Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

Journal: Molecular Oncology

Article Title: The endochondral bone protein CHM 1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

doi: 10.1002/1878-0261.12057

Figure Lengend Snippet: CHM 1 inhibits tube formation and influences osteomimicry. (A) Tube formation assay with constitutively transfected A673 (sh.control and sh. CHM 1) and transiently transfected MHH ‐ ES 1 (si.control and si. CHM 1_1) cells demonstrated CHM 1 to clearly inhibit endothelial differentiation potential (scale bar 0.5 mm). (B) Analysis of osteolysis of A673 sh. CHM 1 and negative controls (sh.control) in an orthotopic bone xenotransplantation model (five to eight mice per group). Affected bones were assessed by histology ( TRAP staining, scale bar 0.25 mm or 0.05 mm). Left panel: quantitative summary of the average number of osteoclasts (mm 2 ) in unaffected bone marrow, tumor samples, and attached to the bone in tumor tissues (bone). Data are mean ± SEM of at least two independent samples (at least 40 segments counted); t ‐test. Right panel: Representative pictures are shown. CHM 1 knockdown significantly enhanced the amount of TRAP ‐positive osteoclasts attached to the bone (b) in the area of tumor (arrow) and thus increased the osteolytic phenotype. (C) Different ES cell lines with constitutive CHM 1 knockdown and respective controls were analyzed by qRT ‐ PCR for expression of osteolytic genes such as HIF 1A , IL 6 , JAG 1 , and VEGF . Data are mean ± SEM of two independent experiments; t‐ test. * P < 0.05; ** P < 0.005; *** P < 0.0005 (see 2.15. Statistical analyses).

Article Snippet: A673 was purchased from ATCC (LGC Standards, Teddington, UK).

Techniques: Tube Formation Assay, Transfection, Control, Staining, Knockdown, Quantitative RT-PCR, Expressing

Journal: Molecular Oncology

Article Title: The endochondral bone protein CHM 1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

doi: 10.1002/1878-0261.12057

Figure Lengend Snippet: CHM 1 delayed proliferation in ES in vitro . (A) Analysis of contact‐dependent growth of constitutively sh. CHM 1‐ and sh.control‐infected ES cell lines with xCELL igence. Left panel: Cellular impedance was measured every four hours (relative cell index). Data are mean ± SEM (hexaplicate/group); t ‐test. Right panel: doubling time of constitutive A673, SK ‐N‐ MC , and TC ‐71 CHM 1 sh RNA infectants. Data are mean ± SEM of two independent experiments/cell line (hexaplicate/group); t ‐test. B. Effect of CHM 1 knockdown on anchorage‐independent growth in A673, SK ‐N‐ MC , and TC ‐71 cells using methylcellulose matrices. Left panel: A representative experiment with SK ‐N‐ MC cells was shown as macrograph. Right panel: The average number of colonies of at least two different experiments with three different ES cell lines was shown after stable CHM 1 suppression. (C) Left panel: evaluation of tumorigenicity of constitutive A673 and TC ‐71 CHM 1 sh RNA infectants in immunodeficient Rag2 −/− γc −/− mice (3–5 mice per group). Right panel: post ex vivo CHM 1 expression using qRT ‐ PCR . Data are mean ± SEM , t ‐test. * P < 0.05; ** P < 0.005; *** P < 0.0005 (see 2.15. Statistical analyses).

Article Snippet: A673 was purchased from ATCC (LGC Standards, Teddington, UK).

Techniques: In Vitro, Control, Infection, Knockdown, Ex Vivo, Expressing, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: The endochondral bone protein CHM 1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

doi: 10.1002/1878-0261.12057

Figure Lengend Snippet: CHM 1 enhances metastasis in ES in vivo . (A) Analysis of invasiveness of ES cell lines through Matrigel after transfection with specific CHM 1 sh RNA constructs. Data are mean ± SEM of two independent experiments; t ‐test. (B) Upper panel: qRT ‐ PCR of MMP 9 expression after stable CHM 1 knockdown. Data are mean ± SEM of two independent experiments; t ‐test. Lower panel: analysis of the invasive potential of A673 and SK ‐N‐ MC cells after transient transfection with two specific MMP 9 si RNA s 48 h before seeding. Data are mean ± SEM ; t ‐test. (C) Analysis of metastasis using A673 and TC ‐71 cells with stable CHM 1 suppression and respective controls (four to five mice per group). Left panel: Representative lungs with corresponding H&E staining of A673‐injected mice are shown (scale bar 5 or 2 mm). Right panel: Average number of apparent metastases per mouse in lung and liver tissues is illustrated; t ‐test. (D) DotBlot of relative CHM 1 expression in ES osseous tumor samples compared to ES lung metastases samples using microarray analysis of 14 patient tumor samples. * P < 0.05; ** P < 0.005; *** P < 0.0005 (see 2.15. Statistical analyses).

