Journal: International Journal of Molecular Sciences

Article Title: Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model

doi: 10.3390/ijms25074130

Figure Lengend Snippet: Expression of ERα gene ( A ) and ERα protein ( B ) in U87 cells cultured under different conditions. Data are representative of each group cultured in control, nutrient-deficient, hypoxic, and necrotic conditions. Statistical analysis was performed using t -test *, p < 0.005.

Article Snippet: Cell culture of the U87 line (obtained from the European Collection of Authenticated Cell Cultures (ECACC)) was performed under standard conditions of 37 °C, 95% humidity, and 5% CO 2 , according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model

doi: 10.3390/ijms25074130

Figure Lengend Snippet: Representative images taken with the FV1000 confocal microscope system (Olympus, Hamburg, Germany) show ERα protein expression in U87 cells cultured under specific conditions: control ( A ), nutrient deficiency ( B ), hypoxia ( C ), and necrotic conditions ( D ). FITC (AR) and DAPI (nuclear) markers were used. Microphotographs were taken at ×20 magnification ( A , B , D ) and ×40 magnification ( C ); scale bar 30 µm.

Article Snippet: Cell culture of the U87 line (obtained from the European Collection of Authenticated Cell Cultures (ECACC)) was performed under standard conditions of 37 °C, 95% humidity, and 5% CO 2 , according to the manufacturer’s instructions.

Techniques: Microscopy, Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model

doi: 10.3390/ijms25074130

Figure Lengend Snippet: Expression of the ERβ gene ( A ) and ERβ protein ( B ) in U87 cells cultured under different conditions. Data are representative of each group cultured in control, nutrient-deficient, hypoxic, and necrotic conditions. Statistical analysis was performed using a t -test * p < 0.05.

Article Snippet: Cell culture of the U87 line (obtained from the European Collection of Authenticated Cell Cultures (ECACC)) was performed under standard conditions of 37 °C, 95% humidity, and 5% CO 2 , according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model

doi: 10.3390/ijms25074130

Figure Lengend Snippet: Representative images taken with the FV1000 confocal microscope system (Olympus, Hamburg, Germany) show ERβ protein expression in U87 cells cultured under specific conditions: control ( A ), nutrient deficiency ( B ), hypoxia ( C ), and necrotic conditions ( D ). FITC (AR) and DAPI (nuclear) markers were used. Microphotographs were taken at ×20 magnification ( A , B , D ) and ×40 magnification ( C ); scale bar 30 µm.

Article Snippet: Cell culture of the U87 line (obtained from the European Collection of Authenticated Cell Cultures (ECACC)) was performed under standard conditions of 37 °C, 95% humidity, and 5% CO 2 , according to the manufacturer’s instructions.

Techniques: Microscopy, Expressing, Cell Culture, Control

Journal: Translational Oncology

Article Title: The role of A-kinase interacting protein 1 in regulating progression and stemness as well as indicating the prognosis in glioblastoma

doi: 10.1016/j.tranon.2022.101463

Figure Lengend Snippet: AKIP1 mRNA and protein expression in GBM and control cell lines. AKIP1 mRNA expression (A) and protein expression (B,C) in GBM cell lines (including GB-1, U-251 MG, LN18, A172, U-87 MG) and control cell lines. Each experiment was replicated three times.

Article Snippet: Human GBM cell lines, including GB1, U-251 MG, LN18, A172, and U-87 MG, were purchased from the American Type Culture Collection (ATCC, USA) or Japanese Collection of Research Bioresources Cell Bank (JCRB, Japan), respectively.

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: Type 5 phosphodiesterase regulates glioblastoma multiforme aggressiveness and clinical outcome

doi: 10.18632/oncotarget.14656

Figure Lengend Snippet: A . Wound healing assay and B . Histogram showing the mean percentage ±SD of closure of sh-scrT98G or sh1 or sh2-PDE5 cells monolayers in the presence or absence of 1 μM sildenafil or 100 μM 8br-cGMP. C . Migration assay on matrigel cushions through transwell filters of sh-scr or sh1-PDE5 T98G cells in the presence or absence of 1 μM sildenafil or 100 μM 8br-cGMP. Scale bars= 50 μm. D . Histogram showing the relative migration capacity (fold increase ± SD) of cells that passed through the filter in the different treatments compared to control cells. E . Migration assay on matrigel cushions through transwell filters of mock- or PDE5-overexpressing U87MG (upper panels) or GBM line 83 cells (lower panels). Scale bar= 20 μm. F . Histogram showing the mean percentage ± SD of cells that passed through the filter. G . Histogram of the mean percentage ±SD of secreted MMP-2 in gelatin zymography (bottom image) in sh-scr or sh1-PDE5 T98G and H . in mock- or PDE5-overexpressing U87MG cells and I . in sh-scr T98G stimulated with CNP, GSNO or sildenafil at the indicated concentrations. Data have been obtained from three independent experiments. (*p < 0.05; **p < 0.001).

