human eif4e3 gene  (OriGene)

Journal: Nature Communications

Article Title: MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

doi: 10.1038/s41467-018-06808-8

Figure Lengend Snippet: MiR-584-5p targets cancer, microtubule dynamics, and translation-associated genes. a Hierarchical clustering of gene expression changes in D425Med and D458Med cells treated with miR-NC or miR-584-5p mimic for 48 h. Data are z -transformed. The cutoff criteria for each gene are fold change > 2, sample intensity > 10, and p -value < 0.05. b Gene set enrichment analysis (GSEA) showing enriched biological processes in miR-584-5p mimic-treated MB cells. c qPCR analysis of eIF4E3 and HDAC1 expression in MB cells (D556Med, D425Med, D458Med, and DAOY) transfected with miR-NC or miR-584-5p mimic. The p -values were calculated using standard Student t -tests. Error bars represent mean ± SEM of three independent experiments (performed in triplicate for each experiment). d Western blot analysis of target genes in miR-NC or miR-584-5p mimic-transfected D556Med, D425Med, D458Med, and DAOY cells. Gel photographs are representative of at least three independent experiments. e Left, luciferase-eIF4E3/HDAC1 3′-UTR constructs. Inset boxes show miR-584-5p -binding sites in 3’-UTRs of eIF4E3 and HDAC1 (wild type). Also shown is miR-584-5p seed sequence mutated sequences. Right, relative luciferase activity in HEK293 cells transfected with miR-NC, miR-584-5p , or miR-584-5p -binding site mutant (mut). Values were normalized to firefly luciferase, which served as an internal control. The p -values were calculated using standard Student t -tests. Error bars represent mean ± SEM of three independent experiments. **** p < 0.0001; *** p < 0.001

Article Snippet: MiR-NC and miR-584-5p were used at 50 nM for all experiments unless otherwise indicated. eIF4E3 plasmid was purchased from Origene (#RC209694).

Techniques: Gene Expression, Transformation Assay, Expressing, Transfection, Western Blot, Luciferase, Construct, Binding Assay, Sequencing, Activity Assay, Mutagenesis, Control

Journal: Nature Communications

Article Title: MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

doi: 10.1038/s41467-018-06808-8

Figure Lengend Snippet: eIF4E3 and HDAC1 promote MB growth and inhibit VCR sensitivity of MB cells. a Proliferation of D556Med cell transfected with scrambled- or eIF4E3/HDAC1-siRNA. Cell proliferation was measured using an IncuCyte phase-only processing module. The p -values were determined by the sum-of-squares F test. Error bars represent mean ± SEM of three independent experiments. b Difference in morphology between D556Med cells transfected with scrambled-siRNA, eIF4E3-siRNA, or HDAC1-siRNA. c Colony-forming assay of D556Med cells transfected with scrambled-siRNA, eIF4E3-siRNA, or HDAC1-siRNA. Bar graph shows crystal violet-stained colonies counted microscopically. The p -value was calculated using a standard Student t -test. Error bars represent mean ± SEM of three independent experiments (performed in triplicate/experiment). d Photomicrograph showing migrated D556Med cells transfected with scrambled-siRNA, eIF4E3-siRNA, or HDAC1-siRNA. Bar graph shows number of migrated cells microscopically counted in ten different fields. The p -value was calculated using a standard Student t -test. Error bars represent mean ± SEM of three independent experiments. e Line graphs show mean tumor volume in nude mice intracranially injected with DAOY–GFP–luciferase cells transfected with scrambled-siRNA or eIF4E3/HDAC1-siRNA ( n = 6 mice/group). The p -values were determined by the sum-of-squares F test. Error bars represent mean ± SEM. f Live bioluminescence (BLI) images of mice injected with DAOY–GFP–luciferase cells transfected with scrambled-siRNA or eIF4E3/HDAC1-siRNA ( n = 6 mice/group). g VCR dose–response curve of D556Med cells transfected with scrambled-siRNA, eIF4E3-siRNA or HDAC1-siRNA and treated with VCR or vehicle. Cell viability was assessed using alamarBlue cell viability assay. The p- values were determined by the sum-of-squares F test. Error bars represent mean ± SEM of three independent experiments (performed in sixtuplicate/experiment). h miR-584-5p rescues cancer growth-promoting effects of eIF4E3 and HDAC1. Bar graphs showing viability of D556Med cells transfected with control plasmid or eIF4E3/HDAC1 expression vector or cotransfected with eIF4E3/HDAC1 expression vector and miR-584-5p mimic. The p -value was calculated using one-way analysis of variance (ANOVA) followed by Sidak’s multiple-comparisons test. Error bars represent mean ± SEM of three independent experiments (performed in sixtuplicate/experiment). **** p < 0.0001; *** p < 0.001; ** p < 0.01. Scale bars: 50 μM ( b , d )

Article Snippet: MiR-NC and miR-584-5p were used at 50 nM for all experiments unless otherwise indicated. eIF4E3 plasmid was purchased from Origene (#RC209694).

