u251 cells  (Thermo Fisher)


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    Matrix TallTip Extended Length Pipette Tips
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    Maximize reach with Thermo Scientific Matrix TallTip Pipette Tips which allow access to the bottom of test tubes reagent bottles flasks and other vessels without touching the barrel of the pipetter against the side of the tube Nonbeveled narrow tip ends provide cleaner touch off of sidewalls for greater precision
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    Structured Review

    Thermo Fisher u251 cells
    KLHDC8A regulates glioma cell proliferation, migration and invasion. A and B, KLHDC8A knockdown inhibits glioma cells proliferation. U87MG and <t>U251</t> cells were transfected with NC and KLHDC8A siRNA for 24 h. Then, 8000 transfected cells per well were incubated in E‐plate and the cell index was detected by using RTCA xCELLigence at 1‐h intervals for 100 h. C, KLHDC8A knockdown inhibits the colony formation of glioma cells. U87MG and U251 cells were transfected with NC and KLHDC8A siRNA for 24 h. Then, 2000 cells per well were incubated in 6‐well plate for 2 wk. Colony was captured after paraformaldehyde fixation and crystal violet staining. D and E, Transwell assays showed that the migration of U87MG and U251 cells was significantly reduced after KLHDC8A knockdown. F and G, Transwell assays showed that the invasion of U87MG and U251 cells was significantly reduced after KLHDC8A knockdown. H, Wound‐healing assays showed that migration of glioma cells was significantly reduced after KLHDC8A knockdown. Data are mean ± SD from three independent experiments. *** P
    Maximize reach with Thermo Scientific Matrix TallTip Pipette Tips which allow access to the bottom of test tubes reagent bottles flasks and other vessels without touching the barrel of the pipetter against the side of the tube Nonbeveled narrow tip ends provide cleaner touch off of sidewalls for greater precision
    https://www.bioz.com/result/u251 cells/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
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    u251 cells - by Bioz Stars, 2021-06
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    1) Product Images from "Lactate induced up‐regulation of KLHDC8A (Kelch domain‐containing 8A) contributes to the proliferation, migration and apoptosis of human glioma cells. Lactate induced up‐regulation of KLHDC8A (Kelch domain‐containing 8A) contributes to the proliferation, migration and apoptosis of human glioma cells"

    Article Title: Lactate induced up‐regulation of KLHDC8A (Kelch domain‐containing 8A) contributes to the proliferation, migration and apoptosis of human glioma cells. Lactate induced up‐regulation of KLHDC8A (Kelch domain‐containing 8A) contributes to the proliferation, migration and apoptosis of human glioma cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15780

    KLHDC8A regulates glioma cell proliferation, migration and invasion. A and B, KLHDC8A knockdown inhibits glioma cells proliferation. U87MG and U251 cells were transfected with NC and KLHDC8A siRNA for 24 h. Then, 8000 transfected cells per well were incubated in E‐plate and the cell index was detected by using RTCA xCELLigence at 1‐h intervals for 100 h. C, KLHDC8A knockdown inhibits the colony formation of glioma cells. U87MG and U251 cells were transfected with NC and KLHDC8A siRNA for 24 h. Then, 2000 cells per well were incubated in 6‐well plate for 2 wk. Colony was captured after paraformaldehyde fixation and crystal violet staining. D and E, Transwell assays showed that the migration of U87MG and U251 cells was significantly reduced after KLHDC8A knockdown. F and G, Transwell assays showed that the invasion of U87MG and U251 cells was significantly reduced after KLHDC8A knockdown. H, Wound‐healing assays showed that migration of glioma cells was significantly reduced after KLHDC8A knockdown. Data are mean ± SD from three independent experiments. *** P
    Figure Legend Snippet: KLHDC8A regulates glioma cell proliferation, migration and invasion. A and B, KLHDC8A knockdown inhibits glioma cells proliferation. U87MG and U251 cells were transfected with NC and KLHDC8A siRNA for 24 h. Then, 8000 transfected cells per well were incubated in E‐plate and the cell index was detected by using RTCA xCELLigence at 1‐h intervals for 100 h. C, KLHDC8A knockdown inhibits the colony formation of glioma cells. U87MG and U251 cells were transfected with NC and KLHDC8A siRNA for 24 h. Then, 2000 cells per well were incubated in 6‐well plate for 2 wk. Colony was captured after paraformaldehyde fixation and crystal violet staining. D and E, Transwell assays showed that the migration of U87MG and U251 cells was significantly reduced after KLHDC8A knockdown. F and G, Transwell assays showed that the invasion of U87MG and U251 cells was significantly reduced after KLHDC8A knockdown. H, Wound‐healing assays showed that migration of glioma cells was significantly reduced after KLHDC8A knockdown. Data are mean ± SD from three independent experiments. *** P

    Techniques Used: Migration, Transfection, Incubation, Staining

    KLHDC8A regulates glioma cell cycle and apoptosis. A and B, KLHDC8A knockdown induces the G0/G1 phase arrest of glioma cells. The U87MG and U251 cells which transfected with NC and KLHDC8A siRNA for 48 h were fixed with 75% ethanol at 4°C overnight before incubated with RNase A and propidium iodide (PI) for 30 min at 37°C. Then, the DNA contents were detected by flow cytometer. The cell cycle was analysed by using FlowJo software. Histograms show the percentage (%) of cell populations at different stages of the cell cycle. C and D, KLHDC8A knockdown induces apoptosis in glioma cells. The U87MG and U251 cells which transfected with NC and KLHDC8A siRNA for 48 h were incubated with annexin V‐FITC and propidium iodide (PI) for 15 min at 4°C. Then, the cell apoptosis was detected by flow cytometer. The cell apoptosis were analysed by using FlowJo software. Histograms show the percentage (%) of cell apoptosis. Data are mean ± SD from three independent experiments. * P
    Figure Legend Snippet: KLHDC8A regulates glioma cell cycle and apoptosis. A and B, KLHDC8A knockdown induces the G0/G1 phase arrest of glioma cells. The U87MG and U251 cells which transfected with NC and KLHDC8A siRNA for 48 h were fixed with 75% ethanol at 4°C overnight before incubated with RNase A and propidium iodide (PI) for 30 min at 37°C. Then, the DNA contents were detected by flow cytometer. The cell cycle was analysed by using FlowJo software. Histograms show the percentage (%) of cell populations at different stages of the cell cycle. C and D, KLHDC8A knockdown induces apoptosis in glioma cells. The U87MG and U251 cells which transfected with NC and KLHDC8A siRNA for 48 h were incubated with annexin V‐FITC and propidium iodide (PI) for 15 min at 4°C. Then, the cell apoptosis was detected by flow cytometer. The cell apoptosis were analysed by using FlowJo software. Histograms show the percentage (%) of cell apoptosis. Data are mean ± SD from three independent experiments. * P

    Techniques Used: Transfection, Incubation, Flow Cytometry, Software

    The genes regulated by KLHDC8A in glioma cells. A, Proteins related to apoptosis (BAX and Bcl2), cell cycle (p21 and CDK2) and migration (MMP2) were detected in U87MG and U251 cells after KLHDC8A knockdown. The U87MG and U251 cells which transfected with NC and KLHDC8A siRNA for 48 h were harvested. Then, Western blot detected the expression of BAX, Bcl2, p21, CDK2 and MMP2 in U251 cells. B, The levels of MAPK signalling proteins (p‐ERK, p‐P38 MAPK and p‐JNK) in KLHDC8A knockdown cells were detected. Numbers on up of bands are fold change of level relative to corresponding negative control. β‐Actin was used as loading control. NC, negative control; siRNA, KLHDC8A gene silencer
    Figure Legend Snippet: The genes regulated by KLHDC8A in glioma cells. A, Proteins related to apoptosis (BAX and Bcl2), cell cycle (p21 and CDK2) and migration (MMP2) were detected in U87MG and U251 cells after KLHDC8A knockdown. The U87MG and U251 cells which transfected with NC and KLHDC8A siRNA for 48 h were harvested. Then, Western blot detected the expression of BAX, Bcl2, p21, CDK2 and MMP2 in U251 cells. B, The levels of MAPK signalling proteins (p‐ERK, p‐P38 MAPK and p‐JNK) in KLHDC8A knockdown cells were detected. Numbers on up of bands are fold change of level relative to corresponding negative control. β‐Actin was used as loading control. NC, negative control; siRNA, KLHDC8A gene silencer

    Techniques Used: Migration, Transfection, Western Blot, Expressing, Negative Control

    Lactate regulates KLHDC8A expression in glioma cells. A, KLHDC8A expression was increased in glioma cells following lactate (0, 10, 20 and 40 mmol/L) treatment for 3 h. RT‐PCR detected the mRNA expression of KLHDC8A in U87MG and U251. B, KLHDC8A expression was increased in glioma cells following glucose (0‐4.5 mg/mL) treatment for 3 h. RT‐PCR detected the mRNA expression of KLHDC8A in U87MG and U251. C, Lactate was accumulated following with glucose treatment. U87MG and U251 cells were stimulated with 0‐3.6 mg/mL glucose for 1 h and lactate was detected. D, LDHA knockdown decreased the expression of KLHDC8A which stimulated by glucose. U251 cells were transfected with NC and LDHA siRNA for 48 h. Then, cells were treated with 0 and 3.6 mg/mL glucose for 3 h before RT‐PCR. Data are mean ± SD from three independent experiments. * P
    Figure Legend Snippet: Lactate regulates KLHDC8A expression in glioma cells. A, KLHDC8A expression was increased in glioma cells following lactate (0, 10, 20 and 40 mmol/L) treatment for 3 h. RT‐PCR detected the mRNA expression of KLHDC8A in U87MG and U251. B, KLHDC8A expression was increased in glioma cells following glucose (0‐4.5 mg/mL) treatment for 3 h. RT‐PCR detected the mRNA expression of KLHDC8A in U87MG and U251. C, Lactate was accumulated following with glucose treatment. U87MG and U251 cells were stimulated with 0‐3.6 mg/mL glucose for 1 h and lactate was detected. D, LDHA knockdown decreased the expression of KLHDC8A which stimulated by glucose. U251 cells were transfected with NC and LDHA siRNA for 48 h. Then, cells were treated with 0 and 3.6 mg/mL glucose for 3 h before RT‐PCR. Data are mean ± SD from three independent experiments. * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

    KLHDC8A overexpression regulates proliferation, migration, invasion and apoptosis, and activates ERK and p38 MAPK signalling. A, KLHDC8A overexpression promotes glioma cells proliferation. The RTCA assay was used to measure U251 proliferation ability. B and C, Transwell using RTCA revealed that KLHDC8A overexpression promotes the migration and invasion of glioma cells. D, Cell apoptosis induced by starvation in U251 were detected using flow cytometry. Histograms show the percentage (%) of cell apoptosis. E, Apoptosis‐ and migration‐related proteins were detected in U251 transfected with the NC or KLHDC8A plasmid. F, U251 were pretreated with KLHDC8A plasmid and the ERK inhibitor U0126 (10 μmol/L) for 24 h Then, Western blot examined for the protein levels. G, U251 were pretreated with KLHDC8A plasmid and the p38 MAPK inhibitor SB203580 (10 μmol/L) for 24 h Then, Western blot examined for the protein levels. Numbers on up of bands are fold change of level relative to corresponding negative control. Data are mean ± SD from three independent experiments. * P
    Figure Legend Snippet: KLHDC8A overexpression regulates proliferation, migration, invasion and apoptosis, and activates ERK and p38 MAPK signalling. A, KLHDC8A overexpression promotes glioma cells proliferation. The RTCA assay was used to measure U251 proliferation ability. B and C, Transwell using RTCA revealed that KLHDC8A overexpression promotes the migration and invasion of glioma cells. D, Cell apoptosis induced by starvation in U251 were detected using flow cytometry. Histograms show the percentage (%) of cell apoptosis. E, Apoptosis‐ and migration‐related proteins were detected in U251 transfected with the NC or KLHDC8A plasmid. F, U251 were pretreated with KLHDC8A plasmid and the ERK inhibitor U0126 (10 μmol/L) for 24 h Then, Western blot examined for the protein levels. G, U251 were pretreated with KLHDC8A plasmid and the p38 MAPK inhibitor SB203580 (10 μmol/L) for 24 h Then, Western blot examined for the protein levels. Numbers on up of bands are fold change of level relative to corresponding negative control. Data are mean ± SD from three independent experiments. * P

    Techniques Used: Over Expression, Migration, Flow Cytometry, Transfection, Plasmid Preparation, Western Blot, Negative Control

    2) Product Images from "NR2C2-uORF targeting UCA1-miR-627-5p-NR2C2 feedback loop to regulate the malignant behaviors of glioma cells"

    Article Title: NR2C2-uORF targeting UCA1-miR-627-5p-NR2C2 feedback loop to regulate the malignant behaviors of glioma cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1149-x

    MiR-627-5p mediated the tumor-suppressive effects of UCA1 knockdown on glioma cell lines. a Cell Counting Kit-8 (CCK-8) assay was used to measure the effect of UCA1 and miR-627-5p on cell proliferation in U87 and U251 cells. b Transwell assay was used to evaluate the effect of UCA1 and miR-627-5p on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis was used to evaluate the effect of effect of UCA1 and miR-627-5p on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P
    Figure Legend Snippet: MiR-627-5p mediated the tumor-suppressive effects of UCA1 knockdown on glioma cell lines. a Cell Counting Kit-8 (CCK-8) assay was used to measure the effect of UCA1 and miR-627-5p on cell proliferation in U87 and U251 cells. b Transwell assay was used to evaluate the effect of UCA1 and miR-627-5p on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis was used to evaluate the effect of effect of UCA1 and miR-627-5p on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P

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

    Interplay of NR2C2 and miR-627-5p. a Cell Counting Kit-8(CCK8) assay to evaluate the effect of miR-627-5p and NR2C2 on cell proliferation in U87 and U251 cells. b Transwell assay to evaluate the effect of miR-627-5p and NR2C2 on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis to evaluate the effect of miR-627-5p and NR2C2 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P
    Figure Legend Snippet: Interplay of NR2C2 and miR-627-5p. a Cell Counting Kit-8(CCK8) assay to evaluate the effect of miR-627-5p and NR2C2 on cell proliferation in U87 and U251 cells. b Transwell assay to evaluate the effect of miR-627-5p and NR2C2 on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis to evaluate the effect of miR-627-5p and NR2C2 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P

    Techniques Used: Cell Counting, Transwell Assay, Migration, Flow Cytometry, Cytometry

    Identification of NR2C2-binding sites on SPOCK1 and UCA1 promoters. a Western blot analysis for SPOCK1 in U87 and U251 cells with the expression of NR2C2 changed. Error bars represent as the mean ± SD ( n = 3, each group). ** P
    Figure Legend Snippet: Identification of NR2C2-binding sites on SPOCK1 and UCA1 promoters. a Western blot analysis for SPOCK1 in U87 and U251 cells with the expression of NR2C2 changed. Error bars represent as the mean ± SD ( n = 3, each group). ** P

    Techniques Used: Binding Assay, Western Blot, Expressing

    3) Product Images from "RNA interference-mediated knockdown of translationally controlled tumor protein induces apoptosis, and inhibits growth and invasion in glioma cells"

    Article Title: RNA interference-mediated knockdown of translationally controlled tumor protein induces apoptosis, and inhibits growth and invasion in glioma cells

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2015.4280

    Establishment of a glioma cell line stably transfected with TCTP shRNA. (A) Expression levels of TCTP in the U373, U251, A172, U87-MG and SHG-44 glioma cell lines were compared using western blot analysis, with β-actin as the internal control. Each experiment was repeated three times. ** P
    Figure Legend Snippet: Establishment of a glioma cell line stably transfected with TCTP shRNA. (A) Expression levels of TCTP in the U373, U251, A172, U87-MG and SHG-44 glioma cell lines were compared using western blot analysis, with β-actin as the internal control. Each experiment was repeated three times. ** P

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

    4) Product Images from "Coiled-coil domain containing 109B is a HIF1α-regulated gene critical for progression of human gliomas"

    Article Title: Coiled-coil domain containing 109B is a HIF1α-regulated gene critical for progression of human gliomas

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-017-1266-9

    CCDC109B expression is induced by hypoxia and regulated by HIF1α. a Representative IHC images of CCDC109B and HIF1α in primary human GBM tissues. Magnification ×100 and ×200. b Analysis of HIF1α and CCDC109B expression in human GBM tissues by IHC staining. Magnification ×200 and ×400. Representative images were labeled as case 1 and case 2. c Western blot analysis of HIF1α in U87MG, T98, U251 and NHA. d qRT-PCR were used to determine mRNA levels of CCDC109B in U87MG or U251 cells cultured under normoxic or hypoxic conditions. e Western blot analysis for HIF1α and CCDC109B protein levels in U87MG and U251 cells cultured under hypoxia for the indicated time. f Western blot analysis for HIF1α and CCDC109B in U87MG and U251 cells transfected with NC, si-HIF1α-1 or si-HIF1α-2 under normoxic or hypoxic conditions for 48 h. g Western blot analysis for HIF1α and CCDC109B in U87MG and U251 cells treated with PX478 (0, 50, 75, 100 μM) and cultured under normoxic or hypoxic conditions for 48 h. h Representative images of implanted nude mice injected intraperitoneally with saline or digoxin (2 mg/kg) every day for one month. Images for corresponding subcutaneous U87MG xenografts after surgical removal are also shown. i Western blot analysis to determine levels of HIF1α and CCDC109B in tumors from nude mice treated with saline or digoxin. Data are presented as the mean ± SEM (*** P
    Figure Legend Snippet: CCDC109B expression is induced by hypoxia and regulated by HIF1α. a Representative IHC images of CCDC109B and HIF1α in primary human GBM tissues. Magnification ×100 and ×200. b Analysis of HIF1α and CCDC109B expression in human GBM tissues by IHC staining. Magnification ×200 and ×400. Representative images were labeled as case 1 and case 2. c Western blot analysis of HIF1α in U87MG, T98, U251 and NHA. d qRT-PCR were used to determine mRNA levels of CCDC109B in U87MG or U251 cells cultured under normoxic or hypoxic conditions. e Western blot analysis for HIF1α and CCDC109B protein levels in U87MG and U251 cells cultured under hypoxia for the indicated time. f Western blot analysis for HIF1α and CCDC109B in U87MG and U251 cells transfected with NC, si-HIF1α-1 or si-HIF1α-2 under normoxic or hypoxic conditions for 48 h. g Western blot analysis for HIF1α and CCDC109B in U87MG and U251 cells treated with PX478 (0, 50, 75, 100 μM) and cultured under normoxic or hypoxic conditions for 48 h. h Representative images of implanted nude mice injected intraperitoneally with saline or digoxin (2 mg/kg) every day for one month. Images for corresponding subcutaneous U87MG xenografts after surgical removal are also shown. i Western blot analysis to determine levels of HIF1α and CCDC109B in tumors from nude mice treated with saline or digoxin. Data are presented as the mean ± SEM (*** P

    Techniques Used: Expressing, Immunohistochemistry, Staining, Labeling, Western Blot, Quantitative RT-PCR, Cell Culture, Transfection, Mouse Assay, Injection

    CCDC109B knockdown inhibits hypoxia-induced migration and invasion of glioma cells. U87MG- and U251-NC or -sh-CCDC109B-1 cells were cultured under normoxic or hypoxic conditions for 48 h and the following assays were performed. a qRT-PCR to detect expression of CCDC109B expression; b Western blot analysis for CCDC109B and HIF1α in cell type indicated. c Representative images of Transwell migration and invasion assays performed on U87MG- and U251-NC and -sh-CCDC109B-1 cells. Cells were seeded into chambers and incubated under normoxia or hypoxia for 24 h. d Graphic representation of cell counts from Transwell assays. Migraded/invaded cells were counted and averaged for each experimental condition. Data are presented as the mean ± SEM (** P
    Figure Legend Snippet: CCDC109B knockdown inhibits hypoxia-induced migration and invasion of glioma cells. U87MG- and U251-NC or -sh-CCDC109B-1 cells were cultured under normoxic or hypoxic conditions for 48 h and the following assays were performed. a qRT-PCR to detect expression of CCDC109B expression; b Western blot analysis for CCDC109B and HIF1α in cell type indicated. c Representative images of Transwell migration and invasion assays performed on U87MG- and U251-NC and -sh-CCDC109B-1 cells. Cells were seeded into chambers and incubated under normoxia or hypoxia for 24 h. d Graphic representation of cell counts from Transwell assays. Migraded/invaded cells were counted and averaged for each experimental condition. Data are presented as the mean ± SEM (** P

    Techniques Used: Migration, Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, Incubation

    CCDC109B is highly expressed in high grade gliomas. a Images of immunofluorescence performed with an antibody against CCDC109B ( red ) in NHA, U87MG, U251, T98 cells and visualized using confocal microscopy. Nuclei were labeled with DAPI ( blue ). Scale bar 20 μm. b Western blot analysis of CCDC109B levels in U87MG, U251, T98 and NHA. c mRNA expression levels of CCDC109B as determined using TCGA, CGGA, GSE4271 and Rembrandt databases. d Representative images of IHC staining with anti-CCDC109B antibody on human glioma and non-neoplastic brain tissue samples. Magnification ×200, upper; ×400, lower. e Graphical representation of scoring performed on IHC staining of glioma and non-neoplastic tissue samples for CCDC109B levels. Bar graphs show the mean ± the standard error of the mean (SEM) for each group. f Western blot analysis of CCDC109B in lysates (20 µg) prepared from different grades of human gliomas (WHO grades II–IV) and normal brain tissues ( ns not significant, * P
    Figure Legend Snippet: CCDC109B is highly expressed in high grade gliomas. a Images of immunofluorescence performed with an antibody against CCDC109B ( red ) in NHA, U87MG, U251, T98 cells and visualized using confocal microscopy. Nuclei were labeled with DAPI ( blue ). Scale bar 20 μm. b Western blot analysis of CCDC109B levels in U87MG, U251, T98 and NHA. c mRNA expression levels of CCDC109B as determined using TCGA, CGGA, GSE4271 and Rembrandt databases. d Representative images of IHC staining with anti-CCDC109B antibody on human glioma and non-neoplastic brain tissue samples. Magnification ×200, upper; ×400, lower. e Graphical representation of scoring performed on IHC staining of glioma and non-neoplastic tissue samples for CCDC109B levels. Bar graphs show the mean ± the standard error of the mean (SEM) for each group. f Western blot analysis of CCDC109B in lysates (20 µg) prepared from different grades of human gliomas (WHO grades II–IV) and normal brain tissues ( ns not significant, * P

    Techniques Used: Immunofluorescence, Confocal Microscopy, Labeling, Western Blot, Expressing, Immunohistochemistry, Staining

    Knockdown of CCDC109B inhibits proliferation, migration, and invasion of glioma cells in vitro. Knockdown efficiency of CCDC109B in U87MG and U251 cells was determined in a by qRT-PCR and in b by western blot analysis; c EdU assays for U87MG- and U251-NC or sh-CCDC109B-1 cells. Magnification ×200. d Graphic representation of ratios of EdU positive cells in U87MG- and U251-NC and sh-CCDC109B-1 cells. Data are presented as the mean ± SEM. e Representative images of colony forming assays for U87MG- and U251-NC ( top ) or shCCDC109B-1 cells ( bottom ). f Graphic representation of colony forming results in U87MG- and U251-NC and -sh-CCDC109B-1. Data are presented as the mean ± SEM. g Images of Transwell migration and invasion assays performed with U87MG- and U251-NC and sh-CCDC109B-1 expressing cells. Magnification ×100. h Graphic representation of cell counts from Transwell assays after a 24 h incubation. Experiments were performed in triplicate and counted from 5 random fields. Data are presented as the mean ± SEM. i Western blot analysis for the expression of MMP2 and MMP9 in NC and sh-CCDC109B-1 U87MG and U251 glioma cell lines (* P
    Figure Legend Snippet: Knockdown of CCDC109B inhibits proliferation, migration, and invasion of glioma cells in vitro. Knockdown efficiency of CCDC109B in U87MG and U251 cells was determined in a by qRT-PCR and in b by western blot analysis; c EdU assays for U87MG- and U251-NC or sh-CCDC109B-1 cells. Magnification ×200. d Graphic representation of ratios of EdU positive cells in U87MG- and U251-NC and sh-CCDC109B-1 cells. Data are presented as the mean ± SEM. e Representative images of colony forming assays for U87MG- and U251-NC ( top ) or shCCDC109B-1 cells ( bottom ). f Graphic representation of colony forming results in U87MG- and U251-NC and -sh-CCDC109B-1. Data are presented as the mean ± SEM. g Images of Transwell migration and invasion assays performed with U87MG- and U251-NC and sh-CCDC109B-1 expressing cells. Magnification ×100. h Graphic representation of cell counts from Transwell assays after a 24 h incubation. Experiments were performed in triplicate and counted from 5 random fields. Data are presented as the mean ± SEM. i Western blot analysis for the expression of MMP2 and MMP9 in NC and sh-CCDC109B-1 U87MG and U251 glioma cell lines (* P

    Techniques Used: Migration, In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Incubation

    5) Product Images from "NR2C2-uORF targeting UCA1-miR-627-5p-NR2C2 feedback loop to regulate the malignant behaviors of glioma cells"

    Article Title: NR2C2-uORF targeting UCA1-miR-627-5p-NR2C2 feedback loop to regulate the malignant behaviors of glioma cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1149-x

    MiR-627-5p mediated the tumor-suppressive effects of UCA1 knockdown on glioma cell lines. a Cell Counting Kit-8 (CCK-8) assay was used to measure the effect of UCA1 and miR-627-5p on cell proliferation in U87 and U251 cells. b Transwell assay was used to evaluate the effect of UCA1 and miR-627-5p on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis was used to evaluate the effect of effect of UCA1 and miR-627-5p on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P
    Figure Legend Snippet: MiR-627-5p mediated the tumor-suppressive effects of UCA1 knockdown on glioma cell lines. a Cell Counting Kit-8 (CCK-8) assay was used to measure the effect of UCA1 and miR-627-5p on cell proliferation in U87 and U251 cells. b Transwell assay was used to evaluate the effect of UCA1 and miR-627-5p on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis was used to evaluate the effect of effect of UCA1 and miR-627-5p on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P

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

    Interplay of NR2C2 and miR-627-5p. a Cell Counting Kit-8(CCK8) assay to evaluate the effect of miR-627-5p and NR2C2 on cell proliferation in U87 and U251 cells. b Transwell assay to evaluate the effect of miR-627-5p and NR2C2 on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis to evaluate the effect of miR-627-5p and NR2C2 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P
    Figure Legend Snippet: Interplay of NR2C2 and miR-627-5p. a Cell Counting Kit-8(CCK8) assay to evaluate the effect of miR-627-5p and NR2C2 on cell proliferation in U87 and U251 cells. b Transwell assay to evaluate the effect of miR-627-5p and NR2C2 on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis to evaluate the effect of miR-627-5p and NR2C2 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P

    Techniques Used: Cell Counting, Transwell Assay, Migration, Flow Cytometry, Cytometry

    Identification of NR2C2-binding sites on SPOCK1 and UCA1 promoters. a Western blot analysis for SPOCK1 in U87 and U251 cells with the expression of NR2C2 changed. Error bars represent as the mean ± SD ( n = 3, each group). ** P
    Figure Legend Snippet: Identification of NR2C2-binding sites on SPOCK1 and UCA1 promoters. a Western blot analysis for SPOCK1 in U87 and U251 cells with the expression of NR2C2 changed. Error bars represent as the mean ± SD ( n = 3, each group). ** P

    Techniques Used: Binding Assay, Western Blot, Expressing

    6) Product Images from "Natural Diterpenoid Isoferritin A (IsoA) Inhibits Glioma Cell Growth and Metastasis via Regulating of TGFβ-Induced EMT Signal Pathway"

    Article Title: Natural Diterpenoid Isoferritin A (IsoA) Inhibits Glioma Cell Growth and Metastasis via Regulating of TGFβ-Induced EMT Signal Pathway

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.910102

    IsoA (4 mg/ml) treatment decreases TGFβ and regulates EMT markers in glioma cells. ( A, B ) IsoA treatment decreased TGFβ mRNA ( A ) and protein ( B ) expression levels in U251 cells. ( C, D ) IsoA treatment decreases Fibronectin, Vimentin, and increased E-cadherin mRNA ( C ) and protein ( D ) expression levels in U251 cells.
    Figure Legend Snippet: IsoA (4 mg/ml) treatment decreases TGFβ and regulates EMT markers in glioma cells. ( A, B ) IsoA treatment decreased TGFβ mRNA ( A ) and protein ( B ) expression levels in U251 cells. ( C, D ) IsoA treatment decreases Fibronectin, Vimentin, and increased E-cadherin mRNA ( C ) and protein ( D ) expression levels in U251 cells.

