GICs and GICs grown in non-coated and fibronectin-coated culture dishes. The cell lysates were immunoblotted for PKC θ, PKC δ, pPKC θ/δ, PKC ζ, integrin β1 and integrin α5.
    Figure Legend Snippet: PKCs regulate the ECM-integrin interaction signal and vice versa in U251 and 5310 non-GICs and GICs. A, SDS-PAGE was conducted with the cell lysates of control, DMSO-treated, rottlerin (200 μ M) and integrin β1 siRNA-treated U251 and 5310 non-GICs and GICs grown in non-coated and collagen-coated culture dishes. The cell lysates were immunoblotted with PKC θ, PKC δ, pPKC θ/δ, PKC ζ, integrin β1 and integrin α2. B, SDS-PAGE was conducted with the cell lysates of control, DMSO-treated, rottlerin (200 μ M) and integrin β1 siRNA-treated U251 and 5310 non-GICs and GICs grown in non-coated and fibronectin-coated culture dishes. The cell lysates were immunoblotted for PKC θ, PKC δ, pPKC θ/δ, PKC ζ, integrin β1 and integrin α5.

    Techniques Used: SDS Page

    Effect of pUC and radiation alone and in combination on PKC and integrin levels in U251 and 5310 non-GICs and GICs. A, Western blot analysis of the cell lysates of U251 and 5310 non-GICs and GICs showing the protein expression levels of PKC θ, PKC δ, pPKC θ/δ, PKC ζ, integrin β1, integrin α2, and integrin α5 after treatment with pUC and radiation alone or in combination. B, Total protein (300 μ g) was collected from pSV, pUC, pSV + 10 Gy and pUC + 10 Gy samples of U251 and 5310 non-GICs and GICs and immunoprecipitated with integrin β1 antibody (2 μ g) and protein A plus G agarose beads (20 μ g) overnight at 4°C. The precipitates were washed with lysis buffer and the integrin β1 pulled down protein was immunoblotted for pPKC θ/δ and PKC ζ. C, Co-localization of PKC ζ and integrin β1 was carried out with pSV and pUC with and without 10 Gy (24 h for non-GICs and 48 h for GICs). The cells were allowed to migrate on 4-well chamber slides for about 16 h after growing them in Ibidi culture inserts for 24 h after treatments. The cells were fixed, stained with primary antibody overnight at 4°C, counter-stained with species-specific Alexa Fluor-conjugated secondary antibodies, nuclear stained with DAPI, mounted, and imaged under a confocal microscope. Arrows indicating the co-localization of PKC ζ and integrin β1.
    Figure Legend Snippet: Effect of pUC and radiation alone and in combination on PKC and integrin levels in U251 and 5310 non-GICs and GICs. A, Western blot analysis of the cell lysates of U251 and 5310 non-GICs and GICs showing the protein expression levels of PKC θ, PKC δ, pPKC θ/δ, PKC ζ, integrin β1, integrin α2, and integrin α5 after treatment with pUC and radiation alone or in combination. B, Total protein (300 μ g) was collected from pSV, pUC, pSV + 10 Gy and pUC + 10 Gy samples of U251 and 5310 non-GICs and GICs and immunoprecipitated with integrin β1 antibody (2 μ g) and protein A plus G agarose beads (20 μ g) overnight at 4°C. The precipitates were washed with lysis buffer and the integrin β1 pulled down protein was immunoblotted for pPKC θ/δ and PKC ζ. C, Co-localization of PKC ζ and integrin β1 was carried out with pSV and pUC with and without 10 Gy (24 h for non-GICs and 48 h for GICs). The cells were allowed to migrate on 4-well chamber slides for about 16 h after growing them in Ibidi culture inserts for 24 h after treatments. The cells were fixed, stained with primary antibody overnight at 4°C, counter-stained with species-specific Alexa Fluor-conjugated secondary antibodies, nuclear stained with DAPI, mounted, and imaged under a confocal microscope. Arrows indicating the co-localization of PKC ζ and integrin β1.

    Techniques Used: Western Blot, Expressing, Immunoprecipitation, Lysis, Staining, Microscopy

    29) Product Images from "microRNA-200a functions as a tumor suppressor by targeting FOXA1 in glioma"

    Article Title: microRNA-200a functions as a tumor suppressor by targeting FOXA1 in glioma

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2018.6895

    T98G and U251 cells were transfected with an miR-NC or miR-200a mimic (A) A reverse transcription-quantitative polymerase chain reaction assay was performed to determine the expression of miR-200a. Non-transfected cells were used as control. **P
    Figure Legend Snippet: T98G and U251 cells were transfected with an miR-NC or miR-200a mimic (A) A reverse transcription-quantitative polymerase chain reaction assay was performed to determine the expression of miR-200a. Non-transfected cells were used as control. **P

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

    T98G and U251 cells were transfected with miR-NCs or miR-200a mimics, respectively. (A) RT-qPCR and (B) western blotting were performed to determine FOXA1 mRNA and protein levels. Non-transfected cells were used as the control group. **P
    Figure Legend Snippet: T98G and U251 cells were transfected with miR-NCs or miR-200a mimics, respectively. (A) RT-qPCR and (B) western blotting were performed to determine FOXA1 mRNA and protein levels. Non-transfected cells were used as the control group. **P

