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Addgene inc plasmid pcdna3 rrm2
Plasmid Pcdna3 Rrm2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 6 article reviews

plasmid pcdna3 rrm2 - by Bioz Stars, 2026-03

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Figure 3. IGF1R regulates RNR activity via AKT, MEK-ERK, and JUN. A, MCF7 cells were siRNA-transfected and after 48 hours harvested for dNTP assay. Graph, mean SEM fold-change in dATP content (n ¼ 3 independent assays). B, HCT15 cells were siRNA-transfected and dNTPs were extracted and assayed as A. Graph, mean SEM fold-change in dNTP content (n ¼ 3 independent assays). C, Serum-starved MCF7 cells IGF-treated for 24 hours; RRM2 mRNA assessed by qPCR (mean SEM of three independent analyses). D, Serum-starved MCF7 cells pretreated with 1 mmol/L xentuzumab for 2 hours, then xentuzumab with 50 nmol/L IGF1 for 24 hours. Graph to right, mean SEM RRM2 protein (n ¼ 3 independent blots, corrected for b-tubulin, expressed as % of levels in serum-starved cells). E and F, MCF7 cells were siRNA transfected, collected after 48 hours for Western blot analysis (E). Graph shows mean SEM RRM2 protein (n ¼ 3 Western blots expressed as % of siControl); qPCR for RRM2 mRNA (mean SEM of three independent analyses; F). G, Breast cancer cells were transfected with Control (siCtrl) or IGF1R siRNAs and lysed after 48 hours for Western blot analysis to assess IGF1R depletion and RRM2 expression. H and I, Serum-starved MCF7 cells treated with IGF1 alone or with 10 nmol/L MEK inhibitor (MEKi) trametinib (H) or 3.5 mmol/L AKT inhibitor (AKTi) AZD5363 (I). Graphs to right, quantiﬁcation of RRM2 from three independent blots in each case. J, MCF7 cells transfected with siIGF1R and/or siJUN were analysed by Western blot analysis, with similar results in a second independent blot. Graph, quantiﬁcation (mean range), showing reduction in RRM2 protein to 67 0.5% of siControl levels with siJUN_1 and 72 17% with siJUN_2. K, Left, schematic of RRM2 promoter reporter showing JUN consensus binding motif TGACTCA. Right, luciferase assay after transient transfection with RRM2 promoter luciferase reporter (n ¼ 3 assays each with triplicate technical replicates). , P < 0.05; , P < 0.01; , P < 0.001; n.s., nonsigniﬁcant.

Journal: Cancer Research

Article Title: Targeting IGF Perturbs Global Replication through Ribonucleotide Reductase Dysfunction

doi: 10.1158/0008-5472.can-20-2860

Figure Lengend Snippet: Figure 3. IGF1R regulates RNR activity via AKT, MEK-ERK, and JUN. A, MCF7 cells were siRNA-transfected and after 48 hours harvested for dNTP assay. Graph, mean SEM fold-change in dATP content (n ¼ 3 independent assays). B, HCT15 cells were siRNA-transfected and dNTPs were extracted and assayed as A. Graph, mean SEM fold-change in dNTP content (n ¼ 3 independent assays). C, Serum-starved MCF7 cells IGF-treated for 24 hours; RRM2 mRNA assessed by qPCR (mean SEM of three independent analyses). D, Serum-starved MCF7 cells pretreated with 1 mmol/L xentuzumab for 2 hours, then xentuzumab with 50 nmol/L IGF1 for 24 hours. Graph to right, mean SEM RRM2 protein (n ¼ 3 independent blots, corrected for b-tubulin, expressed as % of levels in serum-starved cells). E and F, MCF7 cells were siRNA transfected, collected after 48 hours for Western blot analysis (E). Graph shows mean SEM RRM2 protein (n ¼ 3 Western blots expressed as % of siControl); qPCR for RRM2 mRNA (mean SEM of three independent analyses; F). G, Breast cancer cells were transfected with Control (siCtrl) or IGF1R siRNAs and lysed after 48 hours for Western blot analysis to assess IGF1R depletion and RRM2 expression. H and I, Serum-starved MCF7 cells treated with IGF1 alone or with 10 nmol/L MEK inhibitor (MEKi) trametinib (H) or 3.5 mmol/L AKT inhibitor (AKTi) AZD5363 (I). Graphs to right, quantiﬁcation of RRM2 from three independent blots in each case. J, MCF7 cells transfected with siIGF1R and/or siJUN were analysed by Western blot analysis, with similar results in a second independent blot. Graph, quantiﬁcation (mean range), showing reduction in RRM2 protein to 67 0.5% of siControl levels with siJUN_1 and 72 17% with siJUN_2. K, Left, schematic of RRM2 promoter reporter showing JUN consensus binding motif TGACTCA. Right, luciferase assay after transient transfection with RRM2 promoter luciferase reporter (n ¼ 3 assays each with triplicate technical replicates). , P < 0.05; , P < 0.01; , P < 0.001; n.s., nonsigniﬁcant.

Article Snippet: MCF7 stably overexpressing RRM2 MCF7 cells were transfectedwith pcDNA3.1 (#V79520, Invitrogen) or pcDNA3.1 RRM2 (Addgene, Plasmid #13796) using Lipofectamine 3000 (#L3000001, Thermo Fisher Scientific).

