Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Schematic description of the filtering process for identification of therapeutically relevant target candidates. b) Analysis of RRM2 mRNA expression levels in 50 EwS primary tumors compared to 929 normal tissues samples from 71 tissue types. Data are shown as log2 fold increase normalized to expression values of normal tissues. The dotted line indicates the cut-off value of 2 for candidate selection. c) Analysis of overall survival time of 166 EwS patients stratified for candidate gene expression. P -values (–log10) were determined in Kaplan-Meier analyses using a Mantel-Haenszel test (Bonferroni-adjusted for multiple testing). The dotted line indicates a significance value of 1.3. d) Kaplan-Meier survival analysis of 166 EwS patients stratified by the 78th percentile RRM2 expression. P -value determined by log-rank test.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Expressing, Selection, Gene Expression

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Left: Heat map for gene expression which is positively or negatively correlated with RRM2 expression in 166 EwS. Right: Gene ontology (GO) enrichment analysis of RRM2 and its co-expressed genes derived from gene expression data sets of 166 EwS tumors. Pearson correlation coefficients between RRM2 and other genes were determined, of which those with |r Pearson | > 0.5 were further analyzed by GO enrichment analysis. b) Representative images of immunohistochemical RRM2 staining. Scale bar = 50 µm. c) Kaplan-Meier survival analysis of 122 EwS patients stratified by RRM2 protein expression (low IRS≤2, high IRS >2). P -values were determined by log-rank test. d) WGCNA of downregulated genes upon RRM2 silencing in A-673 and ES-7 cells harboring Dox-inducible shRRM2 constructs. NES, normalized enrichment score. e) Analysis of tumor growth of EwS cell lines A-673 and TC-71 harboring Dox-inducible shRRM2 constructs or non-targeting shRNA (shControl) xenografted in NSG mice. Once tumors were palpable, animals were randomized in Dox (+) or Dox (–) group. Tumor growth on time course and f) Tumor weight at the experimental endpoint. Arrows indicate treatment start. Values are normalized to shControl. Horizontal bars represent means and whiskers SEM. FC, fold change. P -values were calculated at the experimental endpoint with two-sided (tumor growth) or one-sided (tumor weight) Mann-Whitney test. g) Representative micrographs of xenografts immunohistochemically stained for RRM2, cleaved caspase-3 (CC3) or γH2A.X (scale bar=250 µm, 50 µm, 250 µm, respectively). h) quantification of positive cells for cleaved caspase-3 (CC3) (left) and γH2A.X (right). Values were normalized to shControl. Horizontal bars represent means and whiskers SEM. FC, fold change. P -values were calculated at the experimental endpoint using a two-sided Mann-Whitney test.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Gene Expression, Expressing, Derivative Assay, Immunohistochemical staining, Staining, Construct, shRNA, MANN-WHITNEY

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Analysis of proliferation assays upon shRNA-mediated RRM2 silencing in EwS cell lines. Upper: Cell proliferation over 120h upon RRM2 silencing in A-673. Viable cells upon RRM2 silencing (middle) and dead cells (lower) in EwS cell lines (A-673, ES7, TC-71) harboring Dox-inducible shRRM2 constructs or non-targeting shRNA (shControl). Values are normalized to shControl. Horizontal bars represent means and whiskers SEM. FC, fold change. Two-sided Mann-Whitney test. b) Analysis of clonogenic growth upon shRNA-mediated RRM2 silencing in EwS cell lines (A-673, ES7, TC-71) harboring Dox-inducible shRRM2 constructs or non-targeting shRNA (shControl). Horizontal bars represent means and whiskers SEM. Two-sided Mann-Whitney test at the experimental endpoint.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: shRNA, Construct, MANN-WHITNEY

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Integrative Gene Ontology (GO) enrichment analysis of gene expression microarray data generated in A-673 and ES7 cells after RRM2 silencing or pharmacological RRM2 inhibition by triapine (corresponding IC50 of 0.44 µM or 0.65 µM, respectively). b) Correlation of gene expression between RRM2 and CHEK1 or WEE1 in 166 EwS. Each dot represents an individual expression value. Solid red lines indicate a trend line created by a simple linear regression. P -values were calculated by a two-tailed t-test. c) Drug interaction and combination efficiency analysis between triapine and CHEK1 inhibitor (CCT245737) or WEE1 inhibitor (MK-1775) in 4 EwS cell lines (A-673, ES7, EW-7, TC-71) assessed by combination index. CI value < 1 indicative of synergistic, CI = 1 additive, and CI > 1 antagonistic d) Drug interaction and combination efficiency estimation between triapine and CHEK1 inhibitor (CCT245737) or WEE1 inhibitor (MK-1775) in A-673 EwS cell line assessed by SynergyFinder 2.0. ZIP synergy score > 10, likely to be synergistic; between -10 and 10, likely to be additive; < –10, likely to be antagonistic.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Gene Expression, Microarray, Generated, Inhibition, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Distribution analysis of RRM2 mRNA expression in 166 EwS patients. Each dot represents individual RRM2 expression. b) Correlation analysis of CNVs at the RRM2 locus with RRM2 mRNA expression levels in primary EwS tumors (n=32). The solid line indicates a trend line estimated by a simple linear regression model. b) Correlation analysis of promoter methylation on five CpG sites with RRM2 expression levels in primary EwS tumors (n=40). The solid lines indicate trend lines estimated by a simple linear regression model.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Expressing, Methylation

Journal: Heliyon

Article Title: Tanshinone IIA attenuates fluoride-induced spinal cord injury by inhibiting ferroptosis and inflammation

doi: 10.1016/j.heliyon.2024.e40549

Figure Lengend Snippet: Primers used in qRT-PCR study.

Article Snippet: The membranes were then incubated overnight at 4 °C with primary antibodies against CXCL2 (26791-1-AP, Proteintech), PCK2 (67676-1-lg, abcam), RRM2 (DF7248, Affinity), SLC7A11 (BM5318, BOSTER), or β-actin (BA2305, BOSTER).

Techniques:

Journal: Heliyon

Article Title: Tanshinone IIA attenuates fluoride-induced spinal cord injury by inhibiting ferroptosis and inflammation

doi: 10.1016/j.heliyon.2024.e40549

Figure Lengend Snippet: Tanshinone IIA improved spinal cord injury by inhibiting ferroptosis (A) Photomicrographs of spinal cord sections from Tanshinone IIA control, fluoride-exposed rats, and Tanshinone IIA + high fluoride group visualized by transmission electron microscopy (5000X, white box). Fibrous myelin integrity (indicated by red arrows) is maintained in both Tanshinone IIA control and Tanshinone IIA-treated groups, while in the fluorosis group, it appears loosely arranged with an increased gap and disordered structure. (B) Scatter plot exhibited the expression of 15 ferroptosis-related genes in Tan IIA control, high fluoride group, and Tan IIA + high fluoride group. (C–D) Western blots showed that Tan IIA treatment restored the reduced protein expression of SLC7A11 and RRM2 observed in the NaF group. Additionally, Tan IIA treatment decreased the elevated protein levels of CXCL2 and PCK2 in the NaF group. Uncropped and unadjusted original images of the Western blots are provided in . Data are represented as the mean ± SEM (n = 6). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: The membranes were then incubated overnight at 4 °C with primary antibodies against CXCL2 (26791-1-AP, Proteintech), PCK2 (67676-1-lg, abcam), RRM2 (DF7248, Affinity), SLC7A11 (BM5318, BOSTER), or β-actin (BA2305, BOSTER).

Techniques: Control, Transmission Assay, Electron Microscopy, Expressing, Western Blot