Techniques: Transfection, Staining, Injection, Luciferase, Viability Assay, Control, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

doi: 10.1038/s41467-018-06808-8

Figure Lengend Snippet: MiR-584-5p –HDAC1/eIF4E3 signaling regulates cell cycle progression of MB cells. a Bar graphs show quantification of DNA histograms (using FlowJo software) reflecting cell cycle progression in D556Med cells transfected with miR-NC or miR-584-5p and treated with vehicle or vincristine (VCR). DNA histograms were obtained from FACS analysis of cells treated with miR-NC or miR-584-5p and treated with vehicle or vincristine (VCR). Error bars represent mean ± SEM of three independent experiments. b Bar graphs show FACS analysis of Histone H3 pSer10-positive D-556 cells transfected with miR-NC or miR-584-5p and treated with vehicle or VCR. The p -values were calculated using standard Student t -tests. Error bars represent mean ± SEM of three independent experiments. c FACS analysis of cell cycle progression in D556Med cells transfected with scrambled-siRNA or eIF4E3-siRNA or HDAC1-siRNA. Bar graphs reflect DNA histograms quantified using the FlowJo software. Error bars represent mean ± SEM of three independent experiments. d FACS analysis of Annexin V–FITC-positive cells in D556Med cells transfected with miR-NC or miR-584-5p and treated with vehicle or VCR. Bar graphs show dot plots quantified using the FlowJo software. Error bars represent mean ± SEM of three independent experiments. e FACS analysis of Annexin V–FITC-positive cells in D556Med cells transfected with scrambled-siRNA or eIF4E3-siRNA or HDAC1-siRNA. Bar graphs show dot plots quantified using the FlowJo software. Error bars represent mean ± SEM of three independent experiments. The p -values for d – e were determined by two-way ANOVA followed by Tukey’s multiple-comparisons test. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05

Article Snippet: MiR-NC and miR-584-5p were used at 50 nM for all experiments unless otherwise indicated. eIF4E3 plasmid was purchased from Origene (#RC209694).

Techniques: Software, Transfection

Journal: Nature Communications

Article Title: MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

doi: 10.1038/s41467-018-06808-8

Figure Lengend Snippet: MiR-584-5p –eIF4E3/HDAC1 signaling regulates microtubule dynamics in MB cells. a qPCR analysis of TUBB4a in D556Med, D425Med, D458Med, and DAOY cells transfected with miR-NC or miR-584-5p mimic. The p -values were calculated using standard Student t -tests. Error bars represent mean ± SEM. b qPCR analysis of β-tubulin isotypes in D556Med cells transfected with miR-NC or miR-584-5p mimic. The p -values were calculated using standard Student t -tests. Error bars represent mean ± SEM of three independent experiments (performed in sixtuplicate). c Above, types of spindle defects. Below, immunofluorescence images of defective mitotic spindles in D556Med cells transfected with miR-NC or miR-584-5p mimic. DNA is stained with DAPI (blue), whereas spindle is stained with β-tubulin (red). Black and white images in the middle and lower panels are same as images shown in the top panel. Black and white images are included to clearly show miR-584-5p effects on chromatid organization (middle) and spindle defects (lower). d Immunofluorescence images of defective spindles in D556Med cells transfected with scrambled-siRNA or eIF4E3-siRNA or HDAC1-siRNA. Black and white images in the middle and lower panels are same as images shown in the top panel. Black and white images are included to clearly show the effects of eIF4E3 or HDAC1 silencing on chromatid organization (middle) and defects in spindles (lower). e Bar graphs show percentage of defective spindles in scrambled-siRNA, miR-584-5p mimic, eIF4E3-siRNA, or HDAC1-siRNA-transfected D556Med cells. The p -values were calculated using standard Student t -tests. Error bars represent mean ± SEM of three independent experiments. f Above, a cell undergoing mitotic catastrophe. Below, immunofluorescence images of miR-584-5p- transfected D556Med cells undergoing mitotic catastrophe. g Above, cells undergoing aneuploidy and cytokinesis failure. Below, immunofluorescence images of miR-584-5p mimic-transfected D556Med cells undergoing cytokinesis failure and exhibiting aneuploidy. h Bar graph shows number of D556Med cells transfected with miR-NC or miR-584-5p mimic undergoing mitotic catastrophe, aneuploidy, and cytokinesis failure. The p -values were calculated using standard Student t -tests. Error bars represent mean ± SEM of three independent experiments. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. Scale bars: 10 μM ( c , d , f , g )