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

    T98G and U251 cells which overexpressed miR-200a were transfected with a FOXA1 expression plasmid or blank vector. Following transfection, (A) western blotting was performed to examine the expression of FOXA1 protein. (B) An MTT, (C) EdU and (D) transwell assay were then performed to assess cell proliferation, viability and invasion, respectively. The magnification used for the EdU and transwell assays were ×40 and ×200, respectively. **P
    Figure Legend Snippet: T98G and U251 cells which overexpressed miR-200a were transfected with a FOXA1 expression plasmid or blank vector. Following transfection, (A) western blotting was performed to examine the expression of FOXA1 protein. (B) An MTT, (C) EdU and (D) transwell assay were then performed to assess cell proliferation, viability and invasion, respectively. The magnification used for the EdU and transwell assays were ×40 and ×200, respectively. **P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, MTT Assay, Transwell Assay

    30) Product Images from "Knockdown of long non-coding RNA SNHG5 inhibits malignant cellular phenotypes of glioma via Wnt/CTNNB1 signaling pathway"

    Article Title: Knockdown of long non-coding RNA SNHG5 inhibits malignant cellular phenotypes of glioma via Wnt/CTNNB1 signaling pathway

    Journal: Journal of Cancer

    doi: 10.7150/jca.29517

    Knockdown of SNHG5 inhibited malignant cellular phenotypes of glioma cells. A: The expression levels of SNHG5 in U251 and U87 cells. B: The cell proliferation of U251 and U87 cells. C: The cell invasiveness of U251 and U87 cells. D: The cell apoptosis rate of U251 and U87 cells. ** P
    Figure Legend Snippet: Knockdown of SNHG5 inhibited malignant cellular phenotypes of glioma cells. A: The expression levels of SNHG5 in U251 and U87 cells. B: The cell proliferation of U251 and U87 cells. C: The cell invasiveness of U251 and U87 cells. D: The cell apoptosis rate of U251 and U87 cells. ** P

    Techniques Used: Expressing

    Knockdown of SNHG5 inactivated Wnt/CTNNB1 signaling pathway. A: The co-expression patterns between SNHG5 and GSK3B in TCGA Pan-Cancer (PANCAN) database with 171 glioma samples. B: The co-expression patterns between SNHG5 and CTNNB1 in TCGA Pan-Cancer (PANCAN) database with 171 glioma samples. C: The ratio of TOP/FOP luciferase values in U251 and U87 cells. D : The expression of GSK3B and CTNNB1 protein in U251 and U87 cells. ** P
    Figure Legend Snippet: Knockdown of SNHG5 inactivated Wnt/CTNNB1 signaling pathway. A: The co-expression patterns between SNHG5 and GSK3B in TCGA Pan-Cancer (PANCAN) database with 171 glioma samples. B: The co-expression patterns between SNHG5 and CTNNB1 in TCGA Pan-Cancer (PANCAN) database with 171 glioma samples. C: The ratio of TOP/FOP luciferase values in U251 and U87 cells. D : The expression of GSK3B and CTNNB1 protein in U251 and U87 cells. ** P

    Techniques Used: Expressing, Luciferase

    Knockdown of SNHG5 inhibited malignant cellular phenotypes of glioma cells via Wnt/CTNNB1 signaling pathway. A: The ratio of TOP/FOP luciferase values in U251 and U87 cells. B: The expression of CTNNB1 protein in U251 and U87 cells. C: The cell proliferation of U251 and U87 cells. D: The cell invasiveness of U251 and U87 cells. E: The cell apoptosis rate of U251 and U87 cells. ** P
    Figure Legend Snippet: Knockdown of SNHG5 inhibited malignant cellular phenotypes of glioma cells via Wnt/CTNNB1 signaling pathway. A: The ratio of TOP/FOP luciferase values in U251 and U87 cells. B: The expression of CTNNB1 protein in U251 and U87 cells. C: The cell proliferation of U251 and U87 cells. D: The cell invasiveness of U251 and U87 cells. E: The cell apoptosis rate of U251 and U87 cells. ** P

    Techniques Used: Luciferase, Expressing

    31) Product Images from "Genome-wide identification of epithelial-mesenchymal transition-associated microRNAs reveals novel targets for glioblastoma therapy"

    Article Title: Genome-wide identification of epithelial-mesenchymal transition-associated microRNAs reveals novel targets for glioblastoma therapy

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8280

    Influences of miR-95 and miR-223 on the expression and invasion of N-cadherin in glioma cells. (A) Representative cell images of the effect of miR-95 and miR-223 on the cellular invasion of U251 and LN229 glioma cells. (B) The quantitative data of the effect of miR-95 and miR-223 on the cellular invasion of U251 and LN229 glioma cells. All experiments were repeated three times. (C) Following the transfection of miR-95 and miR-223 into U251 and LN229 glioma cells, Reverse transcription-quantitative polymerase chain reaction demonstrated that the N-cadherin mRNA level was significantly regulated. The data are obtained from representative experiments repeated in triplicate. *P
    Figure Legend Snippet: Influences of miR-95 and miR-223 on the expression and invasion of N-cadherin in glioma cells. (A) Representative cell images of the effect of miR-95 and miR-223 on the cellular invasion of U251 and LN229 glioma cells. (B) The quantitative data of the effect of miR-95 and miR-223 on the cellular invasion of U251 and LN229 glioma cells. All experiments were repeated three times. (C) Following the transfection of miR-95 and miR-223 into U251 and LN229 glioma cells, Reverse transcription-quantitative polymerase chain reaction demonstrated that the N-cadherin mRNA level was significantly regulated. The data are obtained from representative experiments repeated in triplicate. *P

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

    32) Product Images from "p300- and Myc-mediated regulation of glioblastoma multiforme cell differentiation"

    Article Title: p300- and Myc-mediated regulation of glioblastoma multiforme cell differentiation

    Journal: Oncotarget

    doi:

