Journal: Genome Biology

Article Title: Exon expression profiling reveals stimulus-mediated exon use in neural cells

doi: 10.1186/gb-2007-8-8-r159

Figure Lengend Snippet: Calcium-mediated changes in transcript abundance are enriched for functional gene categories. (a) Hierarchical clustering of the potassium chloride (KCl) transcript dataset identifies six major patterns of gene expression over the 24-hour time course. The number of transcripts contained in each cluster is designated in parentheses. Shown in each plot is the averaged expression behavior of all transcripts in the indicated cluster, with error bars indicating the deviation from the mean for the entire cluster. Expression behavior of each transcript was obtained by subtracting the mean of all points at all times from each expression value in the original time series, and dividing by the standard deviation. KCl-treated time points are indicated in blue. Thapsigargin (TPG) treatment is indicated in red. (b) Gene Ontology (GO) analyses of the transcripts in each cluster reveals functional enrichment in clusters T1 to T4. (c) Polymerase chain reaction performed on cDNA from treated and untreated IMR-32 cells validates changes in transcript abundance predicted by the microarray data. PREX1 , a gene whose abundance was not predicted to change, served as a normalization control. (d) Transcripts regulated by KCl stimulation at 24 hours are similarly affected by TPG treatment. Numbers indicate upregulated and downregulated transcripts. Among those transcripts affected by both KCl and TPG, 116 were upregulated and 573 were downregulated. ARRDC3 , arrestin domain containing 3; BNIP3 , BCL2 adenovirus E1B 19 kDa interacting protein 3; ETV5 , ets variant gene 5; GLI3 , GLI-Kruppel family member 3; KCNK7 , potassium channel, subfamily K, member 7; PREX1 , phosphatidylinositol 3,4,5-trisphosphate-dependent RAC exchanger 1; OVGP1 , oviductal glycoprotein 1; STC1 , stanniocalcin 1; ZNF143 , zinc finger protein 143.

Article Snippet: Human neuroblastoma IMR-32 cells (ATCC, Manassas, VA, USA) were cultured at 37°C in a humidified atmosphere with 5% carbon dioxide in Eagle's minimum essential media (EMEM; ATCC), modified to contain the following: Earle's balanced salt solution, nonessential amino acids, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, and 1.5 g/l sodium bicarbonate, with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and penicillin/streptomycin.

Techniques: Functional Assay, Gene Expression, Expressing, Standard Deviation, Polymerase Chain Reaction, Microarray, Control, Variant Assay

Journal: Cell metabolism

Article Title: Glutamate/metabotropic glutamate receptor-5 signaling in hepatic stellate cells drives endocannabinoid-mediated alcoholic steatosis

doi: 10.1016/j.cmet.2019.08.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell Lines Mouse Neuro-2a cells (ATCC) originally derived from the neuroblastoma of mouse A strain, human HEK293 T cells (ATCC) originated from human fetus, and human Hep3B cells (ATCC) and HepG2 cells (ATCC) obtained from male origin were cultured in DMDM (#LM001–05, Welgene) supplemented with 10% FBS (Welgene) and 1% penicillin-streptomycin (Thermo Fisher Scientific).

Techniques: Recombinant, Modification, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Western Blot, SYBR Green Assay, Expressing, Microarray, Knock-Out, shRNA, Software

Journal: RNA Biology

Article Title: microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest

doi: 10.1080/15476286.2015.1023495

Figure Lengend Snippet: miR-449a functions as an inducer of cell differentiation in neuroblastoma cells. (A–B), Time- (A) and Dose- (B) dependent effect of miR-449a mimic on neurite outgrowth in BE(2)-C cells. 2500 cells were transfected with different concentrations of miR-449a mimic or negative control oligo in triplicates in 96-well plates, and neurite outgrowth was measured every 6 h for 4 d. Relative neurite length is defined as neurite length per cell body area. Shown are the normalized relative neurite lengths, presented as average values of the 3 replicates. *, P < 0.05, comparing to the negative control oligo at the corresponding time or dose points. (C–D), Effect of miR-449a mimic and precursor mimic on neurite outgrowth in BE(2)-C cells. Cells were transfected with 25 nM control oligo, miR-449a mimic or precursor mimic in triplicates, with neurite lengths measured as above after 4 d. Shown are the representative cell images (C) analyzed to define neurites (pink) and cell body areas (yellow), and the quantification of neurite length (D). *, P < 0.05, comparing to control. (E–F), Effect of miR-449a mimic on neurite outgrowth in multiple neuroblastoma cell lines. Cells were transfected with 25 nM miR-449a mimic or control oligo in triplicates. Neurite lengths were measured as above. (E) Representative cell images analyzed to define neurites (pink) and cell body areas (yellow) after 4 d of transfection. (F) Quantification of neurite lengths. *, P < 0.05, comparing to control. G, Effect of miR-449a overexpression on the protein expression levels of cell differentiation markers βIII-tubulin, NSE and GAP43 with calnexin protein levels used as a loading control. Cells were transfected with 25 nM miR-449a mimic or control oligo, and protein levels were determined by Western blot after 4 d. (H) Endogenous expression levels of miR-449a in undifferentiated and differentiated BE(2)-C cells. BE(2)-C cells were treated with 10 µM of RA or the carrier DMSO (Control) for 5 d to induce cell differentiation. RNA was then isolated, and expression of miR-449a in cells were measured by qPCR with levels of 18s rRNA as a loading control. *, P < 0.05, comparing to control.