Article Snippet: MiR-NC and miR-584-5p were used at 50 nM for all experiments unless otherwise indicated. eIF4E3 plasmid was purchased from Origene (#RC209694).

Techniques: Transfection, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

doi: 10.1038/s41467-018-06808-8

Figure Lengend Snippet: MiR-584-5p damages DNA and sensitizes radiation response in MB cells. a Ionizing radiation (IR) dose–response curves of D556Med, D458Med, D425Med, and DAOY cells transfected with miR-NC or miR-584-5p mimic and treated with increasing dose of IR. All values were normalized to 0 Gy. b IR dose–response curves of D556Med cells transfected with scrambled-siRNA or eIF4E3/HDAC1-siRNA. All values were normalized to 0 Gy. The p- values for a and b were determined by the sum-of-squares F test. Error bars in a and b represent mean ± SEM of three independent experiments. c , d Representative immunofluorescence images showing 53BP1 foci in D556Med cells transfected with miR-NC or miR-584-5p mimic ( c ) or scrambled-siRNA or eIF4E3/HDAC1-siRNAs ( d ) and treated with or without 10 Gy of IR. Bar graphs next to images show average number of 53BP1 foci/cell. The p -values were determined by two-way ANOVA followed by Sidak’s multiple-comparisons test. Error bars represent mean ± SEM of three independent experiments. e DR-GFP reporter assay. Flow cytometry analysis showing number of GFP-positive cells reflecting homologous recombination (HR) events. U2OS-DR-GFP cells were transfected with miR-NC or miR-584-5p or scrambled-siRNA or eIF4E3/HDAC1-siRNAs followed by transfection with pCAGGS vector with ISceI/GFP. I-Sce1 expression leads to double-strand breaks that HR repairs by using the wild-type GFP, resulting in GFP + cells. The p -values were calculated using either Student t -tests (mimic) or one-way ANOVA followed by Dunnett’s multiple-comparisons test (siRNAs). Error bars represent mean ± SEM of three independent experiments. f EJ5-GFP reporter assay. Flow cytometry analysis showing number of GFP-positive cells reflecting non-homologous end joining (NHEJ) events. U2OS-EJ5-GFP cells were transfected with miR-NC or miR-584-5p or scrambled-siRNA or eIF4E3/HDAC1-siRNAs followed by transfection with pCAGGS vector with ISceI/GFP. I-Sce1 expression leads to double-strand breaks that NHEJ repairs, resulting in GFP + cells. The p -values were calculated using either Student t -tests (mimic) or one-way ANOVA followed by Dunnett’s multiple-comparisons test (siRNAs). Error bars represent mean ± SEM of three independent experiments. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. Scale bars: 10 μM ( c , d )

Article Snippet: MiR-NC and miR-584-5p were used at 50 nM for all experiments unless otherwise indicated. eIF4E3 plasmid was purchased from Origene (#RC209694).

Techniques: Transfection, Immunofluorescence, Reporter Assay, Flow Cytometry, Homologous Recombination, Plasmid Preparation, Expressing, Non-Homologous End Joining

