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: Slides were incubated with the polyclonal cleaved caspase 3 primary antibody (rabbit, 1:100; 9661, Cell Signaling, Frankfurt am Main, Germany) for 60 min at RT followed by ImmPRESS Reagent Kit.

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: Slides were incubated with the polyclonal cleaved caspase 3 primary antibody (rabbit, 1:100; 9661, Cell Signaling, Frankfurt am Main, Germany) for 60 min at RT followed by ImmPRESS Reagent Kit.

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

Journal: bioRxiv

Article Title: Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

doi: 10.1101/871574

Figure Lengend Snippet: a-c Injury site homogenates harvested from burn/tenotomy mice on day 0, 3, and 7 post burn/tenotomy. a ) Monocyte/Macrophage associated factors. b ) Monocyte/Macrophage and neutrophil maturation factors. c ) Cytokines stimulated by monocyte factors. d ) TGF family members. e ) Stem cell maintaining factors. Levels of cytokine and chemokines in pg/ug of Total protein, data represented as the median with interquartile range. Changes in cytokines and chemokines across day 3 and day 7 vs day 0 were analyzed by an analysis of variance (ANOVA) with post-hoc Dunnett test (n=3 mice/time point) significance. Non-heteroscedastic data identified by Levene’s test for homogeneity of variances were alternatively analyzed by Welch statistic and post-hoc Dunnett T3. Degrees of freedom (df or df1) across samples = 2. F statistic and significant post-hoc p-values respectively: CXCL1: 30.359, p(D0 vs. D7)=0.036, CXCL2: 8.504, CCL2: 268.773, p(D0 vs. D3)=0.000, p(D0 vs. D7)=0.000, CCL3: 16.430, CCL4: 22.441, p(D0 vs. D3)=0.014, G-CSF: 12.579, GM-CSF: 4.988, IL-1b: 3.486, IL-6: 13.019, TNF-α: 38.435, p(D0 vs. D7)=0.019, TGF-β1: 9.156, TGF-β2: 11.376, TGF-β3: 7.362, CCL5: 0.825, CXCL5: 0.825, LIF: 25.368, p(D0 vs. D3)=0.001, p(D0 vs. D7)=0.002 *p<.05 **p<.01. f) t-SNE dimensionality reduction analysis of single cell sequencing from day 3 cells harvested at the extremity injury site revealed 14 distinct cell clusters (representative, performed in triplicate). g,h) Feature plots displaying the single cell gene expression of g) monocyte/macrophage cytokines and chemokines increased in the homogenates and h) their receptors.

Article Snippet: After re-blocking the slides, the slides were incubated with a mouse anti-human TGF-β1 antibody (Cat No. MAB240, R&D Systems) at a dilution of 1:50 overnight.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

doi: 10.1101/871574

Figure Lengend Snippet: a) Left: GSEA analysis of microarray data collected from buffy coat of human burn injury patients at increased risk of HO compared to post-surgical control patients. Right: GSEA analysis of RNAseq performed of tendon injury site 3 weeks after burn/tenotomy in mice. b) Western blot of whole tissue protein collected from the injury site of C57BL/6J mice after burn/tenotomy at indicated time points. A western blot for p-SMAD2 and H3 was performed on the nuclear fraction and SMAD2 and alpha-Tubulin were performed on the cytosolic fraction. n=5 were pooled for each time point. c) Co-localization of F4/80+ and TGF-β1 at tendon injury site 1 week after burn/tenotomy. Scale bars correspond to 100um. d) Left: Co-localization of CD68+ and TGF-β1 in early human HO. Right: Co-localization of p-SMAD2 and PDGFRα in human HO.

Article Snippet: After re-blocking the slides, the slides were incubated with a mouse anti-human TGF-β1 antibody (Cat No. MAB240, R&D Systems) at a dilution of 1:50 overnight.

