rr m2 subunit  (Aviva Systems)

Journal: PLoS ONE

Article Title: The Efficacy of the Ribonucleotide Reductase Inhibitor Didox in Preclinical Models of AML

doi: 10.1371/journal.pone.0112619

Figure Lengend Snippet: Primary patient sample characteristics.

Article Snippet: For RR detection, a primary antibody against the M2 subunit of RR (1∶1000; sc-10846, Santa Cruz Biotechnology) was used followed by secondary anti-goat antibody (1∶1000; ab98826, AbCam).

Techniques:

Journal: Oncogene

Article Title: Oncolytic herpes virus with defective ICP6 specifically replicates in quiescent cells with homozygous genetic mutations in p16

doi: 10.1038/onc.2008.53

Figure Lengend Snippet: Ribonucleotide reductase expression is elevated in noncycling cells with p16 deletions. Reverse transcription (RT)–PCR shows the ratio of M2 subunit of mammalian ribonucleotide reductase (mRR) mRNA in cycling relative to noncycling cells was 0.9–1.3 in p16-deleted cells (U87, T98, and p16 −/− MEFs). However, in p16-expressing cells (that is, wild-type MEFs, human fibroblasts, p53 −/− MEFs and U373), this ratio was much higher (2.5–5.8). A high capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA) was used to generate cDNA from RNA isolated from cells using Trizol (Invitrogen) per protocol. Real-time RT–PCR was performed on an ABI Prism 7000 (Applied Biosystems) machine using human primer-probe combinations for RR subunit M2 (Applied Biosystems part nos. Hs00357247_g1 and Hs00168784_m1) and 18S rRNA (Applied Biosystems part no. 4308329) combined with TaqMan Master Mix (Applied Biosystems). Relative quantification was performed using 18S rRNA as an endogenous control. All reactions began with 10 min at 95 °C for AmpliTaq Gold activation, followed by 40 cycles at 95 °C for 15 s for denaturation and then 60 °C for 1 min for annealing/extension. Abbreviations used: F, fibroblasts; WT, wild-type; U373-mRR, U373 clone selected after stable transfection with plasmid constitutively expressing the M2 subunit of mammalian ribonucleotide reductase.

Article Snippet: Real-time RT–PCR was performed on an ABI Prism 7000 (Applied Biosystems) machine using human primer-probe combinations for RR subunit M2 (Applied Biosystems part nos.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Quantitative RT-PCR, Activation Assay, Stable Transfection, Plasmid Preparation

Journal: Journal of the National Cancer Institute

Article Title: Effect of chemotherapy-induced DNA repair on oncolytic herpes simplex viral replication.

doi: 10.1093/jnci/djj003

Figure Lengend Snippet: Fig. 4. Effect of temozolomide on growth arrest DNA damage 34 (GADD34) and ribonucleotide reductase (RR) mRNA and protein; contribution of GADD34 and RR to DNA repair. A ) Results of real-time reverse transcription – polymerase chain reaction analysis of levels of the mRNAs encoding GADD34 and the M2 subunit of RR in drug-treated human astrocytes (HA), U87, T98, U87/MGMT, and U87/mp53 cells. Cells were treated for 48 hours with various concentrations of temozolomide (Tem), cisplatin, or O 6 -benzylguanine (BG). Values shown are relative amounts of mRNA for GADD34 ( solid bars ) and the M2 subunit of RR ( open bars ) compared with untreated cells. Three replicates were performed. Error bars indicate upper 95% confi dence intervals. B ) Western blot analysis of GADD34 expression in U87 cells that were untreated, treated with GADD34 small interfering (si) RNA, treated with 1 mM temozolomide, or treated with temozolomide plus GADD34 siRNA; and RR M2 subunit expression in T98 cells that were treated with 100 μ M O 6 -benzylguanine, O 6 - benzylguanine plus RR small interfering (si)RNA, O 6 -benzylguanine plus 1 mM temozolomide, or O 6 -benzylguanine plus 1 mM temozolomide plus RR siRNA. Blots were probed for actin as a loading control. C ) Results of alkaline comet assay presented as mean tail moment (in arbitrary units; tail moment = % DNA in the tail multiplied by tail distance) as a function of temozolomide concentration for U87 cells treated with RR or GADD34 siRNA and U87/ MGMT cells treated with O 6 -benzylguanine (BG) plus RR or GADD34 siRNA. Experiment was performed three times, each in triplicate. Means of one representative experiment are shown. Error bars indicate 95% confi dence intervals.

Article Snippet: Total protein from cultured cells was extracted in radioimmunoprecipitation assay buffer (150 mM NaCl, 1.0% IGEPAL CA-630; IPEGAL CA-630 = 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0), and 30 μ g of protein was separated by sodium dodecyl sulfate – 8% polyacrylamide gel electrophoresis, transferred to polyvinylidene difl uoride membranes by electroblotting, and incubated with antibodies to GADD34 (goat polyclonal, 1 : 100; Imgenex Corp., San Diego, CA) or RR M2 subunit (chicken polyclonal, 1 : 200; GenWay Biotech, San Diego, CA) at 4 °C overnight.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Western Blot, Expressing, Control, Alkaline Single Cell Gel Electrophoresis, Concentration Assay