Article Snippet: The GBM cell lines (T98G, U87MG, A172, U251, LN18) used in this study were grown in DMEM, (Euroclone; Milan, Italy) with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Wound Healing Assay, Migration, Control, Zymography

Journal: Oncotarget

Article Title: Type 5 phosphodiesterase regulates glioblastoma multiforme aggressiveness and clinical outcome

doi: 10.18632/oncotarget.14656

Figure Lengend Snippet: A . PKG1 low and B . PKG1 high GBM samples, as revealed by immuno-histochemistry. Black arrows point to PKG1 expression within the vessel walls. White arrow points to tumor cells. Scale bar=20μm. C . Expression levels of PKGII, PKG1α and PKG1β and total PKG1 in sh-scr and sh1-PDE5 T98G cells and in wtT98G, U87MG, A172, LN18, 83 GBM lines. D . pVASP levels in sh-scr or sh1-PDE5 T98G cells before and after Y-27632, 8br-cGMP (50 μM) treatments. Representative image out of 4 independent experiments. E . Phosphorylation levels of MYPT1 (pSer695) in sh-PDE5 or sh1-scr T98G upon the indicated treatments [8br-cGMP, 50 μM; Y-27632 (RI), 10 μM); KT5823 (PKGi), 2 μM]. F . Densitometric analysis of western blots from three independent experiments is shown. Bars represent the mean ±SD. G . sh-scr and sh1-PDE5 T98G invasion assays on matrigel cushions in the presence of Y-27632 or 8br-cGMP or both. H . Histogram showing the relative migration capacity (fold increase ± SD) of cells that reached the bottom of the matrigel cushion in the different treatments compared to control cells. (**p < 0.001).

Article Snippet: The GBM cell lines (T98G, U87MG, A172, U251, LN18) used in this study were grown in DMEM, (Euroclone; Milan, Italy) with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Immunohistochemistry, Expressing, Phospho-proteomics, Western Blot, Migration, Control

Journal: Oncotarget

Article Title: Type 5 phosphodiesterase regulates glioblastoma multiforme aggressiveness and clinical outcome

doi: 10.18632/oncotarget.14656

Figure Lengend Snippet: A . sh-scr (PDE5+) or sh1-PDE5 (PDE5-) T98G cells were treated with increasing X-ray doses from 2 to 4 Gy and probed with anti-PAR and anti-PARP-1 antibodies. B . sh-scr or sh1-PDE5 T98G cells were exposed to X-rays at 4 Gy in the presence or absence (+/-) of the PARP1 inhibitor olaparib (1 μM). Total protein PARylation levels were assayed after 24 h. C . Schematic representation of cGMP signaling pathway promoting migration and DNA repair in GBM cells.

Article Snippet: The GBM cell lines (T98G, U87MG, A172, U251, LN18) used in this study were grown in DMEM, (Euroclone; Milan, Italy) with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Migration

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

Article Title: Lumefantrine, an antimalarial drug, reverses radiation and temozolomide resistance in glioblastoma

doi: 10.1073/pnas.1921531117

Figure Lengend Snippet: Fli-1 drug screening. (A and B) Relative expression of Fli-1 and HSPB1 in five different GBM cell lines determined by Western blot analysis (A) and normalization of expression (based on β-actin expression) (B). (C) Schematic representation of the Fli-1 drug-screening strategy. Fli-1-Lu: multiple copies of the Fli-1 consensus-binding site (FB site) cloned upstream of a minimal promoter immediately upstream of the luciferase gene. MSCV neo-Fli-1: Fli-1 gene cloned into the MSCV vector. Luciferase gene driven by the Luc promoter: Fli-1-Lu was cotransfected with either MSCV neo-Fli-1 or MSCV empty vector into T98G cells. Cells were treated with various drugs at 36 h posttransfection and screened for efficient down-regulation of luciferase activity. (D) Relative expression by Western blot analysis of Fli-1 and HSPB1 in T98G and U87MG cells treated with positive inhibitory candidates identified using the Fli-1 drug screening protocol. (E) Corresponding densitometry plot for D showing normalization of expression (based on β-actin expression). Each bar represents average of three independent experiments. The level of significance is indicated as *P < 0.05, **P < 0.01, or ***P < 0.001.