Article Snippet: 73 Cell lines BE(2)-C, SKNBE and BE(2)-M17 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Cell Differentiation, Transfection, Negative Control, Over Expression, Expressing, Western Blot, Isolation

Journal: RNA Biology

Article Title: microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest

doi: 10.1080/15476286.2015.1023495

Figure Lengend Snippet: The systematic identification of miR-449a targets that regulate neuroblastoma cell differentiation. (A) Expression level of miR-449a in BE(2)-C cells after transfected with miR-449a (25 nM) mimic for 24 h, as measured by qPCR. (B-C) Histogram of changes in mRNA expression levels of genes (B) predicted as miR-449a targets and (C) not predicated as miR-449a targets induced by miR-449a overexpression. The mRNA expression levels were determined by gene expression microarray. Shown is the relative frequency of observed log2 fold changes for the predicted miR-449a targets. (D) The empirical density curves generated based on the above frequency distributions by Gaussian Kernel Smoothing. (E) Effect of knocking down the top 25 target genes on neurite outgrowth in BE(2)-C cells. Cells were transfected with 25 nM of the indicated siRNAs or negative control oligos (Control) in triplicated for 4 d. Neurite outgrowth was measured as above. *, P < 0.05, comparing to Control. (F) Validation of the effect of knocking down the 5 candidate targets on neurite outgrowth using the 2nd set siRNAs. BE(2)-C cells were transfected and neurite outgrowth were measured as above. *, P < 0.05, comparing to control. (G) Effect of gene knockdown on expression of differentiation markers in BE(2)-C cells. Cells were transfected with the indicated siRNAs or control oligo (25 nM). Protein levels were measured by Western blot as above. (H) Effect of gene knockdown on cell viability in BE(2)-C cells. Cells were transfected with the indicated siRNAs or control oligo (25 nM) in triplicates in 96-well plates. After 4 d, cell viability was measured as above. Shown are the relative cell viabilities, presented as averages of the 3 replicates, normalized to the control oligos. *, P < 0.05, comparing to the control. (I) Effect of gene overexpression on expression of differentiation markers. BE(2)-C cells were co-transfected with the indicated expression vectors and oligos. After 4 d, protein levels of expression markers were measured as above. *, P < 0.05, comparing to Control.

Article Snippet: 73 Cell lines BE(2)-C, SKNBE and BE(2)-M17 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Cell Differentiation, Expressing, Transfection, Over Expression, Microarray, Generated, Negative Control, Western Blot

Journal: RNA Biology

Article Title: microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest

doi: 10.1080/15476286.2015.1023495

Figure Lengend Snippet: Effect of miR-449a on cell cycle progression in neuroblastoma cells. (A–C) Effect of miR-449a overexpression on cell cycle distribution in (A) BE(2)-C, (B) SKNBE and (C) KELLY cells. Cells were transfected with 25 nM miR-449a mimic or control oligo, or treated with 5 μM RA. Mock transfected cells were used as an additional negative control. After 3 d, cells were collected and stained with propidium iodide for cell cycle analysis. The fraction of cells in G0/G1, S and G2 phases was quantified using the Jean-Jett-Fox model. Similar results were obtained from 3 independent experiments. (D–G) Effect of miR-449a and RA on DNA synthesis as measured by BrDU incorporation in BE(2)-C (D), SKNBE (E) and KELLY (F) cells. Cells were transfected with miR-449a mimic or control oligos (25 nM), or treated with RA (5 μM) for 4 d. Cells were incubated with BrdU during the final 2 h of culture. Cells were then stained for BrDU incorporation (red) as described in the Materials and Methods. Cell nuclei were identified by nuclear staining with DAPI (blue). (G) Quantification of percentage of cells positive for BrDU staining. *, P < 0.05, comparing to control.

