Journal: Scientific Reports

Article Title: Fabrication of a protein microarray by fluorous-fluorous interactions

doi: 10.1038/s41598-017-07571-4

Figure Lengend Snippet: Fluorous protein microarray for the simultaneous detection of multiple proteins. ( a ) MBP, GST, and anti-RCA 120 were printed on a fluorous slide. ( b ) Confirmation of the specific fluorous-fluorous interactions. Anti-RCA 120 , anti-RCA 120 pre-incubated with F tag -acid (compound 5), and anti-RCA 120 pre-incubated with F tag -BA (compound 2) solutions were spotted on a fluorous slide.

Article Snippet: The activity of fluorescent eGFP on the slide was directly measured with a NovaRay Microarray Scanner using a fluorescein isothiocyanate (FITC) filter.

Techniques: Microarray, Incubation

Journal: bioRxiv

Article Title: Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

doi: 10.1101/578666

Figure Lengend Snippet: a) Analysis of SOX6 mRNA expression levels in EwS tumors, 9 additional sarcomas or pediatric tumors, and 18 normal tissue types as determined on Affymetrix HG-U133Plus2.0 arrays. Data are represented as dot plots and horizontal bars represent medians. The number of samples is given in parentheses. ASPS, alveolar soft part sarcoma; GIST, gastrointestinal stromal tumor. b) Validation of SOX6 expression on protein level by IHC in the same tissue types as shown in ( a ). Immuno Reactive scores (IRS) are presented as dot plots. Horizontal bars represent medians. The number of samples is given in parentheses. c ) Representative micrographs of the IHC stains; scale bars=20 μm.

Article Snippet: The slides were stained with either polyclonal anti-SOX6 antibody raised in rabbit (1:1,600; HPA003908, Atlas Antibodies, Sweden) or with monoclonal anti-Ki67 raised in rabbit (1:200, 275R-15, Cell Marque/Sigma-Aldrich) for 60 min at RT, followed by a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody (ImmPRESS Reagent Kit, MP-7401, Vector Laboratories, Germany).

Techniques: Expressing

Journal: bioRxiv

Article Title: Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

doi: 10.1101/578666

Figure Lengend Snippet: a) Analysis of EWSR1-FLI1 and SOX6 expression by qRT-PCR in A673/TR/shEF1 cells at indicated time points after addition of Dox. Horizontal bars represent means and whiskers SEM, n ≥3. P values determined via two-sided Mann-Whitney test. b) Left: Analysis of EWSR1-FLI1 and SOX6 expression by Affymetrix microarrays in xenografts from A673/TR/shEF1 cells 96h after start of Dox-addition, Horizontal bars represent means and whiskers SEM, n =3. P value determined via independent one-sample t-test. Right: Representative immunohistological stains of xenografts stained for (EWSR1)FLI1 and SOX6. Scale bar=20µm. c) Analysis of SOX6 expression by Affymetrix microarrays in embryoid bodies after ectopic EWSR1-FLI1 expression. Horizontal bars represent means and whiskers SEM, n =3. P value determined via unpaired two-sided t-test with Welch’s correction. d) Integrated genomic view of the SOX6 locus displaying tracks for DNAse 1 hypersensitivity (HS) and ChIP-Seq data for EWSR1-FLI1 and H3K27ac in A673 and SK-N-MC EwS cells transfected with shRNA against EWSR1-FLI1 (shEF1) or control shRNA (shGFP). e) Analysis of relative enhancer activity of the SOX6-associated GGAA-mSat by dual luciferase reporter assays in A673/TR/shEF1 cells (-/+). Horizontal bars represent means and whiskers SEM, n =4. P value determined via two-sided Mann-Whitney test. f) Correlation of the average enhancer activity of both alleles of the SOX6- associated GGAA-mSat and the average SOX6 mRNA expression levels across six EwS cell lines (TC-32 was set as reference). The color code indicates the average number of consecutive GGAA-repeats of both alleles. *** P <0.001, ** P <0.01, * P <0.05

Article Snippet: The slides were stained with either polyclonal anti-SOX6 antibody raised in rabbit (1:1,600; HPA003908, Atlas Antibodies, Sweden) or with monoclonal anti-Ki67 raised in rabbit (1:200, 275R-15, Cell Marque/Sigma-Aldrich) for 60 min at RT, followed by a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody (ImmPRESS Reagent Kit, MP-7401, Vector Laboratories, Germany).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Staining, ChIP-sequencing, Transfection, shRNA, Activity Assay, Luciferase

