Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A novel mechanism driving poor-prognosis prostate cancer: overexpression of the DNA repair gene, ribonucleotide reductase small subunit M2 (RRM2)

doi: 10.1158/1078-0432.CCR-18-4046

Figure Lengend Snippet: (A) dNTP production in small interfering RNA (siRNA)-transfected cells. dNTP was detected at both 48 hours and 72 hours post-transfection of siRNAs in C4-2 cells. siNS, nonspecific siRNA. (B) siRRM2-induced DNA damage. DNA damage marker activation was monitored in LNCaP and C4-2 cells using Muse multi-color DNA damage kit. (C) Activation of H2A.X was confirmed by immunoblotting. (D) and (E) Analysis of cell proliferation (D) and cell cycle (E) in transfected cells. (F) Apoptosis detected by Annexin V assays and immunoblots. (G) dNTP production in empty vector (EV)/RRM2-overexpressing PC-3 cells (PC3-EV; PC3-RRM2). (H) Cell proliferation in stable cells. (I) soft agar assays of stable PC-3 cells. The colony numbers were normalized to those in the control cells. (J) Wound healing assays after the scratch done for 24 hours. (K) Invasion assays after cells were plated for 48 hours. (L) and (M) EMT marker expression detected by qPCR (L) and immunoblots (M) in both EV- and RRM2-expressing PC-3 cells. (N) Invasion assays after multiple siRNAs were transfected in PC3-RRM2 cells. Figure 1 values represent the mean ± S.E. of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 vs control groups treated with empty vector (EV) or with nonspecific (siNS) siRNA.

Article Snippet: For luciferase reporter assays, siRNAs were transfected in cells for 24 hours and 500 ng of RRM2 promoter reporters (GeneCopoeia) containing 0kb or 3kb promoter sequence of human RRM2 were cotransfected with Lipofectamine 2000 (Invitrogen).

Techniques: Small Interfering RNA, Transfection, Marker, Activation Assay, Western Blot, Plasmid Preparation, Control, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A novel mechanism driving poor-prognosis prostate cancer: overexpression of the DNA repair gene, ribonucleotide reductase small subunit M2 (RRM2)

doi: 10.1158/1078-0432.CCR-18-4046

Figure Lengend Snippet: (A)-(C) The correlation of gene alteration with the fraction of genome altered (FGA) and Gleason grades in TCGA cohort was visualized in (A). The statistical quantitation was shown in (B) and (C). (D) The correlation of RRM2 level with Gleason grade in tumor and matched normal tissues in the PHS/HPFS cohorts. (E) and (F) The correlation of RRM2 expression with tumor progression in Taylor and Grasso cohorts. RRM2 levels were analyzed in prostate gland (PG), primary (Pri), and metastasis (Met) tissue samples. (G) The association of RRM2 expression and the disease-free survival in the TCGA, Taylor, and Glinsky cohorts. (H) The correlation of RRM2 with the risk of lethal prostate cancer over long-term follow-up, independent from clinical characteristics and Gleason grade, in the combined HPFS and PHS prostate cancer cohorts. The odd ratios for lethal disease were adjusted for Gleason score. ****, p<0.0001 vs comparator groups.

Article Snippet: For luciferase reporter assays, siRNAs were transfected in cells for 24 hours and 500 ng of RRM2 promoter reporters (GeneCopoeia) containing 0kb or 3kb promoter sequence of human RRM2 were cotransfected with Lipofectamine 2000 (Invitrogen).

Techniques: Quantitation Assay, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A novel mechanism driving poor-prognosis prostate cancer: overexpression of the DNA repair gene, ribonucleotide reductase small subunit M2 (RRM2)