Article Snippet: 73 Cell lines BE(2)-C, SKNBE and BE(2)-M17 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Over Expression, Transfection, Negative Control, Staining, Cell Cycle Assay, DNA Synthesis, BrdU Incorporation Assay, Incubation, BrdU Staining

Journal: RNA Biology

Article Title: microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest

doi: 10.1080/15476286.2015.1023495

Figure Lengend Snippet: Function of the predicted miR-449a cell cycle-regulating targets in regulating cell cycle progression and cell survival. (A–E), Effect of knocking down the predicted miR-449a targets on cell viability in BE(2)-C, SKNBE and KELLY cells using 2 sets of siRNAs for the indicated genes. Cells were transfected with 25 nM of the indicated siRNAs or control oligo (Control). After 4 d, cell viability was measured as above. *, P < 0.05, comparing to Control. (F–H), Effect of knocking down CDK6 and LEF1 on cell cycle distribution in (F) BE(2)-C, (G) SKNBE and (H) KELLY cells. Cells were transfected with 25 nM of the indicated siRNAs or control oligo (Control). Mock-transfected cells were used as an additional negative control. After 3 d, cell cycle distributions were measured as above. (I–K) Effect of knocking down CDK6 and LEF1 on BrDU incorporation in (I) BE(2)-C, (J) SKNBE and (K) KELLY cells. Cells were transfected as above, and the level of BrDU incorporation was measured as above. (L) Western blot analysis of Caspase-3 activation. BE(2)-C cells were transfected with 25 nM of the indicated oligos for 3 d, and caspase-3 levels were measured as above. (M) Effect of gene overexpression on cell cycle distribution. BE(2)-C cells were transfected with the indicated expression vectors and oligos. After 3 d, cell cycle distributions were analyzed as above.

Article Snippet: 73 Cell lines BE(2)-C, SKNBE and BE(2)-M17 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Negative Control, BrdU Incorporation Assay, Western Blot, Activation Assay, Over Expression, Expressing

Journal: RNA Biology

Article Title: microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest

doi: 10.1080/15476286.2015.1023495

Figure Lengend Snippet: Validation of the predicted direct targets of miR-449a. (A) The predicted interactions between miR-449a and the target sites in the 3′UTRs of the indicated mRNAs. The seed sequences are underlined. (B) Validation of the target sites of miR-449a by luciferase reporter assay. BE(2)-C cells were co-transfected with the indicated vectors and miR-449a mimic. After 3 d of transfection, cells were lysed and luciferase activity was measured. Shown are normalized luciferase activities with the luciferase activity associated with wildtype (WT) 3′UTRs normalized to those associated with the corresponding mutant 3′UTRs. *, P < 0.05, comparing to mutant 3′UTRs. (C) Validation of the direct interactions of miR-449a with the target genes by Bio-miR-449a pulldown assay. BE(2)-C cells were transfected with the 25 nM of the Bio-miR-449a or control oligo (Bio-Control). The pulldown assay was performed following the protocol described in the Materials and Methods. Shown are fold of enrichment of the indicated mRNAs by Bio-miR-449a, calculated by normalizing the levels of mRNAs associated with Bio-miR-449a to those associated with the Bio-control. *, P < 0.05, comparing to the Bio-control. (D) BIRC5 and CDT1 mRNA levels in the Biotin pulldown products were measured by qPCR. *, P < 0.05, comparing to the Bio-control.

Article Snippet: 73 Cell lines BE(2)-C, SKNBE and BE(2)-M17 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Luciferase, Reporter Assay, Transfection, Activity Assay, Mutagenesis

Journal: Genomics Data

Article Title: Genome-wide analysis of HOXC9-induced neuronal differentiation of neuroblastoma cells ☆

doi: 10.1016/j.gdata.2014.04.002

Figure Lengend Snippet:

Article Snippet: The human neuroblastoma cell line BE(2)-C was obtained from ATCC (CRL-2268, ATCC).

Techniques: Microarray, Over Expression, Gene Expression

Journal: Oncotarget

Article Title: Targeting P-selectin blocks neuroblastoma growth

doi: 10.18632/oncotarget.21364

Figure Lengend Snippet: (A) Expression of selectin ligand (B) and selectin mRNAs in neuroblastoma patient samples. 1 = ganglioneuroblastoma (n=2), 2 = ganglioneuroma (n=4), 3 = mature ganglioneuroma (n=1), 4 = maturing ganglioneuroma (n=6), and 5 = neuroblastoma (n=15). (C) Kaplan-Meier curves predicting the patient prognoses based on the expression of CD44 , CD24, SELPLG (D) SELP, SELE and SELL (P-, E- and L-selectin). Probes for CD44, CD24, SELPLG, SELP, SELE and SELL were A_32_P215002, 266_s_at, 209880_at, 206049_at, 206211_at and 204563_a respectively. N=52/50 in high/low expression patient groups except with CD44 probe n=126/125, respectively. Presented neuroblastoma patient data was extracted using Oncomine (A-B) and Oncogenomics (C-D) databases.

