human glioblastoma multiforme gbm cell line u87mg  (National Centre for Cell Science)

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: A) Confocal laser scanning microscopy (CLSM) analyses of CXCR4 (green fluorescence) and PC-PLC (red fluorescence) localization on plasma membrane of unfixed cells. Scale bars, 18μm. B) Western blot (WB) of proteins isolated from U87MG cells by immunoprecipitation with anti-CXCR4 Ab (IP-α-CXCR4). Top panels show IP-α-CXCR4 preparations blotted with anti-PC-PLC Ab compared to control (CTRL IgG) (left) and total cell lysate (right). *IgG heavy chains. The bottom panels represent an α-CXCR4-IP preparation blotted with anti-CXCR4 Ab compared with CTRL IgG (left) and total lysate (right). C) CLSM analyses of CXCR4 (green fluorescence) and PC-PLC localization (red fluorescence) on plasma membrane of unfixed U87MG cells following the respective treatments (indicated on the left of the panel rows). Images represent untreated cells (CTRL) and cells exposed to either D609 or Plerixafor at different time-points (24h, 48h, 72h). Scale bars, 16 μm.

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Confocal Laser Scanning Microscopy, Fluorescence, Western Blot, Isolation, Immunoprecipitation

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: Representative WB of CXCR4 and PC-PLC detection in U87MG cells processed after 24h, 48h and 72h of treatment with either D609 or Plerixafor. β-actin was used as loading control. The histograms represent the mean value ± SD of relative fold change of CXCR4 (on the left) and PC-PLC (on the right) optical density (OD) normalized to β-actin, obtained by densitometric analyses of WB bands (Image J software). CTRL values normalized to 1 at each time point; n = 3 independent experiments. Statistical analyses were performed with one-way ANOVA *** P = 0.0008 (CXCR4 expression), ***P = 0.0008 (PC-PLC expression).

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Software, Expressing

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: A) U87MG cell proliferation after 24h, 48h, 72h of treatments with D609 or Plerixafor evaluated by cell counting using the Trypan blue excluding assay. Curves represent mean values ± SD of U87MG cell counts at different time-points (n = eight independent experiments). ● CTRL; ■ (dark grey square) D6099; and ▲ (light grey triangle) Plerixafor. Statistical analyses were performed with two-tailed unpaired t Student’s test. CTRL versus D609: 24h * P = 0.05; 48h *** P = 0.0001; 72h ** P = 0.0005. B) U87MG cell invasion evaluated by transwell chamber assay using Matrigel, in response to D609 or Plerixafor 24h treatment. The histograms represent the percentage of the area occupied by U87MG cells. CTRL values = 100%. Mean values ± SD (n = 6). Statistical analyses were performed with one-way ANOVA. ***P = 0.0001. C) Representative WB of p-EGFR and total EGFR expression in U87MG cells either untreated (CTRL), or treated with D609 or with Plerixafor for 24h, 48h and 72h. β-actin was used as loading control. The histograms represent the mean values of relative fold changes of p-EGFR and EGFR optical density normalized to β-actin, obtained with densitometric analyses of the WB protein bands (Image J software). CTRL values = 1. Means ± SD of n = 3 independent experiments. Statistical analyses were performed with one-way ANOVA * P = 0.0333 (p-EGFR); * P = 0.0141 (EGFR). D) Representative WB of p-AKT and AKT expression in U87MG control cells (CTRL) and in cells treated with either D609 or Plerixafor for 24h, 48h and 72h. β-actin was used as loading control. Histogram represents the mean of relative fold change of p-AKT and AKT optical density normalized to β-actin, obtained with densitometric analyses of WB protein bands (Image J software). CTRL values = 1. Means ± SD of n = 3 independent experiments. Statistical analyses were performed with one-way ANOVA * P = 0.0230 (p-AKT); * P = 0.0165 (AKT).

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Cell Counting, Two Tailed Test, Transwell Chamber Assay, Expressing, Software

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: The histograms represent the means ± SD of percentage values obtained from quantitative 1 HMRS analysis of each metabolite/percentage of total metabolites content in U87MG untreated (CTRL), treated with D609 or Plerixafor at 24h (A), 48h (B), 72h (C) of treatment. Metabolites reported are the compounds that mainly contribute to the U87MG metabolic profile: Gly, glycine; GPC+PCho, glycerophosphocholine+phosphocholine; Cho, choline; tCr, total creatine (creatine+phosphocreatine); Glu, glutamate; Ala, alanine; Lac, Lactate. Total metabolites = 100%. Means ± SD of n = 3 independent experiments. Statistical analyses were performed with two-tailed unpaired t Student’s test. CTRL versus D609: 24h GPC+PCho **P = 0.0096, Lac *P = 0.0170; 72h GPC+PCho *P = 0.0286, Lac *P = 0.047.

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Two Tailed Test

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: A) Histogram represents mean values of percentage of intracellular Lac concentration in U87MG obtained colorimetric assay after 24h, 48h, 72h of treatments. Mean value ± SD of n = 3 independent experiments. Statistical analyses were performed using one-way ANOVA. ***P = 0.0003. B) Histogram represents mean ± SD of percentage values (n = 3 independent experiments) of LDH intracellular activity measured by colorimetric assay at different treatment time-points (24h, 48h, 72h). Statistical analyses were performed using one-way ANOVA. ***P = 0.0006; unpaired two-tailed Student-t test CTRL 72h VS D609 72h P = 0.004. C) Histogram represents mean ± SD of percentage values of Lac extracellular concentration measured by a colorimetric assay on U87MG-conditioned medium in response to treatments (24h-48h-72h) (n = 3 independent experiments). All values were normalized for μg of proteins. CTRL values = 100%.

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Concentration Assay, Colorimetric Assay, Activity Assay, Two Tailed Test