    Techniques Used: Expressing

    IsoA treatment significantly inhibits tumor growth in glioma-bearing mouse model. ( A ) IsoA treatment (4 mg/kg) significantly inhibited tumor growth in the U251-bearing mouse model. ( B ) IsoA treatment decreased TGFβ, Fibronectin, and Vimentin and increases E-cadherin expression levels in tumors as determined by immunohistochemistry.
    Figure Legend Snippet: IsoA treatment significantly inhibits tumor growth in glioma-bearing mouse model. ( A ) IsoA treatment (4 mg/kg) significantly inhibited tumor growth in the U251-bearing mouse model. ( B ) IsoA treatment decreased TGFβ, Fibronectin, and Vimentin and increases E-cadherin expression levels in tumors as determined by immunohistochemistry.

    Techniques Used: Expressing, Immunohistochemistry

    IsoA regulates growth and aggressiveness of glioma cells through TGFβ-induced EMT signal pathway. ( A, B ) TGFβ knockdown (Si-TGFβ) down-regulates Fibronectin and Vimentin and increased E-cadherin mRNA ( A ) and protein ( B ) expression levels in U251 cells. ( C, D ) TGFβ overexpression (pTGFβ) canceled IsoA-regulated (pTGFβ-IsoA) Fibronectin, Vimentin, and E-cadherin mRNA ( C ) and protein ( D ) expression levels in U251 cells. ( E–G ) TGFβ knockdown (Si-TGFβ) significantly inhibits growth ( E ), migration ( F ), and invasion ( G ) of U251 cells. ( H–J ) TGFβ overexpression abolishes IsoA-inhibited growth ( H ), migration ( I ), and invasion ( J ) of U251 cells
    Figure Legend Snippet: IsoA regulates growth and aggressiveness of glioma cells through TGFβ-induced EMT signal pathway. ( A, B ) TGFβ knockdown (Si-TGFβ) down-regulates Fibronectin and Vimentin and increased E-cadherin mRNA ( A ) and protein ( B ) expression levels in U251 cells. ( C, D ) TGFβ overexpression (pTGFβ) canceled IsoA-regulated (pTGFβ-IsoA) Fibronectin, Vimentin, and E-cadherin mRNA ( C ) and protein ( D ) expression levels in U251 cells. ( E–G ) TGFβ knockdown (Si-TGFβ) significantly inhibits growth ( E ), migration ( F ), and invasion ( G ) of U251 cells. ( H–J ) TGFβ overexpression abolishes IsoA-inhibited growth ( H ), migration ( I ), and invasion ( J ) of U251 cells

    Techniques Used: Expressing, Over Expression, Migration

    IsoA treatment significantly inhibits growth and aggressiveness of glioma cells. ( A ) IsoA inhibits U251 growth in a dose-depended manner (2, 4, and 6 mg/ml). ( B ) IsoA inhibits U251 growth a time-dependent (24, 48, and 72 h) manner. ( C, D ) IsoA (4 mg/ml) significantly inhibits migration ( C ) and invasion ( D ) of U251 cells after 48-h incubation.
    Figure Legend Snippet: IsoA treatment significantly inhibits growth and aggressiveness of glioma cells. ( A ) IsoA inhibits U251 growth in a dose-depended manner (2, 4, and 6 mg/ml). ( B ) IsoA inhibits U251 growth a time-dependent (24, 48, and 72 h) manner. ( C, D ) IsoA (4 mg/ml) significantly inhibits migration ( C ) and invasion ( D ) of U251 cells after 48-h incubation.

    Techniques Used: Migration, Incubation

    IsoA (4 mg/ml) treatment induces apoptosis of glioma cells. ( A ) IsoA treatment induces apoptosis of U251 cells after 48-h incubation. ( B ) IsoA treatment decreases viability of U251 cells after 48-h incubation. ( C ) IsoA treatment decreases anti-apoptosis gene Bcl-2 and Bcl-w gene expression in U251 cells. ( D ) IsoA treatment increases pro-apoptosis gene caspase-3 and Bad gene expression levels in U251 cells.
    Figure Legend Snippet: IsoA (4 mg/ml) treatment induces apoptosis of glioma cells. ( A ) IsoA treatment induces apoptosis of U251 cells after 48-h incubation. ( B ) IsoA treatment decreases viability of U251 cells after 48-h incubation. ( C ) IsoA treatment decreases anti-apoptosis gene Bcl-2 and Bcl-w gene expression in U251 cells. ( D ) IsoA treatment increases pro-apoptosis gene caspase-3 and Bad gene expression levels in U251 cells.

    Techniques Used: Incubation, Expressing

    7) Product Images from "Overexpression of miR-29a reduces the oncogenic properties of glioblastoma stem cells by downregulating Quaking gene isoform 6"

    Article Title: Overexpression of miR-29a reduces the oncogenic properties of glioblastoma stem cells by downregulating Quaking gene isoform 6

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15327

    Overexpression of miR-29a inhibits the expression of WTAP and activation of the PI3K/AKT/ERK pathways by downregulating QKI-6 A . Western blot analysis of WTAP expression, as regulated by miR-29a and QKI-6 in GSCs. B . The expression of p-PI3K/PI3K, p-AKT/AKT and p-ERK/ERK in GSCs-U87 with altered expression of miR-29a and QKI-6. C . Western blot analysis of p-PI3K/PI3K, p-AKT/AKT and p-ERK/ERK expression, as regulated by miR-29a and QKI-6 in GSCs-U251. D . Overexpression of miR-29a inhibited the activation of the p-PI3K/PI3K, p-AKT/AKT and p-ERK/ERK pathways by downregulating QKI-6 in GSCs-GTD. GAPDH was used as an internal loading control. Accompanying graphs display the densitometry analysis of protein expression. Values represent the mean ± SD from five independent experiments.* P
    Figure Legend Snippet: Overexpression of miR-29a inhibits the expression of WTAP and activation of the PI3K/AKT/ERK pathways by downregulating QKI-6 A . Western blot analysis of WTAP expression, as regulated by miR-29a and QKI-6 in GSCs. B . The expression of p-PI3K/PI3K, p-AKT/AKT and p-ERK/ERK in GSCs-U87 with altered expression of miR-29a and QKI-6. C . Western blot analysis of p-PI3K/PI3K, p-AKT/AKT and p-ERK/ERK expression, as regulated by miR-29a and QKI-6 in GSCs-U251. D . Overexpression of miR-29a inhibited the activation of the p-PI3K/PI3K, p-AKT/AKT and p-ERK/ERK pathways by downregulating QKI-6 in GSCs-GTD. GAPDH was used as an internal loading control. Accompanying graphs display the densitometry analysis of protein expression. Values represent the mean ± SD from five independent experiments.* P

    Techniques Used: Over Expression, Expressing, Activation Assay, Western Blot

    Expression and function of miR-29a in GSCs A . Relative expression of miR-29a in GSCs (CD133+) and non-GSCs (CD133-) isolated from U87 and U251 cell lines and glioblastoma tissues. * P
    Figure Legend Snippet: Expression and function of miR-29a in GSCs A . Relative expression of miR-29a in GSCs (CD133+) and non-GSCs (CD133-) isolated from U87 and U251 cell lines and glioblastoma tissues. * P

    Techniques Used: Expressing, Isolation

    8) Product Images from "Low microRNA-622 expression predicts poor prognosis and is associated with ZEB2 in glioma"

    Article Title: Low microRNA-622 expression predicts poor prognosis and is associated with ZEB2 in glioma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S218161

    The relative level of microRNA-622 (miR-622) in 108 glioma tissues and 36 non-neoplastic brain tissues, glioma cell line (U251) and normal human astrocyte (NHA) was tested by qRT-PCR analysis. ( A ) The expression level of miR-622 was found to be remarkably decreased in glioma tissues compared to non-neoplastic brain tissues. ( B ) We also found that miR-622 expression level showed a distinctly downward tendency along with the increasing malignancy degree of glioma. Besides, miR-622 expression was much lower in grades III–IV than that in grades I–II. ( C ) Furthermore, the expression level of miR-622 in U251 cells was distinctly decreased compared to that in NHA. ** p
    Figure Legend Snippet: The relative level of microRNA-622 (miR-622) in 108 glioma tissues and 36 non-neoplastic brain tissues, glioma cell line (U251) and normal human astrocyte (NHA) was tested by qRT-PCR analysis. ( A ) The expression level of miR-622 was found to be remarkably decreased in glioma tissues compared to non-neoplastic brain tissues. ( B ) We also found that miR-622 expression level showed a distinctly downward tendency along with the increasing malignancy degree of glioma. Besides, miR-622 expression was much lower in grades III–IV than that in grades I–II. ( C ) Furthermore, the expression level of miR-622 in U251 cells was distinctly decreased compared to that in NHA. ** p

    Techniques Used: Quantitative RT-PCR, Expressing

    Overexpression of miR-622 significantly inhibited cell proliferation, migration, invasion and apoptosis of glioma cells. Whereas the tumor-suppressive effects of miR-622 were reversed after co-transfection with ZEB2. ( A–D ) MTT assay, wound healing assay, transwell assay and flow cytometry analysis were respectively performed to detect the proliferation, migration, invasion and apoptosis of U251 cells after transfected with NC, miR-622 mimics or miR-622 mimics + ZEB2. * p
    Figure Legend Snippet: Overexpression of miR-622 significantly inhibited cell proliferation, migration, invasion and apoptosis of glioma cells. Whereas the tumor-suppressive effects of miR-622 were reversed after co-transfection with ZEB2. ( A–D ) MTT assay, wound healing assay, transwell assay and flow cytometry analysis were respectively performed to detect the proliferation, migration, invasion and apoptosis of U251 cells after transfected with NC, miR-622 mimics or miR-622 mimics + ZEB2. * p

    Techniques Used: Over Expression, Migration, Cotransfection, MTT Assay, Wound Healing Assay, Transwell Assay, Flow Cytometry, Cytometry, Transfection

    miR-622 functioned as a tumor suppressor by suppressing ZEB2 in vitro. ( A ) The expression of ZEB2 was significantly upregulated in U251 cells compared to that in NHA. ( B ) The expression of ZEB2 was significantly upregulated in glioma tissues compared to that in non-neoplastic brain tissues. ( C ) Overexpression of miR-622 could significantly reduce ZEB2 mRNA expression. ( D ) Overexpression of miR-622 could significantly reduce ZEB2 protein expression. ** p
    Figure Legend Snippet: miR-622 functioned as a tumor suppressor by suppressing ZEB2 in vitro. ( A ) The expression of ZEB2 was significantly upregulated in U251 cells compared to that in NHA. ( B ) The expression of ZEB2 was significantly upregulated in glioma tissues compared to that in non-neoplastic brain tissues. ( C ) Overexpression of miR-622 could significantly reduce ZEB2 mRNA expression. ( D ) Overexpression of miR-622 could significantly reduce ZEB2 protein expression. ** p

    Techniques Used: In Vitro, Expressing, Over Expression

    9) Product Images from "Osteopontin increases heme oxygenase-1 expression and subsequently induces cell migration and invasion in glioma cells"

    Article Title: Osteopontin increases heme oxygenase-1 expression and subsequently induces cell migration and invasion in glioma cells

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/nos262

    Upregulation of osteopontin and HO-1 expression in migration-prone cells. (A) After 10 rounds of selection of U251 cells by a cell culture insert system, the migration-prone subline (P10) exhibited higher migration ability than the original U251 cells.
    Figure Legend Snippet: Upregulation of osteopontin and HO-1 expression in migration-prone cells. (A) After 10 rounds of selection of U251 cells by a cell culture insert system, the migration-prone subline (P10) exhibited higher migration ability than the original U251 cells.

    Techniques Used: Expressing, Migration, Selection, Cell Culture

    High migration activity of human glioma cells correlated with cell invasiveness in vivo. After 10 rounds of selection of U251 cells by a cell culture insert system, the migration-prone subline (P10; B) and the original group (P0; A) were implanted into
    Figure Legend Snippet: High migration activity of human glioma cells correlated with cell invasiveness in vivo. After 10 rounds of selection of U251 cells by a cell culture insert system, the migration-prone subline (P10; B) and the original group (P0; A) were implanted into

    Techniques Used: Migration, Activity Assay, In Vivo, Selection, Cell Culture

    Osteopontin increases HO-1 upregulation in U251 glioma cells. Cells were stimulated with osteopontin with various concentrations (1, 3, or 10 ng/mL for 24 h; A) or with a concentration of 10 ng/mL for indicated time periods (4, 8, and 24 h; B). After
    Figure Legend Snippet: Osteopontin increases HO-1 upregulation in U251 glioma cells. Cells were stimulated with osteopontin with various concentrations (1, 3, or 10 ng/mL for 24 h; A) or with a concentration of 10 ng/mL for indicated time periods (4, 8, and 24 h; B). After

    Techniques Used: Concentration Assay

    Involvement of PI3K/Akt in osteopontin-induced HO-1 expression in U251 glioma cells. Cells were incubated with osteopontin for indicated time periods. The phosphorylation of Akt (A) and ERK (B) were determined by Western blot analysis. (C and D) Cells
    Figure Legend Snippet: Involvement of PI3K/Akt in osteopontin-induced HO-1 expression in U251 glioma cells. Cells were incubated with osteopontin for indicated time periods. The phosphorylation of Akt (A) and ERK (B) were determined by Western blot analysis. (C and D) Cells

    Techniques Used: Expressing, Incubation, Western Blot

    Osteopontin-induced HO-1 upregulation involves Nrf2 activation in U251 glioma cells. (A) Cells were treated with osteopontin for the indicated time periods, and the level of nuclear Nrf2 expression was determined by immunoblotting with Nrf2-specific antibody.
    Figure Legend Snippet: Osteopontin-induced HO-1 upregulation involves Nrf2 activation in U251 glioma cells. (A) Cells were treated with osteopontin for the indicated time periods, and the level of nuclear Nrf2 expression was determined by immunoblotting with Nrf2-specific antibody.

    Techniques Used: Activation Assay, Expressing

    10) Product Images from "Two mature products of MIR-491 coordinate to suppress key cancer hallmarks in glioblastoma"

    Article Title: Two mature products of MIR-491 coordinate to suppress key cancer hallmarks in glioblastoma

    Journal: Oncogene

    doi: 10.1038/onc.2014.98

    miR-491-5p and/or miR-491-3p directly target IGFBP2, CDK6, EGFR, and Bcl-xL and regulate GBM cell proliferation (a) miR-491-5p/miR-491-3p regulates IGFBP2, CDK6, EGFR, and Bcl-xL expression at the protein level. Beta-actin was used as a protein loading control. Each band's intensity was quantified by using Image J, and the relative values (beta-actin as internal control) were shown below the bands. +: 25nM; ++: 50 nM. (b) miR-491 inhibits glioma cell growth. Cell viability of U251 and T98G cells transfected with both miR-491-5p and miR-491-3p mimics or with control mimics was monitored by MTT assay (n = 6). (c) miR-491 inhibits soft agar colonization by glioma cell lines. Upon transfection, cells were seeded into the soft agar and the numbers of colonies were determined four weeks later (n = 3); representative colony morphologies of U251 are shown (n = 3). Bar: 200 μm. (d) miR-491 inhibits glioma cell proliferation. BrdU incorporation assay (n=3) was done seventy-two hours after transfection. Data are presented as mean ± SD (*, P
    Figure Legend Snippet: miR-491-5p and/or miR-491-3p directly target IGFBP2, CDK6, EGFR, and Bcl-xL and regulate GBM cell proliferation (a) miR-491-5p/miR-491-3p regulates IGFBP2, CDK6, EGFR, and Bcl-xL expression at the protein level. Beta-actin was used as a protein loading control. Each band's intensity was quantified by using Image J, and the relative values (beta-actin as internal control) were shown below the bands. +: 25nM; ++: 50 nM. (b) miR-491 inhibits glioma cell growth. Cell viability of U251 and T98G cells transfected with both miR-491-5p and miR-491-3p mimics or with control mimics was monitored by MTT assay (n = 6). (c) miR-491 inhibits soft agar colonization by glioma cell lines. Upon transfection, cells were seeded into the soft agar and the numbers of colonies were determined four weeks later (n = 3); representative colony morphologies of U251 are shown (n = 3). Bar: 200 μm. (d) miR-491 inhibits glioma cell proliferation. BrdU incorporation assay (n=3) was done seventy-two hours after transfection. Data are presented as mean ± SD (*, P

    Techniques Used: Expressing, Transfection, MTT Assay, BrdU Incorporation Assay

    miR-491-3p inhibits glioma cell invasion (a, b) Expression of miR-491-3p inversely correlates with IGFBP2 protein level. Transcript stability in each sample was verified by using U6 as an internal control. Representative images of in situ hybridization staining for miR-491-3p and immunohistochemical staining for IGFBP2 are shown in (a). Data in panel b is presented as mean ± SEM (***, P =0.0002, Mann Whitney test). (c, d) miR-491-3p inhibits invasion of U251 (c) and T98G (d) cells. Invading cells were counted in ten randomly chosen fields under the microscope (n = 3). (e) IGFBP2 partly overcomes the inhibitory effect of miR-491-3p on cell invasion in U251 cells. Levels of exogenous (IGFBP2-EGFP) and endogenous IGFBP2 were determined by Western blot (lower panel). Beta-actin was used as a protein loading control. GFP or IGFBP2 represent U251 cells stably expressing either GFP or IGFBP2-EGFP, respectively. Cell invasion was calculated as the number of invasive cells divided by the number of invasive U251-GFP cells that were transfected with control mimics (upper panel) (n = 3). In c, d and e, data are presented as mean ± SD (*, P
    Figure Legend Snippet: miR-491-3p inhibits glioma cell invasion (a, b) Expression of miR-491-3p inversely correlates with IGFBP2 protein level. Transcript stability in each sample was verified by using U6 as an internal control. Representative images of in situ hybridization staining for miR-491-3p and immunohistochemical staining for IGFBP2 are shown in (a). Data in panel b is presented as mean ± SEM (***, P =0.0002, Mann Whitney test). (c, d) miR-491-3p inhibits invasion of U251 (c) and T98G (d) cells. Invading cells were counted in ten randomly chosen fields under the microscope (n = 3). (e) IGFBP2 partly overcomes the inhibitory effect of miR-491-3p on cell invasion in U251 cells. Levels of exogenous (IGFBP2-EGFP) and endogenous IGFBP2 were determined by Western blot (lower panel). Beta-actin was used as a protein loading control. GFP or IGFBP2 represent U251 cells stably expressing either GFP or IGFBP2-EGFP, respectively. Cell invasion was calculated as the number of invasive cells divided by the number of invasive U251-GFP cells that were transfected with control mimics (upper panel) (n = 3). In c, d and e, data are presented as mean ± SD (*, P

    Techniques Used: Expressing, In Situ Hybridization, Staining, Immunohistochemistry, MANN-WHITNEY, Microscopy, Western Blot, Stable Transfection, Transfection

    11) 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 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

    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 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

    12) Product Images from "Human TRA2A determines influenza A virus host adaptation by regulating viral mRNA splicing"

    Article Title: Human TRA2A determines influenza A virus host adaptation by regulating viral mRNA splicing

    Journal: Science Advances

    doi: 10.1126/sciadv.aaz5764

    TRA2A inhibits YS replication but enhances PR8 replication. ( A ) A549 cells were seeded on the 96-well plates, and cell viability was detected by Cell Counting Kit-8 assay at 36, 48, and 60 hours after transfection. NT, non-treated. ( B to H ) A549 (B to E and H) or U251 (F and G) cells were transfected with either HA-TRA2A, HA-chTRA2A, or pCAGGS vector or either siNC (negative control) or siTRA2A for 24 hours and then infected with the YS (B, C, F, and H) or PR8 (D, E, and G) virus at an MOI of 0.01. Cell culture supernatants were collected at 12, 24, and 36 hpi. Virus titers were determined by TCID 50 assay on MDCK (Madin-Darby canine kidney) cells. A549 cell lysates were analyzed by Western blotting, and the silence efficiency of TRA2A and changes of NP were quantified by ImageJ and normalized to GAPDH (C and E). ( I ) A549 cells were transfected with either siNC or siTRA2A for 24 hours and infected with an avian virus H9N2 or H7N9 or a human virus WSN/H1N1 strain at an MOI of 0.01. Cells lysates were subjected to Western blotting analysis. GAPDH was used as loading control. Each protein band was quantified by ImageJ and normalized to GAPDH levels. Means ± SD (error bars) of three independent experiments are indicated (* P
    Figure Legend Snippet: TRA2A inhibits YS replication but enhances PR8 replication. ( A ) A549 cells were seeded on the 96-well plates, and cell viability was detected by Cell Counting Kit-8 assay at 36, 48, and 60 hours after transfection. NT, non-treated. ( B to H ) A549 (B to E and H) or U251 (F and G) cells were transfected with either HA-TRA2A, HA-chTRA2A, or pCAGGS vector or either siNC (negative control) or siTRA2A for 24 hours and then infected with the YS (B, C, F, and H) or PR8 (D, E, and G) virus at an MOI of 0.01. Cell culture supernatants were collected at 12, 24, and 36 hpi. Virus titers were determined by TCID 50 assay on MDCK (Madin-Darby canine kidney) cells. A549 cell lysates were analyzed by Western blotting, and the silence efficiency of TRA2A and changes of NP were quantified by ImageJ and normalized to GAPDH (C and E). ( I ) A549 cells were transfected with either siNC or siTRA2A for 24 hours and infected with an avian virus H9N2 or H7N9 or a human virus WSN/H1N1 strain at an MOI of 0.01. Cells lysates were subjected to Western blotting analysis. GAPDH was used as loading control. Each protein band was quantified by ImageJ and normalized to GAPDH levels. Means ± SD (error bars) of three independent experiments are indicated (* P

    Techniques Used: Cell Counting, Transfection, Plasmid Preparation, Negative Control, Infection, Cell Culture, Western Blot

    13) Product Images from "NR2C2-uORF targeting UCA1-miR-627-5p-NR2C2 feedback loop to regulate the malignant behaviors of glioma cells"

    Article Title: NR2C2-uORF targeting UCA1-miR-627-5p-NR2C2 feedback loop to regulate the malignant behaviors of glioma cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1149-x

    MiR-627-5p mediated the tumor-suppressive effects of UCA1 knockdown on glioma cell lines. a Cell Counting Kit-8 (CCK-8) assay was used to measure the effect of UCA1 and miR-627-5p on cell proliferation in U87 and U251 cells. b Transwell assay was used to evaluate the effect of UCA1 and miR-627-5p on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis was used to evaluate the effect of effect of UCA1 and miR-627-5p on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P
    Figure Legend Snippet: MiR-627-5p mediated the tumor-suppressive effects of UCA1 knockdown on glioma cell lines. a Cell Counting Kit-8 (CCK-8) assay was used to measure the effect of UCA1 and miR-627-5p on cell proliferation in U87 and U251 cells. b Transwell assay was used to evaluate the effect of UCA1 and miR-627-5p on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis was used to evaluate the effect of effect of UCA1 and miR-627-5p on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P

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

    Interplay of NR2C2 and miR-627-5p. a Cell Counting Kit-8(CCK8) assay to evaluate the effect of miR-627-5p and NR2C2 on cell proliferation in U87 and U251 cells. b Transwell assay to evaluate the effect of miR-627-5p and NR2C2 on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis to evaluate the effect of miR-627-5p and NR2C2 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P
    Figure Legend Snippet: Interplay of NR2C2 and miR-627-5p. a Cell Counting Kit-8(CCK8) assay to evaluate the effect of miR-627-5p and NR2C2 on cell proliferation in U87 and U251 cells. b Transwell assay to evaluate the effect of miR-627-5p and NR2C2 on cell migration and invasion of U87 and U251 cells. c Flow cytometry analysis to evaluate the effect of miR-627-5p and NR2C2 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD ( n = 3, each group). * P

    Techniques Used: Cell Counting, Transwell Assay, Migration, Flow Cytometry, Cytometry

    Identification of NR2C2-binding sites on SPOCK1 and UCA1 promoters. a Western blot analysis for SPOCK1 in U87 and U251 cells with the expression of NR2C2 changed. Error bars represent as the mean ± SD ( n = 3, each group). ** P
    Figure Legend Snippet: Identification of NR2C2-binding sites on SPOCK1 and UCA1 promoters. a Western blot analysis for SPOCK1 in U87 and U251 cells with the expression of NR2C2 changed. Error bars represent as the mean ± SD ( n = 3, each group). ** P

    Techniques Used: Binding Assay, Western Blot, Expressing

    14) Product Images from "Inhibition of autophagy enhances apoptosis induced by proteasome inhibitor bortezomib in human glioblastoma U87 and U251 cells"

    Article Title: Inhibition of autophagy enhances apoptosis induced by proteasome inhibitor bortezomib in human glioblastoma U87 and U251 cells

    Journal: Molecular and Cellular Biochemistry

    doi: 10.1007/s11010-013-1835-z

    Inhibition of autophagy by Atg7 siRNA enhances apoptosis induced by bortezomib in U251 cells. a U251 cells were treated with control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). Cells viability was determined by MTT assay. Data are presented as mean ± SD, n = 6. b U251 cells treated with control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). The cells were harvested after treatment and were stained with JC-1. c Western blot analysis for the expressions of Caspase-3, Cleaved caspase-3, and Cytochrome C in U251 cells treated with control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). d Quantitation of Cleaved caspase-3 and Cytochrome C levels in U251 cells treated with control siRNA or Atg7 siRNA with or without bortezomib. Data are presented as mean ± SD, n = 3, * P
    Figure Legend Snippet: Inhibition of autophagy by Atg7 siRNA enhances apoptosis induced by bortezomib in U251 cells. a U251 cells were treated with control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). Cells viability was determined by MTT assay. Data are presented as mean ± SD, n = 6. b U251 cells treated with control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). The cells were harvested after treatment and were stained with JC-1. c Western blot analysis for the expressions of Caspase-3, Cleaved caspase-3, and Cytochrome C in U251 cells treated with control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). d Quantitation of Cleaved caspase-3 and Cytochrome C levels in U251 cells treated with control siRNA or Atg7 siRNA with or without bortezomib. Data are presented as mean ± SD, n = 3, * P

    Techniques Used: Inhibition, MTT Assay, Staining, Western Blot, Quantitation Assay

    Bortezomib induces apoptosis through mitochondrial apoptotic pathway in U251 cells. a Western blot analysis for the expressions of mitochondria Cytochrome C and cytoplasm Cytochrome C in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. b U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. The cells were harvested after treatment and were stained with JC-1. c Western blot analysis for the expressions of Bcl-2 and Bax in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. d Ratio of Bax/Bcl-2 in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. Data are presented as mean ± SD, n = 3. * P
    Figure Legend Snippet: Bortezomib induces apoptosis through mitochondrial apoptotic pathway in U251 cells. a Western blot analysis for the expressions of mitochondria Cytochrome C and cytoplasm Cytochrome C in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. b U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. The cells were harvested after treatment and were stained with JC-1. c Western blot analysis for the expressions of Bcl-2 and Bax in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. d Ratio of Bax/Bcl-2 in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. Data are presented as mean ± SD, n = 3. * P

    Techniques Used: Western Blot, Staining

    Bortezomib induces autophagy in human glioblastoma U87 and U251 cells. a Western blot analysis for the expressions of LC3 and Beclin 1 in U87 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h and the expression of LC3 in U87 cells treated by bortezomib (10 nM) alone or together with Bafilomycin A1 (1 μM) for 24 h. b Quantitation of LC3-II and Beclin 1 proteins levels in U87 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. c Western blot analysis for the expression of LC3 and Beclin 1 in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h and the expression of LC3 in U251 cells treated by bortezomib (10 nM) alone or together with Bafilomycin A1 (1 μM) for 24 h. d Quantitation of LC3-II and Beclin 1 proteins levels in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h
    Figure Legend Snippet: Bortezomib induces autophagy in human glioblastoma U87 and U251 cells. a Western blot analysis for the expressions of LC3 and Beclin 1 in U87 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h and the expression of LC3 in U87 cells treated by bortezomib (10 nM) alone or together with Bafilomycin A1 (1 μM) for 24 h. b Quantitation of LC3-II and Beclin 1 proteins levels in U87 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. c Western blot analysis for the expression of LC3 and Beclin 1 in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h and the expression of LC3 in U251 cells treated by bortezomib (10 nM) alone or together with Bafilomycin A1 (1 μM) for 24 h. d Quantitation of LC3-II and Beclin 1 proteins levels in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h

    Techniques Used: Western Blot, Expressing, Quantitation Assay

    Inhibition of autophagy by autophagy inhibitor 3-MA enhances apoptosis induced by bortezomib in U251 cells. a U251 cells were treated with control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. Cells viability was determined by MTT assay. Data are presented as mean ± SD, n = 6. b U251 cells treated with control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. The cells were harvested after treatment and were stained with JC-1. c Western blot analysis for the expressions of Caspase-3, Cleaved caspase-3, and Cytochrome C in U251 cells treated with control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. d Quantitation of Cleaved caspase-3 and Cytochrome C levels in U251 cells treated with control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. Data are presented as mean ± SD, n = 3, * P
    Figure Legend Snippet: Inhibition of autophagy by autophagy inhibitor 3-MA enhances apoptosis induced by bortezomib in U251 cells. a U251 cells were treated with control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. Cells viability was determined by MTT assay. Data are presented as mean ± SD, n = 6. b U251 cells treated with control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. The cells were harvested after treatment and were stained with JC-1. c Western blot analysis for the expressions of Caspase-3, Cleaved caspase-3, and Cytochrome C in U251 cells treated with control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. d Quantitation of Cleaved caspase-3 and Cytochrome C levels in U251 cells treated with control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. Data are presented as mean ± SD, n = 3, * P