Article Snippet: A673 was purchased from ATCC (LGC Standards, Teddington, UK).

Techniques: In Vivo, Transfection, Construct, Quantitative RT-PCR, Expressing, Knockdown, Staining, Injection, Microarray

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: ( a ) Cell proliferation and ( b ) cell viability alterations of HT-29 and SW480 colorectal cancer cell lines following different folic acid (FA) supplies. Sulforhodamine B (SRB) was used for cell proliferation detection (* p ≤ 0.05, *** p ≤ 0.001), while cell viability data were obtained by alamarBlue assay (** p ≤ 0.01). FA-depleted cells were kept in media containing 0 ng/mL FA, whereas treated cells were exposed to 100 and 10,000 ng/mL FA for 72 h. The percentages of cell proliferation and viability were given relative to samples kept in the normal growth media. FA: folic acid.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Alamar Blue Assay

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: Genomic stability detection of HT-29 and SW480 cells exposed to different folic acid (FA) concentrations (0, 100, 10,000 ng/mL). ( a ) Micronucleus (MN) scoring was performed on DAPI- and anti-γ-H2AX-stained slides. Left: We obtained the results by proportioning the cells with MN with all cells counted (** p ≤ 0.01, *** p ≤ 0.001). Right: Representative γ-H2AX-positive micronuclei are indicated with arrows. ( b ) DNA integrity was evaluated with comet assay, additionally. Left: Graphs show the changes in genomic stability in consideration of comet tail DNA percentage (* p ≤ 0.05). Right: Characteristic comets of both cell lines were captured following different treatments. FA: folic acid.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Staining, Single Cell Gel Electrophoresis

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: DNA methylation analysis of HT-29 and SW480 cell lines exposed to different folic acid (FA) concentrations. The methylation levels of long interspersed nuclear element 1 (LINE-1) CpG positions (pos 1, pos 2, pos 3) were ( a ) summarized and also ( b ) visualized individually to detect global DNA methylation changes. With the use of Reduced Representation Bisulfite Sequencing (RRBS) method, a genome-wide methylome profile of 10,000 ng/mL FA-treated cells was established in the comparison of cells kept in FA-free (0 ng/mL FA) media. ( c ) Firstly, the number of genes with altered methylation in the investigated CpG sites was assessed. “Hyper” and “hypo” sections indicate the number of genes with methylated and unmethylated CpG sites, respectively. The intersection of these two categories refers to the genes that possess both methylated and unmethylated CpG dinucleotides. ( d ) Heatmap shows the top 10 significantly ( p ≤ 0.05) enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with the number of differentially methylated genes. ( e ) Pie charts represent the localization of differentially methylated sites (DMS) in distinct chromatin states. FA: folic acid; pos: CpG position; hyper: hypermethylation; hypo: hypomethylation; DMSs: differentially methylated sites; heterochrom/lo: heterochromatin or low signal region; txn: transcription; CNV: copy number variation; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: DNA Methylation Assay, Methylation, Methylation Sequencing, Genome Wide, Comparison

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: Genome-wide transcriptome alterations of HT-29 and SW480 cells following 10,000 ng/mL folic acid (FA) supplementation detected by Human Transcriptome Array 2.0 (HTA 2.0). ( a ) Pie charts represent the proportion of up- and downregulated genes. ( b ) Visual networks of protein–protein interactions were generated by the StringApp of Cytoscape software based on the list of genes with significant ( p ≤ 0.05) expression alterations and ≥|1.5| fold change (FC). Colors refer to the expression level of protein-coding genes (dark blue: FC ≤ −2, light blue: FC ≥ −2 and ≤−1.5, light red: FC ≥ 1.5 and ≤2, dark red: FC ≥ 2). ( c ) Top 10 genes showing significant ( p ≤ 0.05) up- and downregulation visualized with volcano plots. Gray points represent all the transcripts detected by HTA 2.0 microarray, while significantly ( p ≤ 0.05) altering genes with FC ≥ |1.5| value were marked with red and blue. P-val: p -value.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Genome Wide, Protein-Protein interactions, Generated, Software, Expressing, Microarray