    Myc overrides p300-mediated transcriptional regulation of GFAP and Nestin. U251 (A B) and U87 (C D) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A C) or 2 µg Nestin-Luc (B D) reporter construct along with 1 µg of p300 and 1 µg of Myc expression constructs, either separately or in combination. Luciferase activity was determined at 72 h posttransfection and normalized percent luciferase activities were plotted as mean ± SE (n=3) (* and ** indicate p
    Figure Legend Snippet: Myc overrides p300-mediated transcriptional regulation of GFAP and Nestin. U251 (A B) and U87 (C D) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A C) or 2 µg Nestin-Luc (B D) reporter construct along with 1 µg of p300 and 1 µg of Myc expression constructs, either separately or in combination. Luciferase activity was determined at 72 h posttransfection and normalized percent luciferase activities were plotted as mean ± SE (n=3) (* and ** indicate p

    Techniques Used: Transfection, Construct, Expressing, Luciferase, Activity Assay

    RNAi-mediated knockdown of p300 enhances invasion and migration of U251 cells in vitro. 2.5×10 4 parental U251 cells, U251 stable clones expressing two different p300-shRNA constructs (Sh#1-clone 10 and Sh#3-clone 10) or empty vector were used for modified Boyden chamber invasion assay in serum-free medium. After 16 h, cells invaded to the underside of the membrane were fixed with methanol, stained with hematoxylin blue and (A) visualized using phase contrast microscopy. The number of invaded cells in (A) were counted and plotted as mean ± SE (n=3) (B). The experiment was repeated two times, and similar results were obtained. (C) U251 cells as used in (A) (parental, vector, Sh#1-clone 10, Sh#3-clone 10) were used for wound healing assay in serum-free medium and phase-contrast images were obtained at 0, 24 and 48 h after scraping. (D) Cell proliferation/viability was determined using MTT assay after 0, 24 and 48 h of serum starvation. The values are plotted as mean ± SE (n=3). ** indicates p
    Figure Legend Snippet: RNAi-mediated knockdown of p300 enhances invasion and migration of U251 cells in vitro. 2.5×10 4 parental U251 cells, U251 stable clones expressing two different p300-shRNA constructs (Sh#1-clone 10 and Sh#3-clone 10) or empty vector were used for modified Boyden chamber invasion assay in serum-free medium. After 16 h, cells invaded to the underside of the membrane were fixed with methanol, stained with hematoxylin blue and (A) visualized using phase contrast microscopy. The number of invaded cells in (A) were counted and plotted as mean ± SE (n=3) (B). The experiment was repeated two times, and similar results were obtained. (C) U251 cells as used in (A) (parental, vector, Sh#1-clone 10, Sh#3-clone 10) were used for wound healing assay in serum-free medium and phase-contrast images were obtained at 0, 24 and 48 h after scraping. (D) Cell proliferation/viability was determined using MTT assay after 0, 24 and 48 h of serum starvation. The values are plotted as mean ± SE (n=3). ** indicates p

    Techniques Used: Migration, In Vitro, Clone Assay, Expressing, shRNA, Construct, Plasmid Preparation, Modification, Invasion Assay, Staining, Microscopy, Wound Healing Assay, MTT Assay

    Tumorigenicity of GBM cells correlates with expression of Nestin and GFAP (A) Rat right frontal lobes were implanted with 5×10 5 U251 or U87 cells in 5 µl PBS. After 3 weeks, tumor volumes were determined by pixel imaging analysis. (B) The right flanks of nude mice were injected (s.c.) with 2.5×10 6 U251 or U87 cells in 100 µl PBS (mixed with 100 µl matrigel). After 5 weeks, tumor volumes were calculated using the formula: volume = width 2 × length × 0.4. U251 or U87 cells grown on coverslips were fixed, permeabilized and stained for (C) GFAP (green) and (D) Nestin (red) using respective primary antibodies and secondary antibodies conjugated with Alexafluor488 and Alexafluor568 respectively. (E) Schematic of luciferase (Luc) reporter constructs: GFAP-Luc constitutes a 2.21 kb human GFAP promoter containing a GAS element (−1558 to −1547) in pGL3-Basic vector (upper panel) and Nestin-Luc constitutes a 714 bp enhancer fragment form the second intron of the human Nestin gene cloned upstream of an 81 bp minimal thymidine kinase (TK) promoter driving the luciferase gene in the vector, TP222 (lower panel). Activities of the GFAP promoter (F) and the Nestin enhancer (G) were determined at 72 h post transfection of U251 and U87 cells (1×10 6 ) with indicated reporter plasmids (or empty vectors) by luciferase assay. For (A) (B), each value represents mean ± SE of 5 individual animals of each group. Normalized percent luciferase values for (F G) are plotted as mean ± SE (n = 3). ** indicates p
    Figure Legend Snippet: Tumorigenicity of GBM cells correlates with expression of Nestin and GFAP (A) Rat right frontal lobes were implanted with 5×10 5 U251 or U87 cells in 5 µl PBS. After 3 weeks, tumor volumes were determined by pixel imaging analysis. (B) The right flanks of nude mice were injected (s.c.) with 2.5×10 6 U251 or U87 cells in 100 µl PBS (mixed with 100 µl matrigel). After 5 weeks, tumor volumes were calculated using the formula: volume = width 2 × length × 0.4. U251 or U87 cells grown on coverslips were fixed, permeabilized and stained for (C) GFAP (green) and (D) Nestin (red) using respective primary antibodies and secondary antibodies conjugated with Alexafluor488 and Alexafluor568 respectively. (E) Schematic of luciferase (Luc) reporter constructs: GFAP-Luc constitutes a 2.21 kb human GFAP promoter containing a GAS element (−1558 to −1547) in pGL3-Basic vector (upper panel) and Nestin-Luc constitutes a 714 bp enhancer fragment form the second intron of the human Nestin gene cloned upstream of an 81 bp minimal thymidine kinase (TK) promoter driving the luciferase gene in the vector, TP222 (lower panel). Activities of the GFAP promoter (F) and the Nestin enhancer (G) were determined at 72 h post transfection of U251 and U87 cells (1×10 6 ) with indicated reporter plasmids (or empty vectors) by luciferase assay. For (A) (B), each value represents mean ± SE of 5 individual animals of each group. Normalized percent luciferase values for (F G) are plotted as mean ± SE (n = 3). ** indicates p

