Journal: bioRxiv

Article Title: Quantitative AI-based DNA fiber workflow to study replication stress

doi: 10.1101/2025.03.27.645743

Figure Lengend Snippet: (a) A schematic representation illustrating the concept of how the level of origin activation induced by ATR and CDC7 inhibitors affects the replication fork speed (see text for details). (b) Quantification of QIBC plots in Extended Data Fig. 3a; each bar indicates the median of mean intensity (data are mean ± s.d.; n = 2 independent experiments). (c) Quantification of QIBC plots in Extended Data Fig. 3d; each bar indicates the median of mean intensity (data are mean ± s.d.; n = 2 independent experiments). (d) DNA fiber labeling protocol with indicated DMSO, ATR, and CDC7 inhibitors treatment (5 µM ATRi, 5 µM CDC7i for 1 h). (e) qAID-based quantification of origin firing frequency in U2OS cells treated with indicated inhibitors (data are mean ± s.d.; n = 2 independent experiments). (f) Manual quantification of replication fork speed for ongoing forks in U2OS cells treated with indicated inhibitors. DNA fibers were prepared by spreading technique. Lines represent medians; n ≈ 350 fibers per condition. (g) qAID-based quantification of replication fork speed for ongoing forks in U2OS cells treated with indicated inhibitors. DNA fibers were prepared by spreading technique. Lines represent medians; n ≈ 1000 fibers per condition. (h) Unbiased qAID galleries of DNA fibers in (g) . Both original fiber images and probability images created by the classification network are displayed. Galleries were masked based on segmentation. Scale bar, 10 μm. (i) Quantification of replication fork speed plots in (f) ; data are mean ± s.d.; n = 3 independent experiments. P value was calculated by a two-tailed unpaired t -test (P = 0.0053). (j) Quantification of qAID-based replication fork speed plots in (g) ; data are mean ± s.d.; n = 3 independent experiments. P value was calculated by a two-tailed unpaired t -test (P = 0.0098). (k) qAID-based quantification of replication fork speed for ongoing forks in U2OS cells treated with indicated inhibitors. DNA fibers were prepared by combing technique. Lines represent medians; n ≈ 1000 fibers per condition. (l) Quantification of qAID-based replication fork speed plots in (k) ; data are mean ± s.d.; n = 2 independent experiments. P value was calculated by a two-tailed unpaired t -test (P = 0.0073).

Article Snippet: ATR inhibitor (AZD6738, Selleckchem, S7693), CDC7 inhibitor (BMS-863233, Selleckchem, S7547), hydroxyurea (Merck, H8627), 5-pH-IAA degron (Merck, SML3574), 5-ethynyl-2’-deoxyuridine (EdU, Thermo Fisher Scientific, C10640), Alexa Fluor 647 picolyl azide (Thermo Fisher Scientific, C10640) for EdU Click-IT reaction, 5-Chloro-2′-deoxyuridine (CldU, Merck, C6891), 5-Iodo-2′-deoxyuridine (IdU, Merck, 17125), and S1 nuclease (Thermo Fisher Scientific, ENO321) were used as indicated in corresponding figure legends.

Techniques: Activation Assay, Labeling, Two Tailed Test

Journal: Cancer Cell International

Article Title: Characterization of G2/M checkpoint classifier for personalized treatment in uterine corpus endometrial carcinoma

doi: 10.1186/s12935-025-03667-4

Figure Lengend Snippet: Validation of real-world cohort based on the classifier. A The expression levels of CDC7, ASPM, CENPE, KIF23 and DEPDC1B on qRT-PCR results. B By inputting the mRNA levels of the 5 model genes into the classifier, the real-world UCEC cohort was identified as two G2MC subtypes. Representative immunohistochemistry images of CDC7, ASPM, CENPE, KIF23, and DEPDC1B in the two G2MC subtypes, magnification 40x. C K-M survival analysis of two G2MC subtypes based on optimal cutoff value grouping. The ending events are PFS. D The proportion of progression after treatment in the two groups of G2MC subtypes. E Comparing the proportion of patients with pathological grades G1 to G3 in the two G2MC subtypes in the real-world cohort. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Subsequently, the sections were first incubated with primary antibodies CDC7 (1:200, CUSABIO, China), ASPM (1:200, CUSABIO, China), CENPE (1:500, Sanying, China), KIF23 (1:200, CUSABIO, China), and DEPDC1B (1:200, CUSABIO, China) at 4 °C overnight, and then incubated with multimerized anti-rabbit IgG-HRP secondary antibodies at room temperature for 90 min.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Immunohistochemistry

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Chromatin Protein Dynamics During Replication Origin Firing in Human Cells

doi: 10.1016/j.mcpro.2025.100915

Figure Lengend Snippet: CHK1i induces a CDC7 and CDK-dependent wave of origin firing in the S phase . A , schematic illustration of the OF process showing three crucial steps—preRC, preIC, and the formation of the RF. CHK1 regulates CDK2 activity which is required, along with DDK to phosphorylate and activate OF factors. DDK phosphorylates MCMs to convert pre-RC to pre-IC. CDK2 phosphorylates TRESLIN-MTBP and RECQL4 to recruit TOPBP1 and the CMG forms the replisome. MCM10 assists in the unwinding of DNA to form the RF . RPA is bound to ssDNA generated at the RF. B , representative QIBC scatter plots of cells either untreated (−) or treated with CHK1i for 30 min with or without a CDK inhibitor or a CDC7 inhibitor. Yellow curves represent the peak of RPA (in S phase cells) in the scatter plot. >1000 cells were analyzed in each condition. C , cells were treated for the indicated times with CHK1i inhibitor, and chromatin-enriched fractions were prepared and analyzed by Western blotting with the indicated antibodies. H3 was used as a chromatin loading control. Vinculin was used as a negative control for chromatin. D , U2OS cells were treated with CHK1i for the indicated times. To assess only the chromatin-bound (CB) fractions, cells were pre-extracted and then fixed and stained for RPA, EdU, and DAPI. Mean intensities in S phase (EdU positive) cells are plotted as boxplots. White lines indicate median values and outliers are represented as small circles. Whiskers indicate the 5th and 95th percentile. >2000 cells were analyzed for each condition. E , U2OS cells were treated with CHK1i for the indicated times, fixed, and stained for FOXM1 pT600 (CDK2 activity reporter), DAPI and EdU. Mean intensities in S phase (EdU positive) cells are plotted as boxplots. White lines indicate median values and outliers are represented as small circles . Whiskers indicate the 5th and 95th percentile. >2000 cells were analyzed for each condition. F , Line diagram comparing the increase in CB-RPA ( orange ) vs FOXM1 pT600 (CDK2 activity; purple ) from D and E . Average (n = 2 experiments) of each timepoint (>2000 cells analyzed) is plotted.

Article Snippet: CDC7 inhibitor XL413 (Selleckchem) was used at a concentration of 1 μM.

Techniques: Activity Assay, Generated, Western Blot, Control, Negative Control, Staining