Techniques: Activity Assay, Transfection, Western Blot, Control, Expressing, Binding Assay, Luciferase

Figure 4. RRM2 overexpression rescues IGF1R depleted or inhibited cells from replication stress. A and B, U2OS cells stably transfected with RRM2 or empty vector (EV) were analyzed by Western blot analysis 48 hours after IGF1R siRNA transfection (A) or 72 hours treatment with 100 nmol/L xentuzumab (B). C, Parallel cultures siRNA-transfected as A were processed for 53BP1 immunoﬂuorescence. Scale bar, 10 mm. Bottom graph, mean SEM 53BP1 bodies (n ¼ 30 nuclei). D and E, MCF7 cells stably transfected with empty vector or RRM2 cDNA were harvested 48 hours after transfection with siControl or siIGF1R for Western blot analysis (D). E, DNA ﬁber analysis showing representative images (left); quantiﬁcation of tract length (n ¼ 200; right). F, Stably transfected MCF7 cells were treated with xentuzumab for 72 hours and processed as C for 53BP1 immunoﬂuorescence. Scale bar, 10 mm. G, Graph. Mean SEM 53BP1 bodies (n ≥20 nuclei). , P < 0.01; , P < 0.001; n.s., nonsigniﬁcant.

Journal: Cancer Research

Article Title: Targeting IGF Perturbs Global Replication through Ribonucleotide Reductase Dysfunction

doi: 10.1158/0008-5472.can-20-2860

Figure Lengend Snippet: Figure 4. RRM2 overexpression rescues IGF1R depleted or inhibited cells from replication stress. A and B, U2OS cells stably transfected with RRM2 or empty vector (EV) were analyzed by Western blot analysis 48 hours after IGF1R siRNA transfection (A) or 72 hours treatment with 100 nmol/L xentuzumab (B). C, Parallel cultures siRNA-transfected as A were processed for 53BP1 immunoﬂuorescence. Scale bar, 10 mm. Bottom graph, mean SEM 53BP1 bodies (n ¼ 30 nuclei). D and E, MCF7 cells stably transfected with empty vector or RRM2 cDNA were harvested 48 hours after transfection with siControl or siIGF1R for Western blot analysis (D). E, DNA ﬁber analysis showing representative images (left); quantiﬁcation of tract length (n ¼ 200; right). F, Stably transfected MCF7 cells were treated with xentuzumab for 72 hours and processed as C for 53BP1 immunoﬂuorescence. Scale bar, 10 mm. G, Graph. Mean SEM 53BP1 bodies (n ≥20 nuclei). , P < 0.01; , P < 0.001; n.s., nonsigniﬁcant.

Techniques: Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Western Blot

Figure 6. ATM loss sensitizes to IGF inhibition. A, Pan-cancer analysis (926 cell lines) associating ATM homozygous mutations and sensitivity to IGF1R inhibitor BMS-754807 (cancerxgene.org). B, ATMþ/þ and ATM/ ﬁbroblasts treated with 188 kinase inhibitors (1 mmol/L), viability measured after 3 days, compounds ranked by ratio of log10 viability. Green box, top-ranked compounds; top hit arrowed. C, Fibroblasts in B treated with BI-885578; viability measured after 5 days. Right, serum-starved ﬁbroblasts treated with BI-885578 for 24 hours and in ﬁnal 15 minuteswith IGF1.D, Viability of colorectal cancer cells treated with xentuzumab orBI-885578 for 5 days. Blot conﬁrms ATM-null status of SK-CO-1. E and F, Mice bearing SK-CO-1 xenografts were treated twice weekly (arrows) with solvent or xentuzumab showing representative tumor-bearing mice (E) and tumor volumes (F). G, IHC analysis of SK-CO-1 tumors and mouse skin. Scale bar, 100 mm. H-scores for RRM2 and BrdU represent moderate-strong (2–3þ) staining. There was less striking reduction in RRM2 and BrdU when including weak (1þ) positivity (Supplementary Fig. S6I and S6J), consistent with S-phase arrest induced in MCF7 cells by IGF1R depletion (Supplementary Fig. S1G). ATM-null status of SK-CO-1 xenografts was conﬁrmed by IHC (Supplementary Fig. S6H); mouse epidermis acted as positive control for ATM. H, ATM-proﬁcient or deﬁcient MiaPaCa2 PDAC spheroids were treated with solvent or xentuzumab. Spheroid size was measured every 1 to 3 days and expressed relative to spheroid size immediately prior to treatment. I, ATM proﬁcient MiaPaCa2 spheroids were treated with AZD0156 and/or xentuzumab and spheroid size measured as H. , P < 0.05; , P < 0.01; , P < 0.001.