    Techniques Used: Inhibition, MTT Assay, Staining, Western Blot, Quantitation Assay

    siRNA Atg7 and 3-MA inhibit autophagy induced by bortezomib in U87 and U251 cells. a Western blot analysis for the expression of Atg7 in U87 cells treated by control siRNA or Atg7 siRNA. b Quantitation of Atg7 levels in U87 cells treated by control siRNA or Atg7 siRNA. c Western blot analysis for the expression of Atg7 in U251 cells treated by control siRNA or Atg7 siRNA. d Quantitation of Atg7 levels in U251 cells treated by control siRNA or Atg7 siRNA. e Western blot analysis for the expression of LC3 in U87 cells treated by control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). f Western blot analysis for the expression of LC3 in U87 cells treated by control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. g Western blot analysis for the expression of LC3 in U251 cells treated by control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). h Western blot analysis for the expression of LC3 in U251 cells treated by control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. Data are presented as mean ± SD, n = 3, ** P
    Figure Legend Snippet: siRNA Atg7 and 3-MA inhibit autophagy induced by bortezomib in U87 and U251 cells. a Western blot analysis for the expression of Atg7 in U87 cells treated by control siRNA or Atg7 siRNA. b Quantitation of Atg7 levels in U87 cells treated by control siRNA or Atg7 siRNA. c Western blot analysis for the expression of Atg7 in U251 cells treated by control siRNA or Atg7 siRNA. d Quantitation of Atg7 levels in U251 cells treated by control siRNA or Atg7 siRNA. e Western blot analysis for the expression of LC3 in U87 cells treated by control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). f Western blot analysis for the expression of LC3 in U87 cells treated by control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. g Western blot analysis for the expression of LC3 in U251 cells treated by control siRNA or Atg7 siRNA with or without bortezomib (10 nM, 24 h). h Western blot analysis for the expression of LC3 in U251 cells treated by control (DMSO) or 3-MA (5 mM) with or without bortezomib (10 nM) for 24 h. Data are presented as mean ± SD, n = 3, ** P

    Techniques Used: Western Blot, Expressing, Quantitation Assay

    Bortezomib inhibits growth in human glioblastoma U251 and U87 cells. U251 and U87 cells were treated with 0, 2.5, 5, 10, and 15 nM bortezomib for 24 h. Cells viability was determined by MTT assay. Data are presented as mean ± SD, n = 6
    Figure Legend Snippet: Bortezomib inhibits growth in human glioblastoma U251 and U87 cells. U251 and U87 cells were treated with 0, 2.5, 5, 10, and 15 nM bortezomib for 24 h. Cells viability was determined by MTT assay. Data are presented as mean ± SD, n = 6

    Techniques Used: MTT Assay

    Bortezomib induces apoptosis in human glioblastoma U251 and U87 cells. a U87 cells were treated with 0, 5, 10, and 15 nM bortezomib for 24 h. Cells were stained with PI and Annexin V-FITC. b The positive stained U87 cells were counted using FACScan. c U251 cells were treated with 0, 5, 10, and 15 nM bortezomib for 24 h. Cells were stained with PI and Annexin V-FITC. d The positive stained U251 cells were counted using FACScan. Data are presented as mean ± SD, n = 3, * P
    Figure Legend Snippet: Bortezomib induces apoptosis in human glioblastoma U251 and U87 cells. a U87 cells were treated with 0, 5, 10, and 15 nM bortezomib for 24 h. Cells were stained with PI and Annexin V-FITC. b The positive stained U87 cells were counted using FACScan. c U251 cells were treated with 0, 5, 10, and 15 nM bortezomib for 24 h. Cells were stained with PI and Annexin V-FITC. d The positive stained U251 cells were counted using FACScan. Data are presented as mean ± SD, n = 3, * P

    Techniques Used: Staining

    Bortezomib increased the apoptotic-related proteins in U87 and U251 cells. a Western blot analysis for the expressions of Caspase-3, Cleaved caspase-3, and PARP in U87 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. b Quantitation of Cleaved caspase-3 and Cleaved PARP proteins levels in U87 cells. c Western blot analysis for the expressions of Caspase-3, Cleaved caspase-3, and PARP in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. d Quantitation of Cleaved caspase-3 and Cleaved PARP proteins levels in U251 cells. Data are presented as mean ± SD, n = 3, * P
    Figure Legend Snippet: Bortezomib increased the apoptotic-related proteins in U87 and U251 cells. a Western blot analysis for the expressions of Caspase-3, Cleaved caspase-3, and PARP in U87 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. b Quantitation of Cleaved caspase-3 and Cleaved PARP proteins levels in U87 cells. c Western blot analysis for the expressions of Caspase-3, Cleaved caspase-3, and PARP in U251 cells treated by 0, 5, 10, and 15 nM bortezomib for 24 h. d Quantitation of Cleaved caspase-3 and Cleaved PARP proteins levels in U251 cells. Data are presented as mean ± SD, n = 3, * P

    Techniques Used: Western Blot, Quantitation Assay

    15) Product Images from "LncRNA MIR4435‐2HG potentiates the proliferation and invasion of glioblastoma cells via modulating miR‐1224‐5p/TGFBR2 axis, et al. LncRNA MIR4435‐2HG potentiates the proliferation and invasion of glioblastoma cells via modulating miR‐1224‐5p/TGFBR2 axis"

    Article Title: LncRNA MIR4435‐2HG potentiates the proliferation and invasion of glioblastoma cells via modulating miR‐1224‐5p/TGFBR2 axis, et al. LncRNA MIR4435‐2HG potentiates the proliferation and invasion of glioblastoma cells via modulating miR‐1224‐5p/TGFBR2 axis

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15280

    Overexpression of MIR4435‐2HG promoted GBM cell proliferation and invasion and in vivo tumour growth. A and B, qRT‐PCR showed the up‐regulation of MIR4435‐2HG expression in U87 (A) and U251 cells (B) by transfecting with pcDNA3.1‐MIR4435‐2HG; empty vector = pcDNA3.1 (n = 3). C and D, CCK‐8 assay was utilized to determine the proliferative ability of the transfected U87 (C) and U251 (D) cells (n = 3). E and F, Colony formation assay was utilized to determine the cell growth of the transfected U87 (E) and U251 (F) cells (n = 3). G and H, Transwell invasion assay was utilized to assess the cell invasive ability of the transfected U87 (G) and U251 (H) cells (n = 3). J and K, In vivo tumour growth assay was used to determine the cell growth of the transfected U87 (J) and U251 (K) cells (n = 5). L and M, The weight of the dissected tumours was determined from empty vector (pcDNA3.1) group and pcDNA3.1‐MIR4435‐2HG group (n = 5). * P
    Figure Legend Snippet: Overexpression of MIR4435‐2HG promoted GBM cell proliferation and invasion and in vivo tumour growth. A and B, qRT‐PCR showed the up‐regulation of MIR4435‐2HG expression in U87 (A) and U251 cells (B) by transfecting with pcDNA3.1‐MIR4435‐2HG; empty vector = pcDNA3.1 (n = 3). C and D, CCK‐8 assay was utilized to determine the proliferative ability of the transfected U87 (C) and U251 (D) cells (n = 3). E and F, Colony formation assay was utilized to determine the cell growth of the transfected U87 (E) and U251 (F) cells (n = 3). G and H, Transwell invasion assay was utilized to assess the cell invasive ability of the transfected U87 (G) and U251 (H) cells (n = 3). J and K, In vivo tumour growth assay was used to determine the cell growth of the transfected U87 (J) and U251 (K) cells (n = 5). L and M, The weight of the dissected tumours was determined from empty vector (pcDNA3.1) group and pcDNA3.1‐MIR4435‐2HG group (n = 5). * P

    Techniques Used: Over Expression, In Vivo, Quantitative RT-PCR, Expressing, Plasmid Preparation, CCK-8 Assay, Transfection, Colony Assay, Transwell Invasion Assay, Growth Assay

    MIR4435‐2HG acts as a sponge for miR‐1224‐5p. A, MiR‐1224‐5p had a binding site for MIR4435‐2HG as predicted by starBase database. B, MiR‐1224‐5p expression in normal human astrocytes (NHA) and GBM cell lines including LN229, U87MG, U87, and U251 was determined by qRT‐PCR (n = 3). C, qRT‐PCR showed the up‐regulation of miR‐1224‐5p expression in U87 cells by transfecting with miR‐1224‐5p mimics (mimics) (n = 3). D and E, Luciferase reporter assay determined the relative luciferase activity of U87 cells by co‐transfection with miRNAs (mimics NC or mimics) and reporter vectors (MIR4435‐2HG‐WT or MIR4435‐2HG‐Mut). F, qRT‐PCR determination of miR‐1224‐5p expression in U87 cells by transfecting with pcDNA3.1 (empty vector) or pcDNA3.1‐MIR4435‐2HG. G, qRT‐PCR determination of miR‐1224‐5p expression in U87 cells by transfecting with MIR4435‐2HG siRNA (siRNA#1) or scrambled siRNA (NC). H‐J, CCK‐8, colony formation and transwell invasion assays were performed to determined cell proliferation, growth and invasion of the transfected/co‐transfected U87 cells. N = 3. * P
    Figure Legend Snippet: MIR4435‐2HG acts as a sponge for miR‐1224‐5p. A, MiR‐1224‐5p had a binding site for MIR4435‐2HG as predicted by starBase database. B, MiR‐1224‐5p expression in normal human astrocytes (NHA) and GBM cell lines including LN229, U87MG, U87, and U251 was determined by qRT‐PCR (n = 3). C, qRT‐PCR showed the up‐regulation of miR‐1224‐5p expression in U87 cells by transfecting with miR‐1224‐5p mimics (mimics) (n = 3). D and E, Luciferase reporter assay determined the relative luciferase activity of U87 cells by co‐transfection with miRNAs (mimics NC or mimics) and reporter vectors (MIR4435‐2HG‐WT or MIR4435‐2HG‐Mut). F, qRT‐PCR determination of miR‐1224‐5p expression in U87 cells by transfecting with pcDNA3.1 (empty vector) or pcDNA3.1‐MIR4435‐2HG. G, qRT‐PCR determination of miR‐1224‐5p expression in U87 cells by transfecting with MIR4435‐2HG siRNA (siRNA#1) or scrambled siRNA (NC). H‐J, CCK‐8, colony formation and transwell invasion assays were performed to determined cell proliferation, growth and invasion of the transfected/co‐transfected U87 cells. N = 3. * P

    Techniques Used: Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Cotransfection, Plasmid Preparation, CCK-8 Assay, Transfection

    TGFBR2 is a direct target of miR‐1224‐5p. A, TGFBR2 3’UTR had a binding site for miR‐1224‐5p as predicted by starBase V3.0. B, TGFBR2 mRNA expression in normal human astrocytes (NHA) and GBM cell lines including LN229, U87MG, U87 and U251 was determined by qRT‐PCR (n = 3). C and D, Luciferase reporter assay determined the relative luciferase activity of U87 cells by co‐transfection with miRNAs (mimics NC or mimics) and reporter vectors (TGFBR2 3’UTR‐WT or TGFBR2 3’UTR‐Mut). E and F, qRT‐PCR and Western blot determination of TGFBR2 mRNA and protein expression in U87 cells by transfecting with mimics NC or miR‐1224‐5p mimics. G and H, qRT‐PCR and Western blot determination of TGFBR2 mRNA and protein expression in U87 cells by transfecting with empty vector (pcDNA3.1) or pcDNA3.1‐MIR4435‐2HG. I and J, qRT‐PCR and Western blot determination of TGFBR2 mRNA and protein expression in U87 cells by transfecting with empty vector (pcDNA3.1) or pcDNA3.1‐TGFBR2. K‐M, CCK‐8, colony formation and transwell invasion assays were performed to determined cell proliferation, growth and invasion of the transfected/co‐transfected U87 cells. N = 3. * P
    Figure Legend Snippet: TGFBR2 is a direct target of miR‐1224‐5p. A, TGFBR2 3’UTR had a binding site for miR‐1224‐5p as predicted by starBase V3.0. B, TGFBR2 mRNA expression in normal human astrocytes (NHA) and GBM cell lines including LN229, U87MG, U87 and U251 was determined by qRT‐PCR (n = 3). C and D, Luciferase reporter assay determined the relative luciferase activity of U87 cells by co‐transfection with miRNAs (mimics NC or mimics) and reporter vectors (TGFBR2 3’UTR‐WT or TGFBR2 3’UTR‐Mut). E and F, qRT‐PCR and Western blot determination of TGFBR2 mRNA and protein expression in U87 cells by transfecting with mimics NC or miR‐1224‐5p mimics. G and H, qRT‐PCR and Western blot determination of TGFBR2 mRNA and protein expression in U87 cells by transfecting with empty vector (pcDNA3.1) or pcDNA3.1‐MIR4435‐2HG. I and J, qRT‐PCR and Western blot determination of TGFBR2 mRNA and protein expression in U87 cells by transfecting with empty vector (pcDNA3.1) or pcDNA3.1‐TGFBR2. K‐M, CCK‐8, colony formation and transwell invasion assays were performed to determined cell proliferation, growth and invasion of the transfected/co‐transfected U87 cells. N = 3. * P

    Techniques Used: Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Cotransfection, Western Blot, Plasmid Preparation, CCK-8 Assay, Transfection

    Knockdown of MIR4435‐2HG inhibited GBM cell proliferation and invasion and in vivo tumour growth. A, MIR4435‐2HG expression in normal human astrocytes (NHA) and GBM cell lines including LN229, U87MG, U87, and U251 was determined by qRT‐PCR (n = 3). B and C, qRT‐PCR showed the down‐regulation of MIR4435‐2HG expression in U87 (B) and U251 cells (C) by transfecting with MIR4435‐2HG siRNAs (siRNA#1 and siRNA#2), NC = scrambled siRNA (n = 3). D and E, CCK‐8 assay was utilized to determine the proliferative ability of the transfected U87 (D) and U251 (E) cells (n = 3). F and G, Colony formation assay was utilized to determine the cell growth of the transfected U87 (F) and U251 (G) cells (n = 3). H and I, Transwell invasion assay was utilized to assess the cell invasive ability of the transfected U87 (H) and U251 (I) cells (n = 3). J and K, In vivo tumour growth assay was used to determine the cell growth of the transfected U87 (J) and U251 (K) cells (n = 5). L and M, The weight of the dissected tumours was determined from NC (scrambled shRNA) group and shRNAs group (shRNA#1 and shRNA#2) (n = 5). * P
    Figure Legend Snippet: Knockdown of MIR4435‐2HG inhibited GBM cell proliferation and invasion and in vivo tumour growth. A, MIR4435‐2HG expression in normal human astrocytes (NHA) and GBM cell lines including LN229, U87MG, U87, and U251 was determined by qRT‐PCR (n = 3). B and C, qRT‐PCR showed the down‐regulation of MIR4435‐2HG expression in U87 (B) and U251 cells (C) by transfecting with MIR4435‐2HG siRNAs (siRNA#1 and siRNA#2), NC = scrambled siRNA (n = 3). D and E, CCK‐8 assay was utilized to determine the proliferative ability of the transfected U87 (D) and U251 (E) cells (n = 3). F and G, Colony formation assay was utilized to determine the cell growth of the transfected U87 (F) and U251 (G) cells (n = 3). H and I, Transwell invasion assay was utilized to assess the cell invasive ability of the transfected U87 (H) and U251 (I) cells (n = 3). J and K, In vivo tumour growth assay was used to determine the cell growth of the transfected U87 (J) and U251 (K) cells (n = 5). L and M, The weight of the dissected tumours was determined from NC (scrambled shRNA) group and shRNAs group (shRNA#1 and shRNA#2) (n = 5). * P

    Techniques Used: In Vivo, Expressing, Quantitative RT-PCR, CCK-8 Assay, Transfection, Colony Assay, Transwell Invasion Assay, Growth Assay, shRNA

    16) Product Images from "Human T-Cell Leukemia Virus Type 1 Tax-Deregulated Autophagy Pathway and c-FLIP Expression Contribute to Resistance against Death Receptor-Mediated Apoptosis"

    Article Title: Human T-Cell Leukemia Virus Type 1 Tax-Deregulated Autophagy Pathway and c-FLIP Expression Contribute to Resistance against Death Receptor-Mediated Apoptosis

    Journal: Journal of Virology

    doi: 10.1128/JVI.03025-13

    Tax suppresses TRAIL-induced apoptosis in U251 astroglioma cells. U251 cells were infected with LV-EGFP or LV-Tax (MOI = 3) for 4 days and then treated with recombinant soluble TRAIL or the anti-DR5 agonistic monoclonal antibody AD5-10 at the indicated concentrations for 0 to 12 h. (A) The cell viability after TRAIL or AD5-10 treatment for 12 h was determined using MTS assays (mean ± SD; n = 3). (B and C) Apoptosis in cells treated with 50 ng/ml TRAIL or PBS (control) for 6 h was detected through fluorescence microscopy analysis of Hoechst 33258-stained cells (B) and flow cytometry analysis of PE-conjugated Annexin V-stained cells (C). The mean percentages of Annexin V-positive cells from three independent experiments are shown (mean ± SD; *, P
    Figure Legend Snippet: Tax suppresses TRAIL-induced apoptosis in U251 astroglioma cells. U251 cells were infected with LV-EGFP or LV-Tax (MOI = 3) for 4 days and then treated with recombinant soluble TRAIL or the anti-DR5 agonistic monoclonal antibody AD5-10 at the indicated concentrations for 0 to 12 h. (A) The cell viability after TRAIL or AD5-10 treatment for 12 h was determined using MTS assays (mean ± SD; n = 3). (B and C) Apoptosis in cells treated with 50 ng/ml TRAIL or PBS (control) for 6 h was detected through fluorescence microscopy analysis of Hoechst 33258-stained cells (B) and flow cytometry analysis of PE-conjugated Annexin V-stained cells (C). The mean percentages of Annexin V-positive cells from three independent experiments are shown (mean ± SD; *, P

    Techniques Used: Infection, Recombinant, Fluorescence, Microscopy, Staining, Flow Cytometry, Cytometry

    Activation of NF-κB is necessary for Tax-induced c-FLIP expression but not for Tax-increased autophagosome accumulation. (A to C) U251 cells were infected with LVs encoding no shRNA sequence (shCo), an shRNA targeting IKKα (A), IKKβ (C), or p65 (B), or IκB-DN mutant genes (C) for 3 days and then infected with LV-EGFP or LV-Tax. The knockdown efficacy, IκBα degradation, and c-FLIP and LC3-II expression levels were analyzed by Western blotting. (D and E) IKKα (D), p65 (D), and IκBα (E) contribute to Tax-induced TRAIL resistance. After the infection of the cells with LVs as described for panels A to C, the U251 cells were cultured in the presence or absence of 50 ng/ml TRAIL for 12 h. Cell viability was then determined via MTS assays (mean ± SD; n = 3; *, P
    Figure Legend Snippet: Activation of NF-κB is necessary for Tax-induced c-FLIP expression but not for Tax-increased autophagosome accumulation. (A to C) U251 cells were infected with LVs encoding no shRNA sequence (shCo), an shRNA targeting IKKα (A), IKKβ (C), or p65 (B), or IκB-DN mutant genes (C) for 3 days and then infected with LV-EGFP or LV-Tax. The knockdown efficacy, IκBα degradation, and c-FLIP and LC3-II expression levels were analyzed by Western blotting. (D and E) IKKα (D), p65 (D), and IκBα (E) contribute to Tax-induced TRAIL resistance. After the infection of the cells with LVs as described for panels A to C, the U251 cells were cultured in the presence or absence of 50 ng/ml TRAIL for 12 h. Cell viability was then determined via MTS assays (mean ± SD; n = 3; *, P

    Techniques Used: Activation Assay, Expressing, Infection, shRNA, Sequencing, Mutagenesis, Western Blot, Cell Culture

    Tax-induced c-FLIP expression also protects U251 cells from TRAIL-induced apoptosis. (A) The expression of c-FLIP L and c-FLIP S mRNA in U251 cells infected with LV-EGFP or LV-Tax for the indicated times was assessed via real-time quantitative PCR. Data are presented as fold change in gene expression relative to EGFP-expressing cells (mean ± SD; n = 3). (B) Tax upregulates the level of c-FLIP protein expression. Western blot analysis of c-FLIP L , c-FLIP S , and Tax expression in U251 cells infected with the LVs for 2 to 4 days was carried out. GAPDH expression was used as a loading control. (C) Primary human astrocytoma cells were infected with LV-EGFP or LV-Tax (MOI = 3), and then the cell lysates of the primary human astrocytoma cells were analyzed 4 and 5 days after infection by Western blotting to detect the expressions of the c-FLIP and Tax-Flag proteins. (D and E) U251 cells were infected with LV-EGFP or LV-Tax for 24 h and then with LVs encoding c-FLIP shRNA (shFLIP) or no shRNA (shCo) (MOI = 3) for 3 days, after which they were treated with 50 ng/ml TRAIL for 6 h (D) or 12 h (E). The expression of c-FLIP and Tax and the activation of caspase-8 and caspase-3 were analyzed by Western blotting. The bands of procaspase-8 and cleaved caspase-8 were cut from two films with different exposure times. GAPDH was used as a loading control (D). Cell viability was detected via MTS assays (E) (mean ± SD; n = 3; *, P
    Figure Legend Snippet: Tax-induced c-FLIP expression also protects U251 cells from TRAIL-induced apoptosis. (A) The expression of c-FLIP L and c-FLIP S mRNA in U251 cells infected with LV-EGFP or LV-Tax for the indicated times was assessed via real-time quantitative PCR. Data are presented as fold change in gene expression relative to EGFP-expressing cells (mean ± SD; n = 3). (B) Tax upregulates the level of c-FLIP protein expression. Western blot analysis of c-FLIP L , c-FLIP S , and Tax expression in U251 cells infected with the LVs for 2 to 4 days was carried out. GAPDH expression was used as a loading control. (C) Primary human astrocytoma cells were infected with LV-EGFP or LV-Tax (MOI = 3), and then the cell lysates of the primary human astrocytoma cells were analyzed 4 and 5 days after infection by Western blotting to detect the expressions of the c-FLIP and Tax-Flag proteins. (D and E) U251 cells were infected with LV-EGFP or LV-Tax for 24 h and then with LVs encoding c-FLIP shRNA (shFLIP) or no shRNA (shCo) (MOI = 3) for 3 days, after which they were treated with 50 ng/ml TRAIL for 6 h (D) or 12 h (E). The expression of c-FLIP and Tax and the activation of caspase-8 and caspase-3 were analyzed by Western blotting. The bands of procaspase-8 and cleaved caspase-8 were cut from two films with different exposure times. GAPDH was used as a loading control (D). Cell viability was detected via MTS assays (E) (mean ± SD; n = 3; *, P

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Western Blot, shRNA, Activation Assay

    HTLV-1 Tax increases autophagosome accumulation. (A) Detection of LC3-I and LC3-II expression. U251 cells (2 × 10 5 cells) were infected with LV-EGFP or LV-Tax for 2 to 4 days. Forty micrograms total protein was loaded onto SDS-PAGE gels and further analyzed by Western blotting to detect the conversion of LC3-I to LC3-II and Tax expression. ( B) GFP-LC3 aggregation was visualized via fluorescence microscopy. U251 cells expressing GFP-LC3 were infected with LV-Empty (empty expression cassette) or LV-Tax; after 4 days, the GFP-LC3 aggregations in the cells were assessed via fluorescence microscopy. Representative images are shown (top), and the percentage of cells with GFP-LC3 puncta (% GFP-LC3 vac cells) is presented (bottom; values are means and standard deviations [SD]; *, P
    Figure Legend Snippet: HTLV-1 Tax increases autophagosome accumulation. (A) Detection of LC3-I and LC3-II expression. U251 cells (2 × 10 5 cells) were infected with LV-EGFP or LV-Tax for 2 to 4 days. Forty micrograms total protein was loaded onto SDS-PAGE gels and further analyzed by Western blotting to detect the conversion of LC3-I to LC3-II and Tax expression. ( B) GFP-LC3 aggregation was visualized via fluorescence microscopy. U251 cells expressing GFP-LC3 were infected with LV-Empty (empty expression cassette) or LV-Tax; after 4 days, the GFP-LC3 aggregations in the cells were assessed via fluorescence microscopy. Representative images are shown (top), and the percentage of cells with GFP-LC3 puncta (% GFP-LC3 vac cells) is presented (bottom; values are means and standard deviations [SD]; *, P

    Techniques Used: Expressing, Infection, SDS Page, Western Blot, Fluorescence, Microscopy

    Inhibition of autophagy promotes TRAIL-induced Tax degradation. (A and B) 3-MA promotes TRAIL-induced degradation of Tax and IKKα/β. U251 cells were infected with LV-EGFP (A) or LV-Tax (B) for 4 days, pretreated without (control) or with 10 mM 3-MA for 3 h, and then treated with 50 ng/ml TRAIL for the indicated times. The caspase-3 activation and the expression levels of the IKK complex, IκBα, and Tax were determined using Western blot assays. (C) The pan-caspase inhibitor blocks the Tax degradation induced by the combination of 3-MA and TRAIL. U251 cells were infected with LV-Tax for 4 days, pretreated with or without 10 mM 3-MA, 50 μM MG-132, 1 mM PS-341, 10 μM z-VAD-fmk, or 200 μM chloroquine for 3 h, and then treated with 50 ng/ml TRAIL for 3 h. The level of Tax expression was analyzed using Western blot assays. Protein quantification was performed via densitometry, and the ratios of Tax to GAPDH are shown for the respective blots. (D) Summarized interactions between Tax, autophagy, and TRAIL-mediated apoptosis.
    Figure Legend Snippet: Inhibition of autophagy promotes TRAIL-induced Tax degradation. (A and B) 3-MA promotes TRAIL-induced degradation of Tax and IKKα/β. U251 cells were infected with LV-EGFP (A) or LV-Tax (B) for 4 days, pretreated without (control) or with 10 mM 3-MA for 3 h, and then treated with 50 ng/ml TRAIL for the indicated times. The caspase-3 activation and the expression levels of the IKK complex, IκBα, and Tax were determined using Western blot assays. (C) The pan-caspase inhibitor blocks the Tax degradation induced by the combination of 3-MA and TRAIL. U251 cells were infected with LV-Tax for 4 days, pretreated with or without 10 mM 3-MA, 50 μM MG-132, 1 mM PS-341, 10 μM z-VAD-fmk, or 200 μM chloroquine for 3 h, and then treated with 50 ng/ml TRAIL for 3 h. The level of Tax expression was analyzed using Western blot assays. Protein quantification was performed via densitometry, and the ratios of Tax to GAPDH are shown for the respective blots. (D) Summarized interactions between Tax, autophagy, and TRAIL-mediated apoptosis.