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: The intersection of genome-wide DNA methylation and gene expression data obtained by Reduced Representation Bisulfite Sequencing (RRBS) and Human Transcriptome Array (HTA) 2.0 analyses. Values represent the methylome and transcriptome pattern changes of 10,000 ng/mL folic acid (FA)-treated HT-29 and SW480 cells compared to non-treated samples (0 ng/mL FA). Only genes with promoter methylation status alteration in accordance with their expression level ( p ≤ 0.05 and fold change ≥|1.5|) were listed (left) and also visualized in volcano plots (right). Gray points represent all the transcripts detected by the microarray, while blue ones highlight down- and red ones show upregulating genes from the list. met. status: DNA methylation status; met. diff.: DNA methylation difference; expr. status: gene expression status; P-val: p -value.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Genome Wide, DNA Methylation Assay, Gene Expression, Methylation Sequencing, Methylation, Expressing, Microarray

Journal: Oncotarget

Article Title: MicroRNA-31 suppresses medulloblastoma cell growth by inhibiting DNA replication through minichromosome maintenance 2

doi:

Figure Lengend Snippet: (A) Hematoxylin and Eosin (H&E) staining of sagittal sections of normal cerebellar and medulloblastoma tissues from Ptch +/- mice. N, normal cerebella. T, medulloblastoma. (B) Heat map of differential miRNA expression by NanoString nCounter analysis of Ptch +/- mouse medulloblastoma and age-matched normal cerebellar tissues. (C) Quantitative RT-PCR analysis of miR-31 using RNAs extracted from normal cerebellar and medulloblastoma tissues. Results were normalized to U6 . Error bars indicate standard deviation. (D) Stem-loop RT-PCR analysis of miR-31 using RNAs extracted from HEK293 cells, human medulloblastoma cell lines DAOY and D283, and lung (A549, NCI-H322M), breast (MCF-7, MDA-MB-231), liver (HEPG2, HEP3B), prostate (PC-3, DU-145), and soft tissue rabdomyosarcoma (RD, RH30) cancer lines. U6 was used as an internal control. (E) Genomic PCR analysis of the p16 CDKN2A locus and its surround areas in DAOY cells. Genomic DNA of 293 cells served as a positive control. “-” denotes water control.

Article Snippet: The human medulloblastoma cells (DAOY and D283), non-small cell lung carcinoma cells (A549 and NCI-H322M), breast cancer cells (MCF-7 and MDA-MB-231), rhabdomyosarcoma cells (RD and RH30), prostate cancer cells (PC3 and DU145), hepatocellular carcinoma cells (HEPG2 and HEP3B), and human embryonic kidney cells (HEK 293) were purchased from ATCC, and cultured in DMEM medium (GIBCO) supplemented with 10% FBS according to the supplier's recommendations.

Techniques: Staining, Expressing, Quantitative RT-PCR, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Control, Positive Control

Journal: Oncotarget

Article Title: MicroRNA-31 suppresses medulloblastoma cell growth by inhibiting DNA replication through minichromosome maintenance 2

doi:

Figure Lengend Snippet: (A) A selected group of cell-cycle associated genes differentially expressed in normal cerebellar and medulloblastoma tissues as determined by microarray analysis. (B) Quantitative RT-PCR analysis of putative miR-31 targets, CDK1, CDK2, CDK4, MCM2, CDC6 and MCM6, in vector control and miR-31 expressing DAOY cells. Data were analyzed according to the comparative Ct method, with GAPDH as a reference. The expression level in vector control cells was set to 1. (C) Immunoblot analysis of MCM2 protein levels in vector control and two independent pools of miR-31 expressing DAOY cells. GAPDH served as an internal control. The bar graph represents the relative MCM2 band intensity (on right panel). It was calculated as a ratio of MCM2 and GAPDH. (D) Predicted miR-31 target recognition sites in the 3'-UTR of human MCM2. The wild type luciferase reporter was generated with a DNA fragment covering two putative miR-31 binding sites. Mutations in the two miR-31 target sites are underlined. (E) Luciferase assays for the effect of re-expressing miR-31 response on the MCM2 3'-UTR reporters and its mutant variants in DAOY cells. The relative luciferase activity, defined as the ratio of the activity of MCM2 3'-UTR reporter (firefly) to that of the internal control (Renilla), was determined 48 h after transfection, and data representing the average of three independent experiments were shown.

Article Snippet: The human medulloblastoma cells (DAOY and D283), non-small cell lung carcinoma cells (A549 and NCI-H322M), breast cancer cells (MCF-7 and MDA-MB-231), rhabdomyosarcoma cells (RD and RH30), prostate cancer cells (PC3 and DU145), hepatocellular carcinoma cells (HEPG2 and HEP3B), and human embryonic kidney cells (HEK 293) were purchased from ATCC, and cultured in DMEM medium (GIBCO) supplemented with 10% FBS according to the supplier's recommendations.