Journal: Cancer Research

Article Title: Targeting IGF Perturbs Global Replication through Ribonucleotide Reductase Dysfunction

doi: 10.1158/0008-5472.can-20-2860

Figure Lengend Snippet: Figure 6. ATM loss sensitizes to IGF inhibition. A, Pan-cancer analysis (926 cell lines) associating ATM homozygous mutations and sensitivity to IGF1R inhibitor BMS-754807 (cancerxgene.org). B, ATMþ/þ and ATM/ ﬁbroblasts treated with 188 kinase inhibitors (1 mmol/L), viability measured after 3 days, compounds ranked by ratio of log10 viability. Green box, top-ranked compounds; top hit arrowed. C, Fibroblasts in B treated with BI-885578; viability measured after 5 days. Right, serum-starved ﬁbroblasts treated with BI-885578 for 24 hours and in ﬁnal 15 minuteswith IGF1.D, Viability of colorectal cancer cells treated with xentuzumab orBI-885578 for 5 days. Blot conﬁrms ATM-null status of SK-CO-1. E and F, Mice bearing SK-CO-1 xenografts were treated twice weekly (arrows) with solvent or xentuzumab showing representative tumor-bearing mice (E) and tumor volumes (F). G, IHC analysis of SK-CO-1 tumors and mouse skin. Scale bar, 100 mm. H-scores for RRM2 and BrdU represent moderate-strong (2–3þ) staining. There was less striking reduction in RRM2 and BrdU when including weak (1þ) positivity (Supplementary Fig. S6I and S6J), consistent with S-phase arrest induced in MCF7 cells by IGF1R depletion (Supplementary Fig. S1G). ATM-null status of SK-CO-1 xenografts was conﬁrmed by IHC (Supplementary Fig. S6H); mouse epidermis acted as positive control for ATM. H, ATM-proﬁcient or deﬁcient MiaPaCa2 PDAC spheroids were treated with solvent or xentuzumab. Spheroid size was measured every 1 to 3 days and expressed relative to spheroid size immediately prior to treatment. I, ATM proﬁcient MiaPaCa2 spheroids were treated with AZD0156 and/or xentuzumab and spheroid size measured as H. , P < 0.05; , P < 0.01; , P < 0.001.

Techniques: Inhibition, Solvent, Staining, Positive Control

A- Relative mRNA expression levels of KDM5A, KDM5B, and the E2F target genes RRM2, CDC6 and CCNE1 upon transfection of the indicated siRNA in U2OS cells (siCtle corresponds to a non-targeting pool of siRNA). Expression levels were normalized to the reference gene P0 (ribosomal phosphoprotein P0) and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 3 independent experiments are shown. The star (*) indicates significant difference between the siCtle and the K5A/B siRNA treated cells (pvalue <0.05 calculated using a paired t-test). B- Western-blot analysis of KDM5A, KDM5B, RRM2 and GAPDH as a loading control from U2OS cells transfected with siRNA directed against KDM5A and KDM5B. Two distinct couples of siRNA (siK5A+B-1 and -2) were used. C- Cell cycle distribution of U2OS cells depleted for KDM5A and KDM5B using siK5A+B-1 compared to siCtle treated cells, analyzed by the high content imaging system Operetta following EdU labeling and DAPI staining. D- Percentage of living cells following depletion of KDM5A and B using siK5A+B-1 siRNAs. The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for siCtle treated cells. Paired t-tests indicate a pvalue <0.05 between the first couple of siK5A/B siRNA and siCtle treated cells (*) but not for the second one with pvalue=0.054. E- ChIP analysis of KDM5A on the RRM2 and CDC6 promoter (Prom.) and coding (Cod.) regions. The myogenin gene is not expressed in U2OS cells and its promoter serves as a negative control. A representative experiment out of 4 is shown. F- Western-blot analysis of KDM5A, KDM5B, RRM2 and GAPDH from U2OS cells treated each 24 hours or not with KDM5 inhibitor CPI-455 for 48 hours. G- Relative mRNA expression levels of KDM5A, KDM5B and RRM2 in cells treated, each 24 hours, with 12.5 mM KDM5 inhibitor CPI-455 (+) or DMSO (-) for 48 hours. Expression levels were normalized to the reference gene P0 (ribosomal phosphoprotein P0) and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 3 independent experiments are shown. A paired t-test indicated no significant difference for all tested genes between CPI treated and untreated cells. H- Percentage of living cells following treatment each 24 hours with CPI-455 for 72 hours (+) or DMSO (-). The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for DMSO treated cells. A paired t-test indicated no significant difference between CPI treated and untreated cells.

Journal: bioRxiv

Article Title: KDM5 histone-demethylases contribute to replication stress response and tolerance

doi: 10.1101/2019.12.16.877399

Figure Lengend Snippet: A- Relative mRNA expression levels of KDM5A, KDM5B, and the E2F target genes RRM2, CDC6 and CCNE1 upon transfection of the indicated siRNA in U2OS cells (siCtle corresponds to a non-targeting pool of siRNA). Expression levels were normalized to the reference gene P0 (ribosomal phosphoprotein P0) and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 3 independent experiments are shown. The star (*) indicates significant difference between the siCtle and the K5A/B siRNA treated cells (pvalue <0.05 calculated using a paired t-test). B- Western-blot analysis of KDM5A, KDM5B, RRM2 and GAPDH as a loading control from U2OS cells transfected with siRNA directed against KDM5A and KDM5B. Two distinct couples of siRNA (siK5A+B-1 and -2) were used. C- Cell cycle distribution of U2OS cells depleted for KDM5A and KDM5B using siK5A+B-1 compared to siCtle treated cells, analyzed by the high content imaging system Operetta following EdU labeling and DAPI staining. D- Percentage of living cells following depletion of KDM5A and B using siK5A+B-1 siRNAs. The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for siCtle treated cells. Paired t-tests indicate a pvalue <0.05 between the first couple of siK5A/B siRNA and siCtle treated cells (*) but not for the second one with pvalue=0.054. E- ChIP analysis of KDM5A on the RRM2 and CDC6 promoter (Prom.) and coding (Cod.) regions. The myogenin gene is not expressed in U2OS cells and its promoter serves as a negative control. A representative experiment out of 4 is shown. F- Western-blot analysis of KDM5A, KDM5B, RRM2 and GAPDH from U2OS cells treated each 24 hours or not with KDM5 inhibitor CPI-455 for 48 hours. G- Relative mRNA expression levels of KDM5A, KDM5B and RRM2 in cells treated, each 24 hours, with 12.5 mM KDM5 inhibitor CPI-455 (+) or DMSO (-) for 48 hours. Expression levels were normalized to the reference gene P0 (ribosomal phosphoprotein P0) and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 3 independent experiments are shown. A paired t-test indicated no significant difference for all tested genes between CPI treated and untreated cells. H- Percentage of living cells following treatment each 24 hours with CPI-455 for 72 hours (+) or DMSO (-). The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for DMSO treated cells. A paired t-test indicated no significant difference between CPI treated and untreated cells.

