u118 mg human gbm cell line  (Procell Inc)

Journal: eBioMedicine

Article Title: Metformin and simvastatin exert additive antitumour effects in glioblastoma via senescence-state: clinical and translational evidence

doi: 10.1016/j.ebiom.2023.104484

Figure Lengend Snippet: Treatments with metformin and/or simvastatin significantly decrease proliferation rate in different glioblastoma (GBM) cell models and high grade (III) astrocytomas, but not the viability in non-tumour brain cells in vitro . (a) Metformin dose–response carried out in U-87 MG and U-118 MG cells (n = 4). (b) IC 50 of metformin in vitro in U-87 MG and U-118 MG cells. (c) Simvastatin dose–response carried out in U-87 MG and U-118 MG cells (n = 4). (d) IC 50 of simvastatin in vitro in U-87 MG and U-118 MG cells. Proliferation/viability rates of GBM cell lines [U-87 MG (e) and U-118 MG (f) ; n = 5], primary patient-derived cell cultures from AIII [grade III (g) , n = 4] and grade IV-GBM [ (h); n = 4], primary non-tumour brain cell cultures [ (i) ; n = 4] and mouse primary-astrocytes progenitor derived cells [ (j) ; n = 4] in response to metformin, simvastatin, and their combination compared to vehicle-treated controls. Four technical replicates (tr) were assessed in each condition. Data represent medians (interquartile range) or means ± SEM (error bars). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001, were set as statistically significant differences vs. control conditions.

Article Snippet: Two human GBM cell line models (U-87 MG and U-118 MG) were obtained from the American Type Culture Collection (ATCC, #HTB-14, RRID:CVCL_0022/#HTB-15, respectively; Manassas, VA, USA) and cultured according to the supplier's recommendations.

Techniques: In Vitro, Derivative Assay, Control

Journal: eBioMedicine

Article Title: Metformin and simvastatin exert additive antitumour effects in glioblastoma via senescence-state: clinical and translational evidence

doi: 10.1016/j.ebiom.2023.104484

Figure Lengend Snippet: The combination of metformin and simvastatin altered the expression levels of key elements involved in the Senescence-Associated Secretory Phenotype (SASP), and in the splicing process in GBM cells. (a) Hierarchical heatmap generated using the expression levels of 32 SASP genes in all GBM experimental cell models treated with metformin, simvastatin, and the combination of both drugs (n = 3; tr = 2; mean value U-87 MG + U-118 MG + primary-derived cell cultures). Hierarchical heatmap showing the expression levels of 20 top altered genes (b) and Partial Least Squares Discriminant Analysis (PLS-DA) (c) of the expression of SASP genes in response to metformin, simvastatin, and their combination in GBM cells [cell lines (U-87 MG and U-118 MG) and primary patient-derived GBM cells (PPdC); data represent the mean value of 3 experiments (4 treatment conditions/experiment) in each GBM cell model]. d) Variable Importance in Projection (VIP) score of the expression levels of SASP key genes in GBM cells. e) Heatmap generated using the expression levels of 4 spliceosome components and (f) individual expression levels of these components in response to metformin, simvastatin, and their combination in GBM cells (n = 3; tr = 2; data represent mean value of U-87 MG + U-118 MG). Data represent medians (interquartile range) or means ± SEM (error bars). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001, were set as statistically significant differences vs. control conditions.

Article Snippet: Two human GBM cell line models (U-87 MG and U-118 MG) were obtained from the American Type Culture Collection (ATCC, #HTB-14, RRID:CVCL_0022/#HTB-15, respectively; Manassas, VA, USA) and cultured according to the supplier's recommendations.