    Techniques Used: Expressing, Imaging, Mouse Assay, Injection, Staining, Luciferase, Construct, Plasmid Preparation, Clone Assay, Transfection

    Effect of RNAi-mediated knockdown of p300 on GFAP and Nestin expression. (A B) Five p300-shRNA stable clones (Sh#1-clones: 10, 20 and 27; Sh#3-clones: 10 and 12), one vector control and parental U251 cells (1×10 6 ) were transfected with 2 µg of GFAP-Luc (A), and 2 µg of Nestin-Luc (B). Luciferase activity was measured at 72 h posttransfection and normalized percent luciferase activities were plotted as mean ± SE (n=3) (* and ** indicate p
    Figure Legend Snippet: Effect of RNAi-mediated knockdown of p300 on GFAP and Nestin expression. (A B) Five p300-shRNA stable clones (Sh#1-clones: 10, 20 and 27; Sh#3-clones: 10 and 12), one vector control and parental U251 cells (1×10 6 ) were transfected with 2 µg of GFAP-Luc (A), and 2 µg of Nestin-Luc (B). Luciferase activity was measured at 72 h posttransfection and normalized percent luciferase activities were plotted as mean ± SE (n=3) (* and ** indicate p

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

    Myc differentially regulates the transcription of GFAP and Nestin genes in GBM cells. U251 (A, B D) and U87 (C, E F) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A, D E) or 2 µg Nestin-Luc (B, C, F) reporter construct along with indicated amounts of Myc expression plasmid (or empty vector) (A, B C) or two different Myc-specific shRNAs (Sh #2 and #3) (D, E, F). GFAP promoter (A, D E) and Nestin enhancer (B, C F) activities were determined at 72 h post transfection by luciferase assay and normalized percent luciferase activities are plotted as mean ± SE (n=3). * and ** indicate p
    Figure Legend Snippet: Myc differentially regulates the transcription of GFAP and Nestin genes in GBM cells. U251 (A, B D) and U87 (C, E F) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A, D E) or 2 µg Nestin-Luc (B, C, F) reporter construct along with indicated amounts of Myc expression plasmid (or empty vector) (A, B C) or two different Myc-specific shRNAs (Sh #2 and #3) (D, E, F). GFAP promoter (A, D E) and Nestin enhancer (B, C F) activities were determined at 72 h post transfection by luciferase assay and normalized percent luciferase activities are plotted as mean ± SE (n=3). * and ** indicate p

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

    p300 differentially regulates transcription of GFAP and Nestin genes U251 (A) and U87 (B C) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A B) or 2 µg Nestin-Luc (C) reporter constructs along with indicated amounts of p300 or empty control vector (A, B C). Luciferase activity was measured at 72 h after transfection. (D) 2×10 6 cells treated with 20 ng/ml of IL-6 or 100 ng/ml of EGF for 30 min and subjected to ChIP using anti-p300 or matched IgG antibodies. p300 occupancy to the GFAP promoter was determined by PCR using radio-labeled primers and product densities plotted as ‘percent of input’. U251 (E F) and U87 (G) cells (1×10 6 ) were transfected with two different p300-shRNAs (Sh#1 and Sh#3) or 2 µg empty vector along with 2µg GFAP-Luc (E) or Nestin-Luc (F G) reporter constructs. The promoter/enhancer activity was determined at 72 h posttransfection by luciferase assay. Normalized percent luciferase values are plotted as mean ± SE (n = 3). * and ** indicate p
    Figure Legend Snippet: p300 differentially regulates transcription of GFAP and Nestin genes U251 (A) and U87 (B C) cells (1×10 6 ) were transfected with 2 µg GFAP-Luc (A B) or 2 µg Nestin-Luc (C) reporter constructs along with indicated amounts of p300 or empty control vector (A, B C). Luciferase activity was measured at 72 h after transfection. (D) 2×10 6 cells treated with 20 ng/ml of IL-6 or 100 ng/ml of EGF for 30 min and subjected to ChIP using anti-p300 or matched IgG antibodies. p300 occupancy to the GFAP promoter was determined by PCR using radio-labeled primers and product densities plotted as ‘percent of input’. U251 (E F) and U87 (G) cells (1×10 6 ) were transfected with two different p300-shRNAs (Sh#1 and Sh#3) or 2 µg empty vector along with 2µg GFAP-Luc (E) or Nestin-Luc (F G) reporter constructs. The promoter/enhancer activity was determined at 72 h posttransfection by luciferase assay. Normalized percent luciferase values are plotted as mean ± SE (n = 3). * and ** indicate p

    Techniques Used: Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Labeling

    33) Product Images from "MicroRNA-423-3p promotes glioma growth by targeting PANX2"