Journal: bioRxiv

Article Title: Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

doi: 10.1101/578666

Figure Lengend Snippet: a) Western blot analysis 96h after Dox-induced shRNA-mediated SOX6 knockdown in RDES and TC-32 EwS cells. GAPDH served as loading control. b) Top: Volcano plot of microarray data showing differentially expressed genes (DEGs) after shRNA-mediated SOX6 knockdown compared to a non-targeting shCtrl. A summary of two EwS cell lines is shown. Bottom: Representative enrichment plots from GSEA of transcriptome profiles of RDES and TC-32 EwS cells 96h after induction of shRNA-mediated SOX6 silencing. c) Left: Quantification of the sphere index after 12 days of Dox-treatment in RDES and TC-32 cells. Horizontal bars represent means and whiskers the SEM, n =3. P values determined via two-sided Mann-Whitney test. Right: Representative micrographs of RDES/TR/shSOX6_3 spheres. Scale bar=1 mm. d) Analysis of tumor growth of xenografted RDES and TC-32 cells containing either Dox-inducible specific shRNAs against SOX6 (shSOX6_2/shSOX6_3) or a non-targeting control shRNA (shCtrl). When tumors were palpable (arrow), mice were randomized and henceforth treated with Dox (+) or vehicle (–). Data are represented as means and SEM, n ≥3 mice per condition. P values determined via two-sided Mann-Whitney test. e) Representative micrographs of xenografts from ( d ) showing IHC stains for SOX6, cleaved caspase 3 and Ki67. Scale bar=20µm. f) Quantification of the relative number of mitoses per high-power field (HPF) of xenografts shown in ( d ). Horizontal bars represent means and whiskers the SEM, n ≥3. P values determined via two-sided Mann-Whitney test. g) Quantification of the relative number of cells positive for cleaved caspase 3 of xenografts shown in ( d ). Horizontal bars represent means and whiskers the SEM, n ≥3. *** P <0.001, ** P <0.01, * P <0.05.

Article Snippet: The slides were stained with either polyclonal anti-SOX6 antibody raised in rabbit (1:1,600; HPA003908, Atlas Antibodies, Sweden) or with monoclonal anti-Ki67 raised in rabbit (1:200, 275R-15, Cell Marque/Sigma-Aldrich) for 60 min at RT, followed by a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody (ImmPRESS Reagent Kit, MP-7401, Vector Laboratories, Germany).

Techniques: Western Blot, shRNA, Microarray, MANN-WHITNEY

Journal: bioRxiv

Article Title: Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

doi: 10.1101/578666

Figure Lengend Snippet: a) Analysis of publicly available matched gene expression and drug-response data of up to 22 EwS cell lines per drug. Highlighted in dark grey, top 7 drugs with P <0.02; pink = Elesclomol. b) LN_IC50 (µM) of the top 7 drugs including Federatinib (JAK-2 inhibitor), PHA-793887 (CDK2/5/7 inhibitor), Rucaparib (PARP inhibitor), Serdemetan (p53 activator), Imatinib (tyrosine kinase inhibitor) and Olaparib (PARP1/2 inhibitor) with P <0.02. Horizontal bars represent means and whiskers SEM, n ≥18 EwS cell lines. c ) Quantification of relative viability of indicated cell lines by a Resazurin assay after treatment with Elesclomol at indicated concentrations for 72h. Modeled dose-response curves and calculated IC50 values (nM) are displayed for SOX6-high expressing EwS cells (black and grey) and the SOX6-low expressing osteosarcoma cell line SAOS-2 and the mesenchymal cell line MSC-52 (dark and light green), n ≥3. d ) Analysis of relative SOX6 expression in indicated cell lines by qRT-PCR. Horizontal bars represent means and whiskers SEM, n ≥3. P values determined via two-sided Mann-Whitney test. e ) Analysis of cell viability of indicated cell lines by a Resazurin assay. Horizontal bars represent means and whiskers SEM, n ≥5. P values determined via two-sided Mann-Whitney test. f ) Quantification of relative Elesclomol IC50 values by a Resazurin assay in indicated cell lines after 72h of Elesclomol treatment and concomitant addition of Dox. Horizontal bars represent means and whiskers SEM, n =7. P values determined via two-sided Mann-Whitney test. g ) Quantification of relative Annexin V positivity of indicated EwS cells 48h after treatment with Elesclomol (10 nM). Horizontal bars represent means and whiskers SEM, n =10. P values determined via unpaired two-sided t-test with Welch’s correction. h ) Analysis of tumor growth of TC-32 EwS cells in NSG mice treated once per day (day 0-4 and day 7-9) with Elesclomol (intravenously, 5 mg/kg). Data represent means and SEM, n =5 mice per condition. P values determined via two-sided Mann-Whitney test. i ) Left: Quantification of the average number of cleaved caspase 3 positive cells per 3 HPF in TC-32 xenografts shown in ( h ). Horizontal bars represent means and whiskers SEM, n =5 per condition. P values determined via two-sided Mann-Whitney test. Right: representative micrographs. Scale bar=100 µm. j ) Left: Quantification of necrotic area in TC-32 xenografts shown in ( h ). Horizontal bars represent means and whiskers SEM, n =5 per condition. P values determined via two-sided Mann-Whitney test. Right: Representative micrographs. Scale bar = 900 µm. *** P <0.001, ** P <0.01, * P <0.05.