Techniques: Microarray, Quantitative RT-PCR, Plasmid Preparation, Control, Expressing, Western Blot, Luciferase, Generated, Binding Assay, Mutagenesis, Activity Assay, Transfection

Journal: Scientific Reports

Article Title: Identification of KIAA1199 as a Biomarker for Pancreatic Intraepithelial Neoplasia

doi: 10.1038/srep38273

Figure Lengend Snippet: ( a ) Expression of KIAA1199 in human cancers via Oncomine analysis. ( b , c ) Expression of KIAA1199 in human pancreatic adenocarcinoma via Oncomine analysis. ( d – f ) Expression of KIAA1199 in human pancreatic adenocarcinoma. Fluorescence immunohistochemical analysis of KIAA1199 of human pancreatic cancer tissue microarray (TMA; US Biomax PA207), using anti-KIAA1199 antibody ( d ). Quantitative analysis of KIAA1199 expression in TMA ( e ). Chromogenic immunohistochemical analysis of human pancreatic cancer tissue microarray (US Biomax BIC14011a, PA242b) using-KIAA1199 antibody and DAB substrate ( f ). ( g , h ) mRNA expression of KIAA1199 in PDAC cell lines (AsPC-1, BxPC-3, and Panc-1). Quantitative RT-PCR ( g ) and semi-quantitative RT-PCR ( h ). KIAA1199 expression in PDAC cell lines compared to HPNE (normal pancreas epithelial cell line) and normalized to 18 S rRNA . Gel images shown have been cropped to show the relevant band. Full-length gels are presented in . ( i , j ) Endogenous and secreted protein expression of KIAA1199 in PDAC cell lines (AsPC-1, BxPC-3, and Panc-1). Molecular weight of KIAA1199 was approximately 150 kDa. Blot images shown have been cropped to show the relevant band. Full-length blots are presented in .

Article Snippet: Human PDAC cell lines AsPC-1, BxPC-3, and Panc-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Fluorescence, Immunohistochemical staining, Microarray, Quantitative RT-PCR, Molecular Weight

Journal: Scientific Reports

Article Title: Identification of KIAA1199 as a Biomarker for Pancreatic Intraepithelial Neoplasia

doi: 10.1038/srep38273

Figure Lengend Snippet: ( a ) Detection of KIAA1199 in mouse blood serum. Mouse blood sera from each group (3-, 5-, 7-, and 10-month-old wild-type [WT] or PanIN [ Pdx1-Cre:K-Ras LSLG12D ] mice) were analyzed by immunoblotting for KIAA1199. Immunoglobulin (Ig) served as an internal control. HCT116 colon cancer cells served as a positive control. ( b ) Experimental scheme of KIAA1199 autoantibody detection. Pancreatic ductal adenocarcinoma (PDAC) cells secrete KIAA1199 protein. Host immune system generates autoantibodies against KIAA1199. Blood samples were collected and incubated with KIAA1199 recombinant protein. KIAA1199 autoantibody binds to KIAA1199 recombinant protein. Immune complex is precipitated using magnetic beads. KIAA1199 autoantibody (IgG heavy chain [H.C.] and light chain [L.C.]) is detected by immunoblotting using antibody against the host. ( c ) Purification of KIAA1199 recombinant protein (N- and C-termini). ( d ) Detection of KIAA1199 autoantibody using immunoprecipitation. Mouse blood sera were immunoprecipitated using recombinant KIAA1199 proteins (N- and C-termini). Then, immunoprecipitates were analyzed by immunoblotting using mouse immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP). ( e ) Purification of GST-KIAA1199 protein C-terminus. GST-KIAA1199 (C-terminus) protein was purified using the bacterial protein expression system. Arrow indicates purified KIAA1199 protein (Coomassie staining). ( f ) Detection of KIAA1199 autoantibody using dot blot assays. Different exposure times are indicated. Recombinant KIAA1199 protein (C-terminus) and mouse IgG (a negative control) were transferred onto the membrane for dot blot assays. The sera of mice (WT or PanINs [ Pdx1-Cre:K-Ras LSLG12D ] mice; 5-month-old) were added to probe for the KIAA1199 autoantibody.