Techniques: Expressing, Generated, Derivative Assay, Control

Journal: PLoS ONE

Article Title: Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling

doi: 10.1371/journal.pone.0111343

Figure Lengend Snippet: In ( A ) LN-229 and ( B ) U-118 MG human GBM cells, protein levels of PODX and soluble and total β-cat were determined with western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 µM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective β-cat signaling inhibitor CCT031374 (50 µM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (ΔN151) β-cat (PODX-shRNA+active-cat, lane 8). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. The total β-cat protein level was not significantly altered by PODX in both LN-229 and U-118 MG cells. Density of the PODX and the soluble β-cat blots was normalized against that of the GAPDH blot to obtain a relative blot density, which was expressed as fold changes to that of NC (designated as 1) to represent the relative protein content. Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODX; c p <0.05 vs. PODX+PD; d p <0.05 vs. PODX+CCT.

Article Snippet: LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling

doi: 10.1371/journal.pone.0111343

Figure Lengend Snippet: ( A ) LN-229 and ( B ) U-118 MG GBM cells were transfected with TOPflash, a synthetic β-catenin luciferase reporter, or FOPflash, a negative control reporter for TOPflash. Thirty hours later, the luciferase activity was determined in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 µM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective β-cat signaling inhibitor CCT031374 (50 µM) for 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (ΔN151) β-cat (PODX-shRNA+active-cat). The luciferase activity was expressed as fold changes to that of NC (designated as 1). a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODX; c p <0.05 vs. PODX+PD; d p <0.05 vs. PODX+CCT; e p <0.05 vs. PODX-shRNA.

Article Snippet: LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Transfection, Luciferase, Negative Control, Activity Assay, Stable Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling

doi: 10.1371/journal.pone.0111343

Figure Lengend Snippet: The mRNA levels of β-cat and β-cat signaling target genes C-Myc and C-Jun were determined in ( A ) LN-229 and ( B ) U-118 MG GBM cells. Real-time RT-PCR was performed with normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 µM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective β-cat signaling inhibitor CCT031374 (50 µM) for 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (ΔN151) β-cat (PODX-shRNA+active-cat). The mRNA level was expressed as fold changes to that of NC (designated as 1). * p <0.05 vs. controls (NC, VC and SC).

Article Snippet: LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling

doi: 10.1371/journal.pone.0111343

Figure Lengend Snippet: In vitro cell invasion assays were performed in ( A ) LN-229 and ( B ) U-118 MG GBM cells. Invading cells were fixed and counted in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 µM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective β-cat signaling inhibitor CCT031374 (50 µM) for 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (ΔN151) β-cat (PODX-shRNA+active-cat). Representative cell invasion images are shown. a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODX; c p <0.05 vs. PODX+PD; d p <0.05 vs. PODX+CCT; e p <0.05 vs. PODX-shRNA.

Article Snippet: LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vitro, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling

doi: 10.1371/journal.pone.0111343

Figure Lengend Snippet: The ( A ) mRNA and the ( B ) protein levels of MMP9 in LN-229 ( left panel ) and U-118 MG ( right panel ) GBM cells were respectively determined by real-time RT-PCR and Western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 µM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective β-cat signaling inhibitor CCT031374 (50 µM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (ΔN151) β-cat (PODX-shRNA+active-cat, lane 8). The mRNA and the protein levels of MMP9 were expressed as fold changes to those of NC (designated as 1), respectively. Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODX; c p <0.05 vs. PODX+PD; d p <0.05 vs. PODX+CCT; e p <0.05 vs. PODX-shRNA.

Article Snippet: LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling

doi: 10.1371/journal.pone.0111343

Figure Lengend Snippet: In ( A ) LN-229 and ( B ) U-118 MG GBM cells, MMP9 activities were determined with a SensoLyte 520 MMP9 Assay Kit (AnaSpec) in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 µM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective β-cat signaling inhibitor CCT031374 (50 µM) for 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (ΔN151) β-cat (PODX-shRNA+active-cat). The MMP9 activity was shown as fold changes to that of NC (designated as 1). a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODX; c p <0.05 vs. PODX+PD; d p <0.05 vs. PODX+CCT; e p <0.05 vs. PODX-shRNA.

