Journal: Frontiers in Immunology

Article Title: Development and evaluation of an inhalable nanoemulsion system for enhancing NK cell function against osteosarcoma pulmonary metastases

doi: 10.3389/fimmu.2026.1772375

Figure Lengend Snippet: SIS3-VAP–DAC nanoemulsion enhances in vitro NK-92 cell-mediated cytotoxicity of osteosarcoma in the presence of TGF-β. Fold-change in MG63-GFP cell count measured by IncuCyte over 48 h following co-incubation with irradiated NK-92 cells (E:T 5:1) in media supplemented with 5 ng/mL TGF-β and 50% (v/v) NE1 (blank), NE2 (SIS3), NE3 (VAP–DAC), or NE4 (SIS3-VAP–DAC) nanoemulsions.

Article Snippet: Recombinant human (rh)IL-2 and rhIL-15 were obtained from the Biological Resources Branch (National Cancer Institute, Frederick, MD) and recombinant murine IL-15 was purchased from R&D Systems (Minneapolis, MN). hSAEC primary small airway epithelial cells (Cat #PCS-301-010), NK-92 human NK cell lymphoma (Cat #CRL-2407), MG63 human osteosarcoma (Cat #CRL-1427), and K7M2 murine osteosarcoma (Cat #CRL-2836) cell lines were all obtained from ATCC (Manassas, VA).

Techniques: In Vitro, Cell Characterization, Incubation, Irradiation

Journal: Frontiers in Immunology

Article Title: Development and evaluation of an inhalable nanoemulsion system for enhancing NK cell function against osteosarcoma pulmonary metastases

doi: 10.3389/fimmu.2026.1772375

Figure Lengend Snippet: Combination SIS3-VAP–DAC nanoemulsion with adoptive NK-92 cell therapy reduces pulmonary osteosarcoma metastases in vivo without systemic toxicity. (A) Representative bioluminescence images of mice from each treatment group at week 1 of dosing. (B) Chest-region bioluminescence ROI values at week 1 across treatment groups. Box and whisker plots display quantitative ROI measurements from the chest area of individual mice, with the middle line indicating the median value. Data represent pulmonary tumor burden after one week of treatment. Whiskers represent data within 1.5 times the interquartile range. (C) Representative bioluminescence images of mice from each treatment group at week 2 of dosing. (D) Chest-region bioluminescence ROI values at week 2 across treatment groups. Box and whisker plots display quantitative ROI measurements from the chest area of individual mice, with the middle line indicating the median value. Data represent pulmonary tumor burden after two weeks of treatment. Whiskers represent data within 1.5 times the interquartile range. (E) Representative bioluminescence images of mice from each treatment group at week 3 of dosing. (F) Chest-region bioluminescence ROI values at week 3 across treatment groups. Box and whisker plots display quantitative ROI measurements from the chest area of individual mice, with the middle line indicating the median value. Data represent pulmonary tumor burden after three weeks of treatment. Whiskers represent data within 1.5 times the interquartile range. At weeks 2 and 3 (D, F) , asterisks denote statistically significant differences between NE7 and the control group by Kruskal–Wallis non−parametric analysis of variance followed by Dunn’s post hoc test (*: p < 0.05, Bonferroni corrected). No significant differences were detected at week 1 (B) . (G) Percentage change in mean body weight over time for control and nanoemulsion-treated mice (NE5-NE7) following MG63 cancer cell injection. Error bars represent ± SD of the percentage changes from baseline at each timepoint. (H) Summed clinical scores in control and nanoemulsion-treated mice (NE5-NE7) over 49 days after MG63 cancer cell injection. Points at zero indicate no observable adverse clinical signs.

Article Snippet: Recombinant human (rh)IL-2 and rhIL-15 were obtained from the Biological Resources Branch (National Cancer Institute, Frederick, MD) and recombinant murine IL-15 was purchased from R&D Systems (Minneapolis, MN). hSAEC primary small airway epithelial cells (Cat #PCS-301-010), NK-92 human NK cell lymphoma (Cat #CRL-2407), MG63 human osteosarcoma (Cat #CRL-1427), and K7M2 murine osteosarcoma (Cat #CRL-2836) cell lines were all obtained from ATCC (Manassas, VA).