Article Snippet: The plasmid pCDNA 3 -RRM2 was purchased from Addgene.

Techniques: Expressing, Transfection, Standard Deviation, Western Blot, Control, Imaging, Labeling, Staining, Negative Control

A --Viability of U2OS, H25 and H50, measured by WST assay, 72 hours following treatment with increasing doses of HU, as indicated. B --Western--blot analysis of CHK1 and S354--phospho CHK1 (P--CHK1), in U2OS, H25 and H50 cells before and following 1 hour treatment with 1mM HU. C-- mRNA expression levels of KDM5A, KDM5B and RRM2, in U2OS, H25 and H50 cell lines. mRNA expression are normalized with the reference gene P0, and calculated relative to 1 for the siCtle. The mean and standard deviation from 3 independent experiments are shown. * pvalue<0.05 using a paired t--test. D --levels of KDM5A, KDM5B and RRM2 were analyzed by western--blot in the parental U2OS cells and its HU tolerant derivatives H25 and H50, grown in the presence of HU at 0, 0.25, and 0.5 mM respectively. GAPDH is used as a loading control. A representative experiment is shown.

Journal: bioRxiv

Article Title: KDM5 histone-demethylases contribute to replication stress response and tolerance

doi: 10.1101/2019.12.16.877399

Figure Lengend Snippet: A --Viability of U2OS, H25 and H50, measured by WST assay, 72 hours following treatment with increasing doses of HU, as indicated. B --Western--blot analysis of CHK1 and S354--phospho CHK1 (P--CHK1), in U2OS, H25 and H50 cells before and following 1 hour treatment with 1mM HU. C-- mRNA expression levels of KDM5A, KDM5B and RRM2, in U2OS, H25 and H50 cell lines. mRNA expression are normalized with the reference gene P0, and calculated relative to 1 for the siCtle. The mean and standard deviation from 3 independent experiments are shown. * pvalue<0.05 using a paired t--test. D --levels of KDM5A, KDM5B and RRM2 were analyzed by western--blot in the parental U2OS cells and its HU tolerant derivatives H25 and H50, grown in the presence of HU at 0, 0.25, and 0.5 mM respectively. GAPDH is used as a loading control. A representative experiment is shown.

Article Snippet: The plasmid pCDNA 3 -RRM2 was purchased from Addgene.

Techniques: WST Assay, Western Blot, Expressing, Standard Deviation, Control

A- Relative mRNA expression levels of KDM5A, KDM5B, and RRM2, upon transfection of the indicated siRNA in H50 cells. Expression levels were normalized to the reference gene P0 and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 4 independent experiments are shown, following normalization to 100 for siCtle treated cells. A paired t-test indicates significant difference between siK5A+B or siRRM2 and siCtle treated cells (*: pvalue<0.05, **: pvalue<0.01) B- Western-blot analysis of KDM5A, KDM5B, RRM2 and GAPDH as a loading control from H50 cells transfected with the indicated siRNA. C -Percentage of living cells following transfection of the indicated siRNA. The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for siCtle treated cells. A paired t-test indicates significant difference between siK5A+B or siRRM2 and siCtle treated cells (*: pvalue=0.013, **: pvalue=0.011) D- Western-blot analysis of KDM5A, KDM5B, RRM2, GAPDH, H3K4me3 and histone H3 from U2OS cells treated each 24 hours with 12.5 μM KDM5 inhibitor CPI-455 (+) or DMSO (-) for 48 hours. E- Relative mRNA expression levels of KDM5A, KDM5B and RRM2 in cells treated, each 24 hours, with 12.5 μM KDM5 inhibitor CPI-455 (+) or DMSO (-) for 48 hours. Expression levels were normalized to the reference gene P0 and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 3 independent experiments are shown. The star * indicates significant difference between siK5A+B and siCtle treated cells (pvalue < 0.05, paired t test). F- Percentage of living cells following treatment of H50 cells each 24 hours with 12.5 μM CPI-455 for 72 hours (+) or DMSO (-). The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for DMSO-treated cells. G- H50 cells were treated with the indicated siRNA and 24 hours later transfected with pCDNA 3 -RRM2 p(RRM2) (+) or the empty vector (-). Expression of KDM5A, KDM5B, RRM2 and GAPDH were analyzed by western-blot 24 hours after plasmids transfection. H -Percentage of living cells 72 hours following electroporation of the indicated siRNA, combined to transfection of pCDNA 3 -RRM2 (pRRM2: +) or the empty vector (pCDNA 3: -) 24 hours later. To ensure efficient knockdown of KDM5A/B, cells were transfected once more with siRNA 24 hours following plasmids transfection. The mean and standard deviation from 4 independent experiments are shown, following normalization to 100 for siCtle/pCDNA 3 transfected cells. * Statistical analysis with a paired t-test indicated that RRM2 surexpression significantly rescue the viability of K5A/B depleted cells with a pvalue <0.05.

