Journal: Cells

Article Title: Systematic Surveys of Iron Homeostasis Mechanisms Reveal Ferritin Superfamily and Nucleotide Surveillance Regulation to be Modified by PINK1 Absence

doi: 10.3390/cells9102229

Figure Lengend Snippet: Quantitative immunoblots for ( A ) FTH1, ( B ) HMOX1, ( C ) Ribonucleotide reductase regulatory subunit M2 (RRM2), and ( D ) nuclear receptor coactivator 4 (NCOA4) in WT and Pink1 −/− MEF, under untreated control conditions (Ctrl), after iron overload (FAC) and after two different iron depletion drugs (DFO, 22BP), administered over 48 h. Protein abundance signals were normalized to beta-Actin levels (Actin beta (ACTB)) as a loading control. The panels on the right show their densitometric quantifications, normalized to WT untreated conditions. WT n = 4–6, Pink1 −/− n = 3, one exemplary set is shown. The statistical trends or levels of significance are illustrated by symbols, namely T: 0.1 > p >0.05, *: p < 0.05, **: p < 0.01, ###: p < 0.001, ****: p < 0.0001. Mutant cells are represented by dashed bars, WT cells by plain colors. Asterisks represent significance in WT MEF, treated versus untreated control, while hashtags refer to Pink1 −/− MEF, treated versus untreated control. Genotype-dependent significant differences of Pink1 −/− versus WT MEF are illustrated by horizontal lines below asterisks.

Article Snippet: For quantification of the individual mRNA levels, the following TaqMan assays (Thermo Scientific) were employed for mouse: Abcb6 - Mm00470049_m1, Abcb7- Mm01235258_m1, Abcb8 - Mm00472410_m1, Abcb10 - Mm00497931_m1, Abce1 - Mm00649858_m1, Aco1- Mm00801417_m1, Aco2 - Mm00475673_g1, Alas1- Mm01235914_m1, Bach1- Mm01344527_m1, Bdh2- Mm00459075_m1, Bnip3- Mm00833810_g1, Bola1- Mm01255885_m1, Brip1 - Mm01297848_m1, Cdc42bpa - Mm01322796_m1, Cisd1 - Mm00728581_s1, Cisd2 - Mm00835272_m1, Cp - Mm00432654_m1, Ctsb - Mm01310605_m1, Ctsd - Mm00515586_m1, Ctsf - Mm00490782_m1, Cygb -Mm00446071_m1, Cyp46a1 -Mm00487306_m1, Dna2 - Mm01169107_m1, Dpyd- Mm00468109_m1, Egln1 - Mm00459770_m1, Elp3- Mm00804536_m1, Ercc2 - Mm00514776_m1, Fdx1 - Mm00433246_m1, Fech - Mm00500394_m1, Flvcr1 - Mm01320423_m1, Foxo3 - Mm01185722_m1, Fth1- Mm00850707_m1, Ftl1- Mm03030144_g1, Fxn - Mm00784016_s1, Gabarapl1 - Mm00457880_m1, Glrx5- Mm00511712_m1, Hebp1 - Mm00469161_m1, Hif1a - Mm00468869_m1, Hk1 - Mm00439344_m1, Homer1 - Mm00516275_m1, Hmox1- Mm00516005_m1, Ireb2 - Mm01179595_m1, Jmjd6 - Mm00466679_m1, Jund - Mm04208316_m1, Keap1 - Mm00497268_m1, Mef2d- Mm00504931_m1, Mitf - Mm00434954_m1, Mmp14 - Mm00485054_m1, Myl6 - Mm02342525_g1, Ncoa4 - Mm00451095_m1, Nfe2l2 - Mm00477784_mL, Nfu1- Mm00777068_m1, Nos2- Mm_00440502_m1, Nqo1 - Mm01253561_m1, Nthl1- Mm00476559_m1, P4ha2 - Mm01288628_m1, Prkn- Mm00450186_m1, Pcbp1 - Mm00478712_s1, Pcbp2 - Mm01296174_g1, Pcbp3 - Mm01149750_m1, Pgrmc1 - Mm00443985_m1, Pink1- Mm00550827_m1, Pold1 - Mm00448253_m1, Ppat - Mm00549096_m1, Prdx1 - Mm012619961_s1, Prim2 - Mm00477104_m1, Rbfox2 - Mm01197021_m1, Rsad1 - Mm01296523_m1, Rsad2 - Mm00491265_m1, Rrm2 - Mm00485881_m1, Rtel1 - Mm01220420_m1, Slc11a2- Mm00435363_m1, Slc25a28- Mm00455077_m1, Slc25a37 - Mm00471133_m1, Slc40a1 - Mm01254822_m1, Sqstm1 - Mm00448091_m1, Steap2 - Mm01320129_m1, Steap3 - Mm01287243_m1, Tbp - Mm00446973_m1, Tfeb - Mm00448968_m1, Tfrc- Mm00441941_m1, Trf- Mm00446715_m1, Tyw5- Mm01254171_m1.

Techniques: Western Blot, Control, Quantitative Proteomics, Mutagenesis

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 1. Pivotal role of Glu246 and Asp247 in the conformation of the RRM2 domain of TDP-43. A. a, Scheme showing the alignment of RRM2 subdomains (residues 232–270 in human TDP-43) in multiple species. Glu246 (E246) and Asp247 (D247) are preserved across all species. b, Chemical properties of amino acid substitution mutants of E246 or D247. E246Q and D247N were used to create substitution mutants with minimal alteration of side chains. E246G and D247G were designed so that the effects of the side chains were eliminated, and the flexibility of the amide bonds was increased. B. Western blot analysis of recombinant RRM2 proteins of wild-type (WT), E246Q/D247N (QN), and E246G/D247G (GG), using an anti-TDP-43 rabbit polyclonal antibody that recognizes the RRM2 domain (Proteintech). The E246G/D247G mutant RRM2 showed marked oligomerization even when incubated at 4uC (vertical line). Note that considerable RRM2 dimers or oligomers were dissociated into monomers in the presence of DTT. Arrowhead and double arrowheads indicate RRM2 monomer and dimers, respectively. Asterisk possibly indicates heat-related high molecular complexes comprising RRM2 domain. C. Thioflavin T (ThT) fluorescence assay showing amyloid fibril formation of RRM2 with mutations in E246 and D247. Each value indicates averaged RFU of ThT with standard error of mean from triplicates. *p,0.01 vs. RRM2 WT at RT by one-way ANOVA with Newman-Keuls test. D. Superdex 75 size exclusion chromatography of recombinant RRM2 domain of WT (a), E246Q/D247N (QN, b), and E246G/D247G (GG, c). a, WT RRM2 alone was exclusively monomeric in its native condition (unfilled circle). Heat denaturation at 70uC for 10 min induced higher molecular assembly (filled circle). Arrowhead indicates the RRM2 oligomer. b, E246Q/D247N (QN) mutant RRM2 without stress was predominantly monomeric (unfilled circle). Heat denaturation markedly increased the ratio of oligomers to monomers (filled circle). c, E246G/D247G (GG) mutant existed as a mixture of monomer and oligomers at the baseline condition (unfilled circle). Higher molecular species were prominently

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Western Blot, Recombinant, Mutagenesis, Incubation, Fluorescence, Size-exclusion Chromatography

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 2. D247 predominantly governs the conformation of RRM2. A. Coomassie staining of recombinant RRM2 protein with either single or double mutation(s) at E246 and D247 incubated for 24 hr at 4uC by denaturing SDS-PAGE. The effects of oligomerization were greater with the single mutant at D247 than at E246. Oligomerization is most prominent in the double E246G/D247G mutant. B. Size exclusion chromatography of RRM2 proteins of E246G and D247G incubated at 4uC (a) and 70uC for 10 min with 24 hr post incubation at 4uC. (b). The effect of oligomerization is more pronounced in the D247G mutant RRM2 than in the E246G in both the 4uC and 70uC conditions. Molecular size markers are as follows: bovine serum albumin (66 kDa), ovalbumin (43 kDa), superoxide dismutase 1 (32 kDa), myoglobin (17.6 kDa), and aprotinin (6.5 kDa). doi:10.1371/journal.pone.0052776.g002