doi: 10.1158/1078-0432.CCR-18-4046

Figure Lengend Snippet: (A) Gene set enrichment analysis (GSEA) of transcriptomic changes. The histograms showed the distribution of select top GSEA molecular signatures: MYC targets (MYC_up_V1_up gene set); E2F targets (hallmark_E2F_targets gene set), cell cycle (Module_54 gene set), p53 pathway (hallmark_p53_pathway), and apoptosis (hallmark apoptosis). Up-gene (up-regulated genes); Down-gene (down-regulated genes). (B) Multiple targets of pathways were validated by quantitative reverse transcription PCR (qRT-PCR). (C) siRRM2-regulated gene profiling and the correlation of these genes with disease-free survival (DFS) in Taylor cohort. (D) Significant enrichment in EMT and Angiogenesis gene sets in RRM2-overexpressing PC-3 cells. (E) RRM2-regulated 126-gene profiling and correlation with the clinical outcome in clinical cohorts. 126 genes were revealed by overlapping 1230 up-regulated genes in PC3-RRM2 cells with 627 common genes positively correlated with RRM2 overexpression in three prostate cancer cohorts (TCGA, Kumar, and SU2C/PCF). The correlation of these genes with disease-free survival was analyzed in the Taylor cohort.

Article Snippet: For luciferase reporter assays, siRNAs were transfected in cells for 24 hours and 500 ng of RRM2 promoter reporters (GeneCopoeia) containing 0kb or 3kb promoter sequence of human RRM2 were cotransfected with Lipofectamine 2000 (Invitrogen).

Techniques: Reverse Transcription, Quantitative RT-PCR, Over Expression

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A novel mechanism driving poor-prognosis prostate cancer: overexpression of the DNA repair gene, ribonucleotide reductase small subunit M2 (RRM2)

doi: 10.1158/1078-0432.CCR-18-4046

Figure Lengend Snippet: (A) dNTP production in COH29-treated C4-2 cells. Two doses of COH29 were used in C4-2 cells, and dNTP production was detected after 24 hours of treatment. (B) and (C) Activation of DNA damage markers. (D) Cell proliferation assay. (E) Cell cycle analysis after 48 hours of COH29 treatment. (F) COH29-induced apoptosis. (G) The global mRNA changes induced by COH29 in C4-2 cells, after 48 hours of treatment. (H) GSEA analysis of mRNA profiling in 20 μM of COH29-treated cells. The histograms showed the distribution of select top GSEA molecular signatures. Up-gene (COH29-induced up-regulated genes); Down-gene (COH29-induced down-regulated genes). NES, normalized enrichment score; FDR, false discovery rate. (I) and (J) Identification of targeted genes by inhibition of RRM2. Genes affected by inhibition of RRM2 in cells were overlapped with genes correlated with RRM2 overexpression in Taylor cohort to reveal 33 down-regulated genes and 12 up-regulated genes (I). These gene panels were validated in three additional prostate cancer cohorts (J). The Figure 4 values represent the mean ± S.E. of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 vs control groups.

Article Snippet: For luciferase reporter assays, siRNAs were transfected in cells for 24 hours and 500 ng of RRM2 promoter reporters (GeneCopoeia) containing 0kb or 3kb promoter sequence of human RRM2 were cotransfected with Lipofectamine 2000 (Invitrogen).

Techniques: Activation Assay, Proliferation Assay, Cell Cycle Assay, Inhibition, Over Expression, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A novel mechanism driving poor-prognosis prostate cancer: overexpression of the DNA repair gene, ribonucleotide reductase small subunit M2 (RRM2)

doi: 10.1158/1078-0432.CCR-18-4046

Figure Lengend Snippet: (A)-(B) Phospho-kinase array analysis after 24-hour COH29 treatment in C4-2 cells. The whole-cell lysates were collected for human phospho-kinase array analysis. Each membrane contains kinase-specific antibodies (number indicated). Relative phosphorylation of spots was quantified by Image J software and the value of vehicle (0 μM) was set up as “1” (B). (C) Validation of phospho-kinase array by immunoblots. For siRNA-treated groups, cell lysates were collected after transfection for 48 hours. The representative blots for each condition are shown and the values represent the mean ± S.E. of two independent experiments. (D) Druggable RRM2 signatures. Top three drugs from ToppGene analysis and COH29 are shown (left panel). Numbers of genes down-regulated by docetaxel (in PC-3 cells) and docetaxel + ADT (in patients) are shown (right panel). (E)-(F) Antitumor effects of COH29 in vivo. Tumor volumes and weights were measured following oral administration of COH29 (200 mg/kg) in established C4-2 xenograft tumors (n=6 per group). Values are means ± S.E. ***P<0.001 versus vehicle mice. (G)-(H) Regulation of key genes by COH29 in vivo. Multiple genes regulated by COH29 were assessed in xenograft tumors by immunohistochemistry staining (G) or immunoblotting (H).