Journal: bioRxiv

Article Title: KDM5 histone-demethylases contribute to replication stress response and tolerance

doi: 10.1101/2019.12.16.877399

Figure Lengend Snippet: A- Relative mRNA expression levels of KDM5A, KDM5B, and RRM2, upon transfection of the indicated siRNA in H50 cells. Expression levels were normalized to the reference gene P0 and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 4 independent experiments are shown, following normalization to 100 for siCtle treated cells. A paired t-test indicates significant difference between siK5A+B or siRRM2 and siCtle treated cells (*: pvalue<0.05, **: pvalue<0.01) B- Western-blot analysis of KDM5A, KDM5B, RRM2 and GAPDH as a loading control from H50 cells transfected with the indicated siRNA. C -Percentage of living cells following transfection of the indicated siRNA. The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for siCtle treated cells. A paired t-test indicates significant difference between siK5A+B or siRRM2 and siCtle treated cells (*: pvalue=0.013, **: pvalue=0.011) D- Western-blot analysis of KDM5A, KDM5B, RRM2, GAPDH, H3K4me3 and histone H3 from U2OS cells treated each 24 hours with 12.5 μM KDM5 inhibitor CPI-455 (+) or DMSO (-) for 48 hours. E- Relative mRNA expression levels of KDM5A, KDM5B and RRM2 in cells treated, each 24 hours, with 12.5 μM KDM5 inhibitor CPI-455 (+) or DMSO (-) for 48 hours. Expression levels were normalized to the reference gene P0 and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 3 independent experiments are shown. The star * indicates significant difference between siK5A+B and siCtle treated cells (pvalue < 0.05, paired t test). F- Percentage of living cells following treatment of H50 cells each 24 hours with 12.5 μM CPI-455 for 72 hours (+) or DMSO (-). The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for DMSO-treated cells. G- H50 cells were treated with the indicated siRNA and 24 hours later transfected with pCDNA 3 -RRM2 p(RRM2) (+) or the empty vector (-). Expression of KDM5A, KDM5B, RRM2 and GAPDH were analyzed by western-blot 24 hours after plasmids transfection. H -Percentage of living cells 72 hours following electroporation of the indicated siRNA, combined to transfection of pCDNA 3 -RRM2 (pRRM2: +) or the empty vector (pCDNA 3: -) 24 hours later. To ensure efficient knockdown of KDM5A/B, cells were transfected once more with siRNA 24 hours following plasmids transfection. The mean and standard deviation from 4 independent experiments are shown, following normalization to 100 for siCtle/pCDNA 3 transfected cells. * Statistical analysis with a paired t-test indicated that RRM2 surexpression significantly rescue the viability of K5A/B depleted cells with a pvalue <0.05.

Article Snippet: The plasmid pCDNA 3 -RRM2 was purchased from Addgene.

Techniques: Expressing, Transfection, Standard Deviation, Western Blot, Control, Plasmid Preparation, Electroporation, Knockdown

( a ) DNA fibre assay measuring the replication fork progression speed in GBM cells (GBM01 and GBM02) transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). See also Table 1. ( b ) Replication fork recovery assay showing the quantification of CldU tract length in GBM01 cells transduced with shCTRL or 2 non-overlapping BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4) and treated or not with 2 mM HU (4 h) prior CldU labelling. See also Table 2; . ( c ) Immunoblot analysis of RRM2 protein levels in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). ( d , e ) RT-qPCR analysis of BRCA1 and RRM2 mRNA levels in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). ( f ) FACS analysis of RRM2 protein level changes throughout cell cycle in GBM cells (GBM01) transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). ( g ) Partial sequence of the human RRM2 promoter (GenBank accession number AY032750) , which was used to design Chip primers: P1 primer forward (F)/reverse (R) and P2 primer forward (F)/reverse (R). Positions are numbered from the downstream transcription initiation site (+1). Putative binding sites for transcription factors are color-coded and identified above the sequence. ( h ) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in GBM01-03 cells using primer set P1 and P2. ( i ) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in NHA-DRB and BJ cells using primer set P1 and P2. ( j ) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in PC3, HELA, OVCAR and Cal51 cells using primer set P1 and P2. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test in d – f , h – j or Student’s t test ( a , b ) and all data are shown as means±s.d. and performed as technical triplicates. (* P <0.05, ** P <0.005, *** P <0.005, **** P <0.0001; NS represents non-significance).