Article Snippet: Human PDAC cell lines AsPC-1, BxPC-3, and Panc-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Control, Positive Control, Incubation, Recombinant, Magnetic Beads, Purification, Immunoprecipitation, Expressing, Staining, Dot Blot, Negative Control, Membrane

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: Mitotic spindle checkpoint genes are broadly overexpressed in human urothelial carcinoma. A, Human samples of normal urothelium (N=10) and urothelial carcinoma of the bladder (N=8) were subjected to RNA microarray. A subset of 13 gene transcripts related to the mitotic spindle checkpoint, including Aurora A and B, were upregulated at least 5-fold in the urothelial carcinoma compared to the normal urothelium. B, Upregulation of these genes was validated by two-step quantitative real-time PCR on a separate set of human samples of urothelial carcinoma (N=3) and normal urothelium (N=3). 10 of 13 genes (asterisked) demonstrated statistical significance (T-test P-value < 0.05) for differential expression in urothelial carcinoma compared to normal urothelium.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Quantitative Proteomics

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: MLN8237 induces cell cycle arrest and aneuploidy of bladder cancer cell lines. A, MLN8237 inhibited expression of phospho-Aurora A-T288 at mitotic spindles. B, MLN8237 demonstrated specificity for inhibiting Aurora A, as expression of histone-H3 and phospho-histone-H3, markers of Aurora B function, was maintained. C, Propidium iodide staining with flow cytometry analysis was performed to assess cell cycle changes. T24, UM-UC-3, and RT4 cells were treated with 10 nM to 1 μM MLN8237 for 48 h. All three cell lines demonstrated dramatic cell cycle arrest and increase in the 4N cell population in a dose-dependent manner. T24 and UM-UC-3 cells also demonstrated a considerable increase in aneuploidy, whereas RT4 cells did not.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Expressing, Staining, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: Cellular phenotypes of T24 and RT4 cells differ after MLN8237 treatment. A, MLN8237 induces a dramatic increase in cell size in T24 cells but not RT4 cells. B, Immunocytochemistry and fluorescence microscopy of T24 and RT4 cells revealed the formation of aberrant spindle figures upon MLN8237 treatment, with multipolar spindle apparatuses and failure of localization of chromatids to a single metaphase plate. C, T24 cells demonstrate a phenotype of increased cell size and ploidy, whereas RT4 cells do not. D, T24 cells also exhibit a subpopulation of cells exhibiting marked cytoplasmic aurora A expression, whereas RT4 cells lacked cytoplasmic aurora A expression. E, Real-time imaging of T24 and RT4 cells treated with MLN8237 was performed over 48 h. T24 cells exhibited dramatic increases in cell size as a result of repeated cell cycle progressions without separation of daughter cells. RT4 cells appeared to become arrested after one failed mitotic attempt, preventing repeated cell cycle progressions that could otherwise result in increased ploidy.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Immunocytochemistry, Fluorescence, Microscopy, Expressing, Imaging

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: MLN8237 induces cytotoxicity and differential apoptotic processes. A, MTS assay was used to calculate IC50 values for each cell line following treatment over a range of MLN8237 concentrations for 48 h. MLN8237 exhibited highest potency in T24 and UM-UC-3 cells (IC50 of 31 nM and 45 nM, respectively) and lowest potency in RT4 cells (IC50 of 120 nM). B, Western blot analysis of T24 and RT4 cells for apoptotic markers revealed induction of p53 expression in RT4 cells, and induction of p73, but not p53, expression in T24 cells. Both cell lines showed increased expression of the apoptotic marker cleaved PARP starting 24 h after initiation of treatment. C, Annexin V staining with flow cytometry analysis of T24 and RT4 cells revealed an increased apoptotic cell fraction at 48 and 72 h after initiation of MLN8237 treatment. D, Clonogenic assays of T24 and RT4 cells showed 90% inhibition of long-term clone forming capability at 100 nM MLN8237.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: MTS Assay, Western Blot, Expressing, Marker, Staining, Flow Cytometry, Inhibition

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: Interactions of MLN8237 with paclitaxel and gemcitabine in vitro are schedule-dependent. MLN8237 (MLN) was combined with either paclitaxel (PTX) or gemcitabine (Gem) in T24 cells. Drugs were administered either simultaneously for 48 h (left column), or sequentially, with one drug for 48 h, followed by washout and the other drug for 48 h (middle and right columns). MTS assay was utilized to quantify the effect on cell viability of these combination treatments. MLN8237 demonstrated synergistic effects with paclitaxel and gemcitabine when dosed sequentially (middle and right columns), and antagonistic effects when dosed simultaneously (left column).