Techniques: In Vivo, Whisker Assay, Control, Injection

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: Hypoxia‐induced alterations in mRNA and protein expression of G2 checkpoint regulators. A. Gene expression of positive G2 checkpoint regulators in U2OS cells. The ratio of mRNA expression in cells treated with hypoxia (0.2% O2, 24 h) relative to mRNA expression in cells cultured at normoxia (21% O2) is shown. Data were obtained from genome wide microarray analysis. The positive G2 checkpoint regulators were found from published studies as described in Table 1. B. Gene expression of negative G2 checkpoint regulators similar as in A. C. Immunoblot analysis of protein extracts from U2OS cells exposed to hypoxia or normoxia for 24 h. The samples are from the same experiment as the microarray results shown in A and B. HIF1α was shown to confirm hypoxia. H4 was used as loading control.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Gene Expression, Cell Culture, Genome Wide, Microarray, Western Blot, Control

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: Protein levels of G2 checkpoint regulators in individual G2 cells following hypoxia. A. Cell cycle profiles of U2OS cells after hypoxia treatment as in Figure 1 (24 h 0.2% O2). Flow cytometric analysis was performed after staining with anti phospho‐H3Ser10 (H3P) to mark mitotic cells, and the DNA stain Hoechst. Numbers indicate fraction of mitotic cells. B. Flow cytometric barcoding analysis for accurate measurement of protein levels in G2 phase cells. U2OS cells treated with four different conditions, as indicated in the right column, were labeled with different concentrations of Pacific Blue and combined into a single sample. The single sample of cells was then stained with antibodies to Cyclin B and phospho‐H3 and with the DNA‐stain FxCycle Far Red, and analyzed by flow cytometry. Gating of the Pacific Blue‐SSC plot (left) was used to separate the four original samples. The G1, S, G2 and M cell cycle phase populations were gated from the scatter plot of phospho‐Histone H3(Ser10) (H3P) versus DNA content, and the median signal for Cyclin B levels in each cell cycle phase could thus be obtained. C.Median values of G2 phase levels of the indicated proteins obtained as in B, after subtraction of background values obtained as in Figure S1. U2OS cells were grown at 21% O2, or exposed to 24 h hypoxia at 0.2% O2, or first exposed to 0.2% O2 for 24 h followed by subsequent incubation at 21% O2 for 90 min (90 min reox) or 6 h (6 h reox). Average results from at least 3 independent experiments are shown. Error bars indicate SEM.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Staining, Labeling, Flow Cytometry, Incubation

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: Hypoxia‐induced changes in CDK activity and G2 checkpoint activation. A. CDK activity in G2 phase cells as measured by phosphorylation of BRCA2‐Ser3291. U2OS cells were grown at 21% O2, or incubated at 0.2% O2 for 24 h and harvested inside the hypoxia chamber and at 1 and 4 h after reoxygenation, or treated with Roscovitine for 2 h at 21% O2. Flow cytometry barcoding analysis of phospho‐BRCA2‐Ser3291 was performed as in Figure 2 and S2. B. Immunoblot analysis of U2OS cells treated as in A with antibodies to total BRCA2 and γ‐tubulin (loading control). C. G2 checkpoint activation after IR (0.2%O2 24 h). Flow cytometric analysis of G2 checkpoint arrest after X‐ray irradiation (0, 0.5, 1 Gy) of normoxic U2OS cells (21%O2) or U2OS cells exposed to 24 h of hypoxia at 0.2%O2 and irradiated 15 min after reoxygenation. Nocodazole was added to all samples 1 h after IR, and the samples were harvested 5 h later. The relative mitotic fraction was determined as the fraction of phospho‐H3 positive cells in irradiated samples divided by the fraction of phospho‐H3 positive cells in non‐irradiated samples. Average values from 3 independent experiments are shown. Error bars indicate SEM. D. Phosphorylation of BRCA2‐Ser3291 in H460 cells treated with hypoxia and analyzed as in A. E. G2 checkpoint activation in H460 cells treated with hypoxia and IR and analyzed as in C.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Incubation, Flow Cytometry, Western Blot, Control, Irradiation

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: IR‐induced G2 checkpoint and expression of G2 checkpoint regulators in U2OS cells after severe hypoxia (∼0.03% O2 20 h) and prolonged mild hypoxia (0.2%O2, 72 h). A. Similar G2 checkpoint measurement after IR as in Figure 3C following incubation at severe hypoxia (∼0.03% O2 20 h). B. Similar as in A following incubation at prolonged mild hypoxia (0.2% O2 72 h). C. Similar flow cytometric barcoding analysis of protein levels in G2 phase cells as in Figure 2C following incubation at severe hypoxia (∼0.03% O2 20 h). D. Similar as in C following incubation at 0.2% O2, 72 h.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Incubation

Journal: PLoS ONE

Article Title: High Expression of CD109 Antigen Regulates the Phenotype of Cancer Stem-Like Cells/Cancer-Initiating Cells in the Novel Epithelioid Sarcoma Cell Line ESX and Is Related to Poor Prognosis of Soft Tissue Sarcoma

doi: 10.1371/journal.pone.0084187

Figure Lengend Snippet: A. Expression of CD109 mRNA in ES cell lines. Bars represent mean±SEM. ※※※ p <0.001, determined by the Mann-Whitney test. N.S.: not significant. B. Expression of CD109 mRNA in human sarcoma cell lines. Cell lines of epithelioid sarcoma (FU-EPS-1 and VA-ES-BJ), osteosarcoma (OS2000, KIKU, NY, U2OS, Saos-2, HuO9 and HOS), Ewing sarcoma (SKES, WES and RDES), synovial sarcoma (Fuji and YaFuSS) and malignant fibrous histiocytoma (MFH2003 and MFH2004) were used. C. Expression of CD109 mRNA in human fetal tissues (upper panel) and human adult tissues (lower panel). ESX was used as a positive control. D. Immunohistochemistry of CD109 in normal adult tissues.

Article Snippet: Human osteosarcoma cell lines (NY, U2OS and HOS), human Ewing sarcoma cell lines (SKES, WES, and RDES), the human synovial sarcoma cell line FUJI, and the human ES cell line VA-ES-BJ were purchased from the Japanese Collection of Research Bioresources Cell Bank (Tokyo, Japan) and American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, MANN-WHITNEY, Positive Control, Immunohistochemistry

Journal: PLoS ONE

Article Title: Ligand Bound β1 Integrins Inhibit Procaspase-8 for Mediating Cell Adhesion-Mediated Drug and Radiation Resistance in Human Leukemia Cells

doi: 10.1371/journal.pone.0000269

Figure Lengend Snippet: (a) Expression of procaspase-8, FADD and β-actin was analyzed by Western blotting. Procaspase-8 negative (Casp-8N), FADD negative (FADD-N) and Jurkat A3 cells were irradiated with 10 Gy in suspension or under adhesion to 100 µg/cm 2 FN. (b) Casp-8N, FADD-N and Jurkat A3 cells were exposed to mAb TS2/16 or mAb13 (1 µg/ml; anti rat IgG1 as control) for 1 h or 20 µM caspase-8 (IETD-fmk), caspase-3 (DEVD-fmk), pan-caspase inhibitor (zVAD-fmk) or 10 µM Ly294002 for 30 min when adhered to 100 µg/cm 2 FN. Subsequently, cells were treated with 10 Gy or 300 ng/ml FasL. After 48 h, the number of apoptotic cells was determined by DAPI staining and counting. Columns represent mean±s.d. (n = 3). Statistical analysis was performed by comparing treatment conditions versus controls. * P <0.01.

Article Snippet: Human promyelocytic HL60 leukemia and Jurkat A3 T-lymphoma cells were purchased from ATCC (Bethesda, MD, USA).

Techniques: Expressing, Western Blot, Irradiation, Suspension, Control, Staining

Journal: PLoS ONE

Article Title: Ligand Bound β1 Integrins Inhibit Procaspase-8 for Mediating Cell Adhesion-Mediated Drug and Radiation Resistance in Human Leukemia Cells

doi: 10.1371/journal.pone.0000269

Figure Lengend Snippet: (a) Jurkat cell lines were transfected with two different β1 integrin (β1.1, β1.2) siRNAs or a non-specific Duplex XII (DXII) siRNA. Expression of β1 integrin was inspected by immunoblotting. (b) Following β1 integrin knockdown, 10 Gy or 300 ng/ml FasL were applied to the cells grown on 100 µg/cm 2 FN. Apoptosis was determined 48 h later by DAPI. (c) In parallel, cell lysates were harvested for analysis of procaspase-8, -3 and Akt expression. (d) Subsequent to administration of 20 µM caspase-8 (IETD-fmk) or caspase-3 (DEVD-fmk), 10 µM Ly294002 or 0.25 µl/ml DMSO for 30 min, caspase-8 and -3 activity was measured at 4 h after 10 Gy. Statistics were calculated by comparing the level of apoptosis in β1 integrin knockdown cells versus DXII. * P <0.01.

Article Snippet: Human promyelocytic HL60 leukemia and Jurkat A3 T-lymphoma cells were purchased from ATCC (Bethesda, MD, USA).

Techniques: Transfection, Expressing, Western Blot, Knockdown, Activity Assay

Journal: Neuro-oncology

Article Title: Gadd45a sensitizes medulloblastoma cells to irradiation and suppresses MMP-9-mediated EMT.

doi: 10.1093/neuonc/nor109

Figure Lengend Snippet: Fig. 1. AnalysisofcellcycleproteinsinvolvedinG2-Marrest afterionizingradiation(IR)treatment. (A) Western blotanalysis forCdc-2andpCdc-2 protein using 40 mg of total cell lysates from non–IR- and IR-treated DAOY and D283 cells. Equal loading of these proteins was confirmed by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B) Immuno-flow cytometry analysis of Alexa Fluor-labeled Gadd45a in DAOY and D283 cells. Approximately 1 × 106 non–IR- and IR-treated cells were trypsinized, washed in phosphate-buffered saline (PBS), incubated with anti-Gadd45a antibody overnight at 48C, and labeled with green Alexa Fluor 488, a fluorescent-labeled, species-specific secondary antibody (Invitrogen) for 1 h at room temperature. Normal immunoglobulin (Ig) G was used as a negative control to set the cut-off point for green fluorescence intensity. (C and D) Fluorescence-activated cell sorting (FACS) analysis of cell cycle progression using knockdown and overexpression of Gadd45a with IR treatment was done as described in Materials and Methods. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells in the G1 (M2), S (M3) and G2/M (M4) phases of the cell cycle were calculated using CellQuest Pro software. Graphical representation of FACS data from 3 independent experiments is shown as the mean+SD (P , .05). (E) TUNEL nuclear staining on D283 cells. Control cells with knockdown (siRNA Gadd45a) and overexpression of Gadd45a (Ov-exp Gadd45a) in combination with IR treatment on 8-well chambered slides were subjected to TUNEL nuclear staining (Roche Applied Science) and viewed by fluorescence microscopy. Green fluorescence represents apoptotic cells (For the color figure, please refer to supplementary material, Fig. S1).

Article Snippet: Human medulloblastoma cell lines DAOY and D283 were purchased from ATCC and maintained in serum containing advanced minimum essential medium (MEM) or improved MEM, respectively, in a 378C incubator with 5% CO2 humidified atmosphere.

Techniques: Western Blot, Cytometry, Labeling, Saline, Incubation, Negative Control, Fluorescence, FACS, Knockdown, Over Expression, Cell Counting, Software, TUNEL Assay, Staining, Control, Microscopy

Journal: Neuro-oncology

Article Title: Gadd45a sensitizes medulloblastoma cells to irradiation and suppresses MMP-9-mediated EMT.

doi: 10.1093/neuonc/nor109

Figure Lengend Snippet: Fig. 2. Co-localization of Gadd45a with Cdc2 and nuclear translocation of b-catenin with ionizing radiation (IR) treatment. (A and B) Immunocytochemistry analysis was carried out on DAOY (A) and D283 (B) transfected (siRNA GAdd45a and Ov-exp Gadd45a) and IR-treated cells. Microscopic images depict expression of Gadd45a (green fluorescence) and Cdc2 (red fluorescence) proteins in the cells with knockdown (siRNA Gadd45a) and overexpression of Gadd45a (Ov-exp Gadd45a) in combination with IR treatment. (C1-C2) Nuclear translocation of b-catenin (red fluorescence) into the nucleus (blue fluorescence) of DAOY and D283 cells with Ov-exp-Gadd45a in combination with IR treatment. Immunostaining was done according to the protocol described in Materials and Methods. Pictures were taken using confocal microscopy (Olympus BX61 Fluoview) at a 40× magnification. (For the color figure, please refer to supplementary material, Fig. S4)

Article Snippet: Human medulloblastoma cell lines DAOY and D283 were purchased from ATCC and maintained in serum containing advanced minimum essential medium (MEM) or improved MEM, respectively, in a 378C incubator with 5% CO2 humidified atmosphere.

Techniques: Translocation Assay, Immunocytochemistry, Transfection, Expressing, Knockdown, Over Expression, Immunostaining, Confocal Microscopy

Journal: Neuro-oncology

Article Title: Gadd45a sensitizes medulloblastoma cells to irradiation and suppresses MMP-9-mediated EMT.

doi: 10.1093/neuonc/nor109

Figure Lengend Snippet: Fig. 3. Gadd45a regulates distribution of cellular b-catenin. (A) Western blot analysis of nuclear extracts (NEs) to demonstrate b-catenin, LEF-1, and p-p53 expression in DAOY and D283 cells. Nuclear extracts were prepared using nuclear extraction kit (Panomics). Forty mg of total nuclear protein was used for analysis, and Lamin B was used as a nuclear protein marker to confirm equal loads of proteins. (B) Western blot analysis of cytoplasmic extracts (CEs) to demonstrate the levels of Gadd45a, p-b-catenin (Ser33/37/Thr41) and intracellular levels of MMP-9. GAPDH was used to confirm equal loading of proteins. (C) Western blot analysis of membrane extracts (MEs) to demonstrate accumulation of b-catenin on membranes of DAOY and D283 cells. Membrane proteins were isolated using the Triton X-114 phase separation method. Flotillin-2 was used as a membrane marker to confirm equal loading of proteins. (D) Immunoprecipitation (IP) using b-catenin antibody to show its interaction with N- and E-cadherins of treated DAOY cells. Approximately 400 mg of ME protein was immunoprecipitated with 2 mg of b-catenin primary antibody and immunoblotted using anti-N-cadherin, anti-E-cadherin, and anti-b-catenin antibodies. (E) The protein band intensity of IP studies was measured using densitometry and quantified data from 3 different experiments are represented graphically (mean+SD, P* , .01). (F) Monitoring p53 activation as assessed by p53 DNA binding using TransAM assay (TransAM, p53 Transcription Factor Assay Kit; Active Motif) as described in Materials and Methods. The experiment was performed at least twice in duplicate and results are graphically represented as bar diagrams. Error bars represent mean+SD (P* , .01). (G) Reverse-transcription polymerase chain reaction (RT-PCR) analysis to determine the b-catenin, N-cadherin, E-cadherin, and matrix metallopeptidase (MMP)–9 transcript levels in DAOY and D283 control, Ov-exp Gadd45a alone and in combination with IR-treated cells. (H) RT-PCR analysis of DAOY and D283 cells transfected with MMP-9 plasmid (pM) and irradiated after 48 h of transfection. PCR analysis was performed using primers specific for Gadd45a, MMP-9, and GAPDH. Total RNA was extracted from treated cells, and cDNA was prepared according to the standard protocols.

Article Snippet: Human medulloblastoma cell lines DAOY and D283 were purchased from ATCC and maintained in serum containing advanced minimum essential medium (MEM) or improved MEM, respectively, in a 378C incubator with 5% CO2 humidified atmosphere.

Techniques: Western Blot, Expressing, Extraction, Marker, Membrane, Isolation, Immunoprecipitation, Activation Assay, Binding Assay, Transcription Factor Assay, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, Irradiation

Journal: Neuro-oncology

Article Title: Gadd45a sensitizes medulloblastoma cells to irradiation and suppresses MMP-9-mediated EMT.

doi: 10.1093/neuonc/nor109

Figure Lengend Snippet: Fig. 4. Gadd45a-mediated decrease in the invasive, migratory, and proliferative potential of medulloblastoma cell lines. Approximately 1 × 105 DAOY (A) and 2 × 106 D283 (B) cells treated with ionizing radiation (IR), knockdown (siRNA Gadd45a) or overexpression of Gadd45a (Ov-exp Gadd45a) were suspended in serum-free media and plated onto Matrigel-coated transwell inserts, as described in Materials and Methods. After a 24-hr incubation period, lower invaded cells were stained with HEMA-3. Images of invaded cells were taken under a light microscope (Olympus IX-71). The invasive potential of treated cells was quantified, and the percentage of cells invading from 3 independent experiments are graphically represented as bar diagrams. Error bars represent mean+SD (*P , .05). (C) Wound healing assay, which is indicative of migration potential of cancer cells, was performed using 80%–85% confluent-treated DAOY cells, as described in Materials and Methods. Photographs were taken using standard 200-mm scale bar. Percent wound repair was calculated from the mean of the average width of the wound obtained from 3 independent experiments and graphically represented as bar diagrams. Error bars represent mean+SD (P* , .05). (D) Gelatin zymography was performed to determine matrix metallopeptidase (MMP)–9 activity in the conditioned media of the aforementioned treated cells. The intensity of clear halo band of MMP-9 in zymography gels was measured using densitometry and graphically represented, and the error bars represent mean+SD (P , .05, with IR; P , .01 with control). (E) Clonogenic assay for DAOY cells. The control cells treated with siRNA-Gadd45a, Ov-exp Gadd45a, and in combination with IR treatment were trypsinized to produce single-cell suspension. Approximately 500 cells from each treatment were plated individually in 60-mm plates containing complete media. The plates were incubated for 12 days until they formed sufficiently large colonies. Next, the cells were fixed and stained using HEMA-3 stain. The number of colonies were quantified from 3 independent experiments and are graphically represented as the measure of clonogenecity. Error bars represent mean+SD (P* , .05).