Article Snippet: LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA, Activity Assay

Journal: PLoS ONE

Article Title: Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling

doi: 10.1371/journal.pone.0111343

Figure Lengend Snippet: In ( A ) LN-229 and ( B ) U-118 MG GBM cells, methlythiazoletetrazolium (MTT) cell proliferation assays were performed for 15 or 30 hours in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 µM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective β-cat signaling inhibitor CCT031374 (50 µM) for 15 or 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (ΔN151) β-cat (PODX-shRNA+active-cat). Cell proliferation at 15 and 30 hours was expressed as fold changes to that of NC (designated as 1). * p <0.05 vs. controls (NC, VC and SC) at 30 hours.

Article Snippet: LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: MTT Cell Proliferation, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling

doi: 10.1371/journal.pone.0111343

Figure Lengend Snippet: In ( A ) LN-229 and ( B ) U-118 MG GBM cells, the p38 MAPK activity was determined with a p38 MAPK Assay kit (Cell Signaling Technology) by measuring phosphorylation of ATF2, a substrate of activated p38 MAPK. The levels of phosphorylated ATF2 (p-ATF2) were determined with western blot in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 µM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective β-cat signaling inhibitor CCT031374 (50 µM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (ΔN151) β-cat (PODX-shRNA+active-cat, lane 8). The p-ATF2 content/p38 MAPK activity was shown as fold changes to that of NC (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODX; c p <0.05 vs. PODX+PD; d p <0.05 vs. PODX+CCT.

Article Snippet: LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling

doi: 10.1371/journal.pone.0111343

Figure Lengend Snippet: In ( A ) LN-229 and ( B ) U-118 MG GBM cells, the levels of p-GSK-3β at serine 389 and total GSK-3β were determined with Western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 µM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective β-cat signaling inhibitor CCT031374 (50 µM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (ΔN151) β-cat (PODX-shRNA+active-cat, lane 8). The total GSK-3β protein level was not significantly altered by PODX in both LN-229 and U-118 MG cells. Density of the p-GSK-3β (serine 389) blot was normalized against that of the total GSK-3β blot to obtain a relative blot density, which was expressed as fold changes to that of NC (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODX; c p <0.05 vs. PODX+PD; d p <0.05 vs. PODX+CCT.

Article Snippet: LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: Frontiers in Genetics

Article Title: Comprehensive development and validation of gene signature for predicting survival in patients with glioblastoma

doi: 10.3389/fgene.2022.900911

Figure Lengend Snippet: Biological functional validation of MSH2 in GBM cell. (A) Silencing efficiency of MSH2. CCK8 assay (B) , EDU assay (C) , and colony formation assay (D) showing the effect of MSH2 knockdown on proliferation of U-118 MG cells. (E) Transwell assay indicating that MSH2 depletion markedly weakens migration and invasion by U-118 MG cells (* p < 0.05, ** p < 0.01, and *** p < 0.001, n = 3).

Article Snippet: The U-118 MG human GBM cell line was purchased from the Procell Life Science and Technology Company (Wuhan, China).

Techniques: Functional Assay, CCK-8 Assay, EdU Assay, Colony Assay, Transwell Assay, Migration

Journal: International Journal of Molecular Sciences

Article Title: The Status of EGFR Modulates the Effect of miRNA-200c on ZEB1 Expression and Cell Migration in Glioblastoma Cells

doi: 10.3390/ijms22010368

Figure Lengend Snippet: EGFR mRNA, miR-200c and ZEB1 mRNA expression in the cultures with different levels of EGFR amplification. Results were normalized to actin ( EGFR and ZEB1 ) or U66 (miR-200c) housekeeping genes. Changes in RNA expression are reported as the mean and standard error with respect to the nonamplified EGFR group using the 2-∆∆Ct method. Four independent experiments were performed. Statistical analyses were performed using one-way analysis of variance, followed by Dunnett’s t-test for multiple comparisons. Significant expression changes ( p ≤ 0.05 and p ≤ 0.01) with respect to U-118 are marked with * and **, respectively.