Article Snippet: For luciferase reporter assays, siRNAs were transfected in cells for 24 hours and 500 ng of RRM2 promoter reporters (GeneCopoeia) containing 0kb or 3kb promoter sequence of human RRM2 were cotransfected with Lipofectamine 2000 (Invitrogen).

Techniques: Membrane, Phospho-proteomics, Software, Biomarker Discovery, Western Blot, Transfection, In Vivo, Immunohistochemistry, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A novel mechanism driving poor-prognosis prostate cancer: overexpression of the DNA repair gene, ribonucleotide reductase small subunit M2 (RRM2)

doi: 10.1158/1078-0432.CCR-18-4046

Figure Lengend Snippet: (A) H3K27Ac ChIP-Seq in tissues. The binding signal on RRM2 enhancer (1Mb region) was quantified. (B) The strategy to identify RRM2-targeting transcription factors (TFs). Human TFs were selected in the genes positively correlated with RRM2 expression in prostate cancer cohorts. Yellow/orange/pink bars indicate that TFs appear in four/three/two cohorts. (C) The correlation of FOXM1 and RRM2 in PHS/HPFS cohorts. (D) FOXM1 binding on RRM2 promoter in cancer cells. The overview of multiple ChIP-Seq datasets were extracted from Cistrome Data browser. P1-P3 primers were designed for FOXM1 ChIP-PCR. (E) FOXM1 or H3K4me3-ChIP-PCR on RRM2 promoter. (F) RRM2 promoter activity regulated by FOXM1. The reporters without (R2-0K) or with (R2-3K) 3kb RRM2 promoter sequence were transfected in siRNA-treated 22Rv1 cells. (G) and (H) Inhibition of RRM2 expression by siFOXM1 in 22Rv1 and C4-2 cells. (I) Inhibition of FOXM1 targets by FDI-6 (20 μM) in 22Rv1 cells. The Figure 6 values represent the mean ± S.E. of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 vs control groups.

Article Snippet: For luciferase reporter assays, siRNAs were transfected in cells for 24 hours and 500 ng of RRM2 promoter reporters (GeneCopoeia) containing 0kb or 3kb promoter sequence of human RRM2 were cotransfected with Lipofectamine 2000 (Invitrogen).

Techniques: ChIP-sequencing, Binding Assay, Expressing, Activity Assay, Sequencing, Transfection, Inhibition, Control

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A, RRM2 mRNA expression in pancreatic cancer cell lines relative to expression identified in HPDE. Columns, mean of triplicate; bars, SD. n = 3. B, Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDa) in whole cell lysates of HPDE and pancreatic cancer cell lines. C, Expression of let- 7 family members in pancreatic cancer cell lines relative to expression in HPDE. Columns , mean of triplicate; bars , SD. n = 3; * P <0.05. D, RRM2 is a direct target of let-7 . 293TA and MIA PaCa-2 cells were virally infected for expression of precursors of let-7a-1 , let-7a-2 , let-7a-3 , let-7b , and miR-214 ( negative control ) and subsequently transfected with a RRM2 3′ UTR luciferase reporter construct. Luciferase activities measured 36 h after transfection (normalized relative to renilla activity) were plotted. Columns , mean of triplicate; bars , SD. n = 3. * p <0.05, ** p <0.01.

Article Snippet: RRM2 cDNA without the 3′ UTR region was constructed from RRM2 truclone (RRM2 (NM_001165931) Human cDNA Clone; Product ID: SC326997; Origene, MD) using PCR-based methods.

Techniques: Expressing, Western Blot, Infection, Negative Control, Transfection, Luciferase, Construct, Activity Assay

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A , Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDA) in whole cell lysates of MIA PaCa-2 overexpressing precursors of let-7 family members. Ratios of RRM2 to β-actin band intensities (normalized to control) from three experiments are indicated ( top ). Asterisks indicate significant reductions ( p <0.05) in RRM2 levels compared with control. B , Immunocytochemical detection of RRM2 in exponentially growing MIA PaCa-2 overexpressing pre- let-7 family members. Original magnification, x20. C , MIA PaCa-2 cells stably overexpressing pre- let-7 family members ( red ) or vector alone ( blue ) were treated with gemcitabine (0.1 nM to 100 µM), and percent inhibition of cellular proliferation was measured using an MTT assay. Points , mean of triplicate; bars , SE. n = 3. Gemcitabine IC 50 estimations indicated ( parentheses ).