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Staining, Recombinant, Mutagenesis, Incubation, SDS Page, Size-exclusion Chromatography

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 3. Substitution mutants of full-length TDP-43 at E246 and D247 are readily misfolded. A. Confocal micrographs of HEK293A cells overexpressing EGFP-fused full-length TDP-43 (a, b, wild-type (WT), c, d, E246G, e, f, D247G, g, h, E246G/D247G). i, j, Percentages of transfected HEK293A cells harboring multiple puncta or inclusions (i, arrowheads) or displaying nucleus-excluded TDP-43 (unfilled arrowhead). (j). Scale bar indicates 30 mm. Data were expressed as the mean 6 SEM (N = 7–10). *p,0.05 vs. WT, #p,0.05 vs. E246G by one-way ANOVA with Newman-Keuls test. NS indicates not significant vs. WT. B. a, Western blotting showing the increased detergent-insolubility of TDP-43 with mutations at E246/D247, with defective nucleus localizing signal (mNLS), or devoid of RRM2 domain (DRRM2). Lysates from HEK293A cells transiently transfected with TDP-43- EGFP were separated into 1% TritonX100-soluble or -insoluble fractions. Top panel, anti-GFP; middle panel, anti-actin; bottom panel, anti-GAPDH. The GAPDH blot validates the successful separation between detergent-soluble and -insoluble components. b, Quantified insolubility of TDP-43-EGFP proteins with or without mutation at E246/D247 to glycine. Relative TDP-43-EGFP in the detergent-soluble or -insoluble fraction was obtained from the ratio of the GFP density to actin density from the densitometric value in each fraction (designated as insoluble TDP or soluble TDP, respectively). Insolubility index was obtained from the ratio of insoluble TDP to soluble TDP, and each value was standardized by the average ratio of WT. Data were expressed as the mean 6 SEM of four experiments.*p,0.05 vs. WT TDP-43-EGFP by one-way ANOVA with Newman-Keuls test. C. Size exclusion chromatography and Western blotting indicating the existence of oligomeric and monomeric states of full-length TDP-43 in cells. WT and E246G/ D247G (GG) TDP-43-FLAG genes were expressed in HEK293A cells. Cells were sonicated in PBS, and the supernatants were fractionated by a Superose 12 column (10/300) at a flow rate of 0.5 mL/min in PBS. Fractionated cell extracts were applied to Western blotting by anti-TDP-43 (Proteintech). Mutant TDP-43 (GG) proteins were collected in a larger fraction than 88–440 kDa. The molecular size markers thyroglobulin (669 kDa), ferritin (440 kDa), Mn-SOD (88 kDa), ovalbumin (43 kDa), and RNase (13.7 kDa) were eluted under the same conditions. Abs280 is presented to show the equal amount of proteins between WT and the GG mutant in each fraction. doi:10.1371/journal.pone.0052776.g003

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Transfection, Western Blot, Mutagenesis, Size-exclusion Chromatography, Sonication

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 4. Oligomerization affects the nucleotide interaction and RNA splicing efficiency of TDP-43. A. Exon 9 skipping assay showing that mutations at E246 and D247 affect the RNA splicing activity of full-length TDP-43. a, Agarose gel electrophoresis of PCR products (top). Western blot analysis of the total cell lysates using anti-EGFP (middle) and -actin (bottom) antibodies was also shown. b, Quantification of spliced and unspliced fragments using densitometry. Each value is the ratio of spliced to unspliced PCR products. Data is mean 6 standard error of mean from triplicates. *p,0.01 vs. Wild-type (WT) TDP-43 by one-way ANOVA with Newman-Keuls test. B. Size exclusion chromatography for recombinant RRM2 proteins of WT (a), or mutants with E246Q/D247N (QN, b) or E246G/D247G (GG, c), and for (TG)12 oligonucleotides. Mixtures of RRM2 mutants and (TG)12 oligonucleotides were centrifuged at 15,0006g for 20 min and subjected to a Superdex75 (10/300) column at a flow rate of 0.5 mL/min in PBS. Only the RRM2 monomer showed a molecular shift with the (TG)12 oligonucleotides to a single peak, indicating their association (arrowheads). Molecular size markers are as follows: bovine serum albumin (66 kDa), ovalbumin (43 kDa), superoxide dismutase 1 (32 kDa), myoglobin (17.6 kDa), and aprotinin (6.5 kDa). *1 indicates free monomeric RRM2, *2 indicates oligomeric RRM2. Note that there is no peak for free (TG)12, indicating all the (TG)12 was bound to RRM2 monomers. doi:10.1371/journal.pone.0052776.g004