Article Snippet: The slides were stained with either polyclonal anti-SOX6 antibody raised in rabbit (1:1,600; HPA003908, Atlas Antibodies, Sweden) or with monoclonal anti-Ki67 raised in rabbit (1:200, 275R-15, Cell Marque/Sigma-Aldrich) for 60 min at RT, followed by a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody (ImmPRESS Reagent Kit, MP-7401, Vector Laboratories, Germany).

Techniques: Expressing, Resazurin Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: bioRxiv

Article Title: Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

doi: 10.1101/578666

Figure Lengend Snippet: a) Left: Representative picture of flow cytometric measurement of ROS by DCF-DA fluorescence in TC-32 cells after Elesclomol-treatment (10 nM, pink color) compared to DMSO control (gray color). Right: Quantification of relative DCF-DA fluorescence in TC-32 and RDES cells after Elesclomol-treatment (10 nM, pink color) compared to DMSO control. Horizontal bars indicate means and whiskers SEM, n =8. P values determined via independent one sample t-test. b) Quantification of relative IC50 concentrations in TC-32 and RDES cells by a Resazurin assays after pre-treatment with the antioxidant N-acetylcysteine (Nac(+), pink color) compared to DMSO control (Nac(-), gray color). Horizontal bars indicate means and whiskers SEM, n ≥5. P values determined via two-sided Mann-Whitney test. c) Left: Representative picture of flow cytometric measurement of ROS by DCF-DA fluorescence in TC-32/TR/shSOX6_2 cells after Dox-induced knockdown of SOX6 (blue color) for 96h as compared to control (Dox (–), gray color). Right: Quantification of relative DCF-DA fluorescence in TC-32 and RDES cells after SOX6 knockdown. Dox(–) = gray color, Dox(+) = blue color. Horizontal bars represent means and whiskers SEM, n ≥3. P values determined via one-sample t-test. d) Quantification of relative IC50 Elesclomol concentrations in TC-32 and RDES cells by a Resazurin assays after Dox-induced SOX6 knockdown and treatment with H 2 O 2 (30 µmol/l) for 72h. Horizontal bars represent means and whiskers SEM, n ≥5. P values determined via two-sided Mann-Whitney test. e) Top: Representative Western blot analysis of TXNIP expression 96h after induction of SOX6 knockdown in TC-32 and RDES cells. GAPDH served as loading control. Bottom: Quantification of relative TXNIP mRNA expression in the same cells by qRT-PCR. Horizontal bars represent means and whiskers SEM, n =3. P values determined via two-sided Mann-Whitney test. f) Left: Analysis of relative TXNIP expression by qRT-PCR in TC-32 cells 96h after transfection with siRNA directed against TXNIP . Horizontal bars represent means and whiskers SEM, n =5. P value determined via unpaired two-sided t-test with Welch’s correction. Right: Analysis of ROS levels by flow cytometric measurement of DCF-DA fluorescence in TC-32 cells after siRNA-induced TXNIP knockdown. Horizontal bars represent means and whiskers SEM, n =5. P values determined via independent one sample t-test. g) Schematic illustration of the EWSR1-FLI1-mediated effect on SOX6 expression in EwS. *** P <0.001, ** P <0.01, * P <0.05