Article Snippet: We also used the U-118 GBM human cell line from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Amplification, RNA Expression

Journal: International Journal of Molecular Sciences

Article Title: The Status of EGFR Modulates the Effect of miRNA-200c on ZEB1 Expression and Cell Migration in Glioblastoma Cells

doi: 10.3390/ijms22010368

Figure Lengend Snippet: Cell viability. Cell viability study by luminescence in the four transfected cell cultures (U-118, HC-444, HC-534 and HC-466) with miR-200c mimic, miR-200c inhibitor and EGFR silencer conditions, with respect to control (h: hours). The points for each curve represent the mean values ± SDs of three independent experiments. Statistical analyses were performed using one-way analysis of variance, followed by Dunnett’s t-test for multiple comparisons.

Article Snippet: We also used the U-118 GBM human cell line from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection

Journal: Carcinogenesis

Article Title: Phospho-valproic acid (MDC-1112) suppresses glioblastoma growth in preclinical models through the inhibition of STAT3 phosphorylation

doi: 10.1093/carcin/bgz069

Figure Lengend Snippet: MDC-1112 inhibits GBM cell growth. (A) Chemical structure of MDC-1112 (Phospho-valproic acid). (B) IC50 values for glioblastoma cells treated with MDC-1112 or VPA for 24 h. These values are representative of three experiments, each performed in triplicates; results were within 10%. The table shows the 13.4- to 25.4-fold enhancements in potency of MDC-1112 over VPA in four human GBM cell lines. (C) MDC-1112 reduces human U87 cell growth in a concentration- and time-dependent manner. Results are expressed as % control. (D) MDC-1112 inhibits GBM cell colony formation in a concentration-dependent manner in U87 and LN-18 cells. (*P < 0.05, versus control). (E) Differential cytotoxic effect of MDC-1112 in GBM cells compared with the NHA. MDC-1112 inhibits human GBM cancer cell growth in a concentration-dependent manner. Cell growth was determined in U118, LN-229 and LN-18 GBM cells and in NHA after treatment with escalating concentrations of MDC-1112 for 48 h. Results are expressed as % control. *Significantly different compared with all other cell lines (P < 0.05, one-way analysis of variance test).

Article Snippet: Human GBM cell lines [U87, LN-18, LN-229, U118; American Type Culture Collection (ATCC), Manassas, VA] were grown as monolayers in the medium suggested by ATCC and supplemented with 10% fetal bovine serum (Mediatech, Herndon, VA), penicillin (50 U/ml) and streptomycin (50 µg/ml; Life Technologies, Grand Island, NY).

Techniques: Concentration Assay

Journal: Carcinogenesis

Article Title: Phospho-valproic acid (MDC-1112) suppresses glioblastoma growth in preclinical models through the inhibition of STAT3 phosphorylation