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Activity Assay, Agarose Gel Electrophoresis, Western Blot, Size-exclusion Chromatography, Recombinant

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 5. 3B12A recognizes cytosol-redistributed TDP-43. A–D, SHSY-5Y cells were transiently transfected with TDP-43-EGFP of wild type (WT), mutants with defective NLS (mNLS), or deletion mutant of RRM2 deletion (DRRM2) (EGFP shown as green). At 48 h after transfection, cells were fixed and stained with 3B12A (red). DAPI was used for counterstaining (blue). A. a–f, Transfected or endogenous WT TDP-43 was rarely stained by 3B12A (unfilled arrowheads). Occasionally, cells with very high fluorescence were labeled (arrowhead). B. Cytosolic redistributed TDP-43 (mNLS) was preferentially stained by 3B12A (arrowheads). 3B12A recognized the mNLS mutant of TDP-43-EGFP even at moderate expression levels, regardless of aggregate formation. c–e are high power fields of a–b. C. Nuclear-excluded WT TDP-43 is recognized by 3B12A. WT TDP-43-EGFP expressing SHSY-5Y cells exposed to 5 mM lactacystin were fixed and stained with 3B12A (arrowheads). D. No reactivity of 3B12A to TDP-43-EGFP devoid of RRM2 (DRRM2) (unfilled arrowheads). E. Immunoprecipitation experiment showing that 3B12A preferentially recognized NLS-defective TDP-43 in cell lysates. HEK293A cells were transiently transfected with WT, mNLS, or FALS mutant (A315T and Q331K) forms of TDP-43-FLAG. Total lysates were immunoprecipitated with the 3B12A. Western blot analysis using a rabbit polyclonal anti-FLAG antibody showed that 3B12A predominantly recognized the defective NLS, but more weakly recognized the WT and FALS-linked mutant forms of TDP-43. doi:10.1371/journal.pone.0052776.g005

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Transfection, Mutagenesis, Staining, Fluorescence, Labeling, Expressing, Immunoprecipitation, Western Blot

Journal: Cancers

Article Title: Identification of Monobenzone as a Novel Potential Anti-Acute Myeloid Leukaemia Agent That Inhibits RNR and Suppresses Tumour Growth in Mouse Xenograft Model

doi: 10.3390/cancers14194710

Figure Lengend Snippet: Analyses of RRM2 expression in samples from patients with AML from the GEO and TCGA databases. ( A , B ) Heatmaps showing the expression profiles of RRM1, RRM2, and RRM2B in patients with AML. ( C , D ) Correlation analyses between the expression of RRM2 and RRM1 with the cell proliferation biomarkers KI67 and PCNA in patients with AML. ( E , F ) KEGG enrichment plots constructed using GSEA for the RRM2 high expression group of patients with AML. Data in ( A , C , E ) were obtained from GSE147515 (N = 1534); data in ( B , D , F ) were obtained from TCGA ( n = 151). **** is a mirror image of the chart and has no special meaning.

Article Snippet: Antibodies against RRM1 (sc-11733), RRM2 (sc-398294) and RRM2B (sc-10840) were purchased from Santa Cruz Biotech (Dallas, TX, USA).