Article Snippet: RRM2 cDNA without the 3′ UTR region was constructed from RRM2 truclone (RRM2 (NM_001165931) Human cDNA Clone; Product ID: SC326997; Origene, MD) using PCR-based methods.

Techniques: Western Blot, Stable Transfection, Plasmid Preparation, Inhibition, MTT Assay

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A, Western blotting analysis of hENT1 (∼50–55 kDa), hENT2 (50 kDa), hCNT1 (72 kDa), hCNT3 (77 kDa), CDA (55 kDa), dCK (30 kDa), RRM1 (94 kDa), RRM2 (45 kDa), and β-actin (45 kDA) levels in whole cell lysates of Capan-1 and Capan-1-GR cells. B , RRM2 protein increased in a gemcitabine dose-dependent fashion in Capan-1-GR cells. C, Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDA) levels in cells with acquired gemcitabine resistance. Ratios of RRM2 to β-actin band intensities (normalized to untreated cells) from three experiments are indicated ( top ). Asterisk indicates significantly higher RRM2 expression in gemcitabine-resistant cells ( p <0.05) compared with untreated cells. D and E, Relative expression of precursor and mature let-7a in Capan-1 ( D ) and L3.6pl ( E ) cells induced to acquire gemcitabine resistance. Columns, mean of triplicate; bars, SD. n = 3. F , Differential miRNA expression in Capan-1-GR compared with Capan-1. Putative RRM2-modulating miRNAs and their extent of reduction in Capan-1-GR cells are shown ( right ).

Article Snippet: RRM2 cDNA without the 3′ UTR region was constructed from RRM2 truclone (RRM2 (NM_001165931) Human cDNA Clone; Product ID: SC326997; Origene, MD) using PCR-based methods.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A and B, Relative expression of primary let-7a transcripts ( A ) and mature let-7a ( B ) in 2 normal pancreatic tissues and 10 PDAC samples representing various tumor stages. C , Ratios of mature to precursor let-7a transcripts calculated from data points in A and B . D, Western blotting analysis of RRM2 (45 kDa) in total lysates (50 µg) of 6 matched normal-PDAC pairs. Ratios of RRM2 band intensities in PDACs compared to matched normal tissues indicated ( top ). Asterisk indicates significantly higher RRM2 expression in PDAC tissues ( p <0.05) compared with matched normal tissues. E and F, Relative expression of primary let-7a transcripts ( E ) and mature let-7a ( F ) in matched normal-PDAC pairs. G , Ratios of mature to precursor let-7a transcripts calculated from data points in E and F . Asterisk indicates significantly lower mature to precursor let-7a ratio in PDAC tissues ( p <0.05) compared with matched normal tissues.

Article Snippet: RRM2 cDNA without the 3′ UTR region was constructed from RRM2 truclone (RRM2 (NM_001165931) Human cDNA Clone; Product ID: SC326997; Origene, MD) using PCR-based methods.

Techniques: Expressing, Western Blot

Journal: BMC Cancer

Article Title: Selection of suitable reference genes for accurate normalization of gene expression profile studies in non-small cell lung cancer

doi: 10.1186/1471-2407-6-200

Figure Lengend Snippet: Gene expression assays.

Article Snippet: - , RRM2 , ribonucleotide reductase M2 polypeptide , Hs00357247_g1.

Techniques: Gene Expression

Journal: BMC Cancer

Article Title: Selection of suitable reference genes for accurate normalization of gene expression profile studies in non-small cell lung cancer

doi: 10.1186/1471-2407-6-200

Figure Lengend Snippet: Targets fold change homogeneity . PCA and agglomerative hierarchical clustering were used to describe the homogeneity degree of target fold change variation as a function of the reference used. Expression levels of three target genes (RRM2, BRCA1, ERCC2) were normalized with respect to each of the RGs and expressed as -ΔΔCt using normal paired samples as calibrator. A) Small fluctuations in fold change target detection using reliable RGs results in a very limited spread over the PCA space. As expected, POLR2A, rRNA18S, ESD and YAP1 produce the best homogeneous cluster (filled dots). B) Similar results are obtained by hierarchical clustering, where the smallest Euclidean distance is associated with the previously indicated set of genes.