Article Snippet: The slides were stained with either polyclonal anti-SOX6 antibody raised in rabbit (1:1,600; HPA003908, Atlas Antibodies, Sweden) or with monoclonal anti-Ki67 raised in rabbit (1:200, 275R-15, Cell Marque/Sigma-Aldrich) for 60 min at RT, followed by a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody (ImmPRESS Reagent Kit, MP-7401, Vector Laboratories, Germany).

Techniques: Fluorescence, MANN-WHITNEY, Western Blot, Expressing, Quantitative RT-PCR, Transfection

Journal: Emerging Microbes & Infections

Article Title: Development of an influenza virus protein microarray to measure the humoral response to influenza virus infection in mallards

doi: 10.1038/emi.2017.98

Figure Lengend Snippet: Influenza virus protein microarray pipeline. Recombinant HA is expressed in a baculovirus expression system, purified, characterized and undergoes quality control ( A ). HAs are arrayed onto an epoxysilane-coated glass slide (Schott) using a Versa 110 spotter (Aurora Biomed) ( B ). The HAs are covalently bound to the slide and blocked; the arrays are incubated with sera, followed by fluorescently labeled secondary antibodies in a 96-well microarray gasket (Arrayit) ( C ). The arrays are then imaged ( D ), spots are automatically detected and their fluorescence is measured ( E ). Data from microarray imaging are analyzed in GraphPad Prism 7.0 ( F ). HA, hemagglutinin.

Article Snippet: Slides were allowed to dry at room temperature, and analyzed for mean fluorescence using a Vidia microarray scanner (Indevr, Boulder, CO, USA), using an exposure time of 1300 ms. AUC was measured as total peak area above the mean fluorescence of spots of the same HA incubated with naive mallard sera.

Techniques: Microarray, Recombinant, Expressing, Purification, Incubation, Labeling, Fluorescence, Imaging

Journal: Emerging Microbes & Infections

Article Title: Development of an influenza virus protein microarray to measure the humoral response to influenza virus infection in mallards

doi: 10.1038/emi.2017.98

Figure Lengend Snippet: Establishing ELISA and IVPM for mallards. ELISA-IVPM correlation for serial 1:2 dilutions of mAb KB2, reacting to a conformational epitope on NC99 ( A ) and PR8 ( B ) H1 HA. The PCC and its P -value are indicated in both panels. ( C ) Testing of commercial secondary antibodies. Antibodies purchased from Antibodies Online (AbO), KPL (KPL) and Novus Biologicals (Novus) are shown, reacting against serum from a mallard infected with H3N8 and H6N2, binding H3, H6 and H18 HAs in ELISA. ( D ) Reactivity of different concentrations of pooled sera from mallards infected with H4N5 probed with different concentrations of the AbO secondary antibody in IVPM. The arrayed protein is recombinant H4. enzyme-linked immunosorbent assay, ELISA; influenza virus protein microarray, IVPM; Pearson correlation coefficients, PCC.

Article Snippet: Slides were allowed to dry at room temperature, and analyzed for mean fluorescence using a Vidia microarray scanner (Indevr, Boulder, CO, USA), using an exposure time of 1300 ms. AUC was measured as total peak area above the mean fluorescence of spots of the same HA incubated with naive mallard sera.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Binding Assay, Recombinant, Microarray

Journal: Emerging Microbes & Infections

Article Title: Development of an influenza virus protein microarray to measure the humoral response to influenza virus infection in mallards

doi: 10.1038/emi.2017.98

Figure Lengend Snippet: Reactivity of sera from experimentally infected mallards against recombinant HA in ELISA and IVPM. Infection and sample collection scheme for experimentally inoculated mallards ( A ). Absolute AUC values ( B ) and fold induction ( C ) over the AUC of naive sera against recombinant HA in ELISA are shown, calculated for each HA. ( D ) AUC and ( E ) fold induction data collected via the IVPM. ( F ) Reactivity of sera to chimeric H5/3 (cH5/3), H3 and H5 in ELISA. area under the curve, AUC; enzyme-linked immunosorbent assay, ELISA; hemagglutinin, HA; influenza virus protein microarray, IVPM.