    Article Title: MicroRNA-423-3p promotes glioma growth by targeting PANX2

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8636

    PANX2 was identified as a target of miR-423-3p, and luciferase activity was compared between cells with miR-423-3p, miR-NC and control with PANX2 protein levels compared between cells treated with a miR-423-3p inhibitor and with a NC inhibitor. (A) TargetScan software demonstrated that PANX2 was a putative target of miR-423-3p. (B) WT or MT PANX2 3′UTR was (C) cloned into a luciferase reporter vector. The luciferase activity was significantly decreased in (D) U251 and (E) U87MG Uppsala cells co-transfected with WT-PANX2-3′UTR plasmid and miR-423-3p mimics compared with the control group, which was eliminated during transfection with the MT-PANX2-3′UTR plasmid. Western blotting was used to examine the protein levels of PANX2 in (F) U251 and (G) U87MG Uppsala cells transfected with miR-423-3p inhibitor or NC inhibitor. **P
    Figure Legend Snippet: PANX2 was identified as a target of miR-423-3p, and luciferase activity was compared between cells with miR-423-3p, miR-NC and control with PANX2 protein levels compared between cells treated with a miR-423-3p inhibitor and with a NC inhibitor. (A) TargetScan software demonstrated that PANX2 was a putative target of miR-423-3p. (B) WT or MT PANX2 3′UTR was (C) cloned into a luciferase reporter vector. The luciferase activity was significantly decreased in (D) U251 and (E) U87MG Uppsala cells co-transfected with WT-PANX2-3′UTR plasmid and miR-423-3p mimics compared with the control group, which was eliminated during transfection with the MT-PANX2-3′UTR plasmid. Western blotting was used to examine the protein levels of PANX2 in (F) U251 and (G) U87MG Uppsala cells transfected with miR-423-3p inhibitor or NC inhibitor. **P

    Techniques Used: Luciferase, Activity Assay, Software, Plasmid Preparation, Transfection, Western Blot

    U251 and U87MG Uppsala cells were co-transfected with miR-423-3p inhibitor and PANX2 siRNA, or miR-423-3p inhibitor and NC siRNA. Following transfection, the reverse transcription-quantitative polymerase chain reaction was performed to assess PANX2 mRNA levels in (A) U251 and (B) U87MG Uppsala cells. Western blotting was used to examine the protein levels of PANX2 in (C) U251 and (D) U87MG Uppsala cells. **P
    Figure Legend Snippet: U251 and U87MG Uppsala cells were co-transfected with miR-423-3p inhibitor and PANX2 siRNA, or miR-423-3p inhibitor and NC siRNA. Following transfection, the reverse transcription-quantitative polymerase chain reaction was performed to assess PANX2 mRNA levels in (A) U251 and (B) U87MG Uppsala cells. Western blotting was used to examine the protein levels of PANX2 in (C) U251 and (D) U87MG Uppsala cells. **P

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

    U251 and U87MG Uppsala cells were co-transfected with miR-423-3p inhibitor and PANX2 siRNA, or miR-423-3p inhibitor and NC siRNA. An MTT assay was used to determine proliferation in (A) U251 and (B) U87MG Uppsala cells. Flow cytometry was used to detect apoptosis in (C) U251 and (D) U87MG Uppsala cells. **P
    Figure Legend Snippet: U251 and U87MG Uppsala cells were co-transfected with miR-423-3p inhibitor and PANX2 siRNA, or miR-423-3p inhibitor and NC siRNA. An MTT assay was used to determine proliferation in (A) U251 and (B) U87MG Uppsala cells. Flow cytometry was used to detect apoptosis in (C) U251 and (D) U87MG Uppsala cells. **P

    Techniques Used: Transfection, MTT Assay, Flow Cytometry, Cytometry

    Comparisons between miR-423-3p expression levels, cell proliferation and cell apoptosis in cells with a miR-423-3p inhibitor and an NC inhibitor. The reverse transcription-quantitative polymerase chain reaction was performed to examine the miR-423-3p expression levels in (A) U251 and (B) U87MG Uppsala cells transfected with either miR-423-3p inhibitor or NC inhibitor. An MTT assay was used to determine proliferation in (C) U251 and (D) U87MG Uppsala cells. Flow cytometry was used to detect apoptosis in (E) U251 cells and (F) U87MG Uppsala cells. **P
    Figure Legend Snippet: Comparisons between miR-423-3p expression levels, cell proliferation and cell apoptosis in cells with a miR-423-3p inhibitor and an NC inhibitor. The reverse transcription-quantitative polymerase chain reaction was performed to examine the miR-423-3p expression levels in (A) U251 and (B) U87MG Uppsala cells transfected with either miR-423-3p inhibitor or NC inhibitor. An MTT assay was used to determine proliferation in (C) U251 and (D) U87MG Uppsala cells. Flow cytometry was used to detect apoptosis in (E) U251 cells and (F) U87MG Uppsala cells. **P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, MTT Assay, Flow Cytometry, Cytometry

    Related Articles

    other:

    Article Title: Characterizing the Indoor-Outdoor Relationship of Fine Particulate Matter in Non-Heating Season for Urban Residences in Beijing
    Article Snippet: TEOM 1405F (Thermo Fisher Scientific Inc., MA, US) are employed at each EQM station to measure ambient PM2.5 mass concentrations.

    Article Title: Estimating PM2.5 Concentrations Based on MODIS AOD and NAQPMS Data over Beijing–Tianjin–Hebei
    Article Snippet: PM2.5 was mainly monitored using a ThermoFisher TEOM 1405F.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Thermo Fisher u251
    Projection electrophoresis permits the simultaneous analysis of hundreds of single cells by concurrent separation after simultaneous lysis. ( a ) Maximum intensity projection 3D renderings of example separation lanes read out by tiled light-sheet microscopy. ( b ) xz ( y -summed) contour plots of background-subtracted actinin and GAPDH protein signal within the lanes depicted in ( a ). ( c ) Revolved z -intensity profiles (arbitrary fluorescence units, AFU, vs. z ) for the four separation lanes depicted in ( a , b ). Each plot depicts z -directional intensity profiles (summed fluorescence within each xy ROI vs. z ) for GAPDH (magenta) and actinin (cyan), revolved around the z -axis to generate 3D rendering of fluorescence distribution. ( d ) Histogram quantification shows 86% of <t>U251</t> cells lyse within 5 s of initiating lysis. ( e ) Quantified fluorescence intensity data for n = 159 separation lanes passing quality control for both the GAPDH and actinin channels. Each plot depicts revolved z -intensity profiles. ( f ) Full-gel wide-field fluorescence image of calcein-stained live BT474 breast tumor cells before analysis. ( g ) Subset of the live cells from ( f ), within a 1.75 × 1.75 mm light-sheet microscopy field of view (scale bar depicts 200 μm). ( h ) Post-separation wide-field fluorescence image of probed GAPDH signal within the same field of view as ( g ). ( f – h ) are representative of n > 3 separation gels. ( i ) Maximum intensity projection 3D rendering (representative of duplicate separation gels) of a light-sheet microscopy image (same field of view as ( g , h )), showing 3D separations of GAPDH and actinin from tens of separation lanes, each corresponding to signal from the settled cells in microwells depicted in ( f , g ). ( j ) Overlay image of segmented spots corresponding to live BT474 cells in microwells (green) and probed GAPDH bands after separation (magenta), for the same separation gel depicted in ( f , i ). ( k ) Quantification of correspondence between the segmented live cells and bands (via intensity thresholding) within the same separation lanes as ( j ). ( l ) Quantified migration distances from a total of n = 507 (GAPDH) and n = 303 (actinin) lanes passing quality control in two projection electrophoresis gels. ( m ) Quantified z -direction peak widths for the same bands analyzed in ( l ). (n-o) Map of the variation in GAPDH ( n ) and actinin ( o ) electromigration distances across the xy gel area. Source data are provided as a Source Data file.
    U251, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u251/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u251 - by Bioz Stars, 2021-05
    97/100 stars
      Buy from Supplier

    99
    Thermo Fisher u251 cells
    PDGFRB is a direct target gene of miR-518b. (A) Inverse relationship between the expression of miR-518b and PDGFRB was detected in GBM specimens (magnification, 200x). (B) Western blot analysis of PDGFRB expression in U87 and <t>U251</t> cells 48 h post-transfection with miR-518b and miR-ctrl. (C) PDGFRB mRNA 3′UTR contains binding sites for miR-518b. (D) U87 and U251 cells were co-transfected with the dual-luciferase reporter plasmid carrying the Wt or Mut 3′UTR sequences of PDGFRB and miR-518b or miR-ctrl mimics. A luciferase reporter system analysis was performed. Data are presented as the mean ± standard deviation (n=5). *P
    U251 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u251 cells/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u251 cells - by Bioz Stars, 2021-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    Projection electrophoresis permits the simultaneous analysis of hundreds of single cells by concurrent separation after simultaneous lysis. ( a ) Maximum intensity projection 3D renderings of example separation lanes read out by tiled light-sheet microscopy. ( b ) xz ( y -summed) contour plots of background-subtracted actinin and GAPDH protein signal within the lanes depicted in ( a ). ( c ) Revolved z -intensity profiles (arbitrary fluorescence units, AFU, vs. z ) for the four separation lanes depicted in ( a , b ). Each plot depicts z -directional intensity profiles (summed fluorescence within each xy ROI vs. z ) for GAPDH (magenta) and actinin (cyan), revolved around the z -axis to generate 3D rendering of fluorescence distribution. ( d ) Histogram quantification shows 86% of U251 cells lyse within 5 s of initiating lysis. ( e ) Quantified fluorescence intensity data for n = 159 separation lanes passing quality control for both the GAPDH and actinin channels. Each plot depicts revolved z -intensity profiles. ( f ) Full-gel wide-field fluorescence image of calcein-stained live BT474 breast tumor cells before analysis. ( g ) Subset of the live cells from ( f ), within a 1.75 × 1.75 mm light-sheet microscopy field of view (scale bar depicts 200 μm). ( h ) Post-separation wide-field fluorescence image of probed GAPDH signal within the same field of view as ( g ). ( f – h ) are representative of n > 3 separation gels. ( i ) Maximum intensity projection 3D rendering (representative of duplicate separation gels) of a light-sheet microscopy image (same field of view as ( g , h )), showing 3D separations of GAPDH and actinin from tens of separation lanes, each corresponding to signal from the settled cells in microwells depicted in ( f , g ). ( j ) Overlay image of segmented spots corresponding to live BT474 cells in microwells (green) and probed GAPDH bands after separation (magenta), for the same separation gel depicted in ( f , i ). ( k ) Quantification of correspondence between the segmented live cells and bands (via intensity thresholding) within the same separation lanes as ( j ). ( l ) Quantified migration distances from a total of n = 507 (GAPDH) and n = 303 (actinin) lanes passing quality control in two projection electrophoresis gels. ( m ) Quantified z -direction peak widths for the same bands analyzed in ( l ). (n-o) Map of the variation in GAPDH ( n ) and actinin ( o ) electromigration distances across the xy gel area. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: 3D projection electrophoresis for single-cell immunoblotting