Journal: Nature Communications

Article Title: BRCA1-regulated RRM2 expression protects glioblastoma cells from endogenous replication stress and promotes tumorigenicity

doi: 10.1038/ncomms13398

Figure Lengend Snippet: ( a ) DNA fibre assay measuring the replication fork progression speed in GBM cells (GBM01 and GBM02) transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). See also Table 1. ( b ) Replication fork recovery assay showing the quantification of CldU tract length in GBM01 cells transduced with shCTRL or 2 non-overlapping BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4) and treated or not with 2 mM HU (4 h) prior CldU labelling. See also Table 2; . ( c ) Immunoblot analysis of RRM2 protein levels in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). ( d , e ) RT-qPCR analysis of BRCA1 and RRM2 mRNA levels in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). ( f ) FACS analysis of RRM2 protein level changes throughout cell cycle in GBM cells (GBM01) transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). ( g ) Partial sequence of the human RRM2 promoter (GenBank accession number AY032750) , which was used to design Chip primers: P1 primer forward (F)/reverse (R) and P2 primer forward (F)/reverse (R). Positions are numbered from the downstream transcription initiation site (+1). Putative binding sites for transcription factors are color-coded and identified above the sequence. ( h ) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in GBM01-03 cells using primer set P1 and P2. ( i ) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in NHA-DRB and BJ cells using primer set P1 and P2. ( j ) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in PC3, HELA, OVCAR and Cal51 cells using primer set P1 and P2. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test in d – f , h – j or Student’s t test ( a , b ) and all data are shown as means±s.d. and performed as technical triplicates. (* P <0.05, ** P <0.005, *** P <0.005, **** P <0.0001; NS represents non-significance).

Article Snippet: GBM01 cells stably overexpressing RRM2 were generated by wet reverse transfection using the X-tremeGENE HP DNA Transfection Reagent (Roche, cat. no. 06 366 244 001). pcDNA3 RRM2 was a gift from Edward Whang (Addgene plasmid #13796) and pcDNA3.1 empty vector was used as control (Invitrogen).

Techniques: Transduction, Western Blot, Quantitative RT-PCR, Sequencing, Binding Assay, Immunoprecipitation

( a ) Luciferase assay of transcriptional activation of the RRM2 promoter in GBM01 transduced with shCTRL or shBRCA1-2/shBRCA1-4. ( b ) Luciferase assay of transcriptional activation of the RRM2 promoter in GBM01 after siRNA-mediated knockdown of Sp1; AP-1 and E2F1 in comparison to BRCA1. ( c ) Luciferase assay of transcriptional activation of the RRM2 promoter in GBM01 after siRNA-mediated knockdown of BRCA1 and E2F1 alone or on combination. ( d ) Chip immunoprecipitation of E2F1 binding RRM2 promoter in GBM01 cells using primer set P1 and P2. ( e ) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in GBM01 cells transfected with siCTRL and siE2F1 using primer set P1 and P2. ( f ) DNA fibre assay measuring the replication fork progression speed in GBM cells lacking BRCA1 (shBRCA1-2 and shBRCA1-4) or not (shCTRL) transfected with either control vector (pcDNA) or vector expressing RRM2 (pcDNA-RRM2). See also Table 3. ( g ) Cell viability of GBM cells lacking BRCA1 (shBRCA1-2 and shBRCA1-4) or not (shCTRL) transfected with either control vector (pcDNA) or vector expressing RRM2 (pcDNA-RRM2). ( h ) Immunoblot analysis of BRCA1, p-RPA, RPA and RRM2 in GBM cells lacking BRCA1 (shBRCA1-2 and shBRCA1-4) or not (shCTRL) transfected with either control vector (pcDNA) or vector expressing RRM2 (pcDNA-RRM2). ( i ) Viability-based assessment of EC 50 triapine concentrations in GBM01, GBM02 and GBM03 cells. ( j ) DNA fibre assay measuring the replication fork progression speed in GBM cells treated with DMSO or EC 50 triapine. See also Table 4. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test in a – d , f , h or Student’s t test ( e , i ) and all data are shown as means ±s.d. and performed as technical triplicates. (* P <0.05, ** P <0.005, *** P <0.005, **** P <0.0001; NS represents non-significance).

Journal: Nature Communications

Article Title: BRCA1-regulated RRM2 expression protects glioblastoma cells from endogenous replication stress and promotes tumorigenicity

doi: 10.1038/ncomms13398

Figure Lengend Snippet: ( a ) Luciferase assay of transcriptional activation of the RRM2 promoter in GBM01 transduced with shCTRL or shBRCA1-2/shBRCA1-4. ( b ) Luciferase assay of transcriptional activation of the RRM2 promoter in GBM01 after siRNA-mediated knockdown of Sp1; AP-1 and E2F1 in comparison to BRCA1. ( c ) Luciferase assay of transcriptional activation of the RRM2 promoter in GBM01 after siRNA-mediated knockdown of BRCA1 and E2F1 alone or on combination. ( d ) Chip immunoprecipitation of E2F1 binding RRM2 promoter in GBM01 cells using primer set P1 and P2. ( e ) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in GBM01 cells transfected with siCTRL and siE2F1 using primer set P1 and P2. ( f ) DNA fibre assay measuring the replication fork progression speed in GBM cells lacking BRCA1 (shBRCA1-2 and shBRCA1-4) or not (shCTRL) transfected with either control vector (pcDNA) or vector expressing RRM2 (pcDNA-RRM2). See also Table 3. ( g ) Cell viability of GBM cells lacking BRCA1 (shBRCA1-2 and shBRCA1-4) or not (shCTRL) transfected with either control vector (pcDNA) or vector expressing RRM2 (pcDNA-RRM2). ( h ) Immunoblot analysis of BRCA1, p-RPA, RPA and RRM2 in GBM cells lacking BRCA1 (shBRCA1-2 and shBRCA1-4) or not (shCTRL) transfected with either control vector (pcDNA) or vector expressing RRM2 (pcDNA-RRM2). ( i ) Viability-based assessment of EC 50 triapine concentrations in GBM01, GBM02 and GBM03 cells. ( j ) DNA fibre assay measuring the replication fork progression speed in GBM cells treated with DMSO or EC 50 triapine. See also Table 4. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test in a – d , f , h or Student’s t test ( e , i ) and all data are shown as means ±s.d. and performed as technical triplicates. (* P <0.05, ** P <0.005, *** P <0.005, **** P <0.0001; NS represents non-significance).