Article Snippet: Slides were allowed to dry at room temperature, and analyzed for mean fluorescence using a Vidia microarray scanner (Indevr, Boulder, CO, USA), using an exposure time of 1300 ms. AUC was measured as total peak area above the mean fluorescence of spots of the same HA incubated with naive mallard sera.

Techniques: Infection, Recombinant, Enzyme-linked Immunosorbent Assay, Microarray

Journal: Emerging Microbes & Infections

Article Title: Development of an influenza virus protein microarray to measure the humoral response to influenza virus infection in mallards

doi: 10.1038/emi.2017.98

Figure Lengend Snippet: Correlation analysis of reactivity data measured by IVPM and ELISA. Correlation between IVPM and ELISA fold induction over naive sera ( A ) and IVPM and ELISA absolute AUC values ( B ) are shown, by HA. Correlation of ELISA and IVPM fold induction data for recombinant H3 shown as an example of the correlation analysis ( C ). area under the curve, AUC; enzyme-linked immunosorbent assay, ELISA; hemagglutinin, HA; influenza virus protein microarray, IVPM.

Article Snippet: Slides were allowed to dry at room temperature, and analyzed for mean fluorescence using a Vidia microarray scanner (Indevr, Boulder, CO, USA), using an exposure time of 1300 ms. AUC was measured as total peak area above the mean fluorescence of spots of the same HA incubated with naive mallard sera.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Microarray

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: A Diagnostic Assay Based on MicroRNA Expression Accurately Identifies Malignant Pleural Mesothelioma

doi: 10.2353/jmoldx.2010.090169

Figure Lengend Snippet: Comparison of microRNA expression levels in MPM and carcinoma samples in the discovery phase. A: Median normalized fluorescence values (microarrays) of microRNAs in seven MPM samples are plotted against the median fluorescence values (microarrays) of these microRNAs in 97 carcinoma samples (Table 1). Gray crosses show control probes and microRNAs whose expression level was at background levels, and red circles mark microRNAs that had statistically significant differences in expression values after false discovery rate correction with at least a twofold change in median expression (see Materials and Methods). Yellow squares highlight the median expression levels of hsa-miR-200c, hsa-miR-192, and hsa-miR-193a–3p. B: Box plot of the expression levels of hsa-miR-200c in tumors of different tissue origin. Tumor origins include MPM (n = 7), RCC (n = 16), and 81 adenocarcinomas from: lung (LUN, n = 15), colon (COL, n = 17), breast (BRE, n = 4), endometrium (END, n = 9), pancreas (PAN, n = 6), esophagus (ESO, n = 11), ovary (OVA, n = 10), stomach (STO, n = 6), and prostate (PRO, n = 3). Units show log2 of the normalized fluorescence signal by microarray. C: Box-plots of the expression levels of hsa-miR-200c, hsa-miR-193a–3p, and hsa-miR-192, in these MPM, adenocarcinoma (Adeno.), and RCC samples. Units show log2 of the normalized fluorescence signal by microarray. D: Box plots of the expression levels of these microRNAs in 22 MPM samples, 39 adenocarcinoma samples, and four RCC samples, measured by qRT-PCR (Table 1). Units show the inverted normalized CT (see Materials and Methods). For all box plots, red crosses indicate outlier values that are defined as being over 1.5 times the distance between the 25th and 75th percentiles above the 75th percentile or below the 25th percentile.

Article Snippet: 12 ,19 747 DNA oligonucleotide probes representing human microRNAs were spotted in triplicate on coated microarray slides (Nexterion Slide E, Schott, Mainz, Germany).

Techniques: Comparison, Expressing, Fluorescence, Control, Microarray, Quantitative RT-PCR

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: A Diagnostic Assay Based on MicroRNA Expression Accurately Identifies Malignant Pleural Mesothelioma

doi: 10.2353/jmoldx.2010.090169

Figure Lengend Snippet: Tumor Samples Used in the Study

Article Snippet: 12 ,19 747 DNA oligonucleotide probes representing human microRNAs were spotted in triplicate on coated microarray slides (Nexterion Slide E, Schott, Mainz, Germany).

Techniques: Microarray, Biomarker Discovery