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: BMC cancer

Article Title: RRM2 and CDC6 are novel effectors of XBP1-mediated endocrine resistance and predictive markers of tamoxifen sensitivity.

doi: 10.1186/s12885-023-10745-1

Figure Lengend Snippet: Fig. 6 Expression of RRM2 and CDC6 rescues tamoxifen hypersensitivity in XBP1 knockout MCF7 cells. A Whole cell lysates from indicated RRM2 expressing cells were analysed by western blotting using antibodies against RRM2 and β-Actin. B Indicated RRM2 expressing cells were treated with tamoxifen (10 µM) for required time points. Line graphs show change in O.D at 490 nm. Data presented is mean ± S.D (n = 4). *p < 0.05, two-tailed unpaired t-test comparing respective time points. C Whole cell lysates from indicated (wild type and mutant) CDC6 expressing cells were subjected to western blotting using antibodies against CDC6 and β-Actin. D Indicated mutant CDC6 expressing cells were treated with tamoxifen (10 µM) for required time points. Line graphs show the change in O.D at 490 nm. Data presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 from two-tailed unpaired t-test comparing respective time points

Article Snippet: Mouse ER-α Ab (Cat# sc-8002), Mouse RRM2 Ab (sc-376973), mouse CDC6 Ab (Cat# sc-9964) were purchased from Santa Cruz Biotechnology, Inc. (Bergheimer, Heidelberg, Germany).

Techniques: Expressing, Knock-Out, Western Blot, Two Tailed Test, Mutagenesis

Journal: BMC cancer

Article Title: RRM2 and CDC6 are novel effectors of XBP1-mediated endocrine resistance and predictive markers of tamoxifen sensitivity.

doi: 10.1186/s12885-023-10745-1

Figure Lengend Snippet: Fig. 7 Association of XBP1-gene signature with outcome in ER-positive breast cancer. A Box plot for expression of RRM2, CDC6 and TOP2A in tumour and normal tissues in human breast cancers is shown. Median is shown by horizontal black line, the box is the upper and lower quartiles and the two lines outside the box show the highest and lowest values. B KM Plotter (https://kmplot.com/analysis/) was used to determine the association of XBP1-gene signature (CDC6, RRM2 and TOP2A) with overall survival (OS) and recurrence free survival (RFS) in ER-positive breast cancer. C Web-based algorithm PROGgeneV2 (http://www.progtools.net/gene/index.php) was used to test the association between XBP1-gene signature and RFS in indicated datasets of breast cancer. D Web-based algorithm ROC plot (http://www.rocplot.org/) was used to evaluate the association between XBP1-gene signature and response to tamoxifen treatment in ER-positive breast cancer

Article Snippet: Mouse ER-α Ab (Cat# sc-8002), Mouse RRM2 Ab (sc-376973), mouse CDC6 Ab (Cat# sc-9964) were purchased from Santa Cruz Biotechnology, Inc. (Bergheimer, Heidelberg, Germany).

Techniques: Expressing

Journal: Cancers

Article Title: Transcriptomic, Proteomic, and Genomic Mutational Fraction Differences Based on HPV Status Observed in Patient-Derived Xenograft Models of Penile Squamous Cell Carcinoma

doi: 10.3390/cancers16051066

Figure Lengend Snippet: Differentially expressed proteins based on HPV status. ( a ) Normalized linear expression of RRPA data of CDKN2A, RRM2, and CDC25C depicted in a bar blot with false discovery rate (FDR) adjusted p -value given for each protein per data set. ( b ) Immunoblot of lysates for HPV+ (red bars) and HPV− (black bars) PDX models for RRM2, HPV16-E7, and GAPDH depicted. ( c ) Immunoblot of CDC25C and GAPDH. ( d ) Immunoblot of p16 and GAPDH. The uncropped blots are shown in File S1.

Article Snippet: Membranes were blotted with primary antibodies for RRM2 (1:1000, 65939S, Cell Signaling, Danvers, MA, USA); p16 (1:1000, 80772S, Cell Signaling); CDC25C (1:1000, 4688S, Cell Signaling); and HPV16-E7 (1:1000, GTX133411, GeneTex, Irvine, CA, USA) and a corresponding secondary antibody (1:2000 of Anti-rabbit IgG HRP linked, 7074P2, Cell Signaling or 1:2000 Anti-mouse IgG HRP linked, 7076S, Cell Signaling).

