goat anti ifitm1  (R&D Systems)

Journal: Oncogenesis

Article Title: A mesenchymal glioma stem cell profile is related to clinical outcome

doi: 10.1038/oncsis.2014.5

Figure Lengend Snippet: Hierarchical clustering analysis of GSCs. ( a ) The unsupervised clustering using Ward's linkage method and Euclidean distance individualized H9 HNSCs, H1-H9-ESCs and two groups of GSCs (GSCX-1 and GSCX-2). Note that culture conditions of H9 HNSC do not influence the expression of pluripotency gene samples H9 HNSC-1 and H9 HNSC-2 samples. ( b ) Box plot representation of COL1A1 and IFITM1 expression levels between GSC groups GSCX-1 and GSCX-2. The charts show the log 10 expression of relative quantification (RQ) values normalized to the expression of H9 HNSC cell line. ( c ) Box plot representation of COL1A1 and IFITM1 expression levels between bulk tumor groups TX-1 and TX-2. The top edge of the boxes represents the 75th percentile, the bottom edge, the 25th percentile and × the mean. The range is shown as vertical edge.

Article Snippet: Antibodies used in this study were goat anti-IFITM1 antibody (R&D Systems Europe, Lille, France), rabbit anti-COL1A1 antibody (Abcam, Paris, France) and mouse β-actin antibody (Abcam).

Techniques: Expressing

Journal: Oncogenesis

Article Title: A mesenchymal glioma stem cell profile is related to clinical outcome

doi: 10.1038/oncsis.2014.5

Figure Lengend Snippet: Knockdown efficiency of COL1A1 and IFITM1 and effect on cell invasion. ( a ) COL1A1 and IFITM1 mRNA expression was monitored by quantitative reverse transcriptase–PCR in GSC-3 and GSC-9 ( n =2). Significant inhibition of 90% of mRNA expression was observed. GADPH was used as internal control. * P <0.05. Each value is expressed as mean±s.e.m. ( b ) Western blot analysis showed diminution of IFITM1 and COL1A1 proteins expression, β-actin was used as loading control. ( c ) Densitometry of protein level. The bar graph shows relative IFITM1 and COL1A1 protein expression levels expressed as percentage of IFITM1 and COL1A1 expression in control cells ( n =3). We observed >80% inhibition for both genes in GSC-3 and GSC-9 cells, as compared with control cells. ( d ) Representative fields of Boyden cell invasion assay. After 3 days of invasion, cells that migrated across the membrane were fixed and stained with hematoxylin/eosin and were counted in one hundred different fields. The invaded cells were photographed under the microscope at × 100 magnification. ( e ) A significant reduction of invasion is observed in both GSC-3 and GSC-9 cells lines with sh-IFITM1 as well as for sh-COL1A1. The results represent a percentage of invaded cells. Data are presented as mean±s.e.m. from three independent experiments. * P <0.05, ** P <0.01.

Article Snippet: Antibodies used in this study were goat anti-IFITM1 antibody (R&D Systems Europe, Lille, France), rabbit anti-COL1A1 antibody (Abcam, Paris, France) and mouse β-actin antibody (Abcam).

Techniques: Expressing, Reverse Transcription, Inhibition, Western Blot, Invasion Assay, Membrane, Staining, Microscopy

Journal: Oncogenesis

Article Title: A mesenchymal glioma stem cell profile is related to clinical outcome

doi: 10.1038/oncsis.2014.5

Figure Lengend Snippet: Effects of COL1A1 and IFITM1 inhibition on cell proliferation and on neurosphere-initiating capacity. ( a ) Cell proliferation was measured by using a MTS assay. Each experiment was carried out in replicates of six and repeated twice and expressed as mean±s.d. ( b ) Inhibition of COL1A1 resulted in a significant (* P <0.05) decrease of neurosphere-initiating cell (NS-IC) frequency whereas inhibition of IFITM1 expression only slightly modified the neurosphere-initiating capacity. Final cell dilutions yielding 37% of negative wells correspond to the dilution at which there is one NS-ICs per well. Each experiment was performed in duplicate and repeated twice and expressed as mean±s.d.