    doi: 10.1038/s41467-020-19738-1

    Figure Lengend Snippet: Projection electrophoresis permits the simultaneous analysis of hundreds of single cells by concurrent separation after simultaneous lysis. ( a ) Maximum intensity projection 3D renderings of example separation lanes read out by tiled light-sheet microscopy. ( b ) xz ( y -summed) contour plots of background-subtracted actinin and GAPDH protein signal within the lanes depicted in ( a ). ( c ) Revolved z -intensity profiles (arbitrary fluorescence units, AFU, vs. z ) for the four separation lanes depicted in ( a , b ). Each plot depicts z -directional intensity profiles (summed fluorescence within each xy ROI vs. z ) for GAPDH (magenta) and actinin (cyan), revolved around the z -axis to generate 3D rendering of fluorescence distribution. ( d ) Histogram quantification shows 86% of U251 cells lyse within 5 s of initiating lysis. ( e ) Quantified fluorescence intensity data for n = 159 separation lanes passing quality control for both the GAPDH and actinin channels. Each plot depicts revolved z -intensity profiles. ( f ) Full-gel wide-field fluorescence image of calcein-stained live BT474 breast tumor cells before analysis. ( g ) Subset of the live cells from ( f ), within a 1.75 × 1.75 mm light-sheet microscopy field of view (scale bar depicts 200 μm). ( h ) Post-separation wide-field fluorescence image of probed GAPDH signal within the same field of view as ( g ). ( f – h ) are representative of n > 3 separation gels. ( i ) Maximum intensity projection 3D rendering (representative of duplicate separation gels) of a light-sheet microscopy image (same field of view as ( g , h )), showing 3D separations of GAPDH and actinin from tens of separation lanes, each corresponding to signal from the settled cells in microwells depicted in ( f , g ). ( j ) Overlay image of segmented spots corresponding to live BT474 cells in microwells (green) and probed GAPDH bands after separation (magenta), for the same separation gel depicted in ( f , i ). ( k ) Quantification of correspondence between the segmented live cells and bands (via intensity thresholding) within the same separation lanes as ( j ). ( l ) Quantified migration distances from a total of n = 507 (GAPDH) and n = 303 (actinin) lanes passing quality control in two projection electrophoresis gels. ( m ) Quantified z -direction peak widths for the same bands analyzed in ( l ). (n-o) Map of the variation in GAPDH ( n ) and actinin ( o ) electromigration distances across the xy gel area. Source data are provided as a Source Data file.

    Article Snippet: Single-cell separations Adherent U251 glioblastoma and BT474 breast tumor cells were detached from culture flasks with 0.05% Trypsin-EDTA (Life Technologies 25300120) (U251) or 5 mM EDTA (Invitrogen 15575-038) in PBS (BT474) and resuspended in cold PBS (Life Technologies 10010049) at a concentration of 1.5 × 106 cells/mL.

    Techniques: Electrophoresis, Lysis, Microscopy, Fluorescence, Staining, Migration

    Design and verification of sample preparation for projection electrophoresis of single mammalian cells. ( a ) High-density endogenous protein bands (ii) correspond to single-cell settling in microwells (i). Scale bars represent 1 mm (left full-gel images) and 200 μm (right zoom images). ( b ) Illustration of top-view and side-view geometries shown in protein dilution studies ( c , d ). ( c ) Modeling and experimental quantification of diffusional dilution during lysis. Simulated and experimental top-view images of diffusional protein dilution during lysis, and side-view simulated results are shown. The simulated initial TurboGFP concentration was 2 μM. Experimental image is representative of 12 monitored cells across 3 independent lysis experiments. Scale bars represent 50 μm. ( d ) Modeling the impact of diffusion during electrophoresis on detectable in-gel protein concentration. Side and top view TurboGFP concentration profiles are shown at different times during electrophoresis. Simulated initial TurboGFP concentration (before lysis and electrophoresis) was 2 μM. Scale bars represent 30 μm. ( e ) Quantification of the percent of protein remaining in the microwell region during lysis (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). ( f ) Quantification of the change in the spatial maximum protein concentration as a function of time after protein solubilization (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). ( g ) Simulated maximum protein concentration vs. electrophoresis time, for 3 model proteins. ( h ) Representative β-tubulin separations from U251 glioblastoma cells lysed with different buffer formulations (2× RIPA lysis buffer and 2× RIPA including 8 M urea), both after 10 s electrophoresis. Lysis/EP buffer requires 8 M urea for fast protein solubilization and electromigration. ( i ) Maximum intensity projection 3D renderings and z -intensity profiles of probed GAPDH bands from single BT474 breast tumor cells. ( j ) Microwell packing density (impacting assay throughput) is dependent on protein band diffusion before photocapture. Protein diffusion profiles confirm that a microwell pitch of 200 μm is sufficient to resolve bands from neighboring microwells. After 10 s EP, the mean peak width ( σ xy ) of the xy GAPDH spots is 32 ± 11 μm (mean ± standard deviation of n = 47 single-cell separation lanes across 5 replicate separation gels). The depicted confocal slice micrograph is representative of 20 confocal image stacks, across different regions of 5 replicate separation gels. At a microwell pitch of 192 μm (6 σ xy ),