Article Snippet: - , RRM2 , ribonucleotide reductase M2 polypeptide , Hs00357247_g1.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Integration of scRNA-seq and ST-seq identifies hyperproliferative RRM2+ cells features and therapeutic targets in gastric cancer

doi: 10.1186/s12967-025-06847-y

Figure Lengend Snippet: Single-cell transcriptome map of cell types in GC and normal gastric specimens. ( A ) Schematic of study design. ( B ) UMAP plot of 158,622 gastric cells, colored by different groups and ( C ) different clusters. ( D ) UMAP shows 15 identified cell types in GC and normal gastric tissue, colored by cell annotation. ( E ) Heatmap displaying the top five discriminative genes for 15 different cell types. Red indicates the high expression. The box on the right presents the GO enrichment analysis results for the top-five genes. ( F ) The proportion of 15 cell types in two groups. ( G ) Expression level of RRM2 in GC and normal gastric tissues

Article Snippet: Antibody against Alpha-Tubulin (11224-1-AP), and RRM2 (11661-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Integration of scRNA-seq and ST-seq identifies hyperproliferative RRM2+ cells features and therapeutic targets in gastric cancer

doi: 10.1186/s12967-025-06847-y

Figure Lengend Snippet: Characteristics of GC epithelial cells during GC progression by trajectory analysis. ( A ) and ( B ) UMAP plots of epithelial and RRM2 + cells, colored by cell types (A) and in the two groups (B). ( C ) Trajectory analysis suggests a potential transformation pathway from epithelial cells to RRM2 + cells. ( D ) Representative gene expression in GC epithelial cells during GC progression. The color intensity reflects the normalized expression levels of genes. ( E ) Heatmap showing the change trend of MKI67, RRM2, PCNA, and TOP2A across pseudotime

Article Snippet: Antibody against Alpha-Tubulin (11224-1-AP), and RRM2 (11661-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Transformation Assay, Gene Expression, Expressing

Journal: Journal of Translational Medicine

Article Title: Integration of scRNA-seq and ST-seq identifies hyperproliferative RRM2+ cells features and therapeutic targets in gastric cancer

doi: 10.1186/s12967-025-06847-y

Figure Lengend Snippet: Analysis results combining scRNA-seq and ST. ( A ) Distribution of 15 cell types on ST slices from GC tissues ( N = 6), represented by pie charts showing the proportion of different cell types in each spot. ( B ) Distribution of RRM2 and classical proliferation markers in GC tissue sections ( N = 6), with the color getting yellow indicating a higher expression of the genes in that spot

Article Snippet: Antibody against Alpha-Tubulin (11224-1-AP), and RRM2 (11661-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Integration of scRNA-seq and ST-seq identifies hyperproliferative RRM2+ cells features and therapeutic targets in gastric cancer

doi: 10.1186/s12967-025-06847-y

Figure Lengend Snippet: Osalmid inhibits RRM2 to trigger ferroptosis and inhibits GPX4, SLC7A11, and FTH1 expression in gastric cancer. ( A ) The volcano plot showed the upregulated and downregulated genes in MKN45 cells treated with osalmid, as compared to NC (untreated with osalmid). ( B ) GO analysis demonstrated that the osalmid treatment group was engaged in the rRNA processing, ribosome biogenesis, ribonucleoprotein complex biogenesis, and so on. ( C ) KEGG analysis revealed that ferroptosis and the p53 signaling pathway were significantly enriched in the osalmid group. ( D ) Western blot analysis was performed to detect the expression differences of RRM2, FTH1, SLC7A11, GPX4, p53, and p-p53 in MKN45 and MKN7 cells with or without osalmid treatment. ( E ) CCK-8 assay showed the proliferation curve of the MKN7 and MKN45 cells after osalmid treatment or not. ( F ) Cell proliferation in the MKN7 and MKN45 were assessed by colony formation assay after osalmid treatment or not. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.001

Article Snippet: Antibody against Alpha-Tubulin (11224-1-AP), and RRM2 (11661-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Western Blot, CCK-8 Assay, Colony Assay

Journal: Journal of Translational Medicine

Article Title: Integration of scRNA-seq and ST-seq identifies hyperproliferative RRM2+ cells features and therapeutic targets in gastric cancer

doi: 10.1186/s12967-025-06847-y

Figure Lengend Snippet: The effects of knocking down RRM2 on cell proliferation, colony, and ferroptosis. ( A ) Western blot of RRM2, GPX4, SLC7A11, FTH1, p53, p-p53, and ATM expression levels in RRM2 knockdown and their control cells. ( B ) The CCK-8 assay showed the proliferation curve of the MKN7 and MKN45 cells after transfection with RRM2-targeting siRNA or not. ( C ) Cell proliferation was assessed by colony formation assay in RRM2 knockdown in GC cell lines. ( D ) Comparison of GSH/GSSG ratios between siRRM2 and their control group in MKN45 and MKN7 cells. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.001

Article Snippet: Antibody against Alpha-Tubulin (11224-1-AP), and RRM2 (11661-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Western Blot, Expressing, Knockdown, Control, CCK-8 Assay, Transfection, Colony Assay, Comparison

Journal: Journal of Translational Medicine

Article Title: Integration of scRNA-seq and ST-seq identifies hyperproliferative RRM2+ cells features and therapeutic targets in gastric cancer

doi: 10.1186/s12967-025-06847-y

Figure Lengend Snippet: Silencing p53 alleviates ferroptosis triggered by RRM2 loss in gastric cancer cells. ( A , B ) Knockdown of p53 in the context of RRM2 deficiency reversed the RRM2 knockdown-induced suppression of SLC7A11, FTH1, and GPX4 expression in MKN7 and MKN45. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.001

Article Snippet: Antibody against Alpha-Tubulin (11224-1-AP), and RRM2 (11661-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Knockdown, Expressing

Journal: Journal of Translational Medicine

Article Title: Integration of scRNA-seq and ST-seq identifies hyperproliferative RRM2+ cells features and therapeutic targets in gastric cancer

doi: 10.1186/s12967-025-06847-y

Figure Lengend Snippet: Osalmid-induced ferroptosis in GC in vivo. ( A ) The flow charts design for in vivo experiments. ( B ) Growth curves of subcutaneous tumors in three groups. ( C ) Overview of subcutaneous tumor in three groups after 12 days. ( D ) Tumor weight and volume were assessed in three groups. ( E ) WB quantified the expression of RRM2, GPX4, SLC7A11, FTH1, p53, and p-p53 in tumor tissues in the different groups. ( F ) Comparison of GSH/GSSG ratios between shRRM2 or Osalmid and their control groups. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.001

Article Snippet: Antibody against Alpha-Tubulin (11224-1-AP), and RRM2 (11661-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: In Vivo, Expressing, Comparison, Control

Journal: eLife

Article Title: Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

doi: 10.7554/eLife.27574

Figure Lengend Snippet: ( A ) Line graphs showing mean abundance profiles for Cyclin A2 (CycA), Cyclin B1, Cyclin B2, and GAPDH. Grey ribbons indicate 1 standard deviation from the mean. ( B ) Mean abundance profile for RRM2. ( C ) Flow cytometry analysis RRM2 levels vs. DNA content. ( D ) Flow cytometry-based comparison of beta-tubulin (negative control, left) and RRM2 (right) levels in CycA+ (red) vs. CycA- (blue) prometaphase cells. ( E ) Violin plots showing CycA, histone H3, and RRM2 levels in cells treated with either DMSO or microtubule drugs that activate the spindle assembly checkpoint (nocodazole, monastrol, taxol). ( F ) Violin plots showing levels of RRM2 in cells treated with either vehicle control (DMSO), MG132, apcin + proTAME, or MLN4924.

Article Snippet: DNA, Expression construct , RRM2-GFP , Origene , RG205718 , .