Article Snippet: Human medulloblastoma cell lines DAOY and D283 were purchased from ATCC and maintained in serum containing advanced minimum essential medium (MEM) or improved MEM, respectively, in a 378C incubator with 5% CO2 humidified atmosphere.

Techniques: Knockdown, Over Expression, Incubation, Staining, Light Microscopy, Wound Healing Assay, Migration, Zymography, Activity Assay, Control, Clonogenic Assay, Suspension

Journal: Neuro-oncology

Article Title: Gadd45a sensitizes medulloblastoma cells to irradiation and suppresses MMP-9-mediated EMT.

doi: 10.1093/neuonc/nor109

Figure Lengend Snippet: Fig. 5. Role of Gadd45a in suppression of matrix metallopeptidase (MMP)–9 and epithelial-mesenchymal transition (EMT). The DAOY and D283 cells were treated with pM, with pM + siRNA Gadd45a alone, and with IR treatment (A) and were separately treated with Ov-exp Gadd45a, Ov-exp Gadd45a + rMMP-9 alone and in combination with IR treatment (B). The control cells were treated with scrambled vector (pSV) alone and with IR treatment. Western analysis was done using 40 mg of total lysates from the treated cells to show the levels of Gadd45a, b-catenin, E-cadherin, N-cadherin, and fibronectin. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Gelatin zymography was performed to determine MMP-9 activity using conditioned media from treated samples. (C) Diagramatic representation showing the role of Gadd45a in the negative regulation of MMP-9 induction with IR treatment; the overexpression of Gadd45a accompanied by IR inhibits MMP-9 induction and nuclear translocation of b-catenin. Compared to the control cells and IR treated cells, the downregulation of MMP-9 (pM) or upregulation of Gadd45a (Ov-exp Gadd45a) accompanied by IR shifts the balance towards Gadd45a and thus has significant therapeutic implications.

Article Snippet: Human medulloblastoma cell lines DAOY and D283 were purchased from ATCC and maintained in serum containing advanced minimum essential medium (MEM) or improved MEM, respectively, in a 378C incubator with 5% CO2 humidified atmosphere.

Techniques: Control, Plasmid Preparation, Western Blot, Zymography, Activity Assay, Over Expression, Translocation Assay

Journal: Neuro-oncology

Article Title: Gadd45a sensitizes medulloblastoma cells to irradiation and suppresses MMP-9-mediated EMT.

doi: 10.1093/neuonc/nor109

Figure Lengend Snippet: Fig. 6. Downregulation of MMP-9 (pM) in in vivo tumors increased radioresponse by inducing Gadd45a expression and suppression of epithelial-mesenchymal transition (EMT). Intracerebral tumors were established in nude mice by injecting 1 × 105 DAOY cells. A group of 5 animals was used for each treatment condition (control, IR, pM, and pM + IR). Alzet osmotic pumps (model 2001; Alzet Osmotic Pumps) were implanted into the animals for pM delivery (6–8 mg/kg body weight) followed by IR treatment after 1 week. (A) Brain tissue sections were subjected to hematoxylin and eoisin staining. (B) Immunohistochemical comparison of treated tumor sections using green Alexa Fluor 488 and red Alexa Fluor 594 secondary antibodies for Gadd45a and matrix metallopeptidase (MMP)–9, respectively. (C) Immunohistochemical analysis of the tumor sections to show the levels of EMT markers, N-cadherin (green) and E-cadherin (red). The merged figures are represented here and the individual stained figures are shown in the Supplementary material, Fig. S2. (D) Semiquantification of tumor volume in control, IR, pM, and pM + IR vector-treated groups was done as described in Materials and Methods. Data shown are the mean+SD values from 3 animals from each group (*P , .005).

Article Snippet: Human medulloblastoma cell lines DAOY and D283 were purchased from ATCC and maintained in serum containing advanced minimum essential medium (MEM) or improved MEM, respectively, in a 378C incubator with 5% CO2 humidified atmosphere.

Techniques: In Vivo, Expressing, Control, Staining, Immunohistochemical staining, Comparison, Plasmid Preparation