    Journal: Nature Communications

    Article Title: 3D projection electrophoresis for single-cell immunoblotting

    doi: 10.1038/s41467-020-19738-1

    Figure Lengend Snippet: Design and verification of sample preparation for projection electrophoresis of single mammalian cells. ( a ) High-density endogenous protein bands (ii) correspond to single-cell settling in microwells (i). Scale bars represent 1 mm (left full-gel images) and 200 μm (right zoom images). ( b ) Illustration of top-view and side-view geometries shown in protein dilution studies ( c , d ). ( c ) Modeling and experimental quantification of diffusional dilution during lysis. Simulated and experimental top-view images of diffusional protein dilution during lysis, and side-view simulated results are shown. The simulated initial TurboGFP concentration was 2 μM. Experimental image is representative of 12 monitored cells across 3 independent lysis experiments. Scale bars represent 50 μm. ( d ) Modeling the impact of diffusion during electrophoresis on detectable in-gel protein concentration. Side and top view TurboGFP concentration profiles are shown at different times during electrophoresis. Simulated initial TurboGFP concentration (before lysis and electrophoresis) was 2 μM. Scale bars represent 30 μm. ( e ) Quantification of the percent of protein remaining in the microwell region during lysis (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). ( f ) Quantification of the change in the spatial maximum protein concentration as a function of time after protein solubilization (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). ( g ) Simulated maximum protein concentration vs. electrophoresis time, for 3 model proteins. ( h ) Representative β-tubulin separations from U251 glioblastoma cells lysed with different buffer formulations (2× RIPA lysis buffer and 2× RIPA including 8 M urea), both after 10 s electrophoresis. Lysis/EP buffer requires 8 M urea for fast protein solubilization and electromigration. ( i ) Maximum intensity projection 3D renderings and z -intensity profiles of probed GAPDH bands from single BT474 breast tumor cells. ( j ) Microwell packing density (impacting assay throughput) is dependent on protein band diffusion before photocapture. Protein diffusion profiles confirm that a microwell pitch of 200 μm is sufficient to resolve bands from neighboring microwells. After 10 s EP, the mean peak width ( σ xy ) of the xy GAPDH spots is 32 ± 11 μm (mean ± standard deviation of n = 47 single-cell separation lanes across 5 replicate separation gels). The depicted confocal slice micrograph is representative of 20 confocal image stacks, across different regions of 5 replicate separation gels. At a microwell pitch of 192 μm (6 σ xy ),

    Article Snippet: Single-cell separations Adherent U251 glioblastoma and BT474 breast tumor cells were detached from culture flasks with 0.05% Trypsin-EDTA (Life Technologies 25300120) (U251) or 5 mM EDTA (Invitrogen 15575-038) in PBS (BT474) and resuspended in cold PBS (Life Technologies 10010049) at a concentration of 1.5 × 106 cells/mL.

    Techniques: Sample Prep, Electrophoresis, Lysis, Concentration Assay, Diffusion-based Assay, Protein Concentration, Standard Deviation

    PDGFRB is a direct target gene of miR-518b. (A) Inverse relationship between the expression of miR-518b and PDGFRB was detected in GBM specimens (magnification, 200x). (B) Western blot analysis of PDGFRB expression in U87 and U251 cells 48 h post-transfection with miR-518b and miR-ctrl. (C) PDGFRB mRNA 3′UTR contains binding sites for miR-518b. (D) U87 and U251 cells were co-transfected with the dual-luciferase reporter plasmid carrying the Wt or Mut 3′UTR sequences of PDGFRB and miR-518b or miR-ctrl mimics. A luciferase reporter system analysis was performed. Data are presented as the mean ± standard deviation (n=5). *P

    Journal: Molecular Medicine Reports

    Article Title: MicroRNA-518b functions as a tumor suppressor in glioblastoma by targeting PDGFRB

    doi: 10.3892/mmr.2017.7298

    Figure Lengend Snippet: PDGFRB is a direct target gene of miR-518b. (A) Inverse relationship between the expression of miR-518b and PDGFRB was detected in GBM specimens (magnification, 200x). (B) Western blot analysis of PDGFRB expression in U87 and U251 cells 48 h post-transfection with miR-518b and miR-ctrl. (C) PDGFRB mRNA 3′UTR contains binding sites for miR-518b. (D) U87 and U251 cells were co-transfected with the dual-luciferase reporter plasmid carrying the Wt or Mut 3′UTR sequences of PDGFRB and miR-518b or miR-ctrl mimics. A luciferase reporter system analysis was performed. Data are presented as the mean ± standard deviation (n=5). *P

    Article Snippet: Dual-luciferase reporter assay For the luciferase assay, U87 and U251 cells were grown to 70–80% confluence in 24-well plates and were co-transfected with miR-518b/miR-ctrl and wild type/mutant recombinant luciferase reporter plasmids using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

    Techniques: Expressing, Western Blot, Transfection, Binding Assay, Luciferase, Plasmid Preparation, Standard Deviation

    Effects of miR-518b on cell proliferation, apoptosis and angiogenesis. U87 and U251 cells were transfected with miR-518b or miR-ctrl mimics for 48 h. (A) miR-518b overexpression in U87 and U251 cells was validated by quantitative polymerase chain reaction. (B) Cell Counting Kit-8 assays were performed to evaluate cell proliferation at the indicated time-points. (C) Proportion of cells at each stage of the cell cycle. (D) The rate of apoptosis was detected by flow cytometry. (E and F) Capillary tube formation ability was determined by calculating the branch points of the newly formed tubes. Data are presented as the mean ± standard deviation (n=5). *P

    Journal: Molecular Medicine Reports

    Article Title: MicroRNA-518b functions as a tumor suppressor in glioblastoma by targeting PDGFRB

    doi: 10.3892/mmr.2017.7298

    Figure Lengend Snippet: Effects of miR-518b on cell proliferation, apoptosis and angiogenesis. U87 and U251 cells were transfected with miR-518b or miR-ctrl mimics for 48 h. (A) miR-518b overexpression in U87 and U251 cells was validated by quantitative polymerase chain reaction. (B) Cell Counting Kit-8 assays were performed to evaluate cell proliferation at the indicated time-points. (C) Proportion of cells at each stage of the cell cycle. (D) The rate of apoptosis was detected by flow cytometry. (E and F) Capillary tube formation ability was determined by calculating the branch points of the newly formed tubes. Data are presented as the mean ± standard deviation (n=5). *P

    Article Snippet: Dual-luciferase reporter assay For the luciferase assay, U87 and U251 cells were grown to 70–80% confluence in 24-well plates and were co-transfected with miR-518b/miR-ctrl and wild type/mutant recombinant luciferase reporter plasmids using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

    Techniques: Transfection, Over Expression, Real-time Polymerase Chain Reaction, Cell Counting, Flow Cytometry, Cytometry, Standard Deviation