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: In Vitro, MTS Assay

Journal: BMC Genomics

Article Title: Global regulatory architecture of human, mouse and rat tissue transcriptomes

doi: 10.1186/1471-2164-14-716

Figure Lengend Snippet: Principle Component Analysis (PCA) of human, mouse and rat tissue expression profiles based on two generations of Affymetrix expression arrays shown as SET 1 (upper plot: Affymetrix HG-U133A, MG-U74Av2, and RG-U34 arrays) and SET 2 (lower plot: Affymetrix Human133 Plus 2.0, Mouse430 2.0, and Rat230 2.0 arrays). A common set of 56 and 55 tissue types, respectively, is represented for each organism. Each tissue type is a single mean expression vector processed from all samples annotated as such in the Genevestigator database. Species are represented by symbols and tissue types are numbered. Tissue types were grouped according to organ systems that are represented by different colors. Related tissues clustered together into an overall consistent architecture between the three species. The two generations of microarrays yielded very similar results, despite being composed of independent and differerent sets of published experiments.

Article Snippet: Representative tissue specific datasets were aggregated from more than 33,900 Affymetrix expression microarrays.

Techniques: Expressing, Plasmid Preparation

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Influenza A alters host miRNA expression. (A) PCR analysis of individual miRNAs that showed differential expression in microarray analysis. miRNA isolated after 3 h of exposure to influenza A (A/WS/33 (H1N1), A/Aichi/2/68 (H3N2), A/Swine/1976/31 (H1N1), and A/Swine/Iowa/15/30 (H1N1)), (MOI of 3). The fold change represents the change in miRNA expression in infected cells relative to that obtained in uninfected cells. (B) A549 cells were infected with 3MOIs of influenza A (A/WS/33 (H1N1), A/Aichi/2/68 (H3N2), A/Swine/1976/31 (H1N1), and A/Swine/Iowa/15/30 (H1N1)), for 3 h and matrix copy numbers were determined by RT-PCR. (C) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to 3MOI of influenza. Data are expressed as ± standard error of the mean (SEM). n=4 (independent experiments) with triplicate replicates (*=p<0.05, **=p<0.01, ***=p<0.001). (D) PCR analysis of individual miRNAs in human bronchial epithelial cells (HBEpC) exposed to influenza A (A/WS/33 (H1N1), and A/Aichi/2/68 (H3N2)). miRNA isolated after 3 h of exposure to influenza A (MOI of 3). The fold change represents the change in miRNA expression in infected cells relative to that of uninfected cells. (E) HBEpC cells were infected with 3 MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. (F) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to 3MOI of influenza A. p<0.05 was considered significant. The fold change represents the change in miRNA and mRNA expression in infected cells relative to that of uninfected cells. Data are expressed as ± standard error of the mean (SEM). n=4 (independent experiments) with triplicate replicates (*=p<0.05, **=p<0.01, ***=p<0.001).

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Quantitative Proteomics, Microarray, Isolation, Infection, Reverse Transcription Polymerase Chain Reaction

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: miRNA-548an and NS1ABP mRNA expressions in A549 cells infected with different MOIs of influenza A. Differentially expressed miRNAs identified in the microarray analysis were screened for a potential role in influenza propagation and miRNA-548an was selected for further analysis. A549 cells were infected for 3 h with increasing MOIs of influenza. (A) miRNA-548an expression in uninfected and infected A549 cells was normalized to expression of let-7 as a housekeeping miRNA. (B) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to increasing MOIs of influenza. p<0.01 was considered significant. The fold change represents the change in miRNA and mRNA expression in infected cells relative to that obtained in uninfected cells. (C) A549 cells were infected with 3 MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. Data are expressed as ± SEM. **=p<0.01, ***=p<0.001. n=4 (independent experiments) with triplicate replicates.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Infection, Microarray, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Time course showing the expression levels of miRNA-548an and NS1ABP mRNA in cells infected with influenza A. (A) A549 cells were infected with 1 MOI of Influenza A and expression of miRNA-548an normalized to let-7 miRNA is shown over 0–6 h of infection. (B) A549 cells were infected with 1 MOI of Influenza A and expression of NS1ABP mRNA normalized to GAPDH mRNA is shown over 0–6 h of infection. The fold change represents the change in miRNA and mRNA expression in infected cells relative to that obtained in uninfected cells. (C) HBEpC cells were infected with 3 MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. (D) Protein expression by Western blot analysis in cells infected with influenza A for up to 3 h and α-tubulin was used as a loading control. Data are expressed as ± SEM. ***=p<0.001, **=p<0.01, *=p<0.05; n=3 (independent experiments) with triplicate replicates.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Down or up-regulation of miRNA 548an alters the expression of NS1ABP mRNA. (A) Uninfected A549 cells were transfected for 48 h with 50 nM or 100 nM of either miRNA 548an inhibitor or a scrambled oligonucleotide as control. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (B) After 48 h, transfected cells were infected with 1 MOI of influenza A for 3 h. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. n=3 (independent experiments) with triplicate replicates. (C) A549 cells were transfected for 48 h with 12.5 nM, 25 nM, or 50 nM of miRNA-548an mimic or scrambled oligonucleotide as control. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (D) A549 cells were transfected for 48 h with miRNA-548an mimic or scrambled oligonucleotide. After 48 h, cells were infected with 1 MOI of influenza A for 3 h. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (E) NS1ABP expression profile after 30 and 48 h of transfection with mimics in A549 cells. (F) NS1ABP expression profile after 30 and 48 h of transfection with inhibitor in A549 cells. The fold change represents the change in NS1ABP mRNA expression and influenza A Matrix copy number in cells transfected with the inhibitor relative to that obtained in cells transfected with the scrambled oligonucleotide. Data are expressed as ± SEM, **=p<0.01 and *=p<0.05. n=4 (independent experiments) with triplicate replicates.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Transfection, Control, Infection