Article Snippet: Antibodies used in this study were goat anti-IFITM1 antibody (R&D Systems Europe, Lille, France), rabbit anti-COL1A1 antibody (Abcam, Paris, France) and mouse β-actin antibody (Abcam).

Techniques: Inhibition, MTS Assay, Expressing, Modification

Journal: Oncogenesis

Article Title: A mesenchymal glioma stem cell profile is related to clinical outcome

doi: 10.1038/oncsis.2014.5

Figure Lengend Snippet: COL1A1 and IFITM1 expression levels in an independent cohort of 30 patients' tumors. ( a ) Box plot representation of the mRNA expression levels. The charts show the log 10 expression of relative quantification (RQ) values normalized to the expression of H9 NSC cell line. The top edge of the boxes represents the 75th percentile, the bottom edge, the 25th percentile and × the mean. The range is shown as vertical edge. ( b ) Kaplan–Meier analysis for overall survival of an independent cohort of 30 GBM patients. Survival distribution of patients with low (group A; n =13) versus high (group B; n =17) expression of COL1A1 and IFITM1 ( P <10 −4 ).

Article Snippet: Antibodies used in this study were goat anti-IFITM1 antibody (R&D Systems Europe, Lille, France), rabbit anti-COL1A1 antibody (Abcam, Paris, France) and mouse β-actin antibody (Abcam).

Techniques: Expressing

Journal: The EMBO Journal

Article Title: hCAF1/CNOT7 regulates interferon signalling by targeting STAT1

doi: 10.1038/emboj.2013.11

Figure Lengend Snippet: Characterization of hCAF1-depleted cells. MCF7 cells were transfected with vectors expressing control miRNA (mock) or with two alternative miRNAs (called kd, and kd-1) targeting hCAF1. After vector transfection and selection, mock, hCAF1kd and hCAF1kd-1 cells were obtained. Total RNA or protein extracts were prepared to test hCAF1 knockdown efficiency. (A) Left panel, SYBR green real-time RT–PCR analysis was performed for detection of transcript levels of hCAF1. Results were normalized using 36B4 mRNA level as an internal control. Transcript levels in control cells (mock) were set to 1. Right panel, hCAF1 protein levels were analysed by western blot from 30 μg of protein extracts. (B) Endogenous hCAF1 expression and localization in hCAF1kd and hCAF1kd-1 compared to the mock control, by Immunofluorescent staining using mouse polyclonal anti-CAF1 antibody and goat anti-mouse Alexa 488-conjugated secondary antibody (green). Scale bar=20 μm. (C) Upper: summary of Human Exon 1.0 ST Array analysis results. Diagram of the hierarchical clustering of gene expression profiles of control versus hCAF1-deficient hCAF1kd cell lines. Probes are represented vertically, whereas conditions are shown horizontally. Lower: type I and II interferon distribution by ISG Database. (D) Validation of the DNA array screen. SYBR green RT-qPCR analysis of the upregulated gene products IFI27, IFI6, TAP1, STAT1, IFITM1, HERC6, PLSCR1 and the downregulated gene CSTA. Total RNA isolated from mock and hCAF1kd cells was reverse transcribed, and PCR was performed with primers specific for the transcripts of the indicated genes. Gene expression levels were normalized to internal controls 36B4 and shown as expression levels of hCAF1kd cells relative to expression levels in control cells (arbitrarily set to 1). (E, F) Rescue of hCAF1 functions in knockdown cells. hCAF1kd cells were stably transfected with a plasmid expressing mouse flag CAF1 (insensitive to miRNAs), or with the empty vector used as a control. (E) The expression of the indicated genes was analysed by real-time RT-qPCR as in (D). (F) Efficiency of flag mCAF1 overexpression and the protein expression of the indicated hCAF1-regulated genes was assessed by western blot. The experiments illustrated in (D) and (E) were performed in triplicate and expressed as mean values of three independent experiments. Standard deviations are shown.

Article Snippet: The rabbit polyclonal anti-STAT1αp91 (C-24), the goat polyclonal anti-IFITM1 (P-17) and the mouse monoclonal anti-PML (PG-M3) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Plasmid Preparation, Selection, SYBR Green Assay, Quantitative RT-PCR, Western Blot, Staining, DNA Array, Isolation, Stable Transfection, Over Expression