    Techniques Used: Inhibition, Infection, Activation Assay, Expressing, Western Blot

    The Tax mutant M22 is unable to induce c-FLIP expression and autophagosome accumulation. (A) M22 has no effect on c-FLIP and LC3-II expression. U251 cells were infected with the indicated LVs; 4 days after the infection, the levels of c-FLIP L , c-FLIP S , and LC3 expression were analyzed by Western blotting. Protein quantification was performed via densitometry, and the ratios of LC3-II to GAPDH are shown under the respective blots. (B) U251 cells were infected as for panel A and processed for the detection of autophagic vacuoles through transmission electron microscopy. Representative images are shown (scale bars, 0.5 μm). (C) U251 cells expressing GFP-LC3 were infected with LV-Empty, Tax, M22, or M47 and analyzed using confocal microscopy 4 days after infection. Cell nuclei were stained with Hoechst 33258. Representative images are shown (left), and the number of GFP-LC3 dots per cell is reported (right) (mean ± SD; 20 cells; *, P
    Figure Legend Snippet: The Tax mutant M22 is unable to induce c-FLIP expression and autophagosome accumulation. (A) M22 has no effect on c-FLIP and LC3-II expression. U251 cells were infected with the indicated LVs; 4 days after the infection, the levels of c-FLIP L , c-FLIP S , and LC3 expression were analyzed by Western blotting. Protein quantification was performed via densitometry, and the ratios of LC3-II to GAPDH are shown under the respective blots. (B) U251 cells were infected as for panel A and processed for the detection of autophagic vacuoles through transmission electron microscopy. Representative images are shown (scale bars, 0.5 μm). (C) U251 cells expressing GFP-LC3 were infected with LV-Empty, Tax, M22, or M47 and analyzed using confocal microscopy 4 days after infection. Cell nuclei were stained with Hoechst 33258. Representative images are shown (left), and the number of GFP-LC3 dots per cell is reported (right) (mean ± SD; 20 cells; *, P

    Techniques Used: Mutagenesis, Expressing, Infection, Western Blot, Transmission Assay, Electron Microscopy, Confocal Microscopy, Staining

    17) Product Images from "Lycorine inhibits glioblastoma multiforme growth through EGFR suppression"

    Article Title: Lycorine inhibits glioblastoma multiforme growth through EGFR suppression

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0785-4

    Lycorine binds to EGFR, inhibits EGF-activated EGFR phosphorylation and exhibits an EGFR-dependent manner to suppress GBM cells proliferation. a Biacore assay to reveal the SPR analysis of the binding between Lycorine and EGFR (696–1022) domain. The purified EGFR (696–1022) protein was immobilized on an activated CM5 sensor chip. Lycorine was then flowed across the chip. b U251 cells were pretreated with or without 25 μM Lycorine for 1 h then followed by 100 ng/mL EGF treatment for 0, 15, 30, 45 and 60 min (Lycorine was maintained during the EGF-treated time course), and EGF-dependent EGFR phosphorylation was measured by western blotting. c Statistical result of Fig. 5b. d After successful construction of stable U251 shEGFR cells, the knockdown efficiency of EGFR protein was detected by Western blotting in Parental (normal U251 cells), shControl and shEGFR stably constructed U251 cells, respectively. e Statistical result of Fig. 5d. f Parental (normal U251 cells), shControl and shEGFR stably constructed U251 cells were seeded in 96 well plates for indicated days and cell viability was assessed by SRB assay. g Statistic result of cell proliferation when EGFR was interfered by shRNA. Parental (normal U251 cells), shControl and shEGFR stably constructed U251 cells with shRNA were treated with indicated concentrations of Lycorine (0 μM, 10 μM and 20 μM) for 48 h and cell viability was detected by SRB assay. All data are represented as mean ± S.D. from triplicate wells. *, p
    Figure Legend Snippet: Lycorine binds to EGFR, inhibits EGF-activated EGFR phosphorylation and exhibits an EGFR-dependent manner to suppress GBM cells proliferation. a Biacore assay to reveal the SPR analysis of the binding between Lycorine and EGFR (696–1022) domain. The purified EGFR (696–1022) protein was immobilized on an activated CM5 sensor chip. Lycorine was then flowed across the chip. b U251 cells were pretreated with or without 25 μM Lycorine for 1 h then followed by 100 ng/mL EGF treatment for 0, 15, 30, 45 and 60 min (Lycorine was maintained during the EGF-treated time course), and EGF-dependent EGFR phosphorylation was measured by western blotting. c Statistical result of Fig. 5b. d After successful construction of stable U251 shEGFR cells, the knockdown efficiency of EGFR protein was detected by Western blotting in Parental (normal U251 cells), shControl and shEGFR stably constructed U251 cells, respectively. e Statistical result of Fig. 5d. f Parental (normal U251 cells), shControl and shEGFR stably constructed U251 cells were seeded in 96 well plates for indicated days and cell viability was assessed by SRB assay. g Statistic result of cell proliferation when EGFR was interfered by shRNA. Parental (normal U251 cells), shControl and shEGFR stably constructed U251 cells with shRNA were treated with indicated concentrations of Lycorine (0 μM, 10 μM and 20 μM) for 48 h and cell viability was detected by SRB assay. All data are represented as mean ± S.D. from triplicate wells. *, p

    Techniques Used: SPR Assay, Binding Assay, Purification, Chromatin Immunoprecipitation, Western Blot, Stable Transfection, Construct, Sulforhodamine B Assay, shRNA

    Lycorine inhibits U251-luc orthotopic tumor growth in vivo. a Tumor growth in the orthotopic intracranial cavity over a 40-day period was detected by bioluminescence analysis every 10 days. b Quantitative analysis of growing cells in brain bioluminescence analysis every 10 days. The means and 95% confidence intervals (error bars) are presented;***, P
    Figure Legend Snippet: Lycorine inhibits U251-luc orthotopic tumor growth in vivo. a Tumor growth in the orthotopic intracranial cavity over a 40-day period was detected by bioluminescence analysis every 10 days. b Quantitative analysis of growing cells in brain bioluminescence analysis every 10 days. The means and 95% confidence intervals (error bars) are presented;***, P

    Techniques Used: In Vivo

    Lycorine’s inhibition on GBM growth is dependent on EGFR in vivo . a Photos of tumor-bearing nude mice and their xenografts at indicated days of U251 shControl and shEGFR after inoculation. White triangle indicates the tumors that grow subcutaneously in mice right backs. b The growth curve of U251 shControl and shEGFR tumor volume after their inoculation from day 0 to day 32. c Representative images of tumor tissue in control, shEGFR, shControl+Lycorine 20 mg/kg/day and shEGFR+Lycorine 20 mg/kg/day groups. d The tumor volume of 4 groups was shown through growth curve ( n = 4, **, P
    Figure Legend Snippet: Lycorine’s inhibition on GBM growth is dependent on EGFR in vivo . a Photos of tumor-bearing nude mice and their xenografts at indicated days of U251 shControl and shEGFR after inoculation. White triangle indicates the tumors that grow subcutaneously in mice right backs. b The growth curve of U251 shControl and shEGFR tumor volume after their inoculation from day 0 to day 32. c Representative images of tumor tissue in control, shEGFR, shControl+Lycorine 20 mg/kg/day and shEGFR+Lycorine 20 mg/kg/day groups. d The tumor volume of 4 groups was shown through growth curve ( n = 4, **, P

    Techniques Used: Inhibition, In Vivo, Mouse Assay

    Effects of Lycorine on proliferation, migration and colony formation of GBM cells in vitro. a Chemical structure of Lycorine. b U251 cells were treated with indicated concentrations of Lycorine (0, 1, 5, 10, 25, 50 μM) for 48 h. Cell viability was assessed by SRB assay ( n = 3) and photos of the cell morphology were taken by microscope at the light field. c Statistical result of Fig. 2b. d U251 cells were seeded on the upper chamber of Transwell. After 5 to 7 h incubation with Lycorine (from 0 μM to 10 μM), migrated cells were fixed and stained. The number of migrated cells were calculated. e Statistical result of Fig. 2d. f Cells were seeded in 6-well plates for 7 days after the treatment of Lycorine in according concentrations and fixed with 4% paraformaldehyde, and stained with 0.2% crystal violet. The statistical results of colony numbers and diameters were presented in g and h . All data are represented as mean ± S.D. from triplicate wells. *, p
    Figure Legend Snippet: Effects of Lycorine on proliferation, migration and colony formation of GBM cells in vitro. a Chemical structure of Lycorine. b U251 cells were treated with indicated concentrations of Lycorine (0, 1, 5, 10, 25, 50 μM) for 48 h. Cell viability was assessed by SRB assay ( n = 3) and photos of the cell morphology were taken by microscope at the light field. c Statistical result of Fig. 2b. d U251 cells were seeded on the upper chamber of Transwell. After 5 to 7 h incubation with Lycorine (from 0 μM to 10 μM), migrated cells were fixed and stained. The number of migrated cells were calculated. e Statistical result of Fig. 2d. f Cells were seeded in 6-well plates for 7 days after the treatment of Lycorine in according concentrations and fixed with 4% paraformaldehyde, and stained with 0.2% crystal violet. The statistical results of colony numbers and diameters were presented in g and h . All data are represented as mean ± S.D. from triplicate wells. *, p

    Techniques Used: Migration, In Vitro, Sulforhodamine B Assay, Microscopy, Incubation, Staining

    Lycorine’s suppression effects on EGF-induced EGFR signaling pathway. a In vitro EGFR kinase inhibition by Lycorine. Half maximal inhibitory concentration (IC 50 ) values of Lycorine and positive control Gefitinib. b U251 cells were incubated with various concentrations of Lycorine (from 0 μM to 50 μM) for 48 h. Effects on the expression of CL–caspase 3 and PARP were determined by Western blotting. Human β-actin served as a loading control. c Suppression by Lycorine of EGFR phosphorylation and its downstream AKT, ERK, mTOR, p27, p21, Bcl-2, Cyclin D1, MMP9 in U251 cell lines. For detecting the phosphorylation of EGFR and its downstream signaling pathways, U251 cells were pretreated with 100 ng/mL human recombinant protein EGF for 6 h, then treated with Lycorine for indicated concentration for another 24 h; and cell lysates were subjected to Western blotting analysis with indicated antibodies. d U251 cells exposed to 100 ng/mL human recombinant protein EGF for indicated time points (0, 15, 30, 45 and 60 min) then followed with 25 μM Lycorine for 1 h or with no treatment. The expression of EGFR and p-EGFR were detected by western blotting. e Statistical result of Fig. 4d
    Figure Legend Snippet: Lycorine’s suppression effects on EGF-induced EGFR signaling pathway. a In vitro EGFR kinase inhibition by Lycorine. Half maximal inhibitory concentration (IC 50 ) values of Lycorine and positive control Gefitinib. b U251 cells were incubated with various concentrations of Lycorine (from 0 μM to 50 μM) for 48 h. Effects on the expression of CL–caspase 3 and PARP were determined by Western blotting. Human β-actin served as a loading control. c Suppression by Lycorine of EGFR phosphorylation and its downstream AKT, ERK, mTOR, p27, p21, Bcl-2, Cyclin D1, MMP9 in U251 cell lines. For detecting the phosphorylation of EGFR and its downstream signaling pathways, U251 cells were pretreated with 100 ng/mL human recombinant protein EGF for 6 h, then treated with Lycorine for indicated concentration for another 24 h; and cell lysates were subjected to Western blotting analysis with indicated antibodies. d U251 cells exposed to 100 ng/mL human recombinant protein EGF for indicated time points (0, 15, 30, 45 and 60 min) then followed with 25 μM Lycorine for 1 h or with no treatment. The expression of EGFR and p-EGFR were detected by western blotting. e Statistical result of Fig. 4d

    Techniques Used: In Vitro, Inhibition, Concentration Assay, Positive Control, Incubation, Expressing, Western Blot, Recombinant

    Effects of Lycorine on cell viability of GBM cells holding different EGFR status. a Expression of EGFR mRNA on different GBM cell lines. Surface EGFR expression on glioma cell lines (U87, wild type EGFR; LN229, wild type EGFR amplification, U251, wild type EGFR amplification, A172, EGFRvIII mutant, Gli36vIII, EGFRvIII mutant, GBM6, wild type EGFR and EGFRvIII co-existing) and a healthy normal human IMA2.1 astrocytes was monitored by RT-PCR. b Lycorine suppresses GBM cell’s proliferation independent of EGFR mutation status but dependent of EGFR expression level. The cell viability assay stained by SRB was performed as described in Methods. All data are represented as mean ± S.D. from triplicate wells
    Figure Legend Snippet: Effects of Lycorine on cell viability of GBM cells holding different EGFR status. a Expression of EGFR mRNA on different GBM cell lines. Surface EGFR expression on glioma cell lines (U87, wild type EGFR; LN229, wild type EGFR amplification, U251, wild type EGFR amplification, A172, EGFRvIII mutant, Gli36vIII, EGFRvIII mutant, GBM6, wild type EGFR and EGFRvIII co-existing) and a healthy normal human IMA2.1 astrocytes was monitored by RT-PCR. b Lycorine suppresses GBM cell’s proliferation independent of EGFR mutation status but dependent of EGFR expression level. The cell viability assay stained by SRB was performed as described in Methods. All data are represented as mean ± S.D. from triplicate wells

    Techniques Used: Expressing, Amplification, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Viability Assay, Staining, Sulforhodamine B Assay

    18) Product Images from "Downregulation of α‐l‐fucosidase 1 suppresses glioma progression by enhancing autophagy and inhibiting macrophage infiltration, et al. Downregulation of α‐l‐fucosidase 1 suppresses glioma progression by enhancing autophagy and inhibiting macrophage infiltration"

    Article Title: Downregulation of α‐l‐fucosidase 1 suppresses glioma progression by enhancing autophagy and inhibiting macrophage infiltration, et al. Downregulation of α‐l‐fucosidase 1 suppresses glioma progression by enhancing autophagy and inhibiting macrophage infiltration

    Journal: Cancer Science

    doi: 10.1111/cas.14427

    Inhibition of α‐l‐fucosidase 1 (FUCA1) induces autophagic cell death in U87 and U251 cells. A, Transient FUCA1‐knockdown cells were subjected to acridine orange staining to detect the formation of acidic vesicular organelles (AVOs). Autophagic cell death values were calculated as the percentage of acridine orange‐positive cells relative to the total number of cells in each random field. B, Western blot analyses for LC3‐A/B, Beclin 1, and Atg12 expression in U87 and U251 cells transfected with siFUCA1 for 48 h. Data are mean ± SD. * P
    Figure Legend Snippet: Inhibition of α‐l‐fucosidase 1 (FUCA1) induces autophagic cell death in U87 and U251 cells. A, Transient FUCA1‐knockdown cells were subjected to acridine orange staining to detect the formation of acidic vesicular organelles (AVOs). Autophagic cell death values were calculated as the percentage of acridine orange‐positive cells relative to the total number of cells in each random field. B, Western blot analyses for LC3‐A/B, Beclin 1, and Atg12 expression in U87 and U251 cells transfected with siFUCA1 for 48 h. Data are mean ± SD. * P

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

    Silencing α‐l‐fucosidase 1 (FUCA1) inhibits macrophage infiltration by downregulating CCL2/CCL5 expression. A, Correlation between FUCA1 and CCL2/CCL5 in 3 Chinese Glioma Genome Atlas datasets. B, C, Quantitative RT‐PCR analyses for CCL2 (B) and CCL5 (C) expression in U87 and U251 cells transfected with siFUCA1 or pcDNA3.1‐FUCA1 for 24 h. Data are mean ± SD. * P
    Figure Legend Snippet: Silencing α‐l‐fucosidase 1 (FUCA1) inhibits macrophage infiltration by downregulating CCL2/CCL5 expression. A, Correlation between FUCA1 and CCL2/CCL5 in 3 Chinese Glioma Genome Atlas datasets. B, C, Quantitative RT‐PCR analyses for CCL2 (B) and CCL5 (C) expression in U87 and U251 cells transfected with siFUCA1 or pcDNA3.1‐FUCA1 for 24 h. Data are mean ± SD. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection

    Effects of α‐l‐fucosidase 1 (FUCA1) on glioma growth in vitro and in vivo. A, B, FUCA1 siRNA caused an obvious downregulation of FUCA1 mRNA (A) and protein (B) levels in U87 and U251 cells. Data are mean ± SD. * P
    Figure Legend Snippet: Effects of α‐l‐fucosidase 1 (FUCA1) on glioma growth in vitro and in vivo. A, B, FUCA1 siRNA caused an obvious downregulation of FUCA1 mRNA (A) and protein (B) levels in U87 and U251 cells. Data are mean ± SD. * P

    Techniques Used: In Vitro, In Vivo

    19) Product Images from "CREB1-induced miR-1204 promoted malignant phenotype of glioblastoma through targeting NR3C2"

    Article Title: CREB1-induced miR-1204 promoted malignant phenotype of glioblastoma through targeting NR3C2

    Journal: Cancer Cell International

    doi: 10.1186/s12935-020-01176-0

    MiR-1204 was transcriptionally activated by CREB1 in GBM. a The upregulation of CREB1 in GBM cells was confirmed by RT-qPCR. b The overexpression efficiency of CREB1 in A172 cells and the knockdown efficiency of CREB1 in U251 cells were confirmed by RT-qPCR. c MiR-1204 expression under the overexpression and knockdown of CREB1 in A172 and U251 cells were detected by RT-qPCR. d The DNA motif of CREB1 and the CREB1 binding site on miR-1204 promoter were obtained from miRGen. e Luciferase reporter assay was used to detect the impact of CREB1 on miR-1204 transcription. f ChIP assay was used to confirm the binding capacity between CREB1 and miR-1204 promoter. GAPDH was used as internal control for CREB1 expression detection, and U6 was used as internal control for miR-1204 expression detection. *P
    Figure Legend Snippet: MiR-1204 was transcriptionally activated by CREB1 in GBM. a The upregulation of CREB1 in GBM cells was confirmed by RT-qPCR. b The overexpression efficiency of CREB1 in A172 cells and the knockdown efficiency of CREB1 in U251 cells were confirmed by RT-qPCR. c MiR-1204 expression under the overexpression and knockdown of CREB1 in A172 and U251 cells were detected by RT-qPCR. d The DNA motif of CREB1 and the CREB1 binding site on miR-1204 promoter were obtained from miRGen. e Luciferase reporter assay was used to detect the impact of CREB1 on miR-1204 transcription. f ChIP assay was used to confirm the binding capacity between CREB1 and miR-1204 promoter. GAPDH was used as internal control for CREB1 expression detection, and U6 was used as internal control for miR-1204 expression detection. *P

    Techniques Used: Quantitative RT-PCR, Over Expression, Expressing, Binding Assay, Luciferase, Reporter Assay, Chromatin Immunoprecipitation

    MiR-1204 drove GBM cell proliferation by inhibiting NR3C2 expression. U251 cells were transfected with NC inhibitor, miR-1204 inhibitor, miR-1204 inhibitor + sh-NC, and miR-1204 inhibitor + sh-NR3C2, respectively. a NR3C2 expression in U251 cells was detected by RT-qPCR. b , c The proliferation of U251 cells was examined by CCK-8 and colony formation assays. d , e The apoptosis of U251 cells was examined by caspase-3 activity and TUNEL assays. GAPDH was used as internal control for NR3C2 expression detection. **P
    Figure Legend Snippet: MiR-1204 drove GBM cell proliferation by inhibiting NR3C2 expression. U251 cells were transfected with NC inhibitor, miR-1204 inhibitor, miR-1204 inhibitor + sh-NC, and miR-1204 inhibitor + sh-NR3C2, respectively. a NR3C2 expression in U251 cells was detected by RT-qPCR. b , c The proliferation of U251 cells was examined by CCK-8 and colony formation assays. d , e The apoptosis of U251 cells was examined by caspase-3 activity and TUNEL assays. GAPDH was used as internal control for NR3C2 expression detection. **P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, Activity Assay, TUNEL Assay

    MiR-1204 was highly expressed in GBM and promoted GBM. a The upregulation of miR-1204 in GBM cells was confirmed by RT-qPCR. b The overexpression efficiency of miR-1204 in A172 cells and the knockdown efficiency of miR-1204 in U251 cells were confirmed by RT-qPCR. c , d The proliferation of transfected A172 and U251 cells was examined by CCK-8 and colony formation assays. e , f The apoptosis of transfected cells was analyzed by caspase-3 and TUNEL assays. U6 served as internal control for miR-1204 expression detection. *P
    Figure Legend Snippet: MiR-1204 was highly expressed in GBM and promoted GBM. a The upregulation of miR-1204 in GBM cells was confirmed by RT-qPCR. b The overexpression efficiency of miR-1204 in A172 cells and the knockdown efficiency of miR-1204 in U251 cells were confirmed by RT-qPCR. c , d The proliferation of transfected A172 and U251 cells was examined by CCK-8 and colony formation assays. e , f The apoptosis of transfected cells was analyzed by caspase-3 and TUNEL assays. U6 served as internal control for miR-1204 expression detection. *P

    Techniques Used: Quantitative RT-PCR, Over Expression, Transfection, CCK-8 Assay, TUNEL Assay, Expressing

    20) Product Images from "MicroRNA-518b functions as a tumor suppressor in glioblastoma by targeting PDGFRB"

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

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.7298

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    21) Product Images from "PIWIL3/OIP5-AS1/miR-367-3p/CEBPA feedback loop regulates the biological behavior of glioma cells"

    Article Title: PIWIL3/OIP5-AS1/miR-367-3p/CEBPA feedback loop regulates the biological behavior of glioma cells

    Journal: Theranostics

    doi: 10.7150/thno.21740

    Effect of TRAF4 on the biological behavior of glioma cells. (A) Cell Counting Kit-8 (CCK-8) assay was used to evaluate the effect of TRAF4 on U87 and U251 cells' proliferation. (B) Flow cytometry analysis of U87 and U251 cells affected by TRAF4 (data are presented as the mean ± SD (n =3, each group);** P
    Figure Legend Snippet: Effect of TRAF4 on the biological behavior of glioma cells. (A) Cell Counting Kit-8 (CCK-8) assay was used to evaluate the effect of TRAF4 on U87 and U251 cells' proliferation. (B) Flow cytometry analysis of U87 and U251 cells affected by TRAF4 (data are presented as the mean ± SD (n =3, each group);** P

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

    Targeted binding between PIWIL3 and piR-30188, piR-30188, and OIP5-AS1; effect of miR-367-3p on the biological behavior of glioma cells. (A) Quantitative real-time PCR (qRT-PCR) analysis for PIWIL3 and piR-30188 regulating OIP5-AS1 expression in U87 and U251 cells (data are presented as the mean ± SD (n = 3, each group);* P
    Figure Legend Snippet: Targeted binding between PIWIL3 and piR-30188, piR-30188, and OIP5-AS1; effect of miR-367-3p on the biological behavior of glioma cells. (A) Quantitative real-time PCR (qRT-PCR) analysis for PIWIL3 and piR-30188 regulating OIP5-AS1 expression in U87 and U251 cells (data are presented as the mean ± SD (n = 3, each group);* P

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

    Over-expression of PIWIL3, piR-30188and knockdown of OIP5-AS1 inhibited CEBPA expression by down-regulating miR-367-3p. (A) Quantitative real-time PCR (qRT-PCR) analysis for PIWIL3, piR-30188, and OIP5-AS1-regulated miR-367-3p expression in U87 and U251 cells (data are presented as the mean ± SD (n =3, each group); *#Δ P
    Figure Legend Snippet: Over-expression of PIWIL3, piR-30188and knockdown of OIP5-AS1 inhibited CEBPA expression by down-regulating miR-367-3p. (A) Quantitative real-time PCR (qRT-PCR) analysis for PIWIL3, piR-30188, and OIP5-AS1-regulated miR-367-3p expression in U87 and U251 cells (data are presented as the mean ± SD (n =3, each group); *#Δ P

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

    22) Product Images from "CREB1-induced miR-1204 promoted malignant phenotype of glioblastoma through targeting NR3C2"

    Article Title: CREB1-induced miR-1204 promoted malignant phenotype of glioblastoma through targeting NR3C2

    Journal: Cancer Cell International

    doi: 10.1186/s12935-020-01176-0

    MiR-1204 was transcriptionally activated by CREB1 in GBM. a The upregulation of CREB1 in GBM cells was confirmed by RT-qPCR. b The overexpression efficiency of CREB1 in A172 cells and the knockdown efficiency of CREB1 in U251 cells were confirmed by RT-qPCR. c MiR-1204 expression under the overexpression and knockdown of CREB1 in A172 and U251 cells were detected by RT-qPCR. d The DNA motif of CREB1 and the CREB1 binding site on miR-1204 promoter were obtained from miRGen. e Luciferase reporter assay was used to detect the impact of CREB1 on miR-1204 transcription. f ChIP assay was used to confirm the binding capacity between CREB1 and miR-1204 promoter. GAPDH was used as internal control for CREB1 expression detection, and U6 was used as internal control for miR-1204 expression detection. *P
    Figure Legend Snippet: MiR-1204 was transcriptionally activated by CREB1 in GBM. a The upregulation of CREB1 in GBM cells was confirmed by RT-qPCR. b The overexpression efficiency of CREB1 in A172 cells and the knockdown efficiency of CREB1 in U251 cells were confirmed by RT-qPCR. c MiR-1204 expression under the overexpression and knockdown of CREB1 in A172 and U251 cells were detected by RT-qPCR. d The DNA motif of CREB1 and the CREB1 binding site on miR-1204 promoter were obtained from miRGen. e Luciferase reporter assay was used to detect the impact of CREB1 on miR-1204 transcription. f ChIP assay was used to confirm the binding capacity between CREB1 and miR-1204 promoter. GAPDH was used as internal control for CREB1 expression detection, and U6 was used as internal control for miR-1204 expression detection. *P

    Techniques Used: Quantitative RT-PCR, Over Expression, Expressing, Binding Assay, Luciferase, Reporter Assay, Chromatin Immunoprecipitation

    MiR-1204 drove GBM cell proliferation by inhibiting NR3C2 expression. U251 cells were transfected with NC inhibitor, miR-1204 inhibitor, miR-1204 inhibitor + sh-NC, and miR-1204 inhibitor + sh-NR3C2, respectively. a NR3C2 expression in U251 cells was detected by RT-qPCR. b , c The proliferation of U251 cells was examined by CCK-8 and colony formation assays. d , e The apoptosis of U251 cells was examined by caspase-3 activity and TUNEL assays. GAPDH was used as internal control for NR3C2 expression detection. **P
    Figure Legend Snippet: MiR-1204 drove GBM cell proliferation by inhibiting NR3C2 expression. U251 cells were transfected with NC inhibitor, miR-1204 inhibitor, miR-1204 inhibitor + sh-NC, and miR-1204 inhibitor + sh-NR3C2, respectively. a NR3C2 expression in U251 cells was detected by RT-qPCR. b , c The proliferation of U251 cells was examined by CCK-8 and colony formation assays. d , e The apoptosis of U251 cells was examined by caspase-3 activity and TUNEL assays. GAPDH was used as internal control for NR3C2 expression detection. **P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, Activity Assay, TUNEL Assay

    MiR-1204 was highly expressed in GBM and promoted GBM. a The upregulation of miR-1204 in GBM cells was confirmed by RT-qPCR. b The overexpression efficiency of miR-1204 in A172 cells and the knockdown efficiency of miR-1204 in U251 cells were confirmed by RT-qPCR. c , d The proliferation of transfected A172 and U251 cells was examined by CCK-8 and colony formation assays. e , f The apoptosis of transfected cells was analyzed by caspase-3 and TUNEL assays. U6 served as internal control for miR-1204 expression detection. *P
    Figure Legend Snippet: MiR-1204 was highly expressed in GBM and promoted GBM. a The upregulation of miR-1204 in GBM cells was confirmed by RT-qPCR. b The overexpression efficiency of miR-1204 in A172 cells and the knockdown efficiency of miR-1204 in U251 cells were confirmed by RT-qPCR. c , d The proliferation of transfected A172 and U251 cells was examined by CCK-8 and colony formation assays. e , f The apoptosis of transfected cells was analyzed by caspase-3 and TUNEL assays. U6 served as internal control for miR-1204 expression detection. *P

    Techniques Used: Quantitative RT-PCR, Over Expression, Transfection, CCK-8 Assay, TUNEL Assay, Expressing

    23) Product Images from "Long non-coding RNA SPRY4-IT1 promotes the proliferation and invasion of U251 cells through upregulation of SKA2"

    Article Title: Long non-coding RNA SPRY4-IT1 promotes the proliferation and invasion of U251 cells through upregulation of SKA2

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.7776

    Effect of SKA2 on the expression of PCNA and cyclin D1 in U251 cells. Western blotting was performed to assess the protein level of PCNA, cyclin D1, MMP2 and MMP9 in siRNA-treated U251 cells. *P
    Figure Legend Snippet: Effect of SKA2 on the expression of PCNA and cyclin D1 in U251 cells. Western blotting was performed to assess the protein level of PCNA, cyclin D1, MMP2 and MMP9 in siRNA-treated U251 cells. *P

    Techniques Used: Expressing, Western Blot

    Knockdown of SPRY4-IT1 inhibits cellular proliferation, migration and invasion in U251 cells. (A) si-SPRY4-IT1 cell proliferation was significantly inhibited compared with si-NC cells, as detected by the MTT assay. (B) Effects of SPRY4-IT1 on clone formation in U251 cells. (C) Scratches were made on confluent monolayers si-NC or si-SPRY4-IT1 cells. (D) U251 cells infected with si-SPRY4-IT1 displayed significantly lower invasion capacity compared with those infected with si-NC. The widths of the gaps from 3 experiments were measured, the mean was calculated and the results presented in a bar graph. **P
    Figure Legend Snippet: Knockdown of SPRY4-IT1 inhibits cellular proliferation, migration and invasion in U251 cells. (A) si-SPRY4-IT1 cell proliferation was significantly inhibited compared with si-NC cells, as detected by the MTT assay. (B) Effects of SPRY4-IT1 on clone formation in U251 cells. (C) Scratches were made on confluent monolayers si-NC or si-SPRY4-IT1 cells. (D) U251 cells infected with si-SPRY4-IT1 displayed significantly lower invasion capacity compared with those infected with si-NC. The widths of the gaps from 3 experiments were measured, the mean was calculated and the results presented in a bar graph. **P

    Techniques Used: Migration, MTT Assay, Infection

    Knockdown of SKA2 inhibits cell growth in U251 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis of SKA2 mRNA expression in si-SKA2 and si-NC. (B) Western blot analysis of SKA2 protein expression in si-SKA2 and si-NC. (C) U251 cell proliferation in the si-SKA2 groups was significantly reduced, as detected by the MTT assay. (D) Effects of SKA2 on clone formation in U251 cells. (E) Effects of SKA2 on the invasion of U251 cells. **P
    Figure Legend Snippet: Knockdown of SKA2 inhibits cell growth in U251 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis of SKA2 mRNA expression in si-SKA2 and si-NC. (B) Western blot analysis of SKA2 protein expression in si-SKA2 and si-NC. (C) U251 cell proliferation in the si-SKA2 groups was significantly reduced, as detected by the MTT assay. (D) Effects of SKA2 on clone formation in U251 cells. (E) Effects of SKA2 on the invasion of U251 cells. **P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot, MTT Assay