Techniques: Expressing, Construct

Journal: Cancers

Article Title: Identification of Monobenzone as a Novel Potential Anti-Acute Myeloid Leukaemia Agent That Inhibits RNR and Suppresses Tumour Growth in Mouse Xenograft Model

doi: 10.3390/cancers14194710

Figure Lengend Snippet: MB interacted with RRM2 and inhibited the activity of recombinant RNR in vitro. ( A ) Chemical structure of MB. ( B ) Chemical structure of osalmid. ( C ) The 3-dimensional interaction model between MB and RRM2 generated using molecular docking (Glide SP in Schrodinger/2020) showing the interaction between MB and key residues of RRM2 (PDB ID: 3OLJ). MB is represented in yellow sticks. RRM2 helices and residues are presented as a cyan cartoon and cyan sticks, respectively. Hydrogen bonds are represented as purple dashes. ( D ) In vitro recombinant RNR enzymatic assays. HU and MB dose-dependently inhibited the enzyme activity. The solvent DMSO was used as a negative control. ( E , F ) The binding between RRM2 protein and MB and the binding between RRM1 protein and MB were determined by MST assay. Fluorescence intensity and trend, and reproducibility of MST traces were shown. Data shown here are representative of three independent experiments, and the error is calculated as the standard deviation.

Article Snippet: Antibodies against RRM1 (sc-11733), RRM2 (sc-398294) and RRM2B (sc-10840) were purchased from Santa Cruz Biotech (Dallas, TX, USA).

Techniques: Activity Assay, Recombinant, In Vitro, Generated, Solvent, Negative Control, Binding Assay, Fluorescence, Standard Deviation

Journal: Cancers

Article Title: Identification of Monobenzone as a Novel Potential Anti-Acute Myeloid Leukaemia Agent That Inhibits RNR and Suppresses Tumour Growth in Mouse Xenograft Model

doi: 10.3390/cancers14194710

Figure Lengend Snippet: MB inhibited cell growth and DNA synthesis by inhibiting RNR enzymatic activity in AML cells. ( A ) MTT assays were performed on tumour cells treated with different doses of MB or HU for 72 h, and the solvent was used as negative control. ( B ) AML cells were treated with different doses of MB for 24 h or 48 h, and the EdU incorporation was examined with flow cytometry (FCM). ( C , D ) AML cells were treated with the indicated concentrations of MB for 24 h and then fixed with 70% ethanol, stained with PI, and subjected to an FCM analysis of the cell cycle distribution. The quantitative results of the number of cells in S phase are shown in a bar plot. ( E , F ) AML cells were treated with different doses of MB for 48 h and then analysed using FCM (PI & Annexin V method). Quantitative results of apoptotic cells are shown in bar plots. ( G ) AML cells were treated with MB for 24 h or 48 h. Whole-cell lysates were subjected to Western blotting with anti-γH2AX and anti-H3 antibodies. ( H , I ) AML cells were treated with MB alone or in combination with dNs for 24 h and then subjected to FCM analyses. The quantitative results of the numbers of cells in different phases of the cell cycle are shown in bar plots. ( J ) KG-1A cells were stably transfected with shRNAs targeting RRM2 (shRRM2-1 and shRRM2-2) or a negative control (shNC). The knockdown effects were measured with qRT-PCR. ( K ) The knockdown effects were measured with Western blotting. ( L , M ) Cells were treated with different doses of MB for 24 h and then subjected to FCM assays. The relative number of cells in the S phase is shown in a bar plot. All experiments described above were performed at least three independent times. Error bars represent the SD. * p < 0.05, ** p < 0.005, *** p < 0.0005 compared to the control group. Full Western blot images and western blotting gray value analysis can be found at , .

Article Snippet: Antibodies against RRM1 (sc-11733), RRM2 (sc-398294) and RRM2B (sc-10840) were purchased from Santa Cruz Biotech (Dallas, TX, USA).

Techniques: DNA Synthesis, Activity Assay, Solvent, Negative Control, Flow Cytometry, Staining, Western Blot, Stable Transfection, Transfection, Knockdown, Quantitative RT-PCR, Control

Journal: Gut and Liver

Article Title: High Expression of Ribonucleotide Reductase Subunit M2 Correlates with Poor Prognosis of Hepatocellular Carcinoma

doi: 10.5009/gnl13392

Figure Lengend Snippet: Immunostaining of ribonucleotide reductase subunit M2 in hepatocellular carcinomas showing low expression (A) or high expression (B) (horseradish peroxidase stain, ×200).

Article Snippet: Sections were incubated with mouse monoclonal antibody to RRM2 (clone 1E1, 1:800; Abcam Corp., Cambridge, MA, USA) for 30 minutes at room temperature.

Techniques: Immunostaining, Expressing, Staining

Journal: Gut and Liver

Article Title: High Expression of Ribonucleotide Reductase Subunit M2 Correlates with Poor Prognosis of Hepatocellular Carcinoma

doi: 10.5009/gnl13392

Figure Lengend Snippet: Ribonucleotide reductase subunit M2 immunoreactivity in hepatocellular carcinomas (right lower part) was similar to that in normal hepatocytes (left upper part) (horseradish peroxidase stain, ×100).

Article Snippet: Sections were incubated with mouse monoclonal antibody to RRM2 (clone 1E1, 1:800; Abcam Corp., Cambridge, MA, USA) for 30 minutes at room temperature.

Techniques: Staining

Journal: Gut and Liver

Article Title: High Expression of Ribonucleotide Reductase Subunit M2 Correlates with Poor Prognosis of Hepatocellular Carcinoma

doi: 10.5009/gnl13392

Figure Lengend Snippet: Correlations between Ribonucleotide Reductase Subunit M2 Expression and Clinicopathologic Features in 259 Patients with Hepatocellular Carcinomas

Article Snippet: Sections were incubated with mouse monoclonal antibody to RRM2 (clone 1E1, 1:800; Abcam Corp., Cambridge, MA, USA) for 30 minutes at room temperature.

Techniques: Expressing

Journal: Gut and Liver

Article Title: High Expression of Ribonucleotide Reductase Subunit M2 Correlates with Poor Prognosis of Hepatocellular Carcinoma

doi: 10.5009/gnl13392

Figure Lengend Snippet: Univariate and Multivariate Logistic Regression Models for Predicting Early Tumor Recurrence in 259 Patients with Hepatocellular Carcinomas

Article Snippet: Sections were incubated with mouse monoclonal antibody to RRM2 (clone 1E1, 1:800; Abcam Corp., Cambridge, MA, USA) for 30 minutes at room temperature.

Techniques: Expressing

Journal: Gut and Liver

Article Title: High Expression of Ribonucleotide Reductase Subunit M2 Correlates with Poor Prognosis of Hepatocellular Carcinoma

doi: 10.5009/gnl13392

Figure Lengend Snippet: Univariate Analyses of Recurrence-Free Survival and Disease-Specific Survival in 259 Patients with Hepatocellular Carcinomas

Article Snippet: Sections were incubated with mouse monoclonal antibody to RRM2 (clone 1E1, 1:800; Abcam Corp., Cambridge, MA, USA) for 30 minutes at room temperature.

Techniques: Expressing

Journal: Gut and Liver

Article Title: High Expression of Ribonucleotide Reductase Subunit M2 Correlates with Poor Prognosis of Hepatocellular Carcinoma

doi: 10.5009/gnl13392

Figure Lengend Snippet: Kaplan-Meier survival curves showing recurrence-free survival (A) and disease-specific survival (B) according to ribonucleotide reductase subunit M2 expression in 259 patients with hepatocellular carcinomas.

Article Snippet: Sections were incubated with mouse monoclonal antibody to RRM2 (clone 1E1, 1:800; Abcam Corp., Cambridge, MA, USA) for 30 minutes at room temperature.

Techniques: Expressing

Journal: Gut and Liver

Article Title: High Expression of Ribonucleotide Reductase Subunit M2 Correlates with Poor Prognosis of Hepatocellular Carcinoma

doi: 10.5009/gnl13392

Figure Lengend Snippet: Multivariate Analyses of Recurrence-Free Survival and Disease-Specific Survival in 259 Patients with Hepatocellular Carcinomas

Article Snippet: Sections were incubated with mouse monoclonal antibody to RRM2 (clone 1E1, 1:800; Abcam Corp., Cambridge, MA, USA) for 30 minutes at room temperature.

