pcdna3 rrm2 plasmid Search Results


pcdna3 1 rrm2  (Addgene inc)
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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; <t>RRM2</t> 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.
Pcdna3 1 Rrm2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 cells were transfected with pcDNA 3.1 (#V79520, Invitogen) or pcDNA3.1 RRM2 (Addgene, Plasmid #13796) using Lipofectamine 3000 (Invitrogen).

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.

Article Snippet: MCF7 cells were transfected with pcDNA 3.1 (#V79520, Invitogen) or pcDNA3.1 RRM2 (Addgene, Plasmid #13796) using Lipofectamine 3000 (Invitrogen).

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.

Article Snippet: MCF7 cells were transfected with pcDNA 3.1 (#V79520, Invitogen) or pcDNA3.1 RRM2 (Addgene, Plasmid #13796) using Lipofectamine 3000 (Invitrogen).

Techniques: Inhibition, Solvent, Staining, Positive Control