doi: 10.1093/carcin/bgz069

Figure Lengend Snippet: MDC-1112 reduces GBM xenograft growth. (A) MDC-1112 inhibits the growth of human U87 xenografts. U87 cells were injected s.c. into the flank areas of nude mice, and when palpable tumors were observed, the mice received MDC-1112 (50 mg/kg per day) in PBS or just PBS (control) by intraperitoneal injection for 15 days. U87 tumor volume growth over time for vehicle control- and MDC-1112-treated mice. *Significantly different compared with control group [P < 0.01, one-way analysis of variance (ANOVA) test]. (B) Mice weight progression for control and MDC-1112-treated mice. (C) MDC-1112 inhibits the growth of human U118 xenografts. U118 cells were injected s.c. into the flank areas of nude mice, and when palpable tumors were observed, the mice received MDC-1112 (50 mg/kg per day) in PBS or just PBS (control) by intraperitoneal injection for 30 days. U118 tumor volume growth over time for vehicle control- and MDC-1112-treated mice. *Significantly different compared with control group (P < 0.01, one-way ANOVA test). (D) Tumor weight at sacrifice. *Significantly different compared with control group (P < 0.01, one-way ANOVA test). (E) Body weight over time of mice bearing U118 xenografts treated with vehicle control (PBS) or MDC-1112 50 mg/kg. All values: mean ± SD. (F) Ki-67 and p21 immunostaining were performed on U87 tumor sections and photographs were taken at ×20 magnification. Representative images are shown. The consecutive section was stained with isotype IgG as negative staining control and it is shown in the upper right corner. Quantification is displayed on the right. Results were expressed as percent of Ki-67+ or p21+ cells ± SEM per ×20 field. *Significant compared with control group; P < 0.05. (G) Ki-67 and cleaved caspase 3 (CC3) immunostaining were performed on U118 tumor sections and photographs were taken at ×20 magnification. Representative images are shown. The consecutive section was stained with isotype IgG as negative staining control and it is shown in the upper right corner. Quantification is displayed on the right. Results were expressed as percent of Ki-67+ cells ± SEM per ×20 field. *Significant compared with control group; P < 0.05.

Article Snippet: Human GBM cell lines [U87, LN-18, LN-229, U118; American Type Culture Collection (ATCC), Manassas, VA] were grown as monolayers in the medium suggested by ATCC and supplemented with 10% fetal bovine serum (Mediatech, Herndon, VA), penicillin (50 U/ml) and streptomycin (50 µg/ml; Life Technologies, Grand Island, NY).

Techniques: Injection, Immunostaining, Staining, Negative Staining

Journal: Carcinogenesis

Article Title: Phospho-valproic acid (MDC-1112) suppresses glioblastoma growth in preclinical models through the inhibition of STAT3 phosphorylation

doi: 10.1093/carcin/bgz069

Figure Lengend Snippet: MDC-1112 induces intrinsic apoptosis in GBM cells. (A) MDC-1112 collapses the mitochondrial membrane potential (ΔΨm) in a concentration-dependent manner. Cells were stained with JC-1 and analyzed with flow cytometry after treatment with MDC-1112 for 3 h. Data were quantified and results are shown as mean ± SEM; *P < 0.05 versus control. (B) Immunoblots for full-length caspase 9 in total cell protein extracts from LN-18 or U87 cells treated with MDC-1112, as indicated, for 24 h. Loading control: β-actin. Bands were quantified and results are shown as the ratio cleaved:β-actin; *P < 0.05 versus control. (C) Immunoblots for full length and cleaved caspase 3 in total cell protein extracts from LN-18 or U118 cells treated with MDC-1112, as indicated, for 24 h. Loading control: β-actin. Bands were quantified and results are shown as the ratio cleaved:full length protein; *P < 0.05 versus control. (D) Immunoblots for full length and cleaved PARP in total cell protein extracts from LN-18 or U87 cells treated with MDC-1112, as indicated, for 24 h. Bands were quantified and results are shown as the ratio cleaved/full length protein; *P < 0.05 versus control. (E) Cell death by apoptosis was determined by flow cytometry using the dual staining (Annexin V and PI) in U87 cells treated with increasing concentrations of MDC-1112 for 24 h. Results are expressed as fold-increase compared with the percentage of Annexin V (+) cells in the control group. (F) Cell death by apoptosis was determined by flow cytometry in LN-18 and LN-229 cells incubated without or with MDC-1112 1 × IC50 for 24 h. Results are expressed as fold-increase compared with the percentage of apoptotic cells in the control group.

Article Snippet: Human GBM cell lines [U87, LN-18, LN-229, U118; American Type Culture Collection (ATCC), Manassas, VA] were grown as monolayers in the medium suggested by ATCC and supplemented with 10% fetal bovine serum (Mediatech, Herndon, VA), penicillin (50 U/ml) and streptomycin (50 µg/ml; Life Technologies, Grand Island, NY).