    24) Product Images from "MicroRNA-30a increases the chemosensitivity of U251 glioblastoma cells to temozolomide by directly targeting beclin 1 and inhibiting autophagy"

    Article Title: MicroRNA-30a increases the chemosensitivity of U251 glioblastoma cells to temozolomide by directly targeting beclin 1 and inhibiting autophagy

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2018.6007

    Overexpression of miR-30a increases the cytotoxicity of TMZ to U251 cells. (A) MTT assay was performed to examine the proliferation of TMZ (30 µg/ml)-treated U251 cells transfected with miR-30a mimic or miR-NC, respectively. (B) Flow cytometry was conducted to examine cell apoptosis. Non-transfected U251 cells treated with TMZ (30 µg/ml) were used as control. *P
    Figure Legend Snippet: Overexpression of miR-30a increases the cytotoxicity of TMZ to U251 cells. (A) MTT assay was performed to examine the proliferation of TMZ (30 µg/ml)-treated U251 cells transfected with miR-30a mimic or miR-NC, respectively. (B) Flow cytometry was conducted to examine cell apoptosis. Non-transfected U251 cells treated with TMZ (30 µg/ml) were used as control. *P

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

    Treatment with TMZ decreases the expression of miR-30a in U251 cells. Reverse transcription-quantitative polymerase chain reaction was conducted to examine the relative miR-30a levels (relative to U6) in U251 cells treated with TMZ (1–30 µg/ml). Non-treated U251 cells were used as a control. *P
    Figure Legend Snippet: Treatment with TMZ decreases the expression of miR-30a in U251 cells. Reverse transcription-quantitative polymerase chain reaction was conducted to examine the relative miR-30a levels (relative to U6) in U251 cells treated with TMZ (1–30 µg/ml). Non-treated U251 cells were used as a control. *P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Restoration of miR-30a level suppresses TMZ-induced autophagy in U251 cells via inhibition of beclin 1. (A) Reverse transcription-quantitative polymerase chain reaction was conducted to examine the relative miR-30a levels in U251 cells transfected with miR-30a mimic or miR-NC, respectively. (B) Western blot analysis was conducted to determine the levels of autophagy-related proteins in the transfected U251 cells treated with TMZ (30 µg/ml). (C) The ratio of LC3-II to LC3-I was calculated. Non-transfected U251 cells treated with TMZ were used as the control. *P
    Figure Legend Snippet: Restoration of miR-30a level suppresses TMZ-induced autophagy in U251 cells via inhibition of beclin 1. (A) Reverse transcription-quantitative polymerase chain reaction was conducted to examine the relative miR-30a levels in U251 cells transfected with miR-30a mimic or miR-NC, respectively. (B) Western blot analysis was conducted to determine the levels of autophagy-related proteins in the transfected U251 cells treated with TMZ (30 µg/ml). (C) The ratio of LC3-II to LC3-I was calculated. Non-transfected U251 cells treated with TMZ were used as the control. *P

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

    Beclin 1 is a direct target of miR-30a in U251 cells. (A) Bioinformatic prediction data indicated that beclin 1 was a direct target gene of miR-30a. (B and C) WT or MUT BECN1 3′-UTR was cloned downstream of the firefly luciferase coding region of the pmirGLO™ vector, to form pMIR-WT BECN1 and pMIR-MUT BECN1, respectively. (D) U251 cells were co-transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and miR-30a mimic or miR-NC, and pRL-TK plasmid for internal normalization, respectively. Luciferase reporter assay data showed that co-transfection with pMIR-WT BECN1 and miR-30a mimic significantly decreased the luciferase activity; however, co-transfection with pMIR-MUT BECN1 and miR-30a mimic caused no change in luciferase activity. Control cells were transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and the pRL-TK plasmid. *P
    Figure Legend Snippet: Beclin 1 is a direct target of miR-30a in U251 cells. (A) Bioinformatic prediction data indicated that beclin 1 was a direct target gene of miR-30a. (B and C) WT or MUT BECN1 3′-UTR was cloned downstream of the firefly luciferase coding region of the pmirGLO™ vector, to form pMIR-WT BECN1 and pMIR-MUT BECN1, respectively. (D) U251 cells were co-transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and miR-30a mimic or miR-NC, and pRL-TK plasmid for internal normalization, respectively. Luciferase reporter assay data showed that co-transfection with pMIR-WT BECN1 and miR-30a mimic significantly decreased the luciferase activity; however, co-transfection with pMIR-MUT BECN1 and miR-30a mimic caused no change in luciferase activity. Control cells were transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and the pRL-TK plasmid. *P

    Techniques Used: Clone Assay, Luciferase, Plasmid Preparation, Transfection, Reporter Assay, Cotransfection, Activity Assay

    Treatment with TMZ inhibits proliferation, and induces apoptosis and autophagy in U251 cells. (A) MTT assay was performed to examine the proliferation of U251 cells treated with TMZ (1–30 µg/ml). *P
    Figure Legend Snippet: Treatment with TMZ inhibits proliferation, and induces apoptosis and autophagy in U251 cells. (A) MTT assay was performed to examine the proliferation of U251 cells treated with TMZ (1–30 µg/ml). *P

    Techniques Used: MTT Assay

    25) Product Images from "CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A2B Adenosine Receptor Signaling"

    Article Title: CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A2B Adenosine Receptor Signaling

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1118-18.2019

    GB expresses CD73 in vitro and upregulates GB-CD73 in CD73 −/− mice in vivo . A , B , Representative flow cytometry analysis of CD73 expression on GL261 ( A ) and U251 ( B ) GB cell lines. Data are representative of three experiments. C , Representative flow cytometry analysis of CD44 and CD73 expression on GL261. Data are representative of three experiments. D , G , Representative images of sham-implanted ( D ) and naive ( G ) mice brain sections stained with anti-CD73 antibody (brown) ( n = 3). E , F , H , Quantification of CD73 expression in caudoputamen or cortex of sham-implanted or naive mice was performed by measuring positive pixel count using ImageScope analysis program ( n = 3, three images analyzed/sample, Student's two-tailed t test). I , Representative images of brain sections from GL261-implanted mice stained with anti-CD73 antibody (brown) ( n = 3–4). J , Quantification of CD73 expression inside tumors was performed by measuring positive pixel count using ImageScope analysis program ( n = 3–4, 3 images analyzed/sample, Student's two-tailed t test). K , Densitometry quantification of CD73 protein expression inside tumors using ImageJ, normalizing to GAPDH and sham CTs ( n = 4–5, Student's two-tailed t test). L , M , N , Flow cytometry analysis of CD73 expression percentage ( L ) ( n = 3, Student's two-tailed t test), histogram ( M ), and median fluorescent intensity quantification ( N ) ( n = 3, Student's two-tailed t test) on disassociated tumor from GB-bearing WT mice 3 weeks after implantation ( n = 3, Student's two-tailed t test). Data are shown as mean ± SEM. * p
    Figure Legend Snippet: GB expresses CD73 in vitro and upregulates GB-CD73 in CD73 −/− mice in vivo . A , B , Representative flow cytometry analysis of CD73 expression on GL261 ( A ) and U251 ( B ) GB cell lines. Data are representative of three experiments. C , Representative flow cytometry analysis of CD44 and CD73 expression on GL261. Data are representative of three experiments. D , G , Representative images of sham-implanted ( D ) and naive ( G ) mice brain sections stained with anti-CD73 antibody (brown) ( n = 3). E , F , H , Quantification of CD73 expression in caudoputamen or cortex of sham-implanted or naive mice was performed by measuring positive pixel count using ImageScope analysis program ( n = 3, three images analyzed/sample, Student's two-tailed t test). I , Representative images of brain sections from GL261-implanted mice stained with anti-CD73 antibody (brown) ( n = 3–4). J , Quantification of CD73 expression inside tumors was performed by measuring positive pixel count using ImageScope analysis program ( n = 3–4, 3 images analyzed/sample, Student's two-tailed t test). K , Densitometry quantification of CD73 protein expression inside tumors using ImageJ, normalizing to GAPDH and sham CTs ( n = 4–5, Student's two-tailed t test). L , M , N , Flow cytometry analysis of CD73 expression percentage ( L ) ( n = 3, Student's two-tailed t test), histogram ( M ), and median fluorescent intensity quantification ( N ) ( n = 3, Student's two-tailed t test) on disassociated tumor from GB-bearing WT mice 3 weeks after implantation ( n = 3, Student's two-tailed t test). Data are shown as mean ± SEM. * p

    Techniques Used: In Vitro, Mouse Assay, In Vivo, Flow Cytometry, Expressing, Staining, Two Tailed Test

    GB upregulates A 2B AR, and signaling through A 2B AR promotes MMP2 expression on GB. A , Representative image of primary astrocyte stained with anti-GFAP antibody (red), anti-NeuN (green), and DAPI (blue). B , Densitometry quantification of ARs protein expression in GL261 cells and in primary mouse astrocytes using ImageJ and normalizing to GAPDH ( n = 3, Student's two-tailed t test). C , Western blot analysis of ARs on human U251 cells ( n = 4). D – F , Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-GFAP antibody (red), DAPI (blue), and anti-A 1 AR (green) ( D ), anti-A 3 AR ( E ), or anti-A 2A AR ( F ) ( n = 3–4 per group). G , Number of ARs per microscopic field ( n = 3–8 per group, two-way ANOVA). H , Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-A 2B AR (green), anti-GFAP antibody (red), and DAPI (blue) ( n = 5–6 per GB-implanted group and n = 2 per sham-treated group). I – L , qRT-PCR analysis of MMP2 ( I ), MMP9 ( J ), TIMP1 ( K ), and TIMP2 ( L ) mRNA from GL261 treated with BAY 60–6583 ( n = 6, one-way ANOVA for I – L ). M , MMP FRET activity of GL261 cells treated with BAY 60–6583 ( n = 6, one-way ANOVA). Data are shown as mean ± SEM. * p
    Figure Legend Snippet: GB upregulates A 2B AR, and signaling through A 2B AR promotes MMP2 expression on GB. A , Representative image of primary astrocyte stained with anti-GFAP antibody (red), anti-NeuN (green), and DAPI (blue). B , Densitometry quantification of ARs protein expression in GL261 cells and in primary mouse astrocytes using ImageJ and normalizing to GAPDH ( n = 3, Student's two-tailed t test). C , Western blot analysis of ARs on human U251 cells ( n = 4). D – F , Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-GFAP antibody (red), DAPI (blue), and anti-A 1 AR (green) ( D ), anti-A 3 AR ( E ), or anti-A 2A AR ( F ) ( n = 3–4 per group). G , Number of ARs per microscopic field ( n = 3–8 per group, two-way ANOVA). H , Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-A 2B AR (green), anti-GFAP antibody (red), and DAPI (blue) ( n = 5–6 per GB-implanted group and n = 2 per sham-treated group). I – L , qRT-PCR analysis of MMP2 ( I ), MMP9 ( J ), TIMP1 ( K ), and TIMP2 ( L ) mRNA from GL261 treated with BAY 60–6583 ( n = 6, one-way ANOVA for I – L ). M , MMP FRET activity of GL261 cells treated with BAY 60–6583 ( n = 6, one-way ANOVA). Data are shown as mean ± SEM. * p

    Techniques Used: Expressing, Staining, Two Tailed Test, Western Blot, Mouse Assay, Quantitative RT-PCR, Activity Assay

    26) Product Images from "IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity"

    Article Title: IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

    IDH1 MUT sensitizes cells to cisplatin. A ) Proliferation assay using IDH1 WT and IDH1 MUT HCT116 cells. B ) U251 cell lines transduced with lentiviral vectors harboring the IDH1 WT and IDH1 MUT genes. Cells were counted after 72 h of cisplatin exposure, and the number of cells was normalized to the number of cells without treatment. C , D ) Colony-forming assay after 72 h of cisplatin exposure of IDH1 WT and IDH1 MUT HCT116 cells. The clonogenic fraction is the number of colonies divided by the number of cells plated, corrected for the plating efficiency. The scale on the y axis is logarithmic. E – G ) As in A , but after 72 h exposure of IDH1 WT and IDH1 MUT HCT116 cells to carboplatin and oxaliplatin. H , I ) As in A and B , but in the presence or absence of 5 μM of ROS-scavenging NAC. Significance values were obtained using 1-way ANOVA with the Tukey correction for multiple comparisons. * P
    Figure Legend Snippet: IDH1 MUT sensitizes cells to cisplatin. A ) Proliferation assay using IDH1 WT and IDH1 MUT HCT116 cells. B ) U251 cell lines transduced with lentiviral vectors harboring the IDH1 WT and IDH1 MUT genes. Cells were counted after 72 h of cisplatin exposure, and the number of cells was normalized to the number of cells without treatment. C , D ) Colony-forming assay after 72 h of cisplatin exposure of IDH1 WT and IDH1 MUT HCT116 cells. The clonogenic fraction is the number of colonies divided by the number of cells plated, corrected for the plating efficiency. The scale on the y axis is logarithmic. E – G ) As in A , but after 72 h exposure of IDH1 WT and IDH1 MUT HCT116 cells to carboplatin and oxaliplatin. H , I ) As in A and B , but in the presence or absence of 5 μM of ROS-scavenging NAC. Significance values were obtained using 1-way ANOVA with the Tukey correction for multiple comparisons. * P

    Techniques Used: Proliferation Assay, Transduction

    27) Product Images from "Salinomycin Potentiates the Cytotoxic Effects of TRAIL on Glioblastoma Cell Lines"

    Article Title: Salinomycin Potentiates the Cytotoxic Effects of TRAIL on Glioblastoma Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094438

    Salinomycin in combination with TRAIL markedly inhibits the growth of glioblastoma xenografts. 1×10 6 U251 cells were injected subcutaneously into nude mice as described in Materials and Methods , and treatment was started when the tumors reached 100 mm 3 . TRAIL (5 mg/Kg) and salinomycin (200 ng/kg) were administered 3 times/week, intraperitoneally. Control mice received vehicle only, according to the same schedule. Tumor volume was measured by caliper. Tumor fold increase is reported inn the Figure. Data represent the mean of 5 tumors±SEM, and significance of the results was evaluated by ANOVA and Bonferroni post-tests, as described in the section on statistical methods. A) Tumor growth kinetic; B) Tumor weight post-sacrifice; Representative pictures of tumor mass harvested at sacrifice, for each of the four experimental subgroups (top panels) and tumor weight post-sacrifice (mean of 5±SEM); C) Tumor histological analysis of tissutal sections stained with Hematoxylin Eosin (representative pictures); D) Immunofluorescence analysis of tumor apoptotic cells detected by TUNEL reaction (representative pictures): left panels: Hoechst 3326 staining; middle panels: TUNEL staining; right panels: merged of both stainings.
    Figure Legend Snippet: Salinomycin in combination with TRAIL markedly inhibits the growth of glioblastoma xenografts. 1×10 6 U251 cells were injected subcutaneously into nude mice as described in Materials and Methods , and treatment was started when the tumors reached 100 mm 3 . TRAIL (5 mg/Kg) and salinomycin (200 ng/kg) were administered 3 times/week, intraperitoneally. Control mice received vehicle only, according to the same schedule. Tumor volume was measured by caliper. Tumor fold increase is reported inn the Figure. Data represent the mean of 5 tumors±SEM, and significance of the results was evaluated by ANOVA and Bonferroni post-tests, as described in the section on statistical methods. A) Tumor growth kinetic; B) Tumor weight post-sacrifice; Representative pictures of tumor mass harvested at sacrifice, for each of the four experimental subgroups (top panels) and tumor weight post-sacrifice (mean of 5±SEM); C) Tumor histological analysis of tissutal sections stained with Hematoxylin Eosin (representative pictures); D) Immunofluorescence analysis of tumor apoptotic cells detected by TUNEL reaction (representative pictures): left panels: Hoechst 3326 staining; middle panels: TUNEL staining; right panels: merged of both stainings.

    Techniques Used: Injection, Mouse Assay, Staining, Immunofluorescence, TUNEL Assay

    Synergistic induction of cell death by salinomycin and TRAIL. (A) T98G and U251 cell lines have been grown for various periods of time (indicated in each panel) in the absence (NT) or in the presence of 1.2 µM salinomycin (Sal) added alone or in combination with increasing doses of TRAIL (from 10 to 200 ng/ml). At the end of the incubation the percentage of viable cells was determined by trypan blue exclusion test. The data represent the mean ± SEM values observed in three separate experiments. *** different from control (NT) at significance level p
    Figure Legend Snippet: Synergistic induction of cell death by salinomycin and TRAIL. (A) T98G and U251 cell lines have been grown for various periods of time (indicated in each panel) in the absence (NT) or in the presence of 1.2 µM salinomycin (Sal) added alone or in combination with increasing doses of TRAIL (from 10 to 200 ng/ml). At the end of the incubation the percentage of viable cells was determined by trypan blue exclusion test. The data represent the mean ± SEM values observed in three separate experiments. *** different from control (NT) at significance level p

    Techniques Used: Incubation

    Effect of silencing of RNA encoding TRAIL-R2 on the cell growth inhibition and induction of cell death induced by salinomycin or TRAIL added alone or in combination. T98G (left panels) and U251 (right panels) cells have been incubated for 72 h either with siRNA C or siRNA TRAIL-R1 or siRNA TRAIL-R2 and then incubated for additional 24 hours either in the absence of additives (Control, C) or in the presence of either salinomycin (10 µM) or TRAIL (50 ng/ml) or both compounds at the above doses. At the end of this time the cells have been recovered and evaluated for TRAIL-R2 expression (top panels) by flow cytometry or for the cell survival (middle panels) by cell counting after trypan blue staining or for the evaluation of cell death (assayed by flow cytometry after labelling with annexin V and propidium iodide). The data reported in the figure represent mean values ± SEM observed in three separate experiments. The statistical analysis of the data showed: in top panels a very significant difference between siRNA TRAIL-R2 and si RNA C and siRNA TRAIL-R1 (for both p
    Figure Legend Snippet: Effect of silencing of RNA encoding TRAIL-R2 on the cell growth inhibition and induction of cell death induced by salinomycin or TRAIL added alone or in combination. T98G (left panels) and U251 (right panels) cells have been incubated for 72 h either with siRNA C or siRNA TRAIL-R1 or siRNA TRAIL-R2 and then incubated for additional 24 hours either in the absence of additives (Control, C) or in the presence of either salinomycin (10 µM) or TRAIL (50 ng/ml) or both compounds at the above doses. At the end of this time the cells have been recovered and evaluated for TRAIL-R2 expression (top panels) by flow cytometry or for the cell survival (middle panels) by cell counting after trypan blue staining or for the evaluation of cell death (assayed by flow cytometry after labelling with annexin V and propidium iodide). The data reported in the figure represent mean values ± SEM observed in three separate experiments. The statistical analysis of the data showed: in top panels a very significant difference between siRNA TRAIL-R2 and si RNA C and siRNA TRAIL-R1 (for both p

    Techniques Used: Inhibition, Incubation, Expressing, Flow Cytometry, Cytometry, Cell Counting, Staining

    Effect of agonistic anti-TRAIL-R1 (Mapatumamab) or anti-TRAIL-R2 (Lexatumamab) mAbs on the induction of cell death of T98G, U87MG and U251 cell lines. The cells were incubated for 24(Sal), Mapatumamab (Mapa) and Lexatumamab (Lexa) and then analysed for cell vitality. The results represent the mean values ± SEM observed in three separate experiments. *** different from control (NT) at significance level p
    Figure Legend Snippet: Effect of agonistic anti-TRAIL-R1 (Mapatumamab) or anti-TRAIL-R2 (Lexatumamab) mAbs on the induction of cell death of T98G, U87MG and U251 cell lines. The cells were incubated for 24(Sal), Mapatumamab (Mapa) and Lexatumamab (Lexa) and then analysed for cell vitality. The results represent the mean values ± SEM observed in three separate experiments. *** different from control (NT) at significance level p

    Techniques Used: Incubation

    Effect of salinomycin and TRAIL added alone or in combination on caspase-8 activation and mitochondrial function. (A) Western blot analysis of cellular extracts derived from T98G and U251 cells incubated for 8 h and 24 h respectively, either in the absence (NT) or in the presence of salinomycin (10 µM), TRAIL (50 ng/ml) or salinomycin+TRAIL (at the above concentrations). The cell extracts were first run on SDS-PAGE, transferred to nitrocellulose membranes and blotted with either anti-human caspase-8 or anti beta-actin. One representative experiment out of three performed is shown. (B) Analysis of the mitochondrial membrane potential (Ψm) in T98G and U251 cells grown in the various conditions reported in the figure for 8 h and 24 h (T98G) or 48 h (U251). The proportion of cells exhibiting low Ψm is reported.
    Figure Legend Snippet: Effect of salinomycin and TRAIL added alone or in combination on caspase-8 activation and mitochondrial function. (A) Western blot analysis of cellular extracts derived from T98G and U251 cells incubated for 8 h and 24 h respectively, either in the absence (NT) or in the presence of salinomycin (10 µM), TRAIL (50 ng/ml) or salinomycin+TRAIL (at the above concentrations). The cell extracts were first run on SDS-PAGE, transferred to nitrocellulose membranes and blotted with either anti-human caspase-8 or anti beta-actin. One representative experiment out of three performed is shown. (B) Analysis of the mitochondrial membrane potential (Ψm) in T98G and U251 cells grown in the various conditions reported in the figure for 8 h and 24 h (T98G) or 48 h (U251). The proportion of cells exhibiting low Ψm is reported.

    Techniques Used: Activation Assay, Western Blot, Derivative Assay, Incubation, SDS Page

    Effect of salinomycin and recombinant human TRAIL on viability and cell cycling of GBM cell lines. (A) GBM cells T98G, U87MG and U251 have been plated either in the absence (not treated, NT) or in the presence of increasing concentrations of either salinomycin (Sal) or TRAIL and the percentage of viable cells after 48 h of treatment ( top panel ) and the number of living cells at 24 and 48 h ( bottom panel ) have been determined. Results reported in the top panel represent mean values ± SEM observed in three separate experiments. ** and *** different from control (NT) at significance level p
    Figure Legend Snippet: Effect of salinomycin and recombinant human TRAIL on viability and cell cycling of GBM cell lines. (A) GBM cells T98G, U87MG and U251 have been plated either in the absence (not treated, NT) or in the presence of increasing concentrations of either salinomycin (Sal) or TRAIL and the percentage of viable cells after 48 h of treatment ( top panel ) and the number of living cells at 24 and 48 h ( bottom panel ) have been determined. Results reported in the top panel represent mean values ± SEM observed in three separate experiments. ** and *** different from control (NT) at significance level p

    Techniques Used: Recombinant

    Effect of salinomycin on TRAIL-R2 expression. (A)Flow cytometric analysis of TRAIL-R2 expression of T98G and U251 cells grown for 24 h in the absence (NT) or in the presence of 10 µM salinomycin (Sal). (B) Western blot showing protein levels of TRAIL-R2 in U251 cells after treatment for the indicated time with TRAIL, salinomycin (Sal), TRAIL+salinomycin (TRAIL+Sal) and TRAIL+salinomycin+zVAD.
    Figure Legend Snippet: Effect of salinomycin on TRAIL-R2 expression. (A)Flow cytometric analysis of TRAIL-R2 expression of T98G and U251 cells grown for 24 h in the absence (NT) or in the presence of 10 µM salinomycin (Sal). (B) Western blot showing protein levels of TRAIL-R2 in U251 cells after treatment for the indicated time with TRAIL, salinomycin (Sal), TRAIL+salinomycin (TRAIL+Sal) and TRAIL+salinomycin+zVAD.

    Techniques Used: Expressing, Western Blot

    Effect of salinomycin and TRAIL added alone or in combination on the induction of apoptosis. T98G and U251 cells were incubated for 24(NT) or in the presence of salinomycin (10 µM), TRAIL (50 ng/ml) or salinomycin+TRAIL (at the above concentrations). The induction of apoptosis was evaluated by Annexin V-FITC and propidium iodide (PI) staining.
    Figure Legend Snippet: Effect of salinomycin and TRAIL added alone or in combination on the induction of apoptosis. T98G and U251 cells were incubated for 24(NT) or in the presence of salinomycin (10 µM), TRAIL (50 ng/ml) or salinomycin+TRAIL (at the above concentrations). The induction of apoptosis was evaluated by Annexin V-FITC and propidium iodide (PI) staining.

    Techniques Used: Incubation, Staining

    Mechanism of cell death induced by salinomycin+TRAIL. (A) Western blot analysis of cellular extracts derived from T98G and U251 cells incubated for 24 h either in the absence (NT) or in the presence of either salinomycin 10 µM, TRAIL (50 ng/ml) or salinomycin+TRAIL (at the above concentrations) or salinomycin+TRAIL+the caspase inhibitor zVADfmk (40 µM). The cell extracts were first run on SDS-PAGE, transferred to nitrocellulose membranes and blotted with either anti-human caspase-3, anti-human PARP or anti beta-actin. One representative experiment out of three performed is shown. (B) T98G, U87MG and U251 cells have been grown for 24 h in the different experimental conditions reported in the figure and then analysed for cell vitality. The proportion of viable cells is reported (mean values ± SEM observed in three separate experiments). *** different from control (NT) at significance level p
    Figure Legend Snippet: Mechanism of cell death induced by salinomycin+TRAIL. (A) Western blot analysis of cellular extracts derived from T98G and U251 cells incubated for 24 h either in the absence (NT) or in the presence of either salinomycin 10 µM, TRAIL (50 ng/ml) or salinomycin+TRAIL (at the above concentrations) or salinomycin+TRAIL+the caspase inhibitor zVADfmk (40 µM). The cell extracts were first run on SDS-PAGE, transferred to nitrocellulose membranes and blotted with either anti-human caspase-3, anti-human PARP or anti beta-actin. One representative experiment out of three performed is shown. (B) T98G, U87MG and U251 cells have been grown for 24 h in the different experimental conditions reported in the figure and then analysed for cell vitality. The proportion of viable cells is reported (mean values ± SEM observed in three separate experiments). *** different from control (NT) at significance level p

    Techniques Used: Western Blot, Derivative Assay, Incubation, SDS Page

    28) Product Images from "CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A2B Adenosine Receptor Signaling"

    Article Title: CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A2B Adenosine Receptor Signaling

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1118-18.2019

    GB expresses CD73 in vitro and upregulates GB-CD73 in CD73 −/− mice in vivo . A , B , Representative flow cytometry analysis of CD73 expression on GL261 ( A ) and U251 ( B ) GB cell lines. Data are representative of three experiments. C , Representative flow cytometry analysis of CD44 and CD73 expression on GL261. Data are representative of three experiments. D , G , Representative images of sham-implanted ( D ) and naive ( G ) mice brain sections stained with anti-CD73 antibody (brown) ( n = 3). E , F , H , Quantification of CD73 expression in caudoputamen or cortex of sham-implanted or naive mice was performed by measuring positive pixel count using ImageScope analysis program ( n = 3, three images analyzed/sample, Student's two-tailed t test). I , Representative images of brain sections from GL261-implanted mice stained with anti-CD73 antibody (brown) ( n = 3–4). J , Quantification of CD73 expression inside tumors was performed by measuring positive pixel count using ImageScope analysis program ( n = 3–4, 3 images analyzed/sample, Student's two-tailed t test). K , Densitometry quantification of CD73 protein expression inside tumors using ImageJ, normalizing to GAPDH and sham CTs ( n = 4–5, Student's two-tailed t test). L , M , N , Flow cytometry analysis of CD73 expression percentage ( L ) ( n = 3, Student's two-tailed t test), histogram ( M ), and median fluorescent intensity quantification ( N ) ( n = 3, Student's two-tailed t test) on disassociated tumor from GB-bearing WT mice 3 weeks after implantation ( n = 3, Student's two-tailed t test). Data are shown as mean ± SEM. * p
    Figure Legend Snippet: GB expresses CD73 in vitro and upregulates GB-CD73 in CD73 −/− mice in vivo . A , B , Representative flow cytometry analysis of CD73 expression on GL261 ( A ) and U251 ( B ) GB cell lines. Data are representative of three experiments. C , Representative flow cytometry analysis of CD44 and CD73 expression on GL261. Data are representative of three experiments. D , G , Representative images of sham-implanted ( D ) and naive ( G ) mice brain sections stained with anti-CD73 antibody (brown) ( n = 3). E , F , H , Quantification of CD73 expression in caudoputamen or cortex of sham-implanted or naive mice was performed by measuring positive pixel count using ImageScope analysis program ( n = 3, three images analyzed/sample, Student's two-tailed t test). I , Representative images of brain sections from GL261-implanted mice stained with anti-CD73 antibody (brown) ( n = 3–4). J , Quantification of CD73 expression inside tumors was performed by measuring positive pixel count using ImageScope analysis program ( n = 3–4, 3 images analyzed/sample, Student's two-tailed t test). K , Densitometry quantification of CD73 protein expression inside tumors using ImageJ, normalizing to GAPDH and sham CTs ( n = 4–5, Student's two-tailed t test). L , M , N , Flow cytometry analysis of CD73 expression percentage ( L ) ( n = 3, Student's two-tailed t test), histogram ( M ), and median fluorescent intensity quantification ( N ) ( n = 3, Student's two-tailed t test) on disassociated tumor from GB-bearing WT mice 3 weeks after implantation ( n = 3, Student's two-tailed t test). Data are shown as mean ± SEM. * p