Techniques: Luciferase, Activation Assay, Transduction, Knockdown, Comparison, Immunoprecipitation, Binding Assay, Transfection, Control, Plasmid Preparation, Expressing, Western Blot

Statistics and comparison of data in Fig. 4f.

Journal: Nature Communications

Article Title: BRCA1-regulated RRM2 expression protects glioblastoma cells from endogenous replication stress and promotes tumorigenicity

doi: 10.1038/ncomms13398

Figure Lengend Snippet: Statistics and comparison of data in Fig. 4f.

Techniques: Comparison

( a ) IHC staining of BRCA1 in normal brain (NB), WHO gr. II, III, IV glioma. Shown are representative sections. Scale bar 50 μm. ( b ) Quantification of ( a ) BRCA1 staining. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test. ( c ) A survival curve for BRCA1 negative ( n =32); BRCA1 low (< 14.5% of BRCA1 + cells, n =35) or BRCA1 high (>14.5% of BRCA1 + cells, n =78; median survival of 230 days) glioma patients; Log-rank P value=0.00. Median survival for BRCA1 negative and low patient is not available as >50% of patients were alive at the end of study. ( d ) A survival curve for BRCA1 high (>14.5% of BRCA1 + cells, n =60, median survival of 159 days) and BRCA1 low (<14.5% of BRCA1 + cells, n =15; median survival of 251 days) GBM patients. Log-rank P -value=0.293. ( e ) Quantification of RRM2 staining (% of positive cells). Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test. ( f ) A survival curve for RRM2 negative ( n =64) and RRM2 positive ( n =81; median survival of 222 days) glioma patients, where the median RRM2-positivity is 1% and Log-rank P value=0.00. Median survival for RRM2 negative patients is not available as more than 50% of patients were alive at the end of study. ( g ) A survival curve for RRM2 positive ( n =56, median survival of 148 days) and RRM2 negative ( n =19; median survival of 320 days) GBM patients. Log-rank P value=0.23. ( h ) Spearman correlation test confirmed a positive correlation between the percentage of BRCA1 and RRM2-positive cells in human gliomas (our study dataset). ( i ) Spearman correlation test confirmed a positive correlation between BRCA1 and RRM2 mRNA expression in human gliomas (REMBRANDT dataset). (* P <0.05, ** P <0.005, *** P <0.005, **** P <0.0001; NS represents non-significance).

Journal: Nature Communications

Article Title: BRCA1-regulated RRM2 expression protects glioblastoma cells from endogenous replication stress and promotes tumorigenicity

doi: 10.1038/ncomms13398

Figure Lengend Snippet: ( a ) IHC staining of BRCA1 in normal brain (NB), WHO gr. II, III, IV glioma. Shown are representative sections. Scale bar 50 μm. ( b ) Quantification of ( a ) BRCA1 staining. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test. ( c ) A survival curve for BRCA1 negative ( n =32); BRCA1 low (< 14.5% of BRCA1 + cells, n =35) or BRCA1 high (>14.5% of BRCA1 + cells, n =78; median survival of 230 days) glioma patients; Log-rank P value=0.00. Median survival for BRCA1 negative and low patient is not available as >50% of patients were alive at the end of study. ( d ) A survival curve for BRCA1 high (>14.5% of BRCA1 + cells, n =60, median survival of 159 days) and BRCA1 low (<14.5% of BRCA1 + cells, n =15; median survival of 251 days) GBM patients. Log-rank P -value=0.293. ( e ) Quantification of RRM2 staining (% of positive cells). Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test. ( f ) A survival curve for RRM2 negative ( n =64) and RRM2 positive ( n =81; median survival of 222 days) glioma patients, where the median RRM2-positivity is 1% and Log-rank P value=0.00. Median survival for RRM2 negative patients is not available as more than 50% of patients were alive at the end of study. ( g ) A survival curve for RRM2 positive ( n =56, median survival of 148 days) and RRM2 negative ( n =19; median survival of 320 days) GBM patients. Log-rank P value=0.23. ( h ) Spearman correlation test confirmed a positive correlation between the percentage of BRCA1 and RRM2-positive cells in human gliomas (our study dataset). ( i ) Spearman correlation test confirmed a positive correlation between BRCA1 and RRM2 mRNA expression in human gliomas (REMBRANDT dataset). (* P <0.05, ** P <0.005, *** P <0.005, **** P <0.0001; NS represents non-significance).

Techniques: Immunohistochemistry, Staining, Expressing

Role of RRM2 in CRC. ( a ) Summary view of RRM1, RRM2, and RRM2B expression profiles in human tumors using published human oncology microarray data (Oncomine). The number in each cell under ‘Cancer versus Normal’ corresponds to the amount of cancer types that contains a significantly different level of RRM1, RRM2, or RRM2B compared with normal corresponding tissue. Thresholds for significance are: fold expression>2; P -value<0.05 and ranking of gene in the analyses>top 10%. Red signifies the gene overexpression in the analyses; blue represents the gene underexpression. Intensity of color signifies the best rank of gene in those analyses. ( b ) A microarray dataset (GSE8671) of colorectal adenomas and adjacent normal mucosa was obtained from NCBI GEO database. Probe IDs of RRM2: 209773_s_at. ( c ) A microarray dataset (GSE1710) of patients with Crohn’s disease and ulcerative colitis was obtained from NCBI GEO database. RRM2 expression in these datasets was shown.

