Journal: Nucleic Acids Research

Article Title: Transcription termination counteracts DNA damage after WEE1 inhibition

doi: 10.1093/nar/gkaf1487

Figure Lengend Snippet: WDR82/PNUTS-PP1 is required to suppress DNA damage in S-phase after WEE1 inhibition. (A) Flow cytometry analysis of control siRNA (scr) or WDR82 siRNA (siWDR82) transfected HeLa cells treated with or without 1 µM AZD1775 for 2 or 6 h and stained with the DNA damage marker γH2AX and the DNA stain Hoechst 33258. Dotted lines indicate S-phase. The bar chart shows quantification of median γH2AX levels in S-phase from three independent experiments. (B) Western blot analysis of HeLa cells (HeLa) and HeLa cells stably expressing siRNA-resistant WDR82 (WDR82-res). Cells were siRNA-transfected and treated as in A. (C) Quantification of pATM T1989, pDNA-PK S2056, and pRPA S4/S8 relative to levels of CDK1 from six (HeLa) or three (WDR82-res) independent experiments, as in B. (D) Flow cytometry analysis of scr or PNUTS siRNA (siPNUTS) transfected cells treated with or without 1 µM AZD1775 for 2 h at 48 h after siRNA transfection, and stained with anti-γH2AX and Hoechst 33258. The bar chart shows quantification of median γH2AX levels in S-phase from three independent experiments. (E) Relative viability of scr and siWDR82 transfected cells treated with indicated concentrations of adavosertib (AZD1775) for 24 h, assessed by the Cell Titer Glow assay four days later. Inhibitors were added 48 h post-transfection ( n = 3). Error bars: SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (two tailed Student’s t-test).

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775, Selleck Biochem) and the CPSF73 inhibitor JTE-607 (MedChemExpress) were used at indicated concentrations, the CDK9 inhibitor 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) (Sigma Aldrich) was used at 100 μM, the CDK7-inhibitor THZ1 (ApexBio) at 1 μM, the XPB inhibitor Triptolide (TPL) (Sigma Aldrich) at 1 μM, and the CHK1 inhibitor AZD7762 (Selleckchem) at 300 nM.

Techniques: Inhibition, Flow Cytometry, Control, Transfection, Staining, Marker, Western Blot, Stable Transfection, Expressing, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Transcription termination counteracts DNA damage after WEE1 inhibition

doi: 10.1093/nar/gkaf1487

Figure Lengend Snippet: Inhibitors of active transcription or depletion of transcription elongation factor CDC73 suppress DNA damage after WEE1 inhibition. (A) Western blot analysis of scr and siWDR82 transfected cells with and without 1 µM adavosertib (AZD1775) for 2 or 6 h. Transcription inhibitors THZ1 and DRB were added 30 min prior to AZD1775. (B) Quantifications from experiments as in A, showing pDNA-PK S2056 and pRPA S4S8 versus CDK1. n = 3 for DRB-treated and n = 2 for THZ1-treated samples. (C) Flow cytometry analysis of cells treated with 1 µM AZD1775 at 72h after siRNA transfection and harvested 6 h later. The bar chart shows quantification of S-phase levels of γH2AX from three independent experiments. (D) Readthrough analysis from chromatin RNA sequencing experiment of HeLa cells 48 h after siRNA transfection. Box plot showing Log2 ratio of readthrough versus gene from the top 2000 expressed genes. