Fig 2: Immunohistochemical analysis for ERCC1, RRM1, RRM2, and hENT-1 expression in malignant pleural mesothelioma patients. High and low nuclear expression for ERCC1 (a, b), and high and low cytoplasmic expression for RRM1 (d, e), RRM2 (g, h), and hENT-1 (j, k), respectively. Negative controls (without primary antibody) are shown for each molecule in c, f, i, and l, respectively

Fig 3: Kaplan-Meier curves for the probability of recurrence and overall survival in patients with cancer of the uterine cervix according to RRM2 immunoreactivity of cancer tissue cores.(A) Patients with positive RRM2 tended to have a higher probability of recurrence (N = 99, p = 0.094), compared with the patients with negative RRM2. (B) Patients with positive RRM2 had significantly lower survival (N = 101, p = 0.016), compared with the patients with negative RRM2. RRM2, ribonucleotide reductase M2.

Fig 4: RRM2 protein expressions in the SiHa cancer cells of the uterine cervix with shRRM2 #354 or #962 and their correlations with cell viability.(A) Protein levels of RRM2 were detected in the SiHa cervical cancer cells with shRRM2 #354 or #962 using Western blotting. ß-actin was used as the internal control. The relative expressions of RRM2/ß-actin are showed. The effect of RRM2 gene knockdown was stronger in the SiHa cells with shRRM2 #354 compared to those with shRRM2 #962. (B) A greater toxic effect on cell viability using cisplatin was found in the SiHa cells with shRRM2 #354 than in those with shRRM2 #962 compared to the control SiHa shLuc cells, in which shLuc was transfected into SiHa cells. Cell viabilities were detected using 3-(4,5-cimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide assay. RRM2, ribonucleotide reductase M2; ***p<0.001.

Fig 5: ATRi induces degradation of RRM2 and dCK in proliferating CD8+ T cells(A) CD8+ T cells were treated with 6 µM T at 22 h and either 5 mM AZD6738 (ATRi) or 5 mM HU at 24 h. At 30 h, cells were re-plated with and without 6 µM dT in the absence of ATRi and HU. CTV histograms of live (eFluor 780-CD44hiCD8+) cells at 24 h (gray), and 30 and 48 h (with recovery) for ATRi- and HU-treated (6 h) samples (DMSO-treated histograms in black; thymidine-treated histograms in pink). Number of divisions is indicated.(B) CD8+ T cells were activated and treated with 6 µM T at 22 h and either ATRi or HU at 24 h. At 33 h, cells were re-plated with and without 6 µM T. At 48 h, live CD8+ T cells (eFluor 780-CD8+TCRß+) were quantitated.(C) Nucleotide metabolism.(D–G) Immunoblots of proliferating CD8+ T whole-cell extracts. (D) CD8+ T cells were treated with ATRi and HU for 1 or 4 h. (E) CD8+ T cells were treated with increasing concentrations of Ro-3306 (CDK1i) for 15 min followed by ATRi for 1 h. (F) CD8+ T cells were treated with increasing concentrations of CVT-313 (CDK2i) for 15 min followed by ATRi for 1 h. (G) CD8+ T cells were treated with 5 mM MG132 (protease inhibitor) and 5 mM Ro-3306 (CDK1i) for 15 min prior to ATRi for 2 h.(H and I) Whole-cell extracts were prepared from CD8+ T cells treated with ATRi and CDK1i for 1 h. (H) Proteins immunopurified with anti-pSPXK antibodies were immunoblotted for RRM2. (I) Proteins immunopurified with anti-pSQ antibody were immunoblotted for dCK.(J) Quantitation of cellular dUTP in proliferating CD8+ T cells treated with DMSO, ATRi, or HU.(A and B) Two biological replicates; two technical replicates. (D–I) Two biological replicates. (J) Two biological replicates; six technical replicates.(B and J) Mean and SD bars are shown. (B) **p < 0.01, ****p < 0.0001, by one-way ANOVA with Tukey’s multiple comparisons. (J) *p < 0.05, ****p < 0.0001, by one-way ANOVA with Dunnett’s multiple comparisons.