Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-RRM2 Clone 1E1 , Bio-Rad , Cat# MCA3434Z.

Techniques: Transduction, Recombinant, Protease Inhibitor, SYBR Green Assay, Mutagenesis, Purification, Gel Extraction, Imaging, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Software, Expressing

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

Journal: The Journal of Biological Chemistry

Article Title: Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

doi: 10.1074/jbc.RA118.004397

Figure Lengend Snippet: Effect of RRM2 on HCV replication. Shown are HCV RNA copy numbers in R6FLR-N HCV replicon cells (A) and cells and supernatant of JFH/K4 cells (B) persistently infected with HCV and treated with RRM2 siRNA. Results are presented as means ± S.D. (vertical bars) of three independent experiments (n = 3). *, p < 0.05. C, immunoblot analyses of NS5B and RRM2 expression in R6FLR-N HCV replicons and JFH/K4 cells using specific antibodies against RRM2, NS5B, and β-actin. Cells were transfected with control (cont.) or RRM2 siRNA (5 and 10 nm). The densitometric NS5B/actin ratios are shown below the blots. Blots shown are representative of four independent experiments (n = 4).

Article Snippet: After 24 h, the cells were fixed with 1% paraformaldehyde in PBS (Ca 2+ /Mg 2+ -free) and reacted with anti-RRM2 and anti-NS5B antibodies (Virogen Co.).

Techniques: Infection, Western Blot, Expressing, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

doi: 10.1074/jbc.RA118.004397

Figure Lengend Snippet: Effect of RRM2 silencing on HCV protein expression. RRM2 or control siRNA was transfected into HuH-7 cells with pcDNA6 plasmids carrying the coding sequence of core, E1, E2, NS2, NS3, NS4B, NS5A, or NS5B. At 72 h post-transfection, cells were harvested and subjected to Western blot analysis using the originally established anti-core (515), anti-E1 (384), anti-E2 (544), anti-NS2, anti-NS4B (4B52), anti-NS5A (5A32), and anti-NS5B (5B14) monoclonal antibodies or rabbit anti-NS3 (R212) polyclonal antibodies (18). Protein levels were measured via an imager; the protein to actin ratio was calculated, and its ratio to mock-treated (non) cells is indicated. Blots shown are representative of three independent experiments (n = 3).

Article Snippet: After 24 h, the cells were fixed with 1% paraformaldehyde in PBS (Ca 2+ /Mg 2+ -free) and reacted with anti-RRM2 and anti-NS5B antibodies (Virogen Co.).

Techniques: Expressing, Control, Transfection, Sequencing, Western Blot, Bioprocessing

Journal: The Journal of Biological Chemistry

Article Title: Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

doi: 10.1074/jbc.RA118.004397

Figure Lengend Snippet: Interaction of RRM2 with HCV RNA polymerase NS5B. A–D, HCV-JFH-1–infected HuH-7 cells were transfected with pcDNA6-myc-His-RRM2 (RRM2) or pcDNA6-myc-His (vector) or mock-transfected (Non) and precipitated (IP) with anti-Myc or anti-NS5B antibodies. Proteins were analyzed via Western blotting (WB) with anti-RRM2 (A and D) or anti-NS5B (B and C) antibodies. *, IgG light chain. (E) Inputs of each cell lysate are shown. Blots shown are representative of three independent experiments (n = 3). F, JFH/K4 cells were labeled with anti-RRM2 and -NS5B antibodies, which were detected with Alexa Fluor 568– and Alexa Fluor 488–conjugated secondary antibodies, respectively. Labeled cells were observed via stimulated emission depletion microscopy, and co-localization (merged area) is indicated by the yellow signal (×2400, bar = 0.5 μm). Nuclei were stained with DAPI. G, enlarged view of the merged image (indicated by the white square in F). H, JFH/K4 cells and HuH-7 cells were treated with 0.1% FCS DMEM for 24 h and stained with anti-NS5B and anti-RRM2 antibodies, which were detected using Alexa Fluor 568– and Alexa Fluor 488–conjugated secondary antibodies, respectively. NS, reaction with normal rabbit sera and secondary antibodies conjugated with Alexa Fluor 488 or 568. Labeled cells were observed using a BZ-X700 fluorescence microscope (Keyence Co., Osaka, Japan). Nuclei were stained with DAPI. Bars, 50 μm. Yellow arrowheads, merged image of NS5B and up-regulated RRM2. Upon overexpression of RRM2, RRM2 expression correlated with NS5B expression in the merged image. All confocal images are representative of three independent experiments (n = 3).

Article Snippet: After 24 h, the cells were fixed with 1% paraformaldehyde in PBS (Ca 2+ /Mg 2+ -free) and reacted with anti-RRM2 and anti-NS5B antibodies (Virogen Co.).