Techniques: Concentration Assay, Staining, Flow Cytometry, Western Blot, Incubation

Journal: Carcinogenesis

Article Title: Phospho-valproic acid (MDC-1112) suppresses glioblastoma growth in preclinical models through the inhibition of STAT3 phosphorylation

doi: 10.1093/carcin/bgz069

Figure Lengend Snippet: Treatment with MDC-1112 extends survival in mice bearing GBM intracranial tumors. (A) U87-Luc cells were intracranially injected, and when tumor size was confirmed by bioluminensence, mice received MDC-1112 (50 mg/kg 5×/week) in PBS or just PBS (control) by intraperitoneal injection for up to 35 days. Tumor growth over time until endpoint in vehicle control (red) or MDC-1112 (blue)-treated mice determined by bioluminescent imaging. (B) MDC-1112 therapy significantly prolonged survival of animals as compared with vehicle control group (P < 0.005). Kaplan–Meier survival curve of vehicle control (red) or MDC-1112-treated mice (blue) is shown. Seven of eleven MDC-1112-treated animals were intentionally killed (censored) for histological analysis of brain at day 35 despite being healthy. (C) Tumor growth was monitored by bioluminescent imaging in mice bearing intracranial U87-Luc cells (n = 10–11 per group). Representative IVIS images of U87-Luc-bearing animals over time treated as indicated.

Article Snippet: Human GBM cell lines [U87, LN-18, LN-229, U118; American Type Culture Collection (ATCC), Manassas, VA] were grown as monolayers in the medium suggested by ATCC and supplemented with 10% fetal bovine serum (Mediatech, Herndon, VA), penicillin (50 U/ml) and streptomycin (50 µg/ml; Life Technologies, Grand Island, NY).

Techniques: Injection, Imaging

Journal: Carcinogenesis

Article Title: Phospho-valproic acid (MDC-1112) suppresses glioblastoma growth in preclinical models through the inhibition of STAT3 phosphorylation

doi: 10.1093/carcin/bgz069

Figure Lengend Snippet: MDC-1112 inhibits STAT3 Ser727 signaling in vitro and in vivo. (A) Immunoblots of STAT3, phosphorylated STAT3 at the Ser727 residue (p-STAT3Ser727), from U87 cells treated with MDC-1112, 1 × IC50 for different periods of time. Bands were quantified and results are shown as the ratio p-STAT3Ser727:STAT3. Values are mean ± SEM. *P < 0.05 versus control. (B) Immunoblots of STAT3 and phosphorylated STAT3 at the Ser727 residue (p-STAT3Ser727), from U87 and LN-18 cells treated with various concentrations of MDC-1112 for 4 h. Bands were quantified and results are shown as the ratio p-STAT3Ser727:STAT3. Values are mean ± SEM. *P < 0.05 versus control. (C) Immunostaining for phosphorylated STAT3 at the Ser727 residue (p-STAT3Ser727) or Tyr705 residue (p-STAT3Tyr705) or p-ERK1/2 expression on tissue sections of U87 tumors from control and MDC-1112-treated mice (×20). Representative images are shown. The consecutive section was stained with isotype IgG as negative staining control and it is shown in the upper right corner. Quantification is displayed on the right. Results were expressed as percent of p-STAT3Ser727, p-STAT3Tyr705 or p-ERK1/2 positive cells per field. *Significant compared with control group; P < 0.05. (D) Immunoblots of ERK1/2 and phosphorylated ERK1/2 (p-ERK), from U87 cells treated with various concentrations of MDC-1112 for 4 h. Bands were quantified and results are shown as the ratio p-ERK:ERK. Values are mean ± SEM. *P < 0.05 versus control. (E) Immunoblots of STAT3, p-STAT3Ser727, ERK1/2 and phosphorylated ERK1/2 (p-ERK), from U87 tumor lysates. Loading control: β-actin. Each lane represents a different tumor sample. Bands were quantified and results are expressed as the ratio of phospho over total expression levels for each protein. Values are mean±SEM. *P < 0.05 versus control. (F) STAT3 overexpression ameliorates, in part, the cell growth inhibition by MDC-1112. U118 cells were transfected with a control (cDNA) or STAT3-expressing plasmid for 48 h and then treated with 50 or 100 µM MDC-1112 for 48 h. Cell growth was evaluated by the MTT assay; *P < 0.05 versus control. Top: STAT3 expression status in whole cell protein lysates following transfection. (G) Effect of silencing STAT3 on MDC-1112-induced cell growth reduction. U118 cells were transfected with either control or STAT3 small interfering RNA. After transfection, cells were treated with MDC-1112 for 24 h and cell growth was evaluated; *P < 0.05 versus control. Immunoblots to verify STAT3 silencing were performed on whole cell extracts obtained from these cells (top panel).