Journal: Cell Death Discovery

Article Title: Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer

doi: 10.1038/cddiscovery.2016.27

Figure Lengend Snippet: Role of RRM2 in CRC. ( a ) Summary view of RRM1, RRM2, and RRM2B expression profiles in human tumors using published human oncology microarray data (Oncomine). The number in each cell under ‘Cancer versus Normal’ corresponds to the amount of cancer types that contains a significantly different level of RRM1, RRM2, or RRM2B compared with normal corresponding tissue. Thresholds for significance are: fold expression>2; P -value<0.05 and ranking of gene in the analyses>top 10%. Red signifies the gene overexpression in the analyses; blue represents the gene underexpression. Intensity of color signifies the best rank of gene in those analyses. ( b ) A microarray dataset (GSE8671) of colorectal adenomas and adjacent normal mucosa was obtained from NCBI GEO database. Probe IDs of RRM2: 209773_s_at. ( c ) A microarray dataset (GSE1710) of patients with Crohn’s disease and ulcerative colitis was obtained from NCBI GEO database. RRM2 expression in these datasets was shown.

Article Snippet: Horseradish peroxidase-labeled goat anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). pcDNA3-RRM2 plasmid was purchased from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Microarray, Over Expression

Identification of GW8510 as a potential RRM2 inhibitor. ( a ) The chemical structures of GW8510. ( b ) HCT116 cells were treated with various doses of GW8510 for 72 h. The cell viability was analyzed by an MTT assay. ( c ) HCT116 cells were treated with various doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( d ) HCT116 cells were treated with various doses of GW8510 for 24 h in the absence or presence of 5 μ M MG132. The protein expressions were analyzed by western blots. ( e ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 48 h, and then treated with indicated doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( f ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 24 h, and then treated with indicated doses of GW8510 for 72 h. The cell viability was analyzed by an MTT assay.

Journal: Cell Death Discovery

Article Title: Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer

doi: 10.1038/cddiscovery.2016.27

Figure Lengend Snippet: Identification of GW8510 as a potential RRM2 inhibitor. ( a ) The chemical structures of GW8510. ( b ) HCT116 cells were treated with various doses of GW8510 for 72 h. The cell viability was analyzed by an MTT assay. ( c ) HCT116 cells were treated with various doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( d ) HCT116 cells were treated with various doses of GW8510 for 24 h in the absence or presence of 5 μ M MG132. The protein expressions were analyzed by western blots. ( e ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 48 h, and then treated with indicated doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( f ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 24 h, and then treated with indicated doses of GW8510 for 72 h. The cell viability was analyzed by an MTT assay.

Techniques: MTT Assay, Western Blot, Transfection, Control, Plasmid Preparation

The gene expression signatures of compounds most positively (mean score> 0.7 and P <0.01) correlated with that of RRM2 siRNA

Journal: Cell Death Discovery

Article Title: Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer

doi: 10.1038/cddiscovery.2016.27

Figure Lengend Snippet: The gene expression signatures of compounds most positively (mean score> 0.7 and P <0.01) correlated with that of RRM2 siRNA

Techniques: Gene Expression, Activity Assay

GW8510 induced autophagic cell death of CRC cells. ( a ) HCT116 cells were treated with various doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( b ) HCT116 cells were treated with various doses of GW8510 for 48 h. The protein expressions were analyzed by western blots. ( c ) HCT116 cells were treated with 2 μ M GW8510 for 48 h in the absence or presence of 50 μ M ZVAD-FMK. The protein expressions were analyzed by western blots. ( d ) HCT116 cells were transiently transfected with RRM2 siRNA for 48 h, and then treated with 4 μ M GW8510 for 24 h. The protein expressions were analyzed by western blots. ( e ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 48 h, and then treated with 4 μ M GW8510 for 24 h. The protein expressions were analyzed by western blots.

Journal: Cell Death Discovery

Article Title: Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer

doi: 10.1038/cddiscovery.2016.27

Figure Lengend Snippet: GW8510 induced autophagic cell death of CRC cells. ( a ) HCT116 cells were treated with various doses of GW8510 for 24 h. The protein expressions were analyzed by western blots. ( b ) HCT116 cells were treated with various doses of GW8510 for 48 h. The protein expressions were analyzed by western blots. ( c ) HCT116 cells were treated with 2 μ M GW8510 for 48 h in the absence or presence of 50 μ M ZVAD-FMK. The protein expressions were analyzed by western blots. ( d ) HCT116 cells were transiently transfected with RRM2 siRNA for 48 h, and then treated with 4 μ M GW8510 for 24 h. The protein expressions were analyzed by western blots. ( e ) HCT116 cells were transiently transfected with a RRM2-overexpressing (pcDNA3-RRM2) or a control (pcDNA3) plasmid for 48 h, and then treated with 4 μ M GW8510 for 24 h. The protein expressions were analyzed by western blots.

Techniques: Western Blot, Transfection, Control, Plasmid Preparation

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