Techniques: Infection, Transfection, Plasmid Preparation, Western Blot, Labeling, Microscopy, Staining, Fluorescence, Over Expression, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

doi: 10.1074/jbc.RA118.004397

Figure Lengend Snippet: RRM2 regulates RNA polymerase NS5B protein stability. A, quantification of NS5B mRNA levels relative to those of GAPDH in HuH-7 lenti-NS5B cells treated with RRM2 or control siRNA or mock-treated (Non) cells for 72 h. Data are shown as an average value of three independent experiments. Vertical bars, S.D. (n = 3). B, HuH-7 lenti-NS5B cells were transfected with RRM2 or control siRNA for 48 h and then treated with CHX (100 μg/ml) and puromycin (Puro; 50 μg/ml) for 6 h. Cells were harvested and analyzed via Western blotting using anti-NS5B, anti-RRM2, and anti-actin antibodies. C, lenti-NS5B–expressing HuH-7 cells were transfected with the pcDNA6-myc-His-RRM2 vector. An empty vector, pcDNA6-myc-His, was used as a negative control. At 48 h post-transfection, cells were treated with CHX (100 μg/ml) for the indicated times. The cells were harvested and analyzed via Western blotting for NS5B, RRM2, and β-actin as reported in B. Blots shown are representative of three independent experiments (n = 3).

Article Snippet: After 24 h, the cells were fixed with 1% paraformaldehyde in PBS (Ca 2+ /Mg 2+ -free) and reacted with anti-RRM2 and anti-NS5B antibodies (Virogen Co.).

Techniques: Control, Transfection, Western Blot, Expressing, Plasmid Preparation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

doi: 10.1074/jbc.RA118.004397

Figure Lengend Snippet: RRM2 may regulate RNA polymerase NS5B protein stability through the ubiquitin/proteasome-dependent protein degradation system. A, HuH-7 cells were transfected with pcDNA6-myc-His-NS5B either alone or with FLAG-hPLIC1 in the presence or absence of RRM2 siRNA. Six hours before harvest, cells were treated with the proteasome inhibitor MG132 (5 μm). Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody and probed with anti-NS5B (top left) or anti-FLAG antibodies (bottom right). Each cell lysate was immunoblotted (WB) with anti-NS5B (arrow), anti-FLAG, anti-RRM2, and anti-actin antibodies (right). B, levels of NS5B and hPLIC1 following RRM2 knockdown. JFH/K4 cells were stained with anti-NS5B and anti-hPLIC1 antibodies, which were detected using Alexa Fluor 488– and Alexa Fluor 568–conjugated secondary antibodies, respectively. Labeled cells were observed using a BZ-X700 fluorescence microscope (Keyence Co.). Nuclei were stained with DAPI. Bars, 50 μm. Representative confocal images from three independent experiments are shown (n = 3). C, ubiquitination assay and Western blotting of lysates from HuH-7 cells co-transfected with pcDNA6-myc-His-NS5B and nontreated, control, or RRM2 siRNA for 48 h. MG132 (10 μm) was added to cells for 6 h prior to harvesting. NS5B was immunoprecipitated with anti-Myc antibodies. Ubiquitinated NS5B was detected using rabbit anti-ubiquitin linkage-specific K48 antibodies. Cell lysates were subjected to Western blot analysis with anti-NS5B, anti-RRM2, and anti-actin antibodies. *, nonspecific reaction. Blots shown are representative of three independent experiments (n = 3).

Article Snippet: After 24 h, the cells were fixed with 1% paraformaldehyde in PBS (Ca 2+ /Mg 2+ -free) and reacted with anti-RRM2 and anti-NS5B antibodies (Virogen Co.).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Knockdown, Staining, Labeling, Fluorescence, Microscopy, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

doi: 10.1074/jbc.RA118.004397

Figure Lengend Snippet: Schematic illustration of the proposed role of RRM2 in HCV replication. i, RRM2 is induced by HCV infection in quiescent hepatocytes. ii, RRM2 is essential for NS5B stability. hPLIC1 promotes the ubiquitin/proteasome-mediated degradation of NS5B, which is blocked by interaction with RRM2. iii, silencing of RRM2 activates ubiquitination and decreases the stability of NS5B and its interaction with hPLIC1. On the other hand, when NS5B is not present, degradation of hPLIC1 was suppressed even without RRM2. This may be because no scaffold for the ubiquitination machinery is formed.

Article Snippet: After 24 h, the cells were fixed with 1% paraformaldehyde in PBS (Ca 2+ /Mg 2+ -free) and reacted with anti-RRM2 and anti-NS5B antibodies (Virogen Co.).

Techniques: Infection, Ubiquitin Proteomics