    Techniques Used: In Vitro, Mouse Assay, In Vivo, Flow Cytometry, Cytometry, Expressing, Staining, Two Tailed Test

    GB upregulates A 2B AR, and signaling through A 2B AR promotes MMP2 expression on GB. A , Representative image of primary astrocyte stained with anti-GFAP antibody (red), anti-NeuN (green), and DAPI (blue). B , Densitometry quantification of ARs protein expression in GL261 cells and in primary mouse astrocytes using ImageJ and normalizing to GAPDH ( n = 3, Student's two-tailed t test). C , Western blot analysis of ARs on human U251 cells ( n = 4). D – F , Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-GFAP antibody (red), DAPI (blue), and anti-A 1 AR (green) ( D ), anti-A 3 AR ( E ), or anti-A 2A AR ( F ) ( n = 3–4 per group). G , Number of ARs per microscopic field ( n = 3–8 per group, two-way ANOVA). H , Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-A 2B AR (green), anti-GFAP antibody (red), and DAPI (blue) ( n = 5–6 per GB-implanted group and n = 2 per sham-treated group). I – L , qRT-PCR analysis of MMP2 ( I ), MMP9 ( J ), TIMP1 ( K ), and TIMP2 ( L ) mRNA from GL261 treated with BAY 60–6583 ( n = 6, one-way ANOVA for I – L ). M , MMP FRET activity of GL261 cells treated with BAY 60–6583 ( n = 6, one-way ANOVA). Data are shown as mean ± SEM. * p
    Figure Legend Snippet: GB upregulates A 2B AR, and signaling through A 2B AR promotes MMP2 expression on GB. A , Representative image of primary astrocyte stained with anti-GFAP antibody (red), anti-NeuN (green), and DAPI (blue). B , Densitometry quantification of ARs protein expression in GL261 cells and in primary mouse astrocytes using ImageJ and normalizing to GAPDH ( n = 3, Student's two-tailed t test). C , Western blot analysis of ARs on human U251 cells ( n = 4). D – F , Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-GFAP antibody (red), DAPI (blue), and anti-A 1 AR (green) ( D ), anti-A 3 AR ( E ), or anti-A 2A AR ( F ) ( n = 3–4 per group). G , Number of ARs per microscopic field ( n = 3–8 per group, two-way ANOVA). H , Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-A 2B AR (green), anti-GFAP antibody (red), and DAPI (blue) ( n = 5–6 per GB-implanted group and n = 2 per sham-treated group). I – L , qRT-PCR analysis of MMP2 ( I ), MMP9 ( J ), TIMP1 ( K ), and TIMP2 ( L ) mRNA from GL261 treated with BAY 60–6583 ( n = 6, one-way ANOVA for I – L ). M , MMP FRET activity of GL261 cells treated with BAY 60–6583 ( n = 6, one-way ANOVA). Data are shown as mean ± SEM. * p

    Techniques Used: Expressing, Staining, Two Tailed Test, Western Blot, Mouse Assay, Quantitative RT-PCR, Activity Assay

    29) Product Images from "Overexpression of CLEC18B Associates With the Proliferation, Migration, and Prognosis of Glioblastoma"

    Article Title: Overexpression of CLEC18B Associates With the Proliferation, Migration, and Prognosis of Glioblastoma

    Journal: ASN NEURO

    doi: 10.1177/1759091418781949

    Growth and colony-formation abilities of GBM cells were inhibited by CLEC18B knockdown. (a to c) Transfection efficacy of CLEC18B si-RNA in U87 and U251 cells was analyzed by qPCR and Western blot assay, respectively. (d) Assessing the effects of CLEC18B downregulation on the proliferation ability of U87 and U251 cells using CCK-8 assay. (e) Evaluating the effects of CLEC18B knockdown on the colony-formation ability of U87 and U251 cells. All values are shown as mean ± SD, ** p
    Figure Legend Snippet: Growth and colony-formation abilities of GBM cells were inhibited by CLEC18B knockdown. (a to c) Transfection efficacy of CLEC18B si-RNA in U87 and U251 cells was analyzed by qPCR and Western blot assay, respectively. (d) Assessing the effects of CLEC18B downregulation on the proliferation ability of U87 and U251 cells using CCK-8 assay. (e) Evaluating the effects of CLEC18B knockdown on the colony-formation ability of U87 and U251 cells. All values are shown as mean ± SD, ** p

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay

    Migration and invasion capabilities of U87 and U251 cells were prohibited by CLEC18B knockdown. (a) The mobility of U87 cells was measured by wound-healing assay. (b) The migration and invasion of U87 cells were tested by transwell assay. (c) The mobility of U251 cells was measured by wound-healing assay. (d) The migration and invasion of U251 cells were tested by transwell assay. All values are shown as mean ± SD, ** p
    Figure Legend Snippet: Migration and invasion capabilities of U87 and U251 cells were prohibited by CLEC18B knockdown. (a) The mobility of U87 cells was measured by wound-healing assay. (b) The migration and invasion of U87 cells were tested by transwell assay. (c) The mobility of U251 cells was measured by wound-healing assay. (d) The migration and invasion of U251 cells were tested by transwell assay. All values are shown as mean ± SD, ** p

    Techniques Used: Migration, Wound Healing Assay, Transwell Assay

    30) Product Images from "Baohuoside I via mTOR Apoptotic Signaling to Inhibit Glioma Cell Growth"

    Article Title: Baohuoside I via mTOR Apoptotic Signaling to Inhibit Glioma Cell Growth

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S265803

    Baohuoside I inhibits cell proliferation of U251. ( A ) RTCA proliferation analysis of U251 cells treated with 0, 20 and 50 μM Baohuoside I. ( B ) CCK-8 assays of U251 cells treated with 0, 20 and 50 μM Baohuoside I for 24 h. ( C ) Ki67 immunofluorescence staining of U251 cells treated with 0, 20 and 50 μM Baohuoside I for 24 h. ( D ) Histogram representing the Ki67 positive rate of U251 cells in each group. ( E ) Colony formation experiment of U251 cells treated with 0.20 and 50 μM Baohuoside I. ( F ) Histogram representing the colony formation assays of U251 cells in each group. All experiments were repeated 3 times and data are presented as mean ± SD. * p
    Figure Legend Snippet: Baohuoside I inhibits cell proliferation of U251. ( A ) RTCA proliferation analysis of U251 cells treated with 0, 20 and 50 μM Baohuoside I. ( B ) CCK-8 assays of U251 cells treated with 0, 20 and 50 μM Baohuoside I for 24 h. ( C ) Ki67 immunofluorescence staining of U251 cells treated with 0, 20 and 50 μM Baohuoside I for 24 h. ( D ) Histogram representing the Ki67 positive rate of U251 cells in each group. ( E ) Colony formation experiment of U251 cells treated with 0.20 and 50 μM Baohuoside I. ( F ) Histogram representing the colony formation assays of U251 cells in each group. All experiments were repeated 3 times and data are presented as mean ± SD. * p

    Techniques Used: CCK-8 Assay, Immunofluorescence, Staining

    Baohuoside I promotes apoptosis of U251 cells. ( A ) Flow cytometry analysis of U251 cells after 24 h treated with 0, 20 and 50 μM Baohuoside I. ( B ) Histogram of flow analysis in each group. ( C ) Western blot analysis of apoptosis-related proteins. ( D ) Histogram representing the relative protein expression level of p62. ( E ) Histogram representing the relative protein expression level of LC3-I. ( F ) Histogram representing the relative protein expression level of LC3-II. ( G ) Histogram representing the relative protein expression level of Bcl-2. ( H ) Histogram representing the relative protein expression level of Bax. ( I ) Histogram representing the relative protein expression level of cleaved caspase 3. ( J ) Histogram representing the relative protein expression level of cleaved caspase 8. All experiments were repeated 3 times and data are presented as mean ± SD. * p
    Figure Legend Snippet: Baohuoside I promotes apoptosis of U251 cells. ( A ) Flow cytometry analysis of U251 cells after 24 h treated with 0, 20 and 50 μM Baohuoside I. ( B ) Histogram of flow analysis in each group. ( C ) Western blot analysis of apoptosis-related proteins. ( D ) Histogram representing the relative protein expression level of p62. ( E ) Histogram representing the relative protein expression level of LC3-I. ( F ) Histogram representing the relative protein expression level of LC3-II. ( G ) Histogram representing the relative protein expression level of Bcl-2. ( H ) Histogram representing the relative protein expression level of Bax. ( I ) Histogram representing the relative protein expression level of cleaved caspase 3. ( J ) Histogram representing the relative protein expression level of cleaved caspase 8. All experiments were repeated 3 times and data are presented as mean ± SD. * p

    Techniques Used: Flow Cytometry, Western Blot, Expressing

    Baohuoside I inhibits the invasion and migration of U251 cells. ( A ) Wound-healing assays of U251 cells treated with Baohuoside I for 24 h. ( B ) Histogram representing the wound-healing assays of U251 cells in each group. ( C ) Transwell assays of U251 cells treated with Baohuoside I for 24 h. ( D ) Histogram representing the Transwell assays of U251 cells in each group. All experiments were repeated 3 times and data are presented as mean ± SD. *** p
    Figure Legend Snippet: Baohuoside I inhibits the invasion and migration of U251 cells. ( A ) Wound-healing assays of U251 cells treated with Baohuoside I for 24 h. ( B ) Histogram representing the wound-healing assays of U251 cells in each group. ( C ) Transwell assays of U251 cells treated with Baohuoside I for 24 h. ( D ) Histogram representing the Transwell assays of U251 cells in each group. All experiments were repeated 3 times and data are presented as mean ± SD. *** p

    Techniques Used: Migration

    Baohuoside I inhibits the growth of glioma in vivo. ( A and B ) U251 cells were subcutaneously injected into nude mice. At the end of the assay, tumors were removed and photographed. ( C ) Tumor volume was detected at various time points. ( D ) Tumor weight was determined after tumors were removed. ( E-G ) Western blot analysis of protein expression. All experiments were repeated 3 times and data were presented as mean ± SD. ** P
    Figure Legend Snippet: Baohuoside I inhibits the growth of glioma in vivo. ( A and B ) U251 cells were subcutaneously injected into nude mice. At the end of the assay, tumors were removed and photographed. ( C ) Tumor volume was detected at various time points. ( D ) Tumor weight was determined after tumors were removed. ( E-G ) Western blot analysis of protein expression. All experiments were repeated 3 times and data were presented as mean ± SD. ** P

    Techniques Used: In Vivo, Injection, Mouse Assay, Western Blot, Expressing

    Baohuoside I inhibits the activity of mTOR signaling. ( A and D ) Western blot analysis of protein expression level in U251 cells treated with 0, 20 and 50 μM Baohuoside I for 24 h. ( B ) Histogram representing the ratio of p-JAK2/JAK2. ( C ) Histogram representing the ratio of p-mTOR/mTOR. ( E ) Histogram representing the ratio of p-AMPKα1/AMPKα1. ( F ) Histogram representing the ratio of p-p70/p70. All experiments were repeated 3 times and data are presented as mean ± SD. * p
    Figure Legend Snippet: Baohuoside I inhibits the activity of mTOR signaling. ( A and D ) Western blot analysis of protein expression level in U251 cells treated with 0, 20 and 50 μM Baohuoside I for 24 h. ( B ) Histogram representing the ratio of p-JAK2/JAK2. ( C ) Histogram representing the ratio of p-mTOR/mTOR. ( E ) Histogram representing the ratio of p-AMPKα1/AMPKα1. ( F ) Histogram representing the ratio of p-p70/p70. All experiments were repeated 3 times and data are presented as mean ± SD. * p

    Techniques Used: Activity Assay, Western Blot, Expressing

    31) Product Images from "PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a)"

    Article Title: PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a)

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

    doi: 10.1073/pnas.0706790105

    FOXO3a impedes p300-dependent HIF-1 transcriptional activity. ( A ) U251 cells were exposed to 1.5% O 2 ± 20 nM LMB for 16 h, and, subsequently, nuclear fractions were collected. IP was carried out on the U251 nuclear lysates by using anti-HIF-1α,
    Figure Legend Snippet: FOXO3a impedes p300-dependent HIF-1 transcriptional activity. ( A ) U251 cells were exposed to 1.5% O 2 ± 20 nM LMB for 16 h, and, subsequently, nuclear fractions were collected. IP was carried out on the U251 nuclear lysates by using anti-HIF-1α,

    Techniques Used: Activity Assay

    32) Product Images from "A New Platinum-Based Prodrug Candidate for Chemotherapy and Its Synergistic Effect With Hadrontherapy: Novel Strategy to Treat Glioblastoma"

    Article Title: A New Platinum-Based Prodrug Candidate for Chemotherapy and Its Synergistic Effect With Hadrontherapy: Novel Strategy to Treat Glioblastoma

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2021.589906

    (A) Western blotting analysis of full-length poly[ADP-ribose] polymerase 1 (PARP-1, 116 kDa) and cleaved PARP-1 (89 kDa). Representative Western blotting bands showing the expression levels of full-length PARP-1 (116 kDa) and cleaved PARP-1 in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], after carbon ion irradiation (4 Gy dose) alone, and in combined exposure conditions [40 μM CDDP or 10 μM Pt(IV)Ac-POA 48-h CT + 4 Gy carbon ion irradiation]. The result was quantified from the average of three different samples. The density bands of full-length (116 kDa) and cleaved (89 kDa) PARP-1 were compared to the loading control and actin (43 kDa). (B) Western blotting study of p62/SQSTM1 as a typical marker of autophagy activation. Histograms representing density band quantification of p62/SQSTM1 (61 kDa) in the controls, differently treated U251 cells, i.e., after 48-h CT with 40 μM CDDP or 10 μM Pt(IV)Ac-POA, after carbon ion radiation (4 Gy dose) alone, and in combined exposure conditions [40 μM CDDP or 10 μM Pt(IV)Ac-POA 48-h CT + 4 Gy carbon ion radiation]. The means, obtained from three independent experiments, have been quantified and compared to the loading control and actin (43 kDa). Statistical significance calculated as follows: * control vs . CDDP + 4 Gy carbon ion radiation; # CDDP vs . CDDP + 4 Gy carbon ion radiation; § Pt(IV)Ac-POA vs . CDDP + 4 Gy carbon ion radiation; + 4 Gy carbon ion radiation alone vs . CDDP + 4 Gy carbon ion radiation; CDDP + 4 Gy carbon ion radiation vs . Pt(IV)Ac-POA + 4 Gy carbon ion radiation. ** p
    Figure Legend Snippet: (A) Western blotting analysis of full-length poly[ADP-ribose] polymerase 1 (PARP-1, 116 kDa) and cleaved PARP-1 (89 kDa). Representative Western blotting bands showing the expression levels of full-length PARP-1 (116 kDa) and cleaved PARP-1 in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], after carbon ion irradiation (4 Gy dose) alone, and in combined exposure conditions [40 μM CDDP or 10 μM Pt(IV)Ac-POA 48-h CT + 4 Gy carbon ion irradiation]. The result was quantified from the average of three different samples. The density bands of full-length (116 kDa) and cleaved (89 kDa) PARP-1 were compared to the loading control and actin (43 kDa). (B) Western blotting study of p62/SQSTM1 as a typical marker of autophagy activation. Histograms representing density band quantification of p62/SQSTM1 (61 kDa) in the controls, differently treated U251 cells, i.e., after 48-h CT with 40 μM CDDP or 10 μM Pt(IV)Ac-POA, after carbon ion radiation (4 Gy dose) alone, and in combined exposure conditions [40 μM CDDP or 10 μM Pt(IV)Ac-POA 48-h CT + 4 Gy carbon ion radiation]. The means, obtained from three independent experiments, have been quantified and compared to the loading control and actin (43 kDa). Statistical significance calculated as follows: * control vs . CDDP + 4 Gy carbon ion radiation; # CDDP vs . CDDP + 4 Gy carbon ion radiation; § Pt(IV)Ac-POA vs . CDDP + 4 Gy carbon ion radiation; + 4 Gy carbon ion radiation alone vs . CDDP + 4 Gy carbon ion radiation; CDDP + 4 Gy carbon ion radiation vs . Pt(IV)Ac-POA + 4 Gy carbon ion radiation. ** p

    Techniques Used: Western Blot, Expressing, Irradiation, Marker, Activation Assay

    Left panel: Apoptotic pathway investigated by fluorescence microscopy in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. Double immunolabeling for active caspase-3 and caspase-8 ( red fluorescence ) and actin ( green fluorescence ). Nuclear counterstaining with Hoechst 33258 ( blue fluorescence ). Inserts : high-magnification micrographs showing caspase-immunopositive nuclei and collapsed cytoskeleton. Scale bar , 40 μm. Right panel: Histograms showing the percentage value of caspase-3- and caspase-8-immunopositive cells, respectively. Statistical significance calculated as follows: ∗ control vs . each experimental condition; # CDDP vs . other treatments; § Pt(IV)Ac-POA vs . REC conditions; + CDDP REC conditions vs . Pt(IV)AC-POA REC. ∗∗∗ p
    Figure Legend Snippet: Left panel: Apoptotic pathway investigated by fluorescence microscopy in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. Double immunolabeling for active caspase-3 and caspase-8 ( red fluorescence ) and actin ( green fluorescence ). Nuclear counterstaining with Hoechst 33258 ( blue fluorescence ). Inserts : high-magnification micrographs showing caspase-immunopositive nuclei and collapsed cytoskeleton. Scale bar , 40 μm. Right panel: Histograms showing the percentage value of caspase-3- and caspase-8-immunopositive cells, respectively. Statistical significance calculated as follows: ∗ control vs . each experimental condition; # CDDP vs . other treatments; § Pt(IV)Ac-POA vs . REC conditions; + CDDP REC conditions vs . Pt(IV)AC-POA REC. ∗∗∗ p

    Techniques Used: Fluorescence, Microscopy, Immunolabeling

    Immunofluorescence study of autophagy activation. (A) Double immunolabeling for p62/SQSTM1 ( green fluorescence ) and LC3B ( red fluorescence ). DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm. (B) Western blotting data: p62/SQSTM1. Histograms representing density band quantification of p62/SQSTM1 in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. Statistical significance: * ρ
    Figure Legend Snippet: Immunofluorescence study of autophagy activation. (A) Double immunolabeling for p62/SQSTM1 ( green fluorescence ) and LC3B ( red fluorescence ). DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm. (B) Western blotting data: p62/SQSTM1. Histograms representing density band quantification of p62/SQSTM1 in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. Statistical significance: * ρ

    Techniques Used: Immunofluorescence, Activation Assay, Immunolabeling, Fluorescence, Western Blot

    Different experimental conditions: chemotherapeutics exposure alone and followed by carbon ion irradiation. The left column shows phase-contrast micrographs of U251 cells cultured in flask sterile on slide in different experimental conditions before being processed for immunofluorescence reactions. The right column displays caspase-3 immunolabeling ( red fluorescence ) in the control (CTR), differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA] alone or followed by carbon ion radiation at 2 or 4 Gy dose. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 40 μm. Histograms ( lower right part ) representing the percentage values of caspase-3-immunopositive cells.
    Figure Legend Snippet: Different experimental conditions: chemotherapeutics exposure alone and followed by carbon ion irradiation. The left column shows phase-contrast micrographs of U251 cells cultured in flask sterile on slide in different experimental conditions before being processed for immunofluorescence reactions. The right column displays caspase-3 immunolabeling ( red fluorescence ) in the control (CTR), differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA] alone or followed by carbon ion radiation at 2 or 4 Gy dose. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 40 μm. Histograms ( lower right part ) representing the percentage values of caspase-3-immunopositive cells.

    Techniques Used: Irradiation, Cell Culture, Immunofluorescence, Immunolabeling, Fluorescence

    Intracellular organelle investigation by immunofluorescence. Double immunocytochemical detection of the Golgi apparatus ( red fluorescence ) and actin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)- acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bars , 20 μm.
    Figure Legend Snippet: Intracellular organelle investigation by immunofluorescence. Double immunocytochemical detection of the Golgi apparatus ( red fluorescence ) and actin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)- acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bars , 20 μm.

    Techniques Used: Immunofluorescence, Fluorescence

    Apoptotic pathway investigated using immunocytochemistry after treatment with chemotherapeutics alone and combined with carbon ion irradiation at different doses. Double immunofluorescence detection of poly[ADP-ribose] polymerase 1 (PARP-1, red fluorescence ) and α-tubulin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in combined exposure conditions [40 μM CDDP or 10 μM Pt(IV)Ac-POA 48-h CT + 2 or 4 Gy carbon ion radiation]. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm.
    Figure Legend Snippet: Apoptotic pathway investigated using immunocytochemistry after treatment with chemotherapeutics alone and combined with carbon ion irradiation at different doses. Double immunofluorescence detection of poly[ADP-ribose] polymerase 1 (PARP-1, red fluorescence ) and α-tubulin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in combined exposure conditions [40 μM CDDP or 10 μM Pt(IV)Ac-POA 48-h CT + 2 or 4 Gy carbon ion radiation]. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm.

    Techniques Used: Immunocytochemistry, Irradiation, Immunofluorescence, Fluorescence

    TEM ultrastructural analysis. (A) U251 control cells: arrowhead indicates the mitochondria with a physiological feature; asterisk identifies a well-visible nucleolus with a decondensed chromatin. A well-organized endoplasmic reticulum (ER) is also visible in the perinuclear zone ( thin arrows ). (B–D) U251 cells after 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA] 48-h continuous treatment (CT). (B) Chromatin condensation ( asterisk ) and absence of the nuclear envelope ( thin arrows ) in apoptotic cells. (C) Several vacuoles engulfed with cell debris ( arrowheads ) in autophagic cells. (D) Typical necroptotic features accompanied by a partially preserved cytoplasmic membrane. Scale bar , 5 μm.
    Figure Legend Snippet: TEM ultrastructural analysis. (A) U251 control cells: arrowhead indicates the mitochondria with a physiological feature; asterisk identifies a well-visible nucleolus with a decondensed chromatin. A well-organized endoplasmic reticulum (ER) is also visible in the perinuclear zone ( thin arrows ). (B–D) U251 cells after 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA] 48-h continuous treatment (CT). (B) Chromatin condensation ( asterisk ) and absence of the nuclear envelope ( thin arrows ) in apoptotic cells. (C) Several vacuoles engulfed with cell debris ( arrowheads ) in autophagic cells. (D) Typical necroptotic features accompanied by a partially preserved cytoplasmic membrane. Scale bar , 5 μm.

    Techniques Used: Transmission Electron Microscopy

    Extrinsic apoptotic pathway evaluation by double immunocytochemical detection of receptor-interacting protein kinase 1 (RIP1, red fluorescence ) and α-tubulin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)- acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm.
    Figure Legend Snippet: Extrinsic apoptotic pathway evaluation by double immunocytochemical detection of receptor-interacting protein kinase 1 (RIP1, red fluorescence ) and α-tubulin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)- acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm.

    Techniques Used: Fluorescence

    Apoptotic pathway investigated using fluorescence microscopy. (A) Double immunofluorescence detection of poly[ADP-ribose] polymerase 1 (PARP-1, red fluorescence ) and α-tubulin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Inserts : high-magnification micrographs showing PARP-1 translocation from the nucleus to the cytoplasm. Thin arrow indicates PARP-1 “spot-like” labeling, while arrowhead designates phagocytic cell. Cytoskeletal alterations are also noticeable. Scale bar , 20 μm. (B) Western blotting data showing full-length PARP-1 (116 kDa) and cleaved PARP-1 (89 kDa) bands, respectively, compared to the loading control and actin (43 kDa).
    Figure Legend Snippet: Apoptotic pathway investigated using fluorescence microscopy. (A) Double immunofluorescence detection of poly[ADP-ribose] polymerase 1 (PARP-1, red fluorescence ) and α-tubulin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Inserts : high-magnification micrographs showing PARP-1 translocation from the nucleus to the cytoplasm. Thin arrow indicates PARP-1 “spot-like” labeling, while arrowhead designates phagocytic cell. Cytoskeletal alterations are also noticeable. Scale bar , 20 μm. (B) Western blotting data showing full-length PARP-1 (116 kDa) and cleaved PARP-1 (89 kDa) bands, respectively, compared to the loading control and actin (43 kDa).

    Techniques Used: Fluorescence, Microscopy, Immunofluorescence, Translocation Assay, Labeling, Western Blot

    Investigation of necroptotic pathway using fluorescence microscopy in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato) platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. Double immunolabeling detection of mixed lineage kinase domain-like (MLKL, red fluorescence ) and actin ( green fluorescence ). DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm.
    Figure Legend Snippet: Investigation of necroptotic pathway using fluorescence microscopy in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato) platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. Double immunolabeling detection of mixed lineage kinase domain-like (MLKL, red fluorescence ) and actin ( green fluorescence ). DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm.

    Techniques Used: Fluorescence, Microscopy, Immunolabeling

    (A) Schematic representation of ( OC -6-44)-acetatodiamminedichlorido (2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA] synthesis process and carbon ion irradiation. (a) Cisplatin ( cis -dichlorodiammineplatinum, CDDP), 2-(2-propynyl)octanoic acid (POA), and the related Pt(IV) mixed derivative (Pt(IV)Ac-POA). (b) Flask sterile on slide used for U251 cell culture irradiation. (c) Equipment setup in the irradiation room facility at the CNAO Foundation. (d) Flasks positioned in a water phantom placed at a depth of 15 cm, corresponding to the central position of a 6-cm-wide spread-out Bragg peak (SOBP), even before irradiation with a horizontal beam. (B) Effects of Pt(IV)Ac-POA and CDDP on cell viability and proliferation of human U251 MB cell lineage. Cell viability/proliferation obtained using MTS assay after standard acute exposure, i.e., 48-h continuous treatment (CT), to increasing concentrations (1–40 μM) of either Pt(IV)Ac-POA or CDDP. The relative cell viability is expressed as a percentage relative to the untreated control cells. Data represent the mean ± SD. Statistical analysis by Student’s t -test: different from CDDP ( ∗∗ p
    Figure Legend Snippet: (A) Schematic representation of ( OC -6-44)-acetatodiamminedichlorido (2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA] synthesis process and carbon ion irradiation. (a) Cisplatin ( cis -dichlorodiammineplatinum, CDDP), 2-(2-propynyl)octanoic acid (POA), and the related Pt(IV) mixed derivative (Pt(IV)Ac-POA). (b) Flask sterile on slide used for U251 cell culture irradiation. (c) Equipment setup in the irradiation room facility at the CNAO Foundation. (d) Flasks positioned in a water phantom placed at a depth of 15 cm, corresponding to the central position of a 6-cm-wide spread-out Bragg peak (SOBP), even before irradiation with a horizontal beam. (B) Effects of Pt(IV)Ac-POA and CDDP on cell viability and proliferation of human U251 MB cell lineage. Cell viability/proliferation obtained using MTS assay after standard acute exposure, i.e., 48-h continuous treatment (CT), to increasing concentrations (1–40 μM) of either Pt(IV)Ac-POA or CDDP. The relative cell viability is expressed as a percentage relative to the untreated control cells. Data represent the mean ± SD. Statistical analysis by Student’s t -test: different from CDDP ( ∗∗ p

    Techniques Used: Irradiation, Cell Culture, MTS Assay

    Intracellular organelle investigation by immunofluorescence. Double immunolabeling for the mitochondria ( red fluorescence ) and α-tubulin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl) octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bars , 20 μm.
    Figure Legend Snippet: Intracellular organelle investigation by immunofluorescence. Double immunolabeling for the mitochondria ( red fluorescence ) and α-tubulin ( green fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl) octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bars , 20 μm.

    Techniques Used: Immunofluorescence, Immunolabeling, Fluorescence

    Recovered (REC) conditions after combined experimental protocol (chemotherapeutics exposure + carbon ion radiation). The left column shows phase-contrast micrographs of U251 cells cultured in flask sterile on slide in different experimental conditions before being processed for immunofluorescence reactions. The right column displays caspase-3 immunolabeling ( red fluorescence ) in the controls (CTR) and all different REC conditions, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA] alone, after carbon ion irradiation (4 Gy dose) alone, or in combined exposure conditions [40 μM CDDP or 10 μM Pt(IV)Ac-POA 48-h CT + 2 or 4 Gy carbon ion radiation]. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm. Histograms ( lower right part ) represent the percentage values of caspase-3-immunopositive cells.
    Figure Legend Snippet: Recovered (REC) conditions after combined experimental protocol (chemotherapeutics exposure + carbon ion radiation). The left column shows phase-contrast micrographs of U251 cells cultured in flask sterile on slide in different experimental conditions before being processed for immunofluorescence reactions. The right column displays caspase-3 immunolabeling ( red fluorescence ) in the controls (CTR) and all different REC conditions, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA] alone, after carbon ion irradiation (4 Gy dose) alone, or in combined exposure conditions [40 μM CDDP or 10 μM Pt(IV)Ac-POA 48-h CT + 2 or 4 Gy carbon ion radiation]. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm. Histograms ( lower right part ) represent the percentage values of caspase-3-immunopositive cells.