Techniques: Standard Deviation, Flow Cytometry, Negative Control

Journal: eLife

Article Title: Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

doi: 10.7554/eLife.27574

Figure Lengend Snippet: Flow cytometry analysis of cells treated either with non-targeting siRNA (siJumble), or siRNA against RRM2 and stained either with secondary antibody only ( A ), or anti-RRM2 antibody ( B ). ( C ) Immunoblot analysis of siJumble vs. siRRM2 cell lysates. ( D ) Overexpression of GFP-Fibrillarin (top) and RRM2-GFP (bottom). GFP and anti-RRM2 staining shows high correlation.

Article Snippet: DNA, Expression construct , RRM2-GFP , Origene , RG205718 , .

Techniques: Flow Cytometry, Staining, Western Blot, Over Expression

Journal: eLife

Article Title: Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

doi: 10.7554/eLife.27574

Figure Lengend Snippet: ( A ) A representative image of FUCCI U2OS cells immunostained for RRM2. ( B ) A psuedocolour scatter plot with x- and y- axes representing the two proteins used in the FUCCI cell cycle reporter system (Red: Cdt1, Green: Geminin) with colour indicating anti-RRM2 signal. ( C ) Bar chart summarising ( B ).

Article Snippet: DNA, Expression construct , RRM2-GFP , Origene , RG205718 , .

Techniques:

Journal: eLife

Article Title: Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

doi: 10.7554/eLife.27574

Figure Lengend Snippet: Anti-Cyclin B1 and anti-RRM2 signals were measured in U2OS cells by immunofluorescence microscopy. Representative images of cyclin B (A, left) and RRM2 (A, right) staining. (B) Quantitation of immunofluorescence with cells ranked ordered based on Cyclin B1 signal. The arrow indicates nuclear concentration of Cyclin B1 signal, which marks mitotic entry.

Article Snippet: DNA, Expression construct , RRM2-GFP , Origene , RG205718 , .

Techniques: Immunofluorescence, Microscopy, Staining, Quantitation Assay, Concentration Assay

Journal: eLife

Article Title: Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

doi: 10.7554/eLife.27574

Figure Lengend Snippet: A) Data from proteomic datasets from the Lamond laboratory can be easily visualised for the same proteins using the navigation bubble map. User interface features include: B) specifying type of search, including search for individual proteins, GO term, and CORUM complex membership, (C) an input box for protein and other identifiers (e.g. GO term), (D) a ribbon graph showing plots for input protein identifiers (here for illustration are shown RRM2 and CCNB1) with lines indicating mean profile and ribbons indicating s.e.m., (E) an interactive legend to show more information on individual proteins, and F) options to output visualisation as an SVG file or underlying data in CSV format. ( G ) An example plot correlating protein abundance and phosphorylation changes. Several proteins containing ‘early rising’ phosphorylation sites are highlighted: CENPF, TMPO, and RIF1.

Article Snippet: DNA, Expression construct , RRM2-GFP , Origene , RG205718 , .

Techniques:

Journal: eLife

Article Title: Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

doi: 10.7554/eLife.27574

Figure Lengend Snippet:

Article Snippet: DNA, Expression construct , RRM2-GFP , Origene , RG205718 , .

Techniques: Expressing, Construct

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Schematic description of the filtering process for identification of therapeutically relevant target candidates. b) Analysis of RRM2 mRNA expression levels in 50 EwS primary tumors compared to 929 normal tissues samples from 71 tissue types. Data are shown as log2 fold increase normalized to expression values of normal tissues. The dotted line indicates the cut-off value of 2 for candidate selection. c) Analysis of overall survival time of 166 EwS patients stratified for candidate gene expression. P -values (–log10) were determined in Kaplan-Meier analyses using a Mantel-Haenszel test (Bonferroni-adjusted for multiple testing). The dotted line indicates a significance value of 1.3. d) Kaplan-Meier survival analysis of 166 EwS patients stratified by the 78th percentile RRM2 expression. P -value determined by log-rank test.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Expressing, Selection, Gene Expression