Techniques: Microarray, Western Blot

Journal: bioRxiv

Article Title: Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

doi: 10.1101/871574

Figure Lengend Snippet: a) Representative Safranin O stain of tendon injury site 3 weeks after burn/tenotomy in p7N3 (CD47 agonist) treated and PBS control mice. n=3/group. b) MicroCT analysis of tenotomy site 9 weeks after burn/tenotomy in PBS and p7N3 (CD47 agonist) treated mice. Left: Representative 3D reconstruction. Right: Quantification of total HO, floating HO and proximal HO.n=7/group. Total HO: t=3.415, df=7.840, p=0.009; Floating HO: t=2.201, df=12, p=0.048; Proximal HO: t=2.686, df=8.549, p=0.026. c) Levels of TGF-β1, TGFβ2, and TGFβ3 in pg/ug total protein and represented as median with interquartile range from Top: homogenates from the extremity injury (TGF-β1: t=-0.635, df=4, p=0.560; TGF-β2: t=-0.643, df=4, p=0.555; TGF-β3: t=-1.272, df=2.186, p=0.322) and Bottom: plasma from PBS and p7N3 (CD47) peptide treated mice 3days after burn/tenotomy (TGF-β1: t=1.544, df=2.037, p=0.260; TGF-β2: t=2.747, df=4, p=0.052; TGF-β3: t=-1.492, df=4, p=0.210). n=3 mice per treatment group. d) qPCR analysis of M1 ( iNos ) and M2 ( Arg1 and Mrc1 ) macrophage markers and Tgfb1 in macrophages isolated from the extremity injury site of naive (day 0), burn/tenotomy day 3, burn/tenotomy day 3 treated with PBS, and burn/tenotomy day 3 treated with p7N3 (CD47) peptide. Day 0 vs. Day 3 – iNOS : t=2.020, df=2, p=0.181; Arg1 : t=-6.084, df=3, p=0.009; Mrc1 : t=0.703, df=4, p=0.521; Tgfb1 : t=0.253, df=4, p=0.812. PBS vs. CD47 – iNOS : t=-0.834, df=2.043, p=0.491; Arg1 : t=0.895, df=4, p=0.421; Mrc1 : t=1.176, df=4, p=0.305; Tgfb1 : t=1.186, df=4, p=0.301. e) Representative images of phagocytosis assay using macrophages isolated from the extremity injury at day 3 after burn/tenotomy in mice treated with PBS or p7N3 (CD47). PBS n=3, CD47 n=4 approximately 25 cells/mouse. f) Measurement of cellular circularity Circularity: t=6.119, df=55.537, p=0.000 and quantification of mean fluorescent intensity phagocytosed by each macrophage. MFI: t=-0.357, df=111, p=0.722.

Article Snippet: After re-blocking the slides, the slides were incubated with a mouse anti-human TGF-β1 antibody (Cat No. MAB240, R&D Systems) at a dilution of 1:50 overnight.

Techniques: Staining, Isolation, Phagocytosis Assay

Journal: Molecular Therapy

Article Title: TALEN-edited allogeneic inducible dual CAR T cells enable effective targeting of solid tumors while mitigating off-tumor toxicity

doi: 10.1016/j.ymthe.2024.08.018

Figure Lengend Snippet: Tumor localized FAP + CAFs inhibit CAR T cell intra-tumoral infiltration and anti-tumor cytotoxicity (A) Schematic illustrating T cell infiltration assay in tumor spheroids plated with or without CAFs. (B) Graph depicting flow cytometry quantitation of T cells infiltrated per tumor spheroids with or without CAFs, represented as percentage of T cell input. Bars show means ± SD, n = 3; p values determined by Student t test (two-tailed, unpaired). ns, not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. (C) Schematic of TRAC KO ML CAR T cell cytotoxicity assay against tumor spheroids of TNBC cell line MDA-MB-231-Luc alone or co-cultured with TNBC-derived CAFs. (D) Bar graph representing percentage MDA-MB-231-Luc tumor cell survival post cytotoxicity assay outlined in (C), at Effector:Target ratio = 5:1. Bars show means ± SD, n = 3; p values determined by Student t test (two-tailed, unpaired). ns, not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01. (E) Immunohistochemistry for detection of human FAP protein in tissue microarray of patient samples from different cancers. (F) Heatmap depicting percentage positive area stained for human FAP protein in immunohistochemical analysis of healthy human tissue microarray.

Article Snippet: Human tumor tissue and healthy tissue microarray slides were obtained from Novus Biologicals (NBP2-30234, NBP2-78082, NBP2-30232, NBP2-30233, NBP2-30189).

Techniques: Flow Cytometry, Quantitation Assay, Two Tailed Test, Cytotoxicity Assay, Cell Culture, Derivative Assay, Immunohistochemistry, Microarray, Staining, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Extracellular matrix stiffness dictates Wnt expression through integrin pathway

doi: 10.1038/srep20395

Figure Lengend Snippet: Results from primary chondrocytes 48 hr after seeding on stiff (100 kPa) or soft (0.5–1 kPa) ECM. ( a ) Microarray profiling of Wnt/β-catenin pathway transcripts. Results are normalized by median scaling using Rosetta Resolver System software. ( b ) Wnt1 and Wnt3a levels were analyzed by western blotting. ( c ) Total and phosphorylated ERK1/2 levels were analyzed by western blotting. ( d ) Axin2, CD44, and ( e ) phosphorylated GSK3β levels were analyzed by western blotting. ( f ) Total and phosphorylated β−catenin levels were analyzed by western blotting. ( g ) β−catenin levels in nucleus and cytoplasm were analyzed by western blotting. ( h ) Total and ( i ) activated β-catenin levels and distribution in chondrocytes 2 hr after seeding on stiff or soft ECM were analyzed by in situ fluorescence staining. ( j ) β-catenin and wnt1 levels in chondrocytes 48 hr after seeding on the Matrigel-coated PAAM were analyzed by western blotting. ( k ) β-catenin and wnt1 levels in chondrocytes 48 hr after seeding on the ColII-coated PAAM were analyzed by western blotting. Western results were from 3 independent experiments for each individual protein, with blots exemplifying one experiment and the bar graphs showing the combined results of 3 experiments on stiff matrix expressed as percentages (mean ± SEM) of the corresponding results on the soft matrix. GAPDH was used to normalize for equal loading. *P < 0.05, **P < 0.01. n.s. stands for not statistically significant.

Article Snippet: Microarray analyses were performed using commercial Mouse cDNA Microarray slides (Phalanx Biotech Group; Hsinchu, Taiwan) according to the manufacturer’s instructions.

Techniques: Microarray, Software, Western Blot, In Situ, Fluorescence, Staining

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: BMC Plant Biology

Article Title: Light has a specific role in modulating Arabidopsis gene expression at low temperature

doi: 10.1186/1471-2229-8-13

Figure Lengend Snippet: Phosphothreonine-immunoblot of thylakoid proteins isolated from Arabidopsis leaves after four different treatments : Control (Ctr), Cold/Light (C/L), Dark (D) and Cold/Dark (C/D). Below the immunoblot, 77 K fluorescence emission ratios (F732/F685 ± S.D.) of thylakoids from differentially treated plants are given. F732 stands for the fluorescence peak at 732 nm representing the emission from PSI and F685 for the fluorescence peak at 685 nm from PSII. Differences in F732/F685 ratios are related to reversible phosphorylation of the light-harvesting chl a/b proteins (LHCII) and their attachment with PSI (phosphorylated, high ratio) and PSII (non-phosphorylated, low ratio). P-CP43, P-D2, P-D1 denote the phosphorylated proteins of PSII core, P-LHCII denote the LHCII phosphoproteins.

Article Snippet: Arabidopsis cDNA microarray slides are based on the GEM1 clone set (8000 ESTs) purchased from InCyte Genomics, Palo Alto, CA, USA [ ].

Techniques: Western Blot, Isolation, Control, Fluorescence, Phospho-proteomics

Journal: BMC Plant Biology

Article Title: Light has a specific role in modulating Arabidopsis gene expression at low temperature

doi: 10.1186/1471-2229-8-13

Figure Lengend Snippet: Verification of some microarray results using northern blot analysis after four different treatments: Control (Ctr), Cold/Light (C/L), Cold/Dark (C/D) and Dark (D) . Hybridizations were made with genes encoding: four photosystem II light harvesting proteins (LHCB) and the Early Light Inducible Protein (ELIP1); two photosystem I related (PSI) proteins, PSI-N and plastocyanin (PC); two proteins of carbohydrate metabolism, a plastidic fructose bisphoshate aldolase (Pl-FBA) and a pyruvate decarboxylase (PDC1); a ZEP protein involved in zeaxanthin and ABA biosynthesis; four chloroplast targeted proteins involved in oxygen radical scavenging and three cytoplasmic or peroxisomal catalases (CAT); a cold-responsive protein (LTI78/RD29A) and genes encoding a MYB-like (CCA1) and three AP2 transcription factors. The hybridization of the 16S rRNA probe to total RNA is shown in the bottom of the figure.

Article Snippet: Arabidopsis cDNA microarray slides are based on the GEM1 clone set (8000 ESTs) purchased from InCyte Genomics, Palo Alto, CA, USA [ ].

Techniques: Microarray, Northern Blot, Control, Hybridization

Journal: BMC Plant Biology

Article Title: Light has a specific role in modulating Arabidopsis gene expression at low temperature

doi: 10.1186/1471-2229-8-13

Figure Lengend Snippet: Accumulation of oxidative stress related compounds in Arabidopsis leaves after Control, Cold/Light and Cold/Dark treatments . A reddish-brown colour indicates production of oxidized DAB in leaves.

Article Snippet: Arabidopsis cDNA microarray slides are based on the GEM1 clone set (8000 ESTs) purchased from InCyte Genomics, Palo Alto, CA, USA [ ].

Techniques: Control

Journal: BMC Plant Biology

Article Title: Light has a specific role in modulating Arabidopsis gene expression at low temperature

doi: 10.1186/1471-2229-8-13

Figure Lengend Snippet: Western blot analysis of dehydrin proteins (A) and of LHCB and glutathione reductase proteins (B) . Protein samples isolated from Arabidopsis leaves under growth condition (Ctr), after the Cold/Light (C/L) treatment, subsequent recovery for one hour at normal growth conditions (re-1hL), after 8-hour Darkness (D) and after the Cold/Dark (C/D) treatment. A typical result is presented out of three independent western blot experiments.

Article Snippet: Arabidopsis cDNA microarray slides are based on the GEM1 clone set (8000 ESTs) purchased from InCyte Genomics, Palo Alto, CA, USA [ ].

Techniques: Western Blot, Isolation