Techniques: 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: mRNA expression in quiescent human hepatocytes of chimeric mice with or without HCV infection and the effects of HCV on RRM2 expression. A, timeline of HCV infection in chimeric mice with humanized livers. Human hepatocytes were transplanted into three uPA-Tg/SCID mice, which were then infected with HCV R6 (genotype 1b) after >90% of mouse hepatocytes had been replaced, as determined by serum human albumin levels and histochemical analysis (see “Experimental procedures” for details). B, RRM2 protein expression in quiescent human hepatocytes from uninfected and HCV-infected chimeric mice. Average expression levels in four chimeric mice (mice 16 and 20 were HCV-negative, and mice 4 and 25 were HCV-positive) were normalized to that of actin. HCV core protein was also detected. C, quantification of RRM2 mRNA levels relative to that of GAPDH in HuH-7, cured K4, and JFH/K4 cells. RRM2 protein expression was verified via Western blotting using anti-RRM2 antibodies (bottom). The densitometric RRM2/actin ratios are shown below the blots. D, quantification of RRM2 mRNA as in C in JFH/K4 cells treated with RRM2 siRNA, HCV R7 siRNA, or control (Cont.) siRNA. Values in untreated cells (Non) are shown for comparison. Expression of RRM2 and β-actin proteins was verified via Western blotting (bottom), as described in C. Results in C and D are an average of triplicate samples, and vertical bars indicate S.D. from three independent experiments (n = 3). *, p < 0.05. E, HCV-infected JFH/K4 cells and uninfected HuH-7 cells were analyzed via Western blotting for RRM1, HCV core, and β-actin. F, dCTP pool size was measured in JFH/K4 and HuH-7 cells using a fluorescence-based assay. Results are shown as -fold change in dCTP levels relative to that in uninfected HuH-7 cells. Values represent the average -fold changes ± S.D. (vertical bars) from 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, Infection, Western Blot, Fluorescence

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: Analysis of up-regulated genes in HCV-infected hepatocytes from humanized chimeric mouse Microarray analysis is described under “Experimental procedures.” The left column shows that cRNA targets from HCV-infected and uninfected humanized mouse livers were conjugated with cyanine 3 (Cy3) and cyanine 5 (Cy5), respectively. The right column shows that, contrary to the left column, Cy5 and Cy3 conjugation were used for cRNA targets from HCV-infected and uninfected humanized livers, respectively. The top five most induced genes are shown.

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: Microarray, Conjugation Assay, Binding Assay, Dominant Negative Mutation

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

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: HCV utilizes RRM2 during replication. A, efficacy of RRM2 siRNA compared with that of HCV R5 siRNA and control siRNA. FLR3-1 replicon cells were incubated with the indicated constructs for 3 days, and replication was measured as luciferase activity (ratio of replication inhibition). Cell viability was determined with the WST assay. B, R6FLR-N cells pretreated with RRM2 siRNA (0.1 nm) and transfected with mock (Non) or pcDNA6-myc-His-RRM2 vectors (0.3 μg each), analyzed via Western blotting with anti-Myc or anti-RRM2 antibodies. C, R6FLR-N replicon cells transfected with RRM2 siRNA (0.1 nm) followed by pcDNA6-myc-His-RRM2 expression vector or empty pcDNA6-myc-His vector at 0.1 or 0.3 μg. Luminescence is shown as relative luminescence units (RLUs). Cell viability was measured by WST assay (A450). Results are presented as means ± S.D. (vertical bars) of three independent experiments (n = 3). *, p < 0.05.

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: Incubation, Construct, Luciferase, Activity Assay, Inhibition, WST Assay, Transfection, Western Blot, Expressing, Plasmid Preparation

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, Transfection, Sequencing, 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: 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: 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: Transfection, Immunoprecipitation, Staining, Labeling, Fluorescence, Microscopy, Ubiquitin Assay, 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: 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