    Techniques Used: Cell Culture, Immunofluorescence, Immunolabeling, Fluorescence, Irradiation

    Caspase-independent cell death pathway activation investigated by fluorescence microscopy. Double immunostaining for mitochondria ( green fluorescence ) and apoptosis-inducing factor (AIF, red fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm.
    Figure Legend Snippet: Caspase-independent cell death pathway activation investigated by fluorescence microscopy. Double immunostaining for mitochondria ( green fluorescence ) and apoptosis-inducing factor (AIF, red fluorescence ) in the controls, differently treated U251 cells, i.e., after 48-h continuous treatment (CT) with 40 μM cis -dichlorodiammineplatinum (CDDP) or 10 μM ( OC -6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV) [Pt(IV)Ac-POA], and in the recovered (REC) conditions. DNA counterstaining with Hoechst 33258 ( blue fluorescence ). Scale bar , 20 μm.

    Techniques Used: Activation Assay, Fluorescence, Microscopy, Double Immunostaining

    33) Product Images from "Recurrent somatic mutation of FAT1 in multiple human cancers leads to aberrant Wnt activation"

    Article Title: Recurrent somatic mutation of FAT1 in multiple human cancers leads to aberrant Wnt activation

    Journal: Nature genetics

    doi: 10.1038/ng.2538

    Functional relationship between β-catenin and FAT1 in the regulation of proliferation (a) Western blot showing co-transfection of β-catenin and FAT1 in chinese hamster ovary (CHO) cells. (b) Growth curve demonstrating accelerated cell growth with over-expression of β-catenin, repressed with co-transfection with FAT1. Error bars represent 1 standard deviation. Experiments performed in CHO cells, in quadruplicate. (c) BrdU (left) and cell cycle (right) assays demonstrate enhancement in DNA synthesis and cells entering S phase, after β-catenin over-expression, repressed with FAT1 co-transfection. Experiments performed in CHO cells, in triplicate. (d) Western blot showing co-transfection of siRNAs targeting FAT1 and β-catenin, in U251 glioma cells. (e) Growth curve demonstrating accelerated growth after FAT1 knockdown, reversed by concurrent knockdown of β-catenin. Error bars represent 1 standard deviation. Experiments performed in quadruplicate, in U251 glioma cells. (f) BrdU (left) and cell cycle (right) assays demonstrate enhancement in DNA synthesis and cells entering S phase, after FAT1 knockdown, repressed with concurrent β-catenin knockdown, in U251 glioma cells. Experiments performed in triplicate. These data are shown with a 2 nd set of siRNAs in Supplementary Fig. 3 . *p
    Figure Legend Snippet: Functional relationship between β-catenin and FAT1 in the regulation of proliferation (a) Western blot showing co-transfection of β-catenin and FAT1 in chinese hamster ovary (CHO) cells. (b) Growth curve demonstrating accelerated cell growth with over-expression of β-catenin, repressed with co-transfection with FAT1. Error bars represent 1 standard deviation. Experiments performed in CHO cells, in quadruplicate. (c) BrdU (left) and cell cycle (right) assays demonstrate enhancement in DNA synthesis and cells entering S phase, after β-catenin over-expression, repressed with FAT1 co-transfection. Experiments performed in CHO cells, in triplicate. (d) Western blot showing co-transfection of siRNAs targeting FAT1 and β-catenin, in U251 glioma cells. (e) Growth curve demonstrating accelerated growth after FAT1 knockdown, reversed by concurrent knockdown of β-catenin. Error bars represent 1 standard deviation. Experiments performed in quadruplicate, in U251 glioma cells. (f) BrdU (left) and cell cycle (right) assays demonstrate enhancement in DNA synthesis and cells entering S phase, after FAT1 knockdown, repressed with concurrent β-catenin knockdown, in U251 glioma cells. Experiments performed in triplicate. These data are shown with a 2 nd set of siRNAs in Supplementary Fig. 3 . *p

    Techniques Used: Functional Assay, Western Blot, Cotransfection, Over Expression, Standard Deviation, DNA Synthesis

    34) Product Images from "MiR-210 Up-Regulation Inhibits Proliferation and Induces Apoptosis in Glioma Cells by Targeting SIN3A"

    Article Title: MiR-210 Up-Regulation Inhibits Proliferation and Induces Apoptosis in Glioma Cells by Targeting SIN3A

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.892994

    ( A ) Impact of SIN3A gene expression changes on the proliferation ability of U251 cells. ( B ) Impact of SIN3A gene expression changes the apoptosis rate of U251 cells. ( C ) Western blot analysis for SIN3A and cleaved Caspase3 expression of U251 cells treated with anti-miR-210 and siRNA-SIN3A transfection. GAPDH was used as a reference control. ( D ) quantitative analysis of the relative protein levels of SIN3A and cleaved Caspase3 normalized to those of GAPDH was shown. *, ** P
    Figure Legend Snippet: ( A ) Impact of SIN3A gene expression changes on the proliferation ability of U251 cells. ( B ) Impact of SIN3A gene expression changes the apoptosis rate of U251 cells. ( C ) Western blot analysis for SIN3A and cleaved Caspase3 expression of U251 cells treated with anti-miR-210 and siRNA-SIN3A transfection. GAPDH was used as a reference control. ( D ) quantitative analysis of the relative protein levels of SIN3A and cleaved Caspase3 normalized to those of GAPDH was shown. *, ** P

    Techniques Used: Expressing, Western Blot, Transfection

    qRT-PCR analysis for the expression of miR-210 and SIN3A in brain glioma tissue and U251 cells. Compared to paracancerous tissue, the expression of miR-210 was higher in brain glioma tissue and U251 cells ( P
    Figure Legend Snippet: qRT-PCR analysis for the expression of miR-210 and SIN3A in brain glioma tissue and U251 cells. Compared to paracancerous tissue, the expression of miR-210 was higher in brain glioma tissue and U251 cells ( P

    Techniques Used: Quantitative RT-PCR, Expressing

    35) Product Images from "WW domain-binding protein 2 acts as an oncogene by modulating the activity of the glycolytic enzyme ENO1 in glioma"

    Article Title: WW domain-binding protein 2 acts as an oncogene by modulating the activity of the glycolytic enzyme ENO1 in glioma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0376-5

    Transiently suppressed ENO1 and Homer3 inhibits WBP2-mediated U251 cell overgrowth and metastasis. a and b MTT assay was performed to test the effect of ENO1 and Homer3 silenced by the increased cell proliferation ability induced by WBP2 overexpression. c Ablation of ENO1 and Homer3 reduced the healing rates in EGFP-WBP2 group cells. Scale bar, 200 μm. siNC, cells transfected with negative control siRNA; siENO1, cells transfected with ENO1-siRNA; siHomer3, cells transfected with Homer3-siRNA. * P
    Figure Legend Snippet: Transiently suppressed ENO1 and Homer3 inhibits WBP2-mediated U251 cell overgrowth and metastasis. a and b MTT assay was performed to test the effect of ENO1 and Homer3 silenced by the increased cell proliferation ability induced by WBP2 overexpression. c Ablation of ENO1 and Homer3 reduced the healing rates in EGFP-WBP2 group cells. Scale bar, 200 μm. siNC, cells transfected with negative control siRNA; siENO1, cells transfected with ENO1-siRNA; siHomer3, cells transfected with Homer3-siRNA. * P

    Techniques Used: MTT Assay, Over Expression, Transfection, Negative Control

    WBP2 affects the expression and activity of ENO1 in glioma cells. a WBP2 affects the transcription of ENO1 in U251 and U87 cells. b , c Phosphorylation of Akt and expression of ENO1 measured by western blot using specific antibodies in four cell groups. d Measurements of Enolase-1 activity in different group cells, following an enolase activity assay, e , f Relative glucose uptake and lactate production of eight group cells, assessed by their corresponding detection kits. g , h Relative glucose uptake and lactate production in xenografted tumor. i The proposed model of WBP2-mediated cellular processes in Glioma cells. Tubulin served as the internal control. WBP2 + siNC, EGFP-WBP2 group cells transfected with negative control siRNA; WBP2 + siWBP2, EGFP-WBP2 group cells transfected with WBP2-siRNA; WBP2 + siENO1, EGFP-WBP2 group cells transfected with ENO1-siRNA; WBP2 + siHomer3, EGFP-WBP2 group cells transfected with Homer3-siRNA; WBP2 + W, EGFP-WBP2 group cells treated with wortmannin; WBP2 + siENO1 + W, EGFP-WBP2 group cells transfected with ENO1 siRNA that were pretreated with wortmannin. * P
    Figure Legend Snippet: WBP2 affects the expression and activity of ENO1 in glioma cells. a WBP2 affects the transcription of ENO1 in U251 and U87 cells. b , c Phosphorylation of Akt and expression of ENO1 measured by western blot using specific antibodies in four cell groups. d Measurements of Enolase-1 activity in different group cells, following an enolase activity assay, e , f Relative glucose uptake and lactate production of eight group cells, assessed by their corresponding detection kits. g , h Relative glucose uptake and lactate production in xenografted tumor. i The proposed model of WBP2-mediated cellular processes in Glioma cells. Tubulin served as the internal control. WBP2 + siNC, EGFP-WBP2 group cells transfected with negative control siRNA; WBP2 + siWBP2, EGFP-WBP2 group cells transfected with WBP2-siRNA; WBP2 + siENO1, EGFP-WBP2 group cells transfected with ENO1-siRNA; WBP2 + siHomer3, EGFP-WBP2 group cells transfected with Homer3-siRNA; WBP2 + W, EGFP-WBP2 group cells treated with wortmannin; WBP2 + siENO1 + W, EGFP-WBP2 group cells transfected with ENO1 siRNA that were pretreated with wortmannin. * P

    Techniques Used: Expressing, Activity Assay, Western Blot, Transfection, Negative Control

    Effects of WBP2 on cell growth and cell cycle progress of U251. a–d After seeding for 24 h, U251 and U87 cells (eight groups of cells) were processed with MTT assay (EGFP-Vector, EGFP-WBP2, siNC, and siWBP2) at 0, 24, 48, and 72 h. e Cell cycle analysis of U251 cells transfected with EGFP-WBP2, EGFP-Vector, siNC and siWBP2. f Cell cycle analysis of U87 cells transfected with EGFP-WBP2, EGFP-Vector, siNC and siWBP2. g , h Representative quantity of cell numbers in S-phase in the eight group cells. EGFP-Vector, control stable cell line transfected with pEGFP-C1 plasmid in U251 or U87 cells; pEGFP-WBP2, stable cell lines transfected with pEGFP-WBP2; siNC, cells transfected with negative control siRNA; siWBP2, downregulation of WBP2 expression transfected with siRNA of WBP2 in U251 or U87 cells. *P
    Figure Legend Snippet: Effects of WBP2 on cell growth and cell cycle progress of U251. a–d After seeding for 24 h, U251 and U87 cells (eight groups of cells) were processed with MTT assay (EGFP-Vector, EGFP-WBP2, siNC, and siWBP2) at 0, 24, 48, and 72 h. e Cell cycle analysis of U251 cells transfected with EGFP-WBP2, EGFP-Vector, siNC and siWBP2. f Cell cycle analysis of U87 cells transfected with EGFP-WBP2, EGFP-Vector, siNC and siWBP2. g , h Representative quantity of cell numbers in S-phase in the eight group cells. EGFP-Vector, control stable cell line transfected with pEGFP-C1 plasmid in U251 or U87 cells; pEGFP-WBP2, stable cell lines transfected with pEGFP-WBP2; siNC, cells transfected with negative control siRNA; siWBP2, downregulation of WBP2 expression transfected with siRNA of WBP2 in U251 or U87 cells. *P

    Techniques Used: MTT Assay, Plasmid Preparation, Cell Cycle Assay, Transfection, Stable Transfection, Negative Control, Expressing

    WBP2 directly binds to ENO1 and Homer3 in U251 cells . a Recombinant human GST-WBP2 protein was purified by adsorption on L-glutathione-sepharose. b SDS-PAGE showed several proteins identified by GST large-scale pulldown. c , d Exogenous ENO1 and Homer3 transfected in U251 cells binds to GST-WBP2 fusion protein, as revealed by GST pulldown using immobilized GST-WBP2
    Figure Legend Snippet: WBP2 directly binds to ENO1 and Homer3 in U251 cells . a Recombinant human GST-WBP2 protein was purified by adsorption on L-glutathione-sepharose. b SDS-PAGE showed several proteins identified by GST large-scale pulldown. c , d Exogenous ENO1 and Homer3 transfected in U251 cells binds to GST-WBP2 fusion protein, as revealed by GST pulldown using immobilized GST-WBP2

    Techniques Used: Recombinant, Purification, Adsorption, SDS Page, Transfection

    WBP2 regulates the migration ability of U251. a Photos of the wounded cells were taken at 0, 24, 48, 72 h after wound induction in EGFP-Vector and EGFP-WBP2 group cells. Scale bar, 200 μm. b Quantitative analysis of the healing rates of EGFP-Vector and EGFP-WBP2 group cells. c Images from the wound-healing experiment of the U251 cells after transient downregulation of WBP2. Scale bar, 200 μm. d Quantitative analysis of the healing rates of U251-siNC and U251-siWBP2 group cells. e Efficiency of WBP2 on the migration ability in U251 cells was determined by transwell assay. Scale bar, 50 μm. f Cells on the lower surface of the membrane quantified and analyzed with five randomly selected fields. g Images of EGFP-Vector and EGFP-WBP2 group cells from mice and mice tumor tissue ( n = 5/group). Scale bar, 1 cm. h Tumor weight of EGFP-Vector and EGFP-WBP2 cells. i The tumor growth curves of the EGFP-Vector and EGFP-WBP2 groups. * P
    Figure Legend Snippet: WBP2 regulates the migration ability of U251. a Photos of the wounded cells were taken at 0, 24, 48, 72 h after wound induction in EGFP-Vector and EGFP-WBP2 group cells. Scale bar, 200 μm. b Quantitative analysis of the healing rates of EGFP-Vector and EGFP-WBP2 group cells. c Images from the wound-healing experiment of the U251 cells after transient downregulation of WBP2. Scale bar, 200 μm. d Quantitative analysis of the healing rates of U251-siNC and U251-siWBP2 group cells. e Efficiency of WBP2 on the migration ability in U251 cells was determined by transwell assay. Scale bar, 50 μm. f Cells on the lower surface of the membrane quantified and analyzed with five randomly selected fields. g Images of EGFP-Vector and EGFP-WBP2 group cells from mice and mice tumor tissue ( n = 5/group). Scale bar, 1 cm. h Tumor weight of EGFP-Vector and EGFP-WBP2 cells. i The tumor growth curves of the EGFP-Vector and EGFP-WBP2 groups. * P

    Techniques Used: Migration, Plasmid Preparation, Transwell Assay, Mouse Assay

    36) Product Images from "Knockdown of LGR5 suppresses the proliferation of glioma cells in vitro and in vivo"

    Article Title: Knockdown of LGR5 suppresses the proliferation of glioma cells in vitro and in vivo

    Journal: Oncology Reports

    doi: 10.3892/or.2013.2826

    LGR5 is overexpressed in glioma cell lines, and LGR5 interference reduces its expression in U87 cells. (A) Protein levels in normal cultured primary astrocytes and in U118, U87 and U251 glioma cells as assessed by western blot analysis. (B) RNA interference reduced the expression of LGR5 in U87 cells as assessed by western blot analysis. Con, parental U87 cells; NC, negative control; KD, LGR5 knockdown cells. (C) mRNA levels in normal cultured primary astrocytes and in U118, U87 and U251 glioma cells as assessed by qRT-PCR analysis. N, normal cultured primary astrocytes.
    Figure Legend Snippet: LGR5 is overexpressed in glioma cell lines, and LGR5 interference reduces its expression in U87 cells. (A) Protein levels in normal cultured primary astrocytes and in U118, U87 and U251 glioma cells as assessed by western blot analysis. (B) RNA interference reduced the expression of LGR5 in U87 cells as assessed by western blot analysis. Con, parental U87 cells; NC, negative control; KD, LGR5 knockdown cells. (C) mRNA levels in normal cultured primary astrocytes and in U118, U87 and U251 glioma cells as assessed by qRT-PCR analysis. N, normal cultured primary astrocytes.

    Techniques Used: Expressing, Cell Culture, Western Blot, Negative Control, Quantitative RT-PCR

    37) Product Images from "MicroRNA-770 affects proliferation and cell cycle transition by directly targeting CDK8 in glioma"

    Article Title: MicroRNA-770 affects proliferation and cell cycle transition by directly targeting CDK8 in glioma

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0694-9

    CDK8 siRNA suppresses the proliferation of human glioma U251 cells. a CDK8 siRNA decreased the activity of U251 cells at 48 and 72 h. b CDK8 siRNA inhibited U251 cell proliferation. c Flow cytometric analysis showed the percentage of U251 cells in the G0/G1, S and G2/M phases. G0/G1 phase cells increased after CDK8 siRNA treatment, and S and G2/M phase cells were reduced. d The data showed the percentage of early and late apoptosis after CDK8 siRNA treatment. e The knockdown efficiency of CDK8 siRNA in U251 cells. f CDK8, β-catenin and cyclin D1 protein expression was examined after CDK8 siRNA treatment. *p
    Figure Legend Snippet: CDK8 siRNA suppresses the proliferation of human glioma U251 cells. a CDK8 siRNA decreased the activity of U251 cells at 48 and 72 h. b CDK8 siRNA inhibited U251 cell proliferation. c Flow cytometric analysis showed the percentage of U251 cells in the G0/G1, S and G2/M phases. G0/G1 phase cells increased after CDK8 siRNA treatment, and S and G2/M phase cells were reduced. d The data showed the percentage of early and late apoptosis after CDK8 siRNA treatment. e The knockdown efficiency of CDK8 siRNA in U251 cells. f CDK8, β-catenin and cyclin D1 protein expression was examined after CDK8 siRNA treatment. *p

    Techniques Used: Activity Assay, Flow Cytometry, Expressing

    CDK8 overexpression rescues miR-770-induced cellular phenotypes in glioma cells. a U251 cell activity was measured after cotransfection with CDK8 and miR-770 vectors. b U251 cell proliferation was examined after cotransfection with CDK8 and miR-770 vectors. c Cell cycle was determined in U251 cells at 48 h. d Apoptosis was detected in U251 cells at 48 h. e CDK8 overexpression rescued CDK8 mRNA expression levels reduced by miR-770. f CDK8, β-catenin and cyclin D1 protein expression was examined after cotransfection with CDK8 and miR-770 vectors. *p
    Figure Legend Snippet: CDK8 overexpression rescues miR-770-induced cellular phenotypes in glioma cells. a U251 cell activity was measured after cotransfection with CDK8 and miR-770 vectors. b U251 cell proliferation was examined after cotransfection with CDK8 and miR-770 vectors. c Cell cycle was determined in U251 cells at 48 h. d Apoptosis was detected in U251 cells at 48 h. e CDK8 overexpression rescued CDK8 mRNA expression levels reduced by miR-770. f CDK8, β-catenin and cyclin D1 protein expression was examined after cotransfection with CDK8 and miR-770 vectors. *p

    Techniques Used: Over Expression, Activity Assay, Cotransfection, Expressing

    miR-770 inhibits human glioma U251 cell proliferation and induces G1-S cell cycle arrest and apoptosis. a miR-770 expression was detected in U251 cells after miR-770 overexpression. b miR-770 expression was examined in U251 cells after anti-miR-770 treatment. c miR-770 overexpression decreased cell activity at 48 and 72 h after transfection. d Anti-miR-770 increased cell activity at 48 and 72 h after transfection. e miR-770 overexpression suppressed glioma cell proliferation. f Anti-miR-770 promoted glioma cell growth. g The histogram represents the proportion of cells in the G0/G1, S and G2/M phases after miR-770 overexpression. h The ratio of cells in the G0/G1, S and G2/M phases after anti-miR-770 transfection. i The data revealed the ratios of early and late apoptosis after miR-770 overexpression. j The data showed the proportions of early apoptosis and late apoptosis after anti-miR-770 transfection. *p
    Figure Legend Snippet: miR-770 inhibits human glioma U251 cell proliferation and induces G1-S cell cycle arrest and apoptosis. a miR-770 expression was detected in U251 cells after miR-770 overexpression. b miR-770 expression was examined in U251 cells after anti-miR-770 treatment. c miR-770 overexpression decreased cell activity at 48 and 72 h after transfection. d Anti-miR-770 increased cell activity at 48 and 72 h after transfection. e miR-770 overexpression suppressed glioma cell proliferation. f Anti-miR-770 promoted glioma cell growth. g The histogram represents the proportion of cells in the G0/G1, S and G2/M phases after miR-770 overexpression. h The ratio of cells in the G0/G1, S and G2/M phases after anti-miR-770 transfection. i The data revealed the ratios of early and late apoptosis after miR-770 overexpression. j The data showed the proportions of early apoptosis and late apoptosis after anti-miR-770 transfection. *p

    Techniques Used: Expressing, Over Expression, Activity Assay, Transfection

    miR-770 regulates the Wnt/β-catenin signaling pathway in human glioma cells by targeting CDK8. a CDK8 mRNA was determined in U251 cells after miR-770 overexpression. b CDK8 mRNA was examined in U251 cells after anti-miR-770 treatment. c miR-770 overexpression inhibited the expression of the CDK8, β-catenin and cyclin D1 proteins in U251 cells. d Anti-miR-770 promoted CDK8, β-catenin and cyclin D1 protein expression. *p
    Figure Legend Snippet: miR-770 regulates the Wnt/β-catenin signaling pathway in human glioma cells by targeting CDK8. a CDK8 mRNA was determined in U251 cells after miR-770 overexpression. b CDK8 mRNA was examined in U251 cells after anti-miR-770 treatment. c miR-770 overexpression inhibited the expression of the CDK8, β-catenin and cyclin D1 proteins in U251 cells. d Anti-miR-770 promoted CDK8, β-catenin and cyclin D1 protein expression. *p

    Techniques Used: Over Expression, Expressing

    38) Product Images from "Long non-coding RNA ferritin heavy polypeptide 1 pseudogene 3 controls glioma cell proliferation and apoptosis via regulation of the microRNA-224-5p/tumor protein D52 axis"

    Article Title: Long non-coding RNA ferritin heavy polypeptide 1 pseudogene 3 controls glioma cell proliferation and apoptosis via regulation of the microRNA-224-5p/tumor protein D52 axis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.9491

    LncRNA FTH1P3 is upregulated in glioma tissues, and its upregulation promotes glioma cell proliferation and inhibits apoptosis. (A) The expression of lncRNA FTH1P3 in low- and high-grade glioma tissues and adjacent normal tissues. (B) Expression of lncRNA FTH1P3 in glioma U251 cells transfected with pc-FTH1P3, si-FTH1P3 and the corresponding controls. (C) The cell viability of each group after 24, 48 h and 72 h was determined with an MTT assay. (D) A BrdU incorporation assay was performed to determine cell proliferation (magnification, ×400). (E) The percentage of apoptotic cells in each group was determined by flow cytometry. (F) Western blot analysis of BAX/BCL2 expression. *P
    Figure Legend Snippet: LncRNA FTH1P3 is upregulated in glioma tissues, and its upregulation promotes glioma cell proliferation and inhibits apoptosis. (A) The expression of lncRNA FTH1P3 in low- and high-grade glioma tissues and adjacent normal tissues. (B) Expression of lncRNA FTH1P3 in glioma U251 cells transfected with pc-FTH1P3, si-FTH1P3 and the corresponding controls. (C) The cell viability of each group after 24, 48 h and 72 h was determined with an MTT assay. (D) A BrdU incorporation assay was performed to determine cell proliferation (magnification, ×400). (E) The percentage of apoptotic cells in each group was determined by flow cytometry. (F) Western blot analysis of BAX/BCL2 expression. *P

    Techniques Used: Expressing, Transfection, MTT Assay, BrdU Incorporation Assay, Flow Cytometry, Cytometry, Western Blot

    TPD52 is a target of miR-224-5p. (A) TargetScanHuman was used to predict the binding sequence between TPD52 and miR-224-5p. (B) Luciferase activity of TPD52 3′-UTR-WT and TPD52 3′-UTR-MUT in the presence of miR-224-5p mimic or mimic control. (C) mRNA and (D) protein expression of TPD52 in U251 cells transfected with miR-224-5p mimic or mimic control. **P
    Figure Legend Snippet: TPD52 is a target of miR-224-5p. (A) TargetScanHuman was used to predict the binding sequence between TPD52 and miR-224-5p. (B) Luciferase activity of TPD52 3′-UTR-WT and TPD52 3′-UTR-MUT in the presence of miR-224-5p mimic or mimic control. (C) mRNA and (D) protein expression of TPD52 in U251 cells transfected with miR-224-5p mimic or mimic control. **P

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

    The miR-224-5p/TPD52 axis may be a functional mechanism of FTH1P3 in glioma. (A) TPD52 expression in low- and high-grade glioma tissues and adjacent normal tissues. (B) The expression of TPD52 in glioma U251 cells treated with mimic control, miR-224-5p mimic, miR-224-5p mimic + blank control, miR-224-5p mimic + pc-TPD52. (C) The cell viability of each group after 24, 48 and 72 h was determined by MTT assay. (D) A BrdU incorporation assay showed BrdU-positive cells in different transfected groups (magnification, ×400). (E) The percentage of apoptotic cells in each group was detected by flow cytometry. (F) Western blot analysis showed the expression levels of BAX/BCL2 in different transfected groups. (G) Schematic representation of the potential mechanism of action of lncRNA FTH1P23 in glioma. *P
    Figure Legend Snippet: The miR-224-5p/TPD52 axis may be a functional mechanism of FTH1P3 in glioma. (A) TPD52 expression in low- and high-grade glioma tissues and adjacent normal tissues. (B) The expression of TPD52 in glioma U251 cells treated with mimic control, miR-224-5p mimic, miR-224-5p mimic + blank control, miR-224-5p mimic + pc-TPD52. (C) The cell viability of each group after 24, 48 and 72 h was determined by MTT assay. (D) A BrdU incorporation assay showed BrdU-positive cells in different transfected groups (magnification, ×400). (E) The percentage of apoptotic cells in each group was detected by flow cytometry. (F) Western blot analysis showed the expression levels of BAX/BCL2 in different transfected groups. (G) Schematic representation of the potential mechanism of action of lncRNA FTH1P23 in glioma. *P

    Techniques Used: Functional Assay, Expressing, MTT Assay, BrdU Incorporation Assay, Transfection, Flow Cytometry, Cytometry, Western Blot

    lncRNA FTH1P3 inhibits miR-224-5p expression. (A) miR-224-5p expression in low- and high-grade glioma tissues in addition to adjacent normal tissues. (B) miR-224-5p expression in glioma U251 cells transfected with pc-FTH1P3, si-FTH1P3 and the corresponding controls. (C) The expression of miR-224-5p in glioma U251 cells transfected with miR-224-5p mimic and the corresponding control. *P
    Figure Legend Snippet: lncRNA FTH1P3 inhibits miR-224-5p expression. (A) miR-224-5p expression in low- and high-grade glioma tissues in addition to adjacent normal tissues. (B) miR-224-5p expression in glioma U251 cells transfected with pc-FTH1P3, si-FTH1P3 and the corresponding controls. (C) The expression of miR-224-5p in glioma U251 cells transfected with miR-224-5p mimic and the corresponding control. *P

    Techniques Used: Expressing, Transfection

    39) Product Images from "IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity"

    Article Title: IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

    IDH1 MUT sensitizes cells to cisplatin. A ) Proliferation assay using IDH1 WT and IDH1 MUT HCT116 cells. B ) U251 cell lines transduced with lentiviral vectors harboring the IDH1 WT and IDH1 MUT genes. Cells were counted after 72 h of cisplatin exposure, and the number of cells was normalized to the number of cells without treatment. C , D ) Colony-forming assay after 72 h of cisplatin exposure of IDH1 WT and IDH1 MUT HCT116 cells. The clonogenic fraction is the number of colonies divided by the number of cells plated, corrected for the plating efficiency. The scale on the y axis is logarithmic. E – G ) As in A , but after 72 h exposure of IDH1 WT and IDH1 MUT HCT116 cells to carboplatin and oxaliplatin. H , I ) As in A and B , but in the presence or absence of 5 μM of ROS-scavenging NAC. Significance values were obtained using 1-way ANOVA with the Tukey correction for multiple comparisons. * P
    Figure Legend Snippet: IDH1 MUT sensitizes cells to cisplatin. A ) Proliferation assay using IDH1 WT and IDH1 MUT HCT116 cells. B ) U251 cell lines transduced with lentiviral vectors harboring the IDH1 WT and IDH1 MUT genes. Cells were counted after 72 h of cisplatin exposure, and the number of cells was normalized to the number of cells without treatment. C , D ) Colony-forming assay after 72 h of cisplatin exposure of IDH1 WT and IDH1 MUT HCT116 cells. The clonogenic fraction is the number of colonies divided by the number of cells plated, corrected for the plating efficiency. The scale on the y axis is logarithmic. E – G ) As in A , but after 72 h exposure of IDH1 WT and IDH1 MUT HCT116 cells to carboplatin and oxaliplatin. H , I ) As in A and B , but in the presence or absence of 5 μM of ROS-scavenging NAC. Significance values were obtained using 1-way ANOVA with the Tukey correction for multiple comparisons. * P