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: NS1ABP protein expression is modulated by miRNA 548an. (A) Flow cytometry analysis of A549 cells transfected for 48 h with 50 nM miRNA-548an inhibitor or scrambled oligonucleotide. (B) Flow cytometry analysis of A549 cells transfected for 48 h with 25 nM miRNA-548an mimic or scrambled oligonucleotide. (C) Flow cytometry analysis of A549 cells transfected for 48 h with 50 nM miRNA-548an inhibitor and then infected with 1 MOI of influenza for 3 h. Expression of NS1ABP protein in cells transfected with the miRNA-548an inhibitor (green line), or scrambled oligonucleotide as control (black line) is shown. (D) Flow cytometry analysis in A549 cells transfected for 48 h with miRNA-548an mimic (green line) or scrambled oligonucleotide (black line) and then infected with 1 MOI of influenza for 3 h. The gray shaded or red shaded regions in the graph represent the isotype control. (E) Geometric mean fluorescent intensity (MFI) was measured from 3 independent experiments with triplicate replicates. ###=p<0.001whencomparing cells transfected with inhibitor to cells transfected with the scrambled oligonucleotide; ##=p<0.01when comparing cells transfected with mimic to cells transfected with the scrambled oligonucleotide **=p<0.01 when comparing cells transfected with mimic or inhibitor.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Flow Cytometry, Transfection, Infection, Control

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Visualization of NS1ABP protein expression following modulation of miRNA-548an. A549 cells were transfected for 48 h with either the scrambled oligonucleotide (negative control) (top panel), miRNA-548an mimic (middle panel), or miRNA-548an inhibitor (third panel), and uninfected cells (bottom panel). Transfected cells were infected 1 MOI of influenza A for 3 h and expression of NS1ABP protein (green) and influenza nucleoprotein (red) was determined by immunofluorescence. DAPI (blue) represents the stained nucleus of the cells (not shown separately).

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Transfection, Negative Control, Infection, Immunofluorescence, Staining

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Apoptotic pattern of cells transfected with miRNA-548an mimic or inhibitor and infected with influenza A. (A and C) A549 cells were transfected for 48 h with the scrambled oligonucleotide or (B) miRNA-548an mimic, or (D) miRNA-548an inhibitor. Transfected cells were then infected with 1 MOI of influenza A for 3 h. The cells were immune-fluorescently labeled with annexin V and apoptotic cells were analyzed by flow cytometry. Transfection did not change the proportion of necrotic cells detected by propidium iodide (PI) staining. Graph is a representative plot from 3 independent experiments and data presented as percentage of cells in each quadrant. Left upper quadrant=necrotic cells; left lower quadrant=viable cells; right lower quadrant=apoptotic cells; right upper quadrant=late apoptotic cells in necrotic state.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Transfection, Infection, Labeling, Flow Cytometry, Staining

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Effect of miRNA 548an mimic on viral replication. (A) A549 cells were transfected for 48 h with 25 nM miRNA-548an mimic or a scrambled oligonucleotide. After 48 h, the cells were infected with increasing MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. Expression of influenza Matrix gene copies in cells transfected with the mimic is expressed relative to that obtained in cells transfected with the scrambled oligonucleotide. Data are expressed as ± SEM, **=p<0.01, and *=p<0.05. n=3 (independent experiments) performed in triplicate.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