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Left: Heat map for gene expression which is positively or negatively correlated with RRM2 expression in 166 EwS. Right: Gene ontology (GO) enrichment analysis of RRM2 and its co-expressed genes derived from gene expression data sets of 166 EwS tumors. Pearson correlation coefficients between RRM2 and other genes were determined, of which those with |r Pearson | > 0.5 were further analyzed by GO enrichment analysis. b) Representative images of immunohistochemical RRM2 staining. Scale bar = 50 µm. c) Kaplan-Meier survival analysis of 122 EwS patients stratified by RRM2 protein expression (low IRS≤2, high IRS >2). P -values were determined by log-rank test. d) WGCNA of downregulated genes upon RRM2 silencing in A-673 and ES-7 cells harboring Dox-inducible shRRM2 constructs. NES, normalized enrichment score. e) Analysis of tumor growth of EwS cell lines A-673 and TC-71 harboring Dox-inducible shRRM2 constructs or non-targeting shRNA (shControl) xenografted in NSG mice. Once tumors were palpable, animals were randomized in Dox (+) or Dox (–) group. Tumor growth on time course and f) Tumor weight at the experimental endpoint. Arrows indicate treatment start. Values are normalized to shControl. Horizontal bars represent means and whiskers SEM. FC, fold change. P -values were calculated at the experimental endpoint with two-sided (tumor growth) or one-sided (tumor weight) Mann-Whitney test. g) Representative micrographs of xenografts immunohistochemically stained for RRM2, cleaved caspase-3 (CC3) or γH2A.X (scale bar=250 µm, 50 µm, 250 µm, respectively). h) quantification of positive cells for cleaved caspase-3 (CC3) (left) and γH2A.X (right). Values were normalized to shControl. Horizontal bars represent means and whiskers SEM. FC, fold change. P -values were calculated at the experimental endpoint using a two-sided Mann-Whitney test.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Gene Expression, Expressing, Derivative Assay, Immunohistochemical staining, Staining, Construct, shRNA, MANN-WHITNEY

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Analysis of proliferation assays upon shRNA-mediated RRM2 silencing in EwS cell lines. Upper: Cell proliferation over 120h upon RRM2 silencing in A-673. Viable cells upon RRM2 silencing (middle) and dead cells (lower) in EwS cell lines (A-673, ES7, TC-71) harboring Dox-inducible shRRM2 constructs or non-targeting shRNA (shControl). Values are normalized to shControl. Horizontal bars represent means and whiskers SEM. FC, fold change. Two-sided Mann-Whitney test. b) Analysis of clonogenic growth upon shRNA-mediated RRM2 silencing in EwS cell lines (A-673, ES7, TC-71) harboring Dox-inducible shRRM2 constructs or non-targeting shRNA (shControl). Horizontal bars represent means and whiskers SEM. Two-sided Mann-Whitney test at the experimental endpoint.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: shRNA, Construct, MANN-WHITNEY

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Integrative Gene Ontology (GO) enrichment analysis of gene expression microarray data generated in A-673 and ES7 cells after RRM2 silencing or pharmacological RRM2 inhibition by triapine (corresponding IC50 of 0.44 µM or 0.65 µM, respectively). b) Correlation of gene expression between RRM2 and CHEK1 or WEE1 in 166 EwS. Each dot represents an individual expression value. Solid red lines indicate a trend line created by a simple linear regression. P -values were calculated by a two-tailed t-test. c) Drug interaction and combination efficiency analysis between triapine and CHEK1 inhibitor (CCT245737) or WEE1 inhibitor (MK-1775) in 4 EwS cell lines (A-673, ES7, EW-7, TC-71) assessed by combination index. CI value < 1 indicative of synergistic, CI = 1 additive, and CI > 1 antagonistic d) Drug interaction and combination efficiency estimation between triapine and CHEK1 inhibitor (CCT245737) or WEE1 inhibitor (MK-1775) in A-673 EwS cell line assessed by SynergyFinder 2.0. ZIP synergy score > 10, likely to be synergistic; between -10 and 10, likely to be additive; < –10, likely to be antagonistic.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Gene Expression, Microarray, Generated, Inhibition, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Distribution analysis of RRM2 mRNA expression in 166 EwS patients. Each dot represents individual RRM2 expression. b) Correlation analysis of CNVs at the RRM2 locus with RRM2 mRNA expression levels in primary EwS tumors (n=32). The solid line indicates a trend line estimated by a simple linear regression model. b) Correlation analysis of promoter methylation on five CpG sites with RRM2 expression levels in primary EwS tumors (n=40). The solid lines indicate trend lines estimated by a simple linear regression model.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Expressing, Methylation