Techniques: Expressing

Journal: Biotechnology and bioengineering

Article Title: Impact of tumor-specific targeting and dosing schedule on tumor growth inhibition after intravenous administration of siRNA-containing nanoparticles.

doi: 10.1002/bit.21668

Figure Lengend Snippet: Figure 5. In vivo growth inhibition of established subcutaneous Neuro2A-Luc tumors in A/J mice after treatment with three independent sequences targeting RRM2 mRNA and an irrelevant control sequence. Tf-targeted (1 mol%) nanoparticles containing 2.5 mg/kg of siR2B þ 5 (circles, n ¼ 5), siR2B þ 6 (squares, n ¼ 5), siCON (diamonds, n ¼ 4), or siR2A þ 5 (triangles, n ¼ 5) were injected intravenously on 3 consecutive days (qd 3) once the tumors had reached a size of 100 mm3. Data points represent mean tumor volume, and error bars ¼ SE.

Article Snippet: Immunohistochemistry was performed on 5-mm cryosections using a 1:25 dilution of goat anti-mouse RRM2 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a horseradish peroxidase-conjugated secondary antibody.

Techniques: In Vivo, Inhibition, Control, Sequencing, Injection

Journal: Biotechnology and bioengineering

Article Title: Impact of tumor-specific targeting and dosing schedule on tumor growth inhibition after intravenous administration of siRNA-containing nanoparticles.

doi: 10.1002/bit.21668

Figure Lengend Snippet: Figure 7. RRM2 protein expression in Neuro2A-Luc tumor cryosections after treatment with siRNA. A: RRM2 protein expression in three different regions within tumors treated with siCON. B: RRM2 protein expression in three different regions within tumors treated with siR2B þ 5. Mice bearing subcutaneous Neuro2A-Luc tumors were injected intravenously (qd 3) with Tf-targeted (1 mol%) nanoparticles containing siRNA (2.5 mg/kg). Tumors were harvested 24 h after the third injection. Brown staining indicates RRM2 protein expression, and scale bar ¼ 50 mm.

Article Snippet: Immunohistochemistry was performed on 5-mm cryosections using a 1:25 dilution of goat anti-mouse RRM2 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a horseradish peroxidase-conjugated secondary antibody.

Techniques: Expressing, Injection, Staining

Journal: Oncology reports

Article Title: Establishment of human pancreatic cancer gemcitabine‑resistant cell line with ribonucleotide reductase overexpression.

doi: 10.3892/or.2014.3599

Figure Lengend Snippet: Figure 4. Western blotting for the resistance-related factors. The examined proteins, except BCRP, were expressed in PANC-1 and PANC-1RG7. Compared with those of the parental PANC-1 cells, NT5, RRM1 and RRM2 expression levels were significantly increased in PANC-1RG7 (p<0.05). No changes in other proteins at the protein level were noted. Results represent 3 independent experiments. Gray scales shown at the bottom indicate results compared with β-actin.

Article Snippet: The specific primary antibodies we used were mouse monoclonal antibodies anti-human β-actin (sc-47778), ENT1 (sc-377283), ENT2 (sc-373871), NT5 (sc-32299), POLA (sc-137021), p-gp (sc-55510) (all from Santa Cruz, USA), and MRP (no. ab32574; Abcam, USA); rabbit polyclonal antibodies anti-human DCK (no. ab151966; Abcam, USA), CDA (sc-134754), BCRP (sc-130933) (both from Santa Cruz), Akt (no. BS1810) and mTOR (no. BS3611) (both from BioWorld, USA); rabbit monoclonal antibodies anti-human PI3K (no. 4249; Cell Signaling Technology, USA); and goat polyclonal antibodies anti-human RRM1 (sc-11733) and RRM2 (sc-10846) (both from Santa Cruz).

Techniques: Western Blot, Expressing