Article Snippet: Human GBM cell lines [U87, LN-18, LN-229, U118; American Type Culture Collection (ATCC), Manassas, VA] were grown as monolayers in the medium suggested by ATCC and supplemented with 10% fetal bovine serum (Mediatech, Herndon, VA), penicillin (50 U/ml) and streptomycin (50 µg/ml; Life Technologies, Grand Island, NY).

Techniques: In Vitro, In Vivo, Western Blot, Immunostaining, Expressing, Staining, Negative Staining, Over Expression, Inhibition, Transfection, Plasmid Preparation, MTT Assay, Small Interfering RNA

Journal: Carcinogenesis

Article Title: Phospho-valproic acid (MDC-1112) suppresses glioblastoma growth in preclinical models through the inhibition of STAT3 phosphorylation

doi: 10.1093/carcin/bgz069

Figure Lengend Snippet: MDC-1112 reduces mitochondrial STAT3 levels and induces mitochondrial ROS in GBM cells. (A) Immunoblots for STAT3, Hsp60 or COX IV in mitochondrial (MF) fractions from U87 cells treated with MDC-1112 for 3 h. Bands were quantified and results expressed as percent control for each protein. Values are mean ± SEM. *P < 0.05 versus control. (B) Immunoblots for STAT3, phosphorylated STAT3 at the Ser727 residue (p-STAT3Ser727), GRIM-19 and COX IV in MF from U118 and LN-18 cells treated with MDC-1112 for 3 h. Bands were quantified and results expressed as percent control for each protein. Values are mean ± SEM. *P < 0.05 versus control. (C) Immunoblots of GRIM-19 in total fractions from U87 cells treated with various concentrations of MDC-1112 for 4 h. Bands were quantified and results are shown as the ratio GRIM-19:β-actin. (D) U87 and LN-18 cells were treated with MDC-1112 for 1 h as indicated. The levels of superoxide anion in the mitochondria were determined by flow cytometry using the MitoSOX-Red fluorescent probe. Bar graph analysis of Geometric means of cells that underwent MitoSOX Red Staining. (E) U87 and LN-18 cells were treated without (Control) or with MDC-1112 1 × IC50 or VPA (1 × IC50) for 1 h. Superoxide anion levels in the mitochondria was determined by confocal microscopy. Representative images are shown. (F) Treatment with Mito-TEMPO prevents the increase in MitoSOX-Red induced by MDC-1112 in various GBM cells. Values are mean ± SEM. *P < 0.05 versus control.

Article Snippet: Human GBM cell lines [U87, LN-18, LN-229, U118; American Type Culture Collection (ATCC), Manassas, VA] were grown as monolayers in the medium suggested by ATCC and supplemented with 10% fetal bovine serum (Mediatech, Herndon, VA), penicillin (50 U/ml) and streptomycin (50 µg/ml; Life Technologies, Grand Island, NY).

Techniques: Western Blot, Flow Cytometry, Staining, Confocal Microscopy