Article Snippet: Human neuroblastoma cell lines SK-N-BE(2), SK-N-AS, SK-N-SH, SH-SY5Y were purchased from American Type Culture Collection.

Techniques: Expressing

Journal: Oncotarget

Article Title: Targeting P-selectin blocks neuroblastoma growth

doi: 10.18632/oncotarget.21364

Figure Lengend Snippet: Relative binding of selectins to neuroblastoma cells. Mean fluorescence intensity (MFI) of selectin staining divided by MFI of control (Fc) staining

Article Snippet: Human neuroblastoma cell lines SK-N-BE(2), SK-N-AS, SK-N-SH, SH-SY5Y were purchased from American Type Culture Collection.

Techniques: Binding Assay, Fluorescence, Staining, Control

Journal: Oncotarget

Article Title: Targeting P-selectin blocks neuroblastoma growth

doi: 10.18632/oncotarget.21364

Figure Lengend Snippet: (A) Binding of neuroblastoma cells to selectins. Human IgG Fc and recombinant E-, P- or L-selectin-Fc (10 μg/ml) were incubated each with 300 000 neuroblastoma cells for 30 min and detected with APC ɑ-human IgG Fc antibodies. (B) Examples from neuroblastoma tissue microarray showing H&E, P-selectin and PSGL-1 staining. 1) tonsils, a positive control for PSGL-1 staining, small arrows point to endothelial P-selectin staining 2) metastatic neuroblastoma, post treatment, arrow points to P-selectin staining 3) stage 2B neuroblastoma and 4) unfavorable stage 4 neuroblastoma. (C) Endogenous expression of PSGL-1, CD44 and CD24 in neuroblastoma cells. PSGL-1 expression was detected by incubating 300 000 cells per sample first with monoclonal ɑ-PSGL-1 and then with APC ɑ-mouse Ig. PE CD44, APC CD24 and isotype controls were incubated each with 500 000 cells. SH-EP and SH-SY5Y cells are sublines of SK-N-SH cells. (D-E) Redundancy of selectin ligand usage in P-selectin binding. (D) P-selectin binding is decreased in SK-N-BE(2) cells and unchanged in SH-EP cells due to PSGL-1 silencing. (E) Western blot analysis shows the extent of CD24 decrease in SH-EP CD24 knockout (KO) cells compared to parental SH-EP cells (ctrl). On left flow analysis shows CD24 expression and P-selectin binding of SH-EP CD24 knockout and control cells. Controls in all flow charts are shaded.

Article Snippet: Human neuroblastoma cell lines SK-N-BE(2), SK-N-AS, SK-N-SH, SH-SY5Y were purchased from American Type Culture Collection.

Techniques: Binding Assay, Recombinant, Incubation, Microarray, Staining, Positive Control, Expressing, Western Blot, Knock-Out, Control

Journal: Oncotarget

Article Title: Targeting P-selectin blocks neuroblastoma growth

doi: 10.18632/oncotarget.21364

Figure Lengend Snippet: (A) Western blot analysis shows P-selectin treatment inducing dynamic changes in p-Src family protein levels in neuroblastoma cells. Equal numbers of dissociated cells were treated various times with control human IgG Fc fragment or recombinant human P-selectin chimera (both 10 μg/ml), lysed and run on gel. 40 μg of protein was loaded per lane. Western blots were incubated with ɑ-p-Src family protein antibodies that detect all phosphorylated Src family members and β-actin was used as a loading control. (B) Mass cytometry analysis of Fc and P-selectin treated NBL1 and SK-N-BE(2) cells further confirm the upregulation of Src and p-Src family proteins. Flow charts show the effect of 30 and 60 min Fc (shaded) and P-selectin-Fc treatment on Src and p-Src family expression in NBL1 and SK-N-BE(2) cells. Heat maps in the middle show the relative expression of all measured proteins and viSNE plots on right show in single-cell level the expression of Src and phosphorylated Src family proteins.

Article Snippet: Human neuroblastoma cell lines SK-N-BE(2), SK-N-AS, SK-N-SH, SH-SY5Y were purchased from American Type Culture Collection.

Techniques: Western Blot, Control, Recombinant, Incubation, Mass Cytometry, Expressing