Article Snippet: The GBM cell lines were authenticated by cell repository (National Centre for Cell Science).

Techniques: Drug discovery, Expressing, Western Blot, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay

Journal: PNAS Nexus

Article Title: The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma

doi: 10.1093/pnasnexus/pgac013

Figure Lengend Snippet: NHE9 increases activation and expression of RTKs. (A) Schematic of hypothesis linking endosomal NHE9 to RTK (RTK) recycling and oncogenic signaling. (B) Relative mRNA expression of NHE9 measured by qPCR for 10 GBM cell lines established from samples obtained from Johns Hopkins neurosurgery operating room. GBM 276 and GBM 612 were chosen to represent low and high expressors, without being outliers. (C) Portion of phospho-RTK dot blot (left) probed with lysates from GBM 276 ectopically expressing GFP or NHE9-GFP. Densitometry quantification (in arbitrary units) of p-PDGFRα, p-IGF-1Rβ, and p-IR from the dot blot (right). (D) and (F) Representative western blot showing p-IGF-1R, p-IR, and p-PDGFRα (top row) and IGF-1R, IR, and PDGFRα (bottom row) for GBM 276 GFP or NHE9-GFP and densitometry quantification (E) and (G). (H) and (J) Representative western blot showing p-IGF-1R, p-IR, and p-PDGFRα (top row) and IGF-1R, IR, and PDGFRα (bottom row) for GBM 612 scramble or shNHE9 and densitometry quantification (I) and (K). Averages are from 3 independently generated transfections.

Article Snippet: Therefore, to test our hypothesis, we screened for NHE9 expression a diverse panel of 10 patient-derived GBM cell lines, established from samples obtained from the operative room at the Johns Hopkins Hospital and propagated in serum-free medium (Fig. ).

Techniques: Activation Assay, Expressing, Dot Blot, Western Blot, Generated, Transfection

Journal: PNAS Nexus

Article Title: The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma

doi: 10.1093/pnasnexus/pgac013

Figure Lengend Snippet: NHE9 increases surface expression and decreases lysosomal degradation of RTKs. (A) and (B) Representative flow cytometry analysis of nonpermeabilized GBM 276 cells expressing GFP or NHE9-GFP, treated with monoclonal antibodies for IGF-1R (A) and PDGFRα (B) in comparison with IgG1 kappa isotope control. Mean fluorescence intensity of peak areas are from triplicates of independently transfected cultures. (C) Immunofluorescence images of GBM 276 cells showing IGF-1R (Red), NHE9-GFP (Green), and DAPI (blue). Merge of all 3 labels shows co-localization of IGF-1R and NHE9 (yellow). Scale bar: 10 µm. (D) Western blotting analysis of IGF-1R and PDGFRα in GBM 612 scramble or NHE9KD cells treated with 25 nM MG132 or 25 nM bafilomycin for 6 h. Note that only bafilomycin diminishes the effect of shNHE9 on receptor levels.

Article Snippet: Therefore, to test our hypothesis, we screened for NHE9 expression a diverse panel of 10 patient-derived GBM cell lines, established from samples obtained from the operative room at the Johns Hopkins Hospital and propagated in serum-free medium (Fig. ).

Techniques: Expressing, Flow Cytometry, Bioprocessing, Comparison, Control, Fluorescence, Transfection, Immunofluorescence, Western Blot