Journal: Nature Communications

Article Title: MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

doi: 10.1038/s41467-018-06808-8

Figure Lengend Snippet: MiR-584-5p –eIF4E3/HDAC1 regulates c-MYC and MB stem cell proliferation. a , b Western blot analysis of DAOY cells treated with miR-NC or miR-584-5p mimic ( a ) or scrambled or eIF4E3/HDAC1-siRNA ( b ) using antibodies against indicated proteins. Gel pictures are representative of three independent experiments. c Medullospheres grown from D556Med cells transfected with miR-NC or miR-584-5p mimic. Bar graphs show number of medullospheres obtained from D556Med cells transfected with miR-NC or miR-584-5p mimic. The p- value was calculated using a standard Student t -test. Error bars represent mean ± SEM of three independent experiments (performed in triplicate for each experiment). d Medullospheres grown from D556Med cells transfected with scrambled-siRNA or eIF4E3/HDAC1-siRNAs. e qPCR analysis of stem cell markers in D556Med cells transfected with miR-NC or miR-584-5p mimic. The p -value was calculated using one-way ANOVA followed by Sidak’s multiple-comparisons test. Error bars represent mean ± SEM of three independent experiments (performed in triplicate for each experiment). f qPCR analysis of MYC expression in D556Med, D425Med, D458Med, and DAOY cell lines. The p -values were calculated using standard Student t -tests. Error bars represent mean ± SEM of three independent experiments (performed in triplicate for each experiment). g Western blot analysis of c-Myc in D556Med, DAOY, D425Med, and D458Med cells transfected with miR-NC or miR-584-5p mimic. h Western blot analysis of MYC in D556Med cells transfected with siRNA against scrambled, eIF4E3, HDAC1, or c-Myc. Gel pictures in g and h are representative of three independent experiments. i miR-584-5p and eIF4E3 rescue c-Myc-dependent MB cell growth. Bar graphs show percentage of cell viability in control vector, c-Myc expression vector, or miR-584-5p mimic plus c-Myc expression vector or eIF4E3-siRNA plus c-Myc expression vector - transfected D556Med cells. Cell viability was assessed using alamarBlue. The p -value was calculated using one-way ANOVA followed by Sidak’s multiple-comparisons test. Error bars represent mean ± SEM of three independent experiments (performed in sixtuplicate for each experiment). **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. Scale bars: 100 μM ( c , d )

Article Snippet: MiR-NC and miR-584-5p were used at 50 nM for all experiments unless otherwise indicated. eIF4E3 plasmid was purchased from Origene (#RC209694).

Techniques: Western Blot, Transfection, Expressing, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

doi: 10.1038/s41467-018-06808-8

Figure Lengend Snippet: Lower miR-584-5p expression parallels with higher eIF4E3/HDAC1 levels in MB. a Meta-analysis of GSE42657 data set showing miR-584-5p expression levels in human MB samples and control cerebellum. Data represent Log2 ratio of tumor/cerebellum. b Meta-analysis of miRIAD: intragenic microRNA database showing correlation between miR-584-5p expression and the expression of its host gene SH3TC2. c Meta-analysis of GSE28245 data set showing SH3TC2 expression levels in different sub-types of MB samples (WNT: n = 16; SHH: n = 20; Gp3: n = 20; Gp4: n = 20) and control cerebellum. Data represent Log2 ratio of tumor/cerebellum control. p -values are calculated using the Wilcoxon-signed rank test (theoretical median = zero). d Meta-analysis of GSE28245 data set showing SH3TC2, eIF4E3, and HDAC1 expression levels in different sub-types of MB. Data represent Log2 ratio of tumor/cerebellum control. e , f Meta-analysis of GSE28245 data set showing eIF4E3 ( e ) and HDAC1 ( f ) expression levels in different sub-types of MB. Data represent Log2 ratio tumor/cerebellum control. p -values are calculated using the Wilcoxon-signed rank test (theoretical median = zero). g , h Meta-analysis of GSE85217 data set showing eIF4E3 ( g ) and HDAC1 ( h ) expression levels in different sub-types of medulloblastoma. Data represent Log2 ratio of tumor/cerebellum control (WNT: n = 763; SHH: n = 223; Gp3: n = 144; Gp4: n = 326. Whiskers in the box plots represent minimum to maximum value. The center line in the box represents the median value. Black open circles in the box represent individual MB patient tumor sample. **** p < 0.0001; *** p < 0.001; ** p < 0.01

Article Snippet: MiR-NC and miR-584-5p were used at 50 nM for all experiments unless otherwise indicated. eIF4E3 plasmid was purchased from Origene (#RC209694).

Techniques: Expressing, Control

Journal: Nature Communications

Article Title: MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

doi: 10.1038/s41467-018-06808-8

Figure Lengend Snippet: List of primers

Article Snippet: MiR-NC and miR-584-5p were used at 50 nM for all experiments unless otherwise indicated. eIF4E3 plasmid was purchased from Origene (#RC209694).

Techniques: