Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-29b Mediates Lung Mesenchymal-Epithelial Transition and Prevents Lung Fibrosis in the Silicosis Model

doi: 10.1016/j.omtn.2018.10.017

Figure Lengend Snippet: Silica Induced EMT and Alterations of miRNA Expression in A549 Cells (A) Cytotoxicity of silica in A549 cells. The cells were treated with different concentrations of silica (25, 50, and 100 μg/mL). Cell viability was defined as 100% when cells were cultured with DMEM only. (B) Western blot analyses revealed that the protein expression of vimentin and Snail was upregulated gradually along with increased levels of silica in A549 cells. (C) Immunofluorescence staining for E-cadherin (red) and vimentin (green) confirmed that silica induced EMT in A549 cells. (D) The wound-healing assay showed that silica induced migration of A549 cells. (E) miRNA microarray showed that the expressions of miR-200c, miR-149, and miR-29b were significantly downregulated in A549 cells treated with 50 μg/mL silica. (F) The results of qPCR confirmed that the level of miR-29b was significantly decreased in A549 cells. All results were replicated at least three times independently. The data are presented as means ± SD. * p < 0.05 and ** p < 0.01 versus the control group.

Article Snippet: After being washed by PBS, it was fixed with 0.1% Triton X-100 for 20 min. After being rinsed in PBS and blocked with 3% BSA for 10 min, slides were incubated with primary anti-bodies against E-cadherin (1:100; Abcam, CA, USA) or vimentin (1:100; Abcam, CA, USA) overnight at 4°C.

Techniques: Expressing, Cell Culture, Western Blot, Immunofluorescence, Staining, Wound Healing Assay, Migration, Microarray

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-29b Mediates Lung Mesenchymal-Epithelial Transition and Prevents Lung Fibrosis in the Silicosis Model

doi: 10.1016/j.omtn.2018.10.017

Figure Lengend Snippet: Silica (50 μg/mL) Induced EMT and the Downregulation of miR-29b in RLE-6TN Cells (A) The RLE-6TN cells showed a typical epithelial cuboidal shape in the control group and spindle-shaped morphology in the silica group. (B) Western blot analyses showed that, in the silica group, the protein expression of E-cadherin was downregulated and vimentin expression was upregulated. (C) The results of qPCR revealed that silica obviously decreased the level of CDH1 and increased the levels of VIM and α-SMA. (D) The results of qPCR verified that the level of miR-29b was decreased in the silica group. All results were replicated at least three times independently. The data are presented as means ± SD. * p < 0.05 and ** p < 0.01 versus the control group.

Article Snippet: After being washed by PBS, it was fixed with 0.1% Triton X-100 for 20 min. After being rinsed in PBS and blocked with 3% BSA for 10 min, slides were incubated with primary anti-bodies against E-cadherin (1:100; Abcam, CA, USA) or vimentin (1:100; Abcam, CA, USA) overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-29b Mediates Lung Mesenchymal-Epithelial Transition and Prevents Lung Fibrosis in the Silicosis Model

doi: 10.1016/j.omtn.2018.10.017

Figure Lengend Snippet: miR-29b Mimics Promoted MET and Reversed EMT in RLE-6TN Cells After being incubated with silica (TGF-β1) for 24 hr, the RLE-6TN cells were incubated with silica (TGF-β1) in addition to miR-29b mimics or mimic negative control (NC) for an additional 24 hr. (A) The transfection efficiency was greater than 80% of the RLE-6TN cells transfected with Cy3-labeled miR-29b mimics, and qPCR analysis revealed that miR-29b mimics markedly increased the level of miR-29b in RLE-6TN cells. (B) qPCR analysis showed that miR-29b mimics increased the level of CDH1 and decreased the levels of VIM and α-SMA. (C) The results of qPCR suggested that miR-29b mimics did not alter the level of snai1. (D) Western blot analysis revealed that miR-29b mimics increased the ratio of E-cadherin:vimentin significantly in RLE-6TN cells. (E) TGF-β1 induced the downregulation of CDH1 expression and upregulation of VIM and α-SMA in RLE-6TN cells. (F) TGF-β1 induced the downregulation of miR-29b. (G) qPCR analysis showed that miR-29b mimics decreased the levels of VIM, α-SMA, COL1A1, and Tgfb1 compared with the TGF-β1 + mimic NC group. (H) Western blot analysis confirmed that miR-29b mimics increased the ratio of E-cadherin:vimentin significantly in the classical EMT model. All results were replicated at least three times independently. The data are presented as means ± SD. * p < 0.05 and ** p < 0.01 versus the control group; # p < 0.05 and ## p < 0.01 compared with the silica or TGF-β1 + mimic NC group.

Article Snippet: After being washed by PBS, it was fixed with 0.1% Triton X-100 for 20 min. After being rinsed in PBS and blocked with 3% BSA for 10 min, slides were incubated with primary anti-bodies against E-cadherin (1:100; Abcam, CA, USA) or vimentin (1:100; Abcam, CA, USA) overnight at 4°C.

Techniques: Incubation, Negative Control, Transfection, Labeling, Western Blot, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-29b Mediates Lung Mesenchymal-Epithelial Transition and Prevents Lung Fibrosis in the Silicosis Model

doi: 10.1016/j.omtn.2018.10.017

Figure Lengend Snippet: miR-29b Inhibitor Enhanced the Development of EMT in RLE-6TN Cells After silica (TGF-β1) treatment for 24 hr, the RLE-6TN cells were incubated with silica (TGF-β1) plus miR-29b inhibitor or inhibitor NC for another 24 hr. (A) The transfection efficiency was greater than 80% of the RLE-6TN cells transfected with Cy3-labeled miR-29b inhibitor, and qPCR analysis revealed that miR-29b inhibitor markedly decreased the level of miR-29b in RLE-6TN cells. (B) qPCR analysis showed that miR-29b inhibitor increased the level of COL1A1 compared with the silica + inhibitor NC group but did not alter the levels of CDH1 and VIM. (C) qPCR for snai1 revealed similar results as with COL1A1. (D) miR-29b inhibitor decreased the ratio of E-cadherin:vimentin. (E) The results of qPCR showed that miR-29b inhibitor downregulated the expression of CDH1 but upregulated the expression of VIM, α-SMA, and COL1A1 in the TGF-β1 EMT model. (F) miR-29b inhibitor also decreased the ratio of E-cadherin:vimentin in the TGF-β1 EMT model. All results were replicated at least three times independently. The data are presented as means ± SD. * p < 0.05 and ** p < 0.01 versus the control group; # p < 0.05 and ## p < 0.01 compared with the silica or TGF-β1 inhibitor NC group.

Article Snippet: After being washed by PBS, it was fixed with 0.1% Triton X-100 for 20 min. After being rinsed in PBS and blocked with 3% BSA for 10 min, slides were incubated with primary anti-bodies against E-cadherin (1:100; Abcam, CA, USA) or vimentin (1:100; Abcam, CA, USA) overnight at 4°C.

Techniques: Incubation, Transfection, Labeling, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-29b Mediates Lung Mesenchymal-Epithelial Transition and Prevents Lung Fibrosis in the Silicosis Model

doi: 10.1016/j.omtn.2018.10.017

Figure Lengend Snippet: Agomir-29b Inhibited EMT and Decreased the Levels of ECM Genes in a Mouse Model of Silicosis Fibrosis (A) The results of qPCR showed that agomir-29b upregulated the expression of CDH1 but downregulated the expression of VIM, α-SMA, and ECM genes (COL1A1 and FN). (B) Agomir-29b had no effect on the level of snai1. (C) Immunofluorescence staining suggested that the expression of vimentin was significantly increased following silica instillation but was decreased by agomir-29b treatment in alveolar epithelial cells (arrows), while the change of E-cadherin expression was not evident. (D) Amplified fluorescence images in the lungs of the mice. The data are presented as means ± SD (n = 6). * p < 0.05 and ** p < 0.01 compared with the control group; # p < 0.05 and ## p < 0.01 compared with the silica + NC group.

Article Snippet: After being washed by PBS, it was fixed with 0.1% Triton X-100 for 20 min. After being rinsed in PBS and blocked with 3% BSA for 10 min, slides were incubated with primary anti-bodies against E-cadherin (1:100; Abcam, CA, USA) or vimentin (1:100; Abcam, CA, USA) overnight at 4°C.

Techniques: Expressing, Immunofluorescence, Staining, Amplification, Fluorescence

Journal: Experimental Biology and Medicine

Article Title: Annona atemoya leaf extract ameliorates cognitive impairment in amyloid-β injected Alzheimer’s disease-like mouse model

doi: 10.1177/1535370219886269

Figure Lengend Snippet: Molecular mechanisms responsible for anti-AD effects of AAL extract. (a) HT22 cells were exposed to H2O2 in the absence or presence of AAL extract (50 μg/mL) for 6 h. Antibody microarray assay was performed using the phospho-specific antibody microarray slide (Full Moon BioSystems). For data acquisition, GenePix 4100A scanner (Axon Instrument, USA) was used. The normalization data were analyzed using Genowiz 4.0™ (Ocimum Biosolutions). The phosphorylation ratio was calculated and represented as fold changes of indicated phosphoproteins after H2O2 treatment normalized to total protein expression (upper panel). Total protein quantification is shown (lower panel). (b) HT22 cells were exposed to H2O2 with or without various concentrations of AAL extract (0, 12.5, 25, or 50 μg/mL) for 6 h. Cell lysates were prepared from HT22 cells and equal amounts of protein were subjected to Western blotting using anti-phospho-CaMK2 β/ν/δ, GRK2, EGFR, Myc, FER, caveolin-1, NFκB p65, and MLRN 2 antibodies to validate the Ab microarray. (c) Protein extracts were prepared from hippocampal tissues in an Aβ-induced AD mouse model. Vehicle or various concentrations of AAL extract (50, 100, or 200 mg/kg) were administered to Aβ mice for 23 days. Western blotting was performed for anti-phospho-EGFR and phospho-GRK2. The validity of the two phospho-antibodies was determined using pre-stained protein marker (Bio-Rad). GAPDH was used as an internal control. Shown blots are representative results from three independent experiments.

Article Snippet: Molecular mechanisms responsible for anti-AD effects of AAL extract. (a) HT22 cells were exposed to H 2 O 2 in the absence or presence of AAL extract (50 μg/mL) for 6 h. Antibody microarray assay was performed using the phospho-specific antibody microarray slide (Full Moon BioSystems).

Techniques: Microarray, Phospho-proteomics, Expressing, Western Blot, Staining, Marker, Control

Journal: Cancer Science

Article Title: Antibody‐based proteomics for esophageal cancer: Identification of proteins in the nuclear factor‐κB pathway and mitotic checkpoint

doi: 10.1111/j.1349-7006.2009.01230.x

Figure Lengend Snippet: List of the proteins found to be overexpressed in the microarray analysis and examined by western blotting and immunohistochemistry

Article Snippet: Labeled protein extracts were incubated on Panorama Ab Microarray slides (Sigma‐Aldrich) for 30 min with moderate shaking.

Techniques: Microarray, Western Blot, Immunohistochemistry

Journal: Cancer Science

Article Title: Antibody‐based proteomics for esophageal cancer: Identification of proteins in the nuclear factor‐κB pathway and mitotic checkpoint

doi: 10.1111/j.1349-7006.2009.01230.x

Figure Lengend Snippet: Scheme showing the experimental flow: the antibody microarray analysis results were validated using western blotting and immunohistochemistry. N, normal; T, tumor.

Article Snippet: Labeled protein extracts were incubated on Panorama Ab Microarray slides (Sigma‐Aldrich) for 30 min with moderate shaking.

Techniques: Microarray, Western Blot, Immunohistochemistry

Journal: Cancer Science

Article Title: Antibody‐based proteomics for esophageal cancer: Identification of proteins in the nuclear factor‐κB pathway and mitotic checkpoint

doi: 10.1111/j.1349-7006.2009.01230.x

Figure Lengend Snippet: Reproducibility of the antibody microarray analysis. The scatter plot shows that the intensity values of 63.1–94.1% of the antibodies contained in the microarrays used were scattered within a twofold difference, and that the correlation coefficient (R) was 0.7172–0.9332. X‐axis, intensity of one spot of duplicate spots; Y‐axis, intensity of other spot of duplicate spots.

Article Snippet: Labeled protein extracts were incubated on Panorama Ab Microarray slides (Sigma‐Aldrich) for 30 min with moderate shaking.

Techniques: Microarray

Journal: Cancer Science

Article Title: Antibody‐based proteomics for esophageal cancer: Identification of proteins in the nuclear factor‐κB pathway and mitotic checkpoint

doi: 10.1111/j.1349-7006.2009.01230.x

Figure Lengend Snippet: Western blotting results. (a) Fourteen of the 24 proteins identified in the microarray analysis as being overexpressed in tumor tissues showed concordant results in western blotting analysis. (b) Five proteins showed discordant results.

Article Snippet: Labeled protein extracts were incubated on Panorama Ab Microarray slides (Sigma‐Aldrich) for 30 min with moderate shaking.

Techniques: Western Blot, Microarray

Journal: Cancer Science

Article Title: Antibody‐based proteomics for esophageal cancer: Identification of proteins in the nuclear factor‐κB pathway and mitotic checkpoint

doi: 10.1111/j.1349-7006.2009.01230.x

Figure Lengend Snippet: The scattergram demonstrates the correlation between the intensity of duplicate antibody signals of the 24 proteins found to be overexpressed in the microarray analysis. The intensity values of the proteins found to be overexpressed in tumor tissues in the microarrays showed high reproducibility, irrespective of whether these microarray results were discordant (R = 0.7666) or concordant (R = 0.6921) with western blotting results, or whether these proteins were not detected by western blotting (R = 0.6317). X‐axis, intensity of one spot of duplicate spots; Y‐axis, intensity of other spot of duplicate spots.

Article Snippet: Labeled protein extracts were incubated on Panorama Ab Microarray slides (Sigma‐Aldrich) for 30 min with moderate shaking.

Techniques: Microarray, Western Blot

Journal: Cancer Science

Article Title: Antibody‐based proteomics for esophageal cancer: Identification of proteins in the nuclear factor‐κB pathway and mitotic checkpoint

doi: 10.1111/j.1349-7006.2009.01230.x

Figure Lengend Snippet: Comparison of the microarray with the western blotting data. The correlation coefficient of the 14 proteins found to be overexpressed in tumor tissues in western blotting was calculated. Normalization of each tumor tissue's data was carried out using the normal counterpart's data.

Article Snippet: Labeled protein extracts were incubated on Panorama Ab Microarray slides (Sigma‐Aldrich) for 30 min with moderate shaking.

Techniques: Comparison, Microarray, Western Blot

Journal: Cancer Science

Article Title: Antibody‐based proteomics for esophageal cancer: Identification of proteins in the nuclear factor‐κB pathway and mitotic checkpoint

doi: 10.1111/j.1349-7006.2009.01230.x

Figure Lengend Snippet: Tissue microarray results

Article Snippet: Labeled protein extracts were incubated on Panorama Ab Microarray slides (Sigma‐Aldrich) for 30 min with moderate shaking.

Techniques: Microarray

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor signaling regulates oligodendrocyte differentiation. a Mean number of oligodendrocyte lineage cells (Olig2+) per field ± s.e.m. in corpus callosum of P16 Acvr1b fl/fl ( n = 3 mice), PDGFRa-Cre; Acvr1b fl/+ ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 7 mice). b Mean proportion of oligodendrocyte lineage cells (Olig2+) which are mature oligodendrocytes (CC1+) versus immature cells (CC1−), per field ± s.e.m. in corpus callosum of P16 Acvr1b fl/fl ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 7 mice). Multiple t tests with false discovery rate of 1%, *** P = 0.000026. c Images of differentiating oligodendrocytes (cytoplasmic Olig1+ and nuclear Olig2+) in corpus callosum of P16 Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice. Scale bar 25 μm. d Mean number of cytoplasmic Olig1 and Olig2 double positive cells per field ± s.e.m. in corpus callosum of P16 Acvr1b fl/fl ( n = 3 mice), PDGFRa-Cre; Acvr1b fl/+ ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 7 mice). Two-tailed Student’s t test, ** P = 0.0047, 0.0026, respectively. e Images of maturing oligodendrocytes (MAG+ MBP−) at P1 in corpus callosum of Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice. Scale bar 25 μm. f Mean number of MAG+ cultured oligodendrocytes per field in vehicle control-treated or activin-A-treated conditions (10 ng ml −1 ) in vitro. n = 3 biological replicates. Two-tailed paired Student’s t test, * P = 0.0484. g Images of cultured OPCs treated with vehicle or 10 ng ml −1 activin-A and immunostained for MAG (green), counterstained with Hoechst (blue). Scale bar 25 μm. h Data-mining of microarray of human fetal brain at 9 and 12 gestational weeks (gw) represented as fold change in expression (normalized to 9 gw), showing paralleled expression changes between activin-A ( INHBA ) and oligodendrocyte differentiation-associated genes ( MAG , MOG ) in development

Article Snippet: Samples were applied to TGF-β phospho-antibody microarray slides (Full Moon Biosystems) which have 176 immobilized antibodies against phosphorylated and unphosphorylated specific residues in proteins associated with the 5 TGFβ signaling pathways, with 6 technical replicates per antibody.

Techniques: Two Tailed Test, Cell Culture, Control, In Vitro, Microarray, Expressing

Journal: eLife

Article Title: Mucosal effects of tenofovir 1% gel

doi: 10.7554/eLife.04525

Figure Lengend Snippet: ( A and B ) Quantification of mRNA copy numbers measured in 9 cm biopsies by reverse transcription (RT) droplet digital PCR (ddPCR) relative to the housekeeping gene hemoglobin beta (HBB) copy numbers in seven additional study participants. ( A ) Nine selected genes induced in the microarrays: CCL19, CCL21, CCL23, CXCL9, CCR7, CD7, CD19, matrix metallopeptidase 12 (MMP12), and serine peptidase inhibitor of the Kazal type 4 (SPINK4). Copy numbers at baseline (0), after a single tenofovir gel application (I) and after seven consecutive once-daily applications (VII) are shown. Line colors signify each of the seven study participants. ( B ) Six selected suppressed genes: p21-activated kinase (PAK2), nuclear factor of activated T cells 5 (NFAT5), desmoplakin (DSP), TGF-β receptor associated protein (TGFBRAP), interleukin 10 (IL-10), and tripartite motif-containing protein 5 (TRIM5). ( C ) Normalized fold changes of gene expression at Day VII over baseline in all 15 individuals treated with tenofovir 1% gel. Red dots depict fold changes measured by microarray, blue dots depict fold changes measured by RT-ddPCR. The boxes indicate median and 25th–75th percentiles and the whiskers indicate 10th–90th percentile. Asterisks indicate statistical significance level relative to baseline (one asterisk p < 0.05; two asterisks p < 0.01, by one-sided Wilcoxon tests, adjusted for multiple testing). ( D ) Immunostaining of formalin-fixed 9 cm rectal biopsies from 10 participants for the proteins CD7 (immunohistochemistry [IHC]), CD3 (immunofluorescence) and ubiquitin D (UBD; IHC), predicted to be induced by the microarrays, and for IL-10 (IHC), predicted to be suppressed. For CD7 and CD3, tissue sections were evaluated in their entirety and positive cells per mm 2 are shown at baseline (0) and after seven consecutive once-daily applications (VII). Representative images are shown in . For UBD and IL-10, only columnar epithelial cells were evaluated. For UBD, the average mean staining intensities (MSI) per cell are shown. Representative images are shown in . Colors signify each of the 10 study participants. The boxes indicate median and 25th–75th percentiles and the whiskers indicate the range. Paired Wilcoxon signed-rank test p values for differences between 0 and VII are listed. DOI: http://dx.doi.org/10.7554/eLife.04525.006

Article Snippet: For CD3 staining, the slides were incubated with anti-CD3 for 60 min at a dilution of 1:25, washed in Wash Buffer, incubated for 30 min with sheep-anti-rabbit Dylight 649 (611-643-122; Rockland Immunochemicals, Limerick, PA), washed, and incubated for 30 min with donkey-anti-sheep Dylight 649 (613-743-168; Rockland).

Techniques: Digital PCR, Expressing, Microarray, Immunostaining, Immunohistochemistry, Immunofluorescence, Staining

Journal: eLife

Article Title: Mucosal effects of tenofovir 1% gel

doi: 10.7554/eLife.04525

Figure Lengend Snippet: Individual study participants are designated by letters. Blue indicates nuclei, red indicates CD3 or CD7. DOI: http://dx.doi.org/10.7554/eLife.04525.007

Article Snippet: For CD3 staining, the slides were incubated with anti-CD3 for 60 min at a dilution of 1:25, washed in Wash Buffer, incubated for 30 min with sheep-anti-rabbit Dylight 649 (611-643-122; Rockland Immunochemicals, Limerick, PA), washed, and incubated for 30 min with donkey-anti-sheep Dylight 649 (613-743-168; Rockland).

Techniques:

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