Journal: PNAS Nexus

Article Title: The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma

doi: 10.1093/pnasnexus/pgac013

Figure Lengend Snippet: NHE9 activates the STAT3 signaling axis. (A) Heatmap of Spearman coefficient correlating protein expression levels of various oncogenic signaling proteins and NHE9 transcript in the TCGA pan-cancer dataset. (B) Correlative analysis between p-STAT3 (Y705) protein level and NHE9 transcript in the TCGA glioma patient cohort. (Correlation coefficient: 0.19; **** P- value < 0.0001) (C) Schematic of the IGF-1R-JAK2-STAT3 signaling axis. (D) Representative western blot of p-STAT3 and STAT3, and densitometry quantification (E). (F) Representative western blot GBM 612 scramble or NHE9KD probing for p-STAT3 and STAT3 and averages of densitometry quantification of 3 independent transfections are shown (G). (H)–(J) Western blotting analysis with time course for the indicated time points in minutes, and 25 h postligand treatment with insulin in GBM 276 GFP or NHE9-GFP as described in Methods. Blots were probed for (H) pro-IGF-1R, IGF-1R, p-IR/IGF-1R, p-IGF-1R (Y1216), p-IGF-1R (Y1135), (I) p-JAK2, JAK2, p-STAT3, STAT3, (J) Nanog (D73G4), Nanog (1E6C4), OCT4A, and GAPDH. (K) qPCR analysis for Oct4 and Nanog transcript in GBM 276 GFP or NHE9-GFP with or without STAT3 inhibitors, Cryptotanshinone (Crypto) or Stattic.

Article Snippet: Therefore, to test our hypothesis, we screened for NHE9 expression a diverse panel of 10 patient-derived GBM cell lines, established from samples obtained from the operative room at the Johns Hopkins Hospital and propagated in serum-free medium (Fig. ).

Techniques: Expressing, Western Blot, Transfection

Journal: PNAS Nexus

Article Title: The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma

doi: 10.1093/pnasnexus/pgac013

Figure Lengend Snippet: NHE9 increases stemness gene expression. (A) Schematic showing that Oct4 and Nanog shift distribution of cell state toward cancer stem cells (red), relative to differentiated cells (blue). (B)–(D) qPCR analysis of Oct4, Nanog, and Sox2 transcripts, as indicated. (B) GBM 276 GFP and NHE9-GFP (C) GBM 612 scramble and shNHE9 (NHE9 KD) (D) GBM 612 control siRNA or siNHE9-coated PBAE nanoparticles (NHE9 KD). Representative western blots for (E) Oct4 and (G) Nanog in GBM 276 GFP or NHE9-GFP and (F) and (H) averages of densitometry quantifications are shown. Figure shows different sections of the same gel as Fig. and has the same loading control, tubulin. (I)–(L) Representative western blots for (I) Oct4 and (L) Nanog in GBM 612 scramble or NHE9 KD and densitometry quantifications (J) and (L) are shown. Figure and (K) are also different sections of the same gel and have the same tubulin loading controls. In all cases, averages of 3 independently transfected sets of cells are shown.

Article Snippet: Therefore, to test our hypothesis, we screened for NHE9 expression a diverse panel of 10 patient-derived GBM cell lines, established from samples obtained from the operative room at the Johns Hopkins Hospital and propagated in serum-free medium (Fig. ).

Techniques: Gene Expression, Control, Western Blot, Transfection

Journal: PNAS Nexus

Article Title: The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma

doi: 10.1093/pnasnexus/pgac013

Figure Lengend Snippet: NHE9 decreases expression of differentiation markers. (A) Schematic showing that lineage markers such as GFAP shift cancer stem cells toward a differentiated state. (B)–(D) qPCR analysis for the indicated lineage markers, averaged from 3 biological replicates. (B) GBM 612 and GBM 275 (C) GBM 276 GFP or NHE9-GFP (D) GBM 612 scramble or NHE9 KD. (E) Representative western blot for GFAP in GBM 276 GFP or NHE9-GFP and (F) averages of densitometry quantification from 3 independent transfections are shown. (G) and (H) Representative immunofluorescence image for (G) GFAP (red) and DAPI (blue) in GBM 276 GFP and NHE9-GFP and (H) fluorescence signal quantification. (I) Representative western blot for GFAP in GBM 612 scramble or NHE9-GFP and (J) densitometry quantification. (K) and (L) Representative immunofluorescence image for (K) GFAP (light blue) and DAPI (blue) in GBM 612 scramble or NHE9 KD and (L) mean fluorescence signal quantification.

Article Snippet: Therefore, to test our hypothesis, we screened for NHE9 expression a diverse panel of 10 patient-derived GBM cell lines, established from samples obtained from the operative room at the Johns Hopkins Hospital and propagated in serum-free medium (Fig. ).