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

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: (A) HepG2 cells were treated with various doses of Vel as indicated in the presence (2.5, 5.0 mM) or the absence of LC for 24 h, cell viability was detected by MTS assay. Mean+SD (n = 3). Combination index (CI) was shown. Vel: bortezomib; DM: DMSO; LC: L-carnitine. (B) Human hepatic SMMC-7721 cancer cells were treated with LC and/or Vel for 48 h, cell viability was detected by MTS assay and PARP cleavage was detected by Western blot. CI was shown. (C) HepG2 cells were treated with the combination of Vel (50 nM) and LC (5 mM) for 48 h and cell apoptosis was labeled with Annexin V and propidium iodide (PI), and detected by flow cytometry. Representative flow images were shown and cell death data was summarized. Mean+SD (n = 3). * P <0.05, compared with Vel or LC treatment alone; # P <0.05, compared with Veh control. (D) as treated in C, living cells were directly stained with PI and imaged under an inverted fluorescent miscoscope. The phase contrast and fluorescent images were taken and merged. Red indicates PI-positive. Typical images were shown.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: MTS Assay, Western Blot, Labeling, Flow Cytometry, Control, Staining

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: ( A ) p21 cip1 gene expression. HepG2 cancer cells were either treated with various doses of LC (2.5, 5, 10 mM) for 24 h, or with 50 nM of Vel for 9 h and 24 h. A mixture of three cell samples were collected and DNA microarray assay was performed. Fold increase of the LC-treated versus control was shown. ( B ) p21 cip1 mRNA expression. HepG2 cells were exposed to either LC (5 mM) and Vel (50 nM) or the combination for 18 h; a mixture of three cell samples were extracted for mRNA assay by real-time-PCR. Fold increases of p21 cip1 and p27 kip1 were shown. ( C ) p21 cip1 protein expression and histone acetylation. HepG2 cells were treated with the combination of LC (5 mM) and various doses of Vel as indicated for 24 h, protein levels were detected by Western blot. Antibodies against p21 cip1 , p27 kip1 , histone and acetylated histones were used. GAPDH was used as a loading control. At least three repeats were performed and representative images were shown. ( D ) ChIP assay. HepG2 cells were treated with either vehicle or Vel (50 nM) for 24 h; cells were collected for ChIP assay. Fold enrichment of p21 cip1 and p27 kip1 promoter gene was summarized.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Gene Expression, Microarray, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: ( A ) HepG2 cells were treated with LC (5 mM) and/or Vel (25, 50, 75 nM) for 24 h, and ubiquitinated proteins were detected by Western blot. ( B ) HepG2 cells were treated with either vehicle or LC (5 mM) for 24 h, then various doses of Vel were added to the culture medium for 8 h and chymotrypsin-like (CT-like) activity in situ was detected. Mean+SD (n = 3). * P <0.05, compared with the control at each point. ( C ) The natural bond Orbital (NBO) charge and geometric optimization were calculated by the DFT method and the NBO charges in the hydroxyl O atom of threonine or acetylthreonine was shown.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Western Blot, Activity Assay, In Situ, Control

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: (A) HepG2 cells were treated with LC (5 mM), Vel (25, 50, 75 nM) and the combination of them for 24 h. Western blot was performed for the detection of the related proteins including caspases, PARP and ER-related proteins. GAPDH was used as a loading control. (B) As treated and analyzed in , ER-stress-related gene expression as indicated was shown.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Western Blot, Control, Gene Expression

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: ( A ) As treated and analyzed in , Bax and Bcl-2 gene expression was shown. ( B ) As treated and analyzed in , Bax mRNA was detected by real-time PCR and fold increase of Bax mRNA was shown. ( C ) As treated in , Bax and Bcl-2 protein levels were detected. Representative images were shown. ( D ) HepG2 cells were transfected with Bax-siRNA (#1) for 48 h and then treated with the combination of LC (5 mM) and Vel (50 nM) for 24 h, Western blot was performed to detect Bax and PARP cleavage. GAPDH was used as a loading control.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Control

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: Nude mice bearing HepG2 tumor were i.p injected with vehicle or LC (400 mg/kg, i.p. once/day except day 8), Vel (0.75 mg/kg, i.v. once/3 days) alone and the combination respectively for 15 days. A, Tumor images, tumor weight, body weight, tumor growth curve including Box-plot image and the relative tumor volume in all the four groups (Vehicle-, LC-, Vel- and LC+Vel-) were shown. * P <0.05, compared with the Veh control; # P <0.05, compared with the treatment alone, ## P >0.05, compared with the treatment alone. B, p21 cip1 and acetylated H3 proteins in tumor tissues of various groups were detected by immunochemistry and the results were shown respectively. All the immunostaining were repeated in three mouse tumor tissues and the typical images were shown.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Injection, Control, Immunostaining