    Techniques Used: Proliferation Assay, Transduction

    40) Product Images from "STAT3 Activation Promotes Oncolytic HSV1 Replication in Glioma Cells"

    Article Title: STAT3 Activation Promotes Oncolytic HSV1 Replication in Glioma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071932

    oHSV replication in human glioblastoma U251 cells, stably expressing control shRNA (pLKO/control shRNAi) or STAT3 knock down (pLKO/shSTAT3 RNAi). a; Western blot analysis of STAT3 in the stably transfected control shRNAi or shSTAT3 RNAi. b; GFP reporter signal from oHSV (rQNestin34.5) was detected 1 day after virus infection (MOI 0.05) of U251 cell lines stably knocked down for STAT3 gene expression. c; In vitro viral replication plaque assay. Cells were infected (MOI 0.05) with oHSV (rQNestin34.5) or wild type HSV-1 (F strain) and yields of progeny virus were determined on Vero cells. Black bars represent oHSV. White bars represent F strain. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. ** P
    Figure Legend Snippet: oHSV replication in human glioblastoma U251 cells, stably expressing control shRNA (pLKO/control shRNAi) or STAT3 knock down (pLKO/shSTAT3 RNAi). a; Western blot analysis of STAT3 in the stably transfected control shRNAi or shSTAT3 RNAi. b; GFP reporter signal from oHSV (rQNestin34.5) was detected 1 day after virus infection (MOI 0.05) of U251 cell lines stably knocked down for STAT3 gene expression. c; In vitro viral replication plaque assay. Cells were infected (MOI 0.05) with oHSV (rQNestin34.5) or wild type HSV-1 (F strain) and yields of progeny virus were determined on Vero cells. Black bars represent oHSV. White bars represent F strain. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. ** P

    Techniques Used: Stable Transfection, Expressing, shRNA, Western Blot, Transfection, Infection, In Vitro, Plaque Assay, Standard Deviation

    Effect of pharmacologic inhibition of STAT3 on oHSV replication. A . U251 cells transfected with an expression vector, encoding a STAT3-responsive transcriptional element driving luciferase were infected with oHSV (MOI 1.0) (Black bars) or control (White bars) following treatment with VPA or the STAT3 inhibitor, LLL12, for 20 hours. Cells were harvested and STAT3 promoter transcriptional activity was assayed. Each data point represents the mean of triplicate samples. Error bars represent the standard deviation. *** P
    Figure Legend Snippet: Effect of pharmacologic inhibition of STAT3 on oHSV replication. A . U251 cells transfected with an expression vector, encoding a STAT3-responsive transcriptional element driving luciferase were infected with oHSV (MOI 1.0) (Black bars) or control (White bars) following treatment with VPA or the STAT3 inhibitor, LLL12, for 20 hours. Cells were harvested and STAT3 promoter transcriptional activity was assayed. Each data point represents the mean of triplicate samples. Error bars represent the standard deviation. *** P

    Techniques Used: Inhibition, Transfection, Expressing, Plasmid Preparation, Luciferase, Infection, Activity Assay, Standard Deviation

    Cytotoxicity of oHSV against glioma cells with altered STAT3 gene expression. a; Control transfected cells (open circle) were compared to STAT3 over-expressing cells HA-STAT3 (closed circle) and FLAG-STAT3 (closed triangle). ED 50 of pCR/CMV, HA-STAT3, and FLAG-STAT3 were calculated to occur at MOI 0.04, 0.019, and 0.022, respectively. b; lentivirus control transfected cells (closed square) were compared with STAT3 knock down cells (open square). ED 50 of shControl and shSTAT3 were calculated to be at MOIs 0.025 and > 1.0, respectively. Cell viability (measured by MTT) of U251 glioma cells was assayed 3 days after infection of oHSV (rQNestin34.5) at different MOI. Data shown represents the mean ± SD of three replicates for each sample.
    Figure Legend Snippet: Cytotoxicity of oHSV against glioma cells with altered STAT3 gene expression. a; Control transfected cells (open circle) were compared to STAT3 over-expressing cells HA-STAT3 (closed circle) and FLAG-STAT3 (closed triangle). ED 50 of pCR/CMV, HA-STAT3, and FLAG-STAT3 were calculated to occur at MOI 0.04, 0.019, and 0.022, respectively. b; lentivirus control transfected cells (closed square) were compared with STAT3 knock down cells (open square). ED 50 of shControl and shSTAT3 were calculated to be at MOIs 0.025 and > 1.0, respectively. Cell viability (measured by MTT) of U251 glioma cells was assayed 3 days after infection of oHSV (rQNestin34.5) at different MOI. Data shown represents the mean ± SD of three replicates for each sample.

    Techniques Used: Expressing, Transfection, Polymerase Chain Reaction, MTT Assay, Infection

    Expression of Type-I IFN responsive genes, PKR, STAT1, IRF3, and IRF7, 8 hours following oHSV (rQNestin34.5) infection (MOI 1.0) in A: U251 cells stably expressing HA-STAT3 or FLAG-STAT3 compared to control pCR/CMV and B: U251 cells stably expressing shSTAT3 RNAi or control. Each data point represents the mean of triplicate samples. Error bars indicate standard deviation. * P
    Figure Legend Snippet: Expression of Type-I IFN responsive genes, PKR, STAT1, IRF3, and IRF7, 8 hours following oHSV (rQNestin34.5) infection (MOI 1.0) in A: U251 cells stably expressing HA-STAT3 or FLAG-STAT3 compared to control pCR/CMV and B: U251 cells stably expressing shSTAT3 RNAi or control. Each data point represents the mean of triplicate samples. Error bars indicate standard deviation. * P

    Techniques Used: Expressing, Infection, Stable Transfection, Polymerase Chain Reaction, Standard Deviation

    Effect of VPA on STAT3 expression in glioma cells in the presence or absence of oHSV infection. A : Expression of nuclear phosho-STAT3 (p-STAT3), total cellular STAT3, activated NF-kappaB (p52), and Histone H3. Twenty hours after incubation with or without 3 mM VPA, U251 cells were infected with oHSV (MOI 1.0). At the indicated times following infection, total cellular or nuclear fractions were collected and analyzed by Western blot. The 0, 0.5 and 1.0 hour (hr) times indicates time after oHSV infection. Before infection, cells were incubated with VPA or vehicle for 20 hours. The results of blot densitometry are also provided in tabular format in the lower part of the panel. B : Gene expression level of STAT3 was detected using quantitative RT-PCR at the indicated time points following VPA treatment (3 mM) of U251 cells. Each data point represents the mean of triplicate samples. Error bars indicate standard deviation. ** P
    Figure Legend Snippet: Effect of VPA on STAT3 expression in glioma cells in the presence or absence of oHSV infection. A : Expression of nuclear phosho-STAT3 (p-STAT3), total cellular STAT3, activated NF-kappaB (p52), and Histone H3. Twenty hours after incubation with or without 3 mM VPA, U251 cells were infected with oHSV (MOI 1.0). At the indicated times following infection, total cellular or nuclear fractions were collected and analyzed by Western blot. The 0, 0.5 and 1.0 hour (hr) times indicates time after oHSV infection. Before infection, cells were incubated with VPA or vehicle for 20 hours. The results of blot densitometry are also provided in tabular format in the lower part of the panel. B : Gene expression level of STAT3 was detected using quantitative RT-PCR at the indicated time points following VPA treatment (3 mM) of U251 cells. Each data point represents the mean of triplicate samples. Error bars indicate standard deviation. ** P

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

    Effect of STAT3 gene expression on oHSV replication in human glioblastoma U251 cells. Control transfected cells (pCR/CMV) were compared to STAT3 over-expressing cells (HA-STAT3 and FLAG-STAT3). a; Western blot analysis of HA-tagged or FLAG-tagged STAT3 in stable transfectants of human U251 glioma cells. Blots of STAT3 and GAPDH were scanned and analyzed by densitometry and are shown in tabular format in the lower part of the panel. The ratios were normalized to pCR/CMV control. b; GFP reporter signal from oHSV (rQNestin34.5) was detected 1 day after oHSV infection (MOI 0.05) of U251 cell lines stably transfected with STAT3. c; In vitro viral replication plaque assay. Cells were infected (MOI 0.05) with oHSV (rQNestin34.5) or wild type HSV-1 (F strain) and yields of progeny virus were determined on Vero cells. Black bars represent oHSV. White bars rpresent F strain. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. *** P
    Figure Legend Snippet: Effect of STAT3 gene expression on oHSV replication in human glioblastoma U251 cells. Control transfected cells (pCR/CMV) were compared to STAT3 over-expressing cells (HA-STAT3 and FLAG-STAT3). a; Western blot analysis of HA-tagged or FLAG-tagged STAT3 in stable transfectants of human U251 glioma cells. Blots of STAT3 and GAPDH were scanned and analyzed by densitometry and are shown in tabular format in the lower part of the panel. The ratios were normalized to pCR/CMV control. b; GFP reporter signal from oHSV (rQNestin34.5) was detected 1 day after oHSV infection (MOI 0.05) of U251 cell lines stably transfected with STAT3. c; In vitro viral replication plaque assay. Cells were infected (MOI 0.05) with oHSV (rQNestin34.5) or wild type HSV-1 (F strain) and yields of progeny virus were determined on Vero cells. Black bars represent oHSV. White bars rpresent F strain. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. *** P

    Techniques Used: Expressing, Transfection, Polymerase Chain Reaction, Western Blot, Infection, Stable Transfection, In Vitro, Plaque Assay, Standard Deviation

    Effect of exogenous IFNalpha with and without IFNalphaR antibody on oHSV replication in cells over-expressing STAT3 compared to control. A : Cells were incubated with the indicated concentration of INFalpha in the presence or absence of anti-IFNalpha/beta receptor antibody (IFNR Ab) for 24h, and then infected with oHSV (MOI 0.05). Two days later, GFP expression from oHSV infected cells was visualized. B : In vitro viral replication assay. U251 transfected cells were treated with (black) or without (white) anti-IFNalpha/betaR (IFNR Ab) antibody for 24 hours. Three days later yields of progeny virus were assayed on Vero cells. Each data point represents the mean of triplicate samples. Error bars indicate standard deviation. *** P
    Figure Legend Snippet: Effect of exogenous IFNalpha with and without IFNalphaR antibody on oHSV replication in cells over-expressing STAT3 compared to control. A : Cells were incubated with the indicated concentration of INFalpha in the presence or absence of anti-IFNalpha/beta receptor antibody (IFNR Ab) for 24h, and then infected with oHSV (MOI 0.05). Two days later, GFP expression from oHSV infected cells was visualized. B : In vitro viral replication assay. U251 transfected cells were treated with (black) or without (white) anti-IFNalpha/betaR (IFNR Ab) antibody for 24 hours. Three days later yields of progeny virus were assayed on Vero cells. Each data point represents the mean of triplicate samples. Error bars indicate standard deviation. *** P

    Techniques Used: Expressing, Incubation, Concentration Assay, Infection, In Vitro, Viral Replication Assay, Transfection, Standard Deviation

    Effect of IL-6 on STAT3 activation and oHSV replication. A : Activity of the STAT3 binding promoter was assayed following IL-6 stimulation (10 ng/ml) of U251 cells transfected with an expression vector, encoding a STAT3-responsive transcriptional element driving luciferase. Eight hours following treatment, cells were harvested and luciferase activity was assayed. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. *** P
    Figure Legend Snippet: Effect of IL-6 on STAT3 activation and oHSV replication. A : Activity of the STAT3 binding promoter was assayed following IL-6 stimulation (10 ng/ml) of U251 cells transfected with an expression vector, encoding a STAT3-responsive transcriptional element driving luciferase. Eight hours following treatment, cells were harvested and luciferase activity was assayed. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. *** P

    Techniques Used: Activation Assay, Activity Assay, Binding Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Standard Deviation

    Related Articles

    Transfection:

    Article Title: Tobacco Exposure Enhances Human Papillomavirus 16 Oncogene Expression via EGFR/PI3K/Akt/c-Jun Signaling Pathway in Cervical Cancer Cells
    Article Snippet: .. SiHa and CaSki cells were plated in triplicate in 24-well plate at 70–80% confluency and were transfected with 500 ng pmir-Glo LCR luciferase reporter plasmid with 1 μL Lipofectamine 2000 per well (Invitrogen). ..

    Article Title: Pax6 Expressed in Osteocytes Inhibits Canonical Wnt Signaling
    Article Snippet: The PCR products were cloned into the T & A cloning vector (RBC Life Sciences, Real Biotech Corporation, Taiwan) and subcloned into a pcDNA3.1 mammalian expression vector (Invitrogen, USA) after digestion with Hin dIII restriction enzyme to generate expression constructs [i.e., pcDNA3.1-Pax6 (+5a) and pcDNA3.1-Pax6 (−5a)]. .. Cells were then seeded in 6-well plates and transfected with 0.8 μg of pax6 plasmid at 70–80% confluency using Lipofectamine Plus™ reagent (Invitrogen, USA). .. At 48 h after transfection, the cells were processed to determine mRNA and protein expression levels of Pax6 and other osteocyte marker genes.

    Luciferase:

    Article Title: Tobacco Exposure Enhances Human Papillomavirus 16 Oncogene Expression via EGFR/PI3K/Akt/c-Jun Signaling Pathway in Cervical Cancer Cells
    Article Snippet: .. SiHa and CaSki cells were plated in triplicate in 24-well plate at 70–80% confluency and were transfected with 500 ng pmir-Glo LCR luciferase reporter plasmid with 1 μL Lipofectamine 2000 per well (Invitrogen). ..

    Article Title: MicroRNA-518b functions as a tumor suppressor in glioblastoma by targeting PDGFRB
    Article Snippet: The PCR amplicons were then cloned downstream of the luciferase open reading frame in the pMIR-REPORT vector (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. .. 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. .. After 24 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer's protocol.

    Plasmid Preparation:

    Article Title: Tobacco Exposure Enhances Human Papillomavirus 16 Oncogene Expression via EGFR/PI3K/Akt/c-Jun Signaling Pathway in Cervical Cancer Cells
    Article Snippet: .. SiHa and CaSki cells were plated in triplicate in 24-well plate at 70–80% confluency and were transfected with 500 ng pmir-Glo LCR luciferase reporter plasmid with 1 μL Lipofectamine 2000 per well (Invitrogen). ..

    Article Title: Pax6 Expressed in Osteocytes Inhibits Canonical Wnt Signaling
    Article Snippet: The PCR products were cloned into the T & A cloning vector (RBC Life Sciences, Real Biotech Corporation, Taiwan) and subcloned into a pcDNA3.1 mammalian expression vector (Invitrogen, USA) after digestion with Hin dIII restriction enzyme to generate expression constructs [i.e., pcDNA3.1-Pax6 (+5a) and pcDNA3.1-Pax6 (−5a)]. .. Cells were then seeded in 6-well plates and transfected with 0.8 μg of pax6 plasmid at 70–80% confluency using Lipofectamine Plus™ reagent (Invitrogen, USA). .. At 48 h after transfection, the cells were processed to determine mRNA and protein expression levels of Pax6 and other osteocyte marker genes.

    Reporter Assay:

    Article Title: MicroRNA-518b functions as a tumor suppressor in glioblastoma by targeting PDGFRB
    Article Snippet: The PCR amplicons were then cloned downstream of the luciferase open reading frame in the pMIR-REPORT vector (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. .. 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. .. After 24 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer's protocol.

    Recombinant:

    Article Title: MicroRNA-518b functions as a tumor suppressor in glioblastoma by targeting PDGFRB
    Article Snippet: The PCR amplicons were then cloned downstream of the luciferase open reading frame in the pMIR-REPORT vector (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. .. 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. .. After 24 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer's protocol.

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    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
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    SS’s potential way of action was studied by evaluating ( a ) oxidative stress by thiol group levels in R2J and <t>U251</t> 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
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    The electrotactic directedness of T98G <t>U-251MG</t> glioblastoma cells under 300 V m −1 dcEF after 6 hours with pharmacological inhibition on various ion channels. † indicates the electrotaxis tested against those without EF stimulation; ‡ indicate the electrotaxis group tested against those with cytochrome C which prevents adsorption of short peptides to experimental apparatus; All other groups are statistically compared to their respective controls in cell culture media with 10% FBS; n.s. indicates not significant; * indicates P
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    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

    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

    Journal: Cancers

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

    doi: 10.3390/cancers11010012

    Figure Lengend 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

    Article Snippet: U251 were harvested using 0.5%-Trypsin-EDTA (10×) (#15400-054, LifeTechnologies).

    Techniques: 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

    Journal: Cancers

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

    doi: 10.3390/cancers11010012

    Figure Lengend 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

    Article Snippet: U251 were harvested using 0.5%-Trypsin-EDTA (10×) (#15400-054, LifeTechnologies).

    Techniques: MTT Assay

    The electrotactic directedness of T98G U-251MG glioblastoma cells under 300 V m −1 dcEF after 6 hours with pharmacological inhibition on various ion channels. † indicates the electrotaxis tested against those without EF stimulation; ‡ indicate the electrotaxis group tested against those with cytochrome C which prevents adsorption of short peptides to experimental apparatus; All other groups are statistically compared to their respective controls in cell culture media with 10% FBS; n.s. indicates not significant; * indicates P

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: The electrotactic directedness of T98G U-251MG glioblastoma cells under 300 V m −1 dcEF after 6 hours with pharmacological inhibition on various ion channels. † indicates the electrotaxis tested against those without EF stimulation; ‡ indicate the electrotaxis group tested against those with cytochrome C which prevents adsorption of short peptides to experimental apparatus; All other groups are statistically compared to their respective controls in cell culture media with 10% FBS; n.s. indicates not significant; * indicates P

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques: Inhibition, Adsorption, Cell Culture

    Phase contrast images of T98G and U-251MG cells before and after 6 hours 300 V m −1 stimulation. The electric field is from left to right. Only T98G cells demonstrate prominent perpendicular allignment after electrical stimulation.

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: Phase contrast images of T98G and U-251MG cells before and after 6 hours 300 V m −1 stimulation. The electric field is from left to right. Only T98G cells demonstrate prominent perpendicular allignment after electrical stimulation.

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques:

    Time series plot of orientation in electrically stimulated glioblastoma cells. T98G cell results are indicated in closed symbols. U-251MG cell results are indicated in open symbols. Only T98G cells demonstrate prominent perpendicular alignment after electrical stimulation. The dashed lines indicate the 95% confidence interval for respective groups.

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: Time series plot of orientation in electrically stimulated glioblastoma cells. T98G cell results are indicated in closed symbols. U-251MG cell results are indicated in open symbols. Only T98G cells demonstrate prominent perpendicular alignment after electrical stimulation. The dashed lines indicate the 95% confidence interval for respective groups.

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques:

    Phase contrast images of T98G U-251MG cells under EF stimulations in different microenvironments after 6 hours. The scale bars represent 100 μ m. 4-AP: 4-aminopyridine. T98G cells demonstrate perpendicular alignment after electric field stimulation in cell culture media with or without FBS. However, without calcium and FBS in the cell culture medium, T98G cells’ viability decreases. Furthermore, in cell culture medium supplemented with 4 mM 4-AP, T98G also lose viability and start to detach from the substrate. These trends were not observed in the U-251MG cells, suggesting heterogeneity between cell lines.

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: Phase contrast images of T98G U-251MG cells under EF stimulations in different microenvironments after 6 hours. The scale bars represent 100 μ m. 4-AP: 4-aminopyridine. T98G cells demonstrate perpendicular alignment after electric field stimulation in cell culture media with or without FBS. However, without calcium and FBS in the cell culture medium, T98G cells’ viability decreases. Furthermore, in cell culture medium supplemented with 4 mM 4-AP, T98G also lose viability and start to detach from the substrate. These trends were not observed in the U-251MG cells, suggesting heterogeneity between cell lines.

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques: Cell Culture

    The electrotaxis of T98G U-251MG glioblastoma cells in dependence of the extracellular serum and calcium by varying the medium recipe. (a) The electrotaxis directedness; (b) The electrotaxis speed; n.s. indicates not significant; **** indicate P

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: The electrotaxis of T98G U-251MG glioblastoma cells in dependence of the extracellular serum and calcium by varying the medium recipe. (a) The electrotaxis directedness; (b) The electrotaxis speed; n.s. indicates not significant; **** indicate P

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques:

    Video clip showing the electrotaxis of U-251MG glioblastoma cells suppressed with 200 nM Agatoxin IVA on P/Q-type VGCC under 300 V m −1 dcEF for six hours and the respective tracking results using Usiigaci software.

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: Video clip showing the electrotaxis of U-251MG glioblastoma cells suppressed with 200 nM Agatoxin IVA on P/Q-type VGCC under 300 V m −1 dcEF for six hours and the respective tracking results using Usiigaci software.

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques: Cross-linking Immunoprecipitation, Software

    Phase contrast images of T98G U-251MG cells on various ECMs. Scale bar: 100 μ m.

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: Phase contrast images of T98G U-251MG cells on various ECMs. Scale bar: 100 μ m.

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques:

    The electrotaxis of T98G U-251MG glioblastoma cells on various ECMs after 6 hours under 300 V m −1 EF stimulation. (a) The electrotaxis directedness; (b) The electrotaxis speed; † indicates the electrotaxis groups tested against those without EF stimulation; All other groups are statistically compared to their respective controls on Geltrex™ ECM; n.s. indicates not significant; * indicates P

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: The electrotaxis of T98G U-251MG glioblastoma cells on various ECMs after 6 hours under 300 V m −1 EF stimulation. (a) The electrotaxis directedness; (b) The electrotaxis speed; † indicates the electrotaxis groups tested against those without EF stimulation; All other groups are statistically compared to their respective controls on Geltrex™ ECM; n.s. indicates not significant; * indicates P

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques:

    The electrotaxis of T98G U-251MG glioblastoma cells in dependence of extracellular calcium by varying concentration of calcium chelators, EDTA and EGTA. (a) The electrotaxis directedness; (b) The electrotaxis speed; † indicate the electrotaxis groups tested against those without EF stimulation; All groups are statistically compared to controls in cell culture media with 10% FBS; n.s. indicates not significant; **** indicate P

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: The electrotaxis of T98G U-251MG glioblastoma cells in dependence of extracellular calcium by varying concentration of calcium chelators, EDTA and EGTA. (a) The electrotaxis directedness; (b) The electrotaxis speed; † indicate the electrotaxis groups tested against those without EF stimulation; All groups are statistically compared to controls in cell culture media with 10% FBS; n.s. indicates not significant; **** indicate P

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques: Concentration Assay, Cell Culture

    Video clip showing the electrotaxis of U-251MG glioblastoma cells suppressed with 4-AP on A-type VGKC under 300 V m −1 dcEF for six hours and the respective tracking results using Usiigaci software.

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: Video clip showing the electrotaxis of U-251MG glioblastoma cells suppressed with 4-AP on A-type VGKC under 300 V m −1 dcEF for six hours and the respective tracking results using Usiigaci software.

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques: Cross-linking Immunoprecipitation, Software

    The results of U-251MG cell seeding inside microchannels by various methods. (a, d, g, j) In tip loading method, cells are introduced by using gravitational flow with micropipette tips. The cells can flocculate inside the tips and in microchannels as illustrated in (d). The microscopy image of seeded cells is shown in (g) and magnified in (j); (b, e, h, k) In tip injection method, cells are injected into the channels and tips are removed. The small hydrostatic pressure differences between the inlet/outlet (shown as Δh) will contribute to hydrodynamic flow and disturb the cell distribution, causing non-uniform cell distribution and aggregates as shown in (e). The microscopy image of seeded cells is shown in (h) and magnified in (k); (c, f, i, l) In our pressure-balanced sub-merged cell seeding method, the hydrostatic pressure difference is eliminated. The injected cells remain uniform through-out the channel as shown in (f). The microscopy image of seeded cells is shown in (i) and magnified in (l). The uniform and sparse cell seeding method is suitable for many different applications from single cell tracking, ensembled cell studies to cell assembly. The scale bars in (g, h, i) represent 500 μ m. The scale bars in (j, k, l) represent 200 μ m.

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: The results of U-251MG cell seeding inside microchannels by various methods. (a, d, g, j) In tip loading method, cells are introduced by using gravitational flow with micropipette tips. The cells can flocculate inside the tips and in microchannels as illustrated in (d). The microscopy image of seeded cells is shown in (g) and magnified in (j); (b, e, h, k) In tip injection method, cells are injected into the channels and tips are removed. The small hydrostatic pressure differences between the inlet/outlet (shown as Δh) will contribute to hydrodynamic flow and disturb the cell distribution, causing non-uniform cell distribution and aggregates as shown in (e). The microscopy image of seeded cells is shown in (h) and magnified in (k); (c, f, i, l) In our pressure-balanced sub-merged cell seeding method, the hydrostatic pressure difference is eliminated. The injected cells remain uniform through-out the channel as shown in (f). The microscopy image of seeded cells is shown in (i) and magnified in (l). The uniform and sparse cell seeding method is suitable for many different applications from single cell tracking, ensembled cell studies to cell assembly. The scale bars in (g, h, i) represent 500 μ m. The scale bars in (j, k, l) represent 200 μ m.

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques: Microscopy, Injection, Single Cell Tracking

    The electrotactic speed of T98G U-251MG glioblastoma cells under 300 V m −1 dcEF after 6 hours with pharmacological inhibition on various ion channels. † indicates the electrotaxis tested against those without EF stimulation; † indicate the electrotaxis group tested against those with cytochrome C which prevents adsorption of short peptides to experimental apparatus; All other groups are statistically compared to their respective controls in cell culture media with 10% FBS; n.s. indicates not significant; ** indicate P

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: The electrotactic speed of T98G U-251MG glioblastoma cells under 300 V m −1 dcEF after 6 hours with pharmacological inhibition on various ion channels. † indicates the electrotaxis tested against those without EF stimulation; † indicate the electrotaxis group tested against those with cytochrome C which prevents adsorption of short peptides to experimental apparatus; All other groups are statistically compared to their respective controls in cell culture media with 10% FBS; n.s. indicates not significant; ** indicate P

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques: Inhibition, Adsorption, Cell Culture

    Video clip showing the random migration of U-251MG glioblastoma cells for six hours and the respective tracking results using Usiigaci software.

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: Video clip showing the random migration of U-251MG glioblastoma cells for six hours and the respective tracking results using Usiigaci software.

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques: Cross-linking Immunoprecipitation, Migration, Software

    The electrotaxis of T98G U-251MG glioblastoma cells under 300 V m −1 dcEF after 6 hours with pharmacological inhibition on L-type (50 μ M Gadolinium [Gd] and Nicardipine) and N-type VGCCs (Conotoxin GVIA). a) The electrotactic directedness of the two cells. (b) The electrotactic speed of the two cells. ‡ indicate the electrotaxis group tested against those with cytochrome C which prevents adsorption of short peptides to experimental apparatus; All other groups are statistically compared to their respective controls in cell culture media with 10% FBS; n.s. indicates not significant; **** indicate P

    Journal: bioRxiv

    Article Title: Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

    doi: 10.1101/2020.02.14.948638

    Figure Lengend Snippet: The electrotaxis of T98G U-251MG glioblastoma cells under 300 V m −1 dcEF after 6 hours with pharmacological inhibition on L-type (50 μ M Gadolinium [Gd] and Nicardipine) and N-type VGCCs (Conotoxin GVIA). a) The electrotactic directedness of the two cells. (b) The electrotactic speed of the two cells. ‡ indicate the electrotaxis group tested against those with cytochrome C which prevents adsorption of short peptides to experimental apparatus; All other groups are statistically compared to their respective controls in cell culture media with 10% FBS; n.s. indicates not significant; **** indicate P

    Article Snippet: 1.3 Diffusion-limited chemical transport validation in HMEFC The diffusion-limited chemical transport in HMEFC was visualized by flowing 40 kDa fluorescein-dextran (FD40, Sigma-Aldrich, USA) with U-251MG cells or 40 kDa tetramethylrhodamine-dextran (D1842, Thermo Fisher Scientific, USA) in Fluorobrite DMEM (Gibco, USA).

    Techniques: Inhibition, Adsorption, Cell Culture