Techniques: Expressing, Western Blot, Transfection, Immunofluorescence, Fluorescence

Journal: PNAS Nexus

Article Title: The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma

doi: 10.1093/pnasnexus/pgac013

Figure Lengend Snippet: Endosomal pH regulates stemness in GBM cells. (A) Schematic of the 3 approaches used to query the role of endosomal pH. Loss of function (LOF) mutations in the Na + /H + exchanger prevent endosomal alkalinization. Acidification by Vacuolar H + -ATPase is blocked by bafilomycin. The ionophore monensin mimics Na + /H + exchange activity of NHE9. (B) qPCR analysis of Oct4 and Nanog transcripts in GBM 276 expressing vector (GFP), or the following GFP-tagged constructs: NHE9, S438P, or V176I. (C) Western blot of GBM 276 lysates expressing GFP (Ctrl) or the NHE9 constructs indicated, for IGF-1R, Oct4, and Nanog. (D) qPCR for Oct4 and Nanog transcripts in GBM 276 with no-treatment, 0.5 µM monensin, or 1 µM monensin for 16 h. (E) Western blot of PDGFRα, IGF-1R, and Oct4 in GBM 276 treated with monensin at the concentrations shown for 16 h. (F) qPCR analysis for Oct4 and Nanog transcripts in GBM 276 treated with bafilomycin at the concentrations shown for 6 h. (G) Western blot for PDGFRα and IGF-1R in GBM 276 treated with bafilomycin as shown for 6 h.

Article Snippet: Therefore, to test our hypothesis, we screened for NHE9 expression a diverse panel of 10 patient-derived GBM cell lines, established from samples obtained from the operative room at the Johns Hopkins Hospital and propagated in serum-free medium (Fig. ).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Construct, Western Blot

Journal: PNAS Nexus

Article Title: The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma

doi: 10.1093/pnasnexus/pgac013

Figure Lengend Snippet: NHE9 increases self-renewal in GBM cells. (A) Light microscopy and fluorescence images of tumorspheres formed from GBM 276 GFP or NHE9-GFP cells in suspension in serum-free media on a noncoated petri dish after 2 weeks of culturing (scale bars, 50 µm). (B)–(D) Number of tumorspheres per well in a 96-well plate at various seeding dilutions as indicated in cell numbers for (B) GBM 612 or GBM 276 (C) GBM 612 scramble or NHE9 KD (D) GBM 276 GFP or NHE9-GFP. (E)–(H) ELDA for estimated stem cell frequency and CI for (E) GBM 612 or GBM 276, (F) GBM 612 scramble or NHE9KD, (G) GBM 276 GFP or NHE9-GFP, and (H) GBM 276 GFP, NHE9-GFP, NHE9-GFP (S438P), or NHE9-GFP (V176I).

Article Snippet: Therefore, to test our hypothesis, we screened for NHE9 expression a diverse panel of 10 patient-derived GBM cell lines, established from samples obtained from the operative room at the Johns Hopkins Hospital and propagated in serum-free medium (Fig. ).

Techniques: Light Microscopy, Fluorescence, Suspension

Journal: PNAS Nexus

Article Title: The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma

doi: 10.1093/pnasnexus/pgac013

Figure Lengend Snippet: NHE9 increases tumor initiation in vivo. (A) Comparison of NHE9 ( SLC9A9 ) mRNA expression between GBM tumor and normal samples from cBioPortal database (* P -value: 0.0423; Student's t test). (B) Representative immunohistochemistry staining for NHE9 protein from normal, LGG, and HGG, obtained from the Human Protein Atlas. (C) Bioluminescence signal from mice with orthotopic brain xenograft of luciferase-expressing 612 GBM tumor cells with or without NHE9-shRNA seeded with 3,000 or 1,000 cells. (D) Quantification of total bioluminescence flux from mice with 3,000 cells (** P -value: 0.0084; Student's t test). (E) Representative immunostaining images for Human Nuclei (HuNu) and Ki-67 from brain tissues injected with control or NHE9 KD cells with 3,000 cells. (F) Tumor frequency in mice injected with GBM 612 control or NHE9 KD cells with 1,000 and 3,000 cells.

Article Snippet: Therefore, to test our hypothesis, we screened for NHE9 expression a diverse panel of 10 patient-derived GBM cell lines, established from samples obtained from the operative room at the Johns Hopkins Hospital and propagated in serum-free medium (Fig. ).

Techniques: In Vivo, Comparison, Expressing, Immunohistochemistry, Staining, Luciferase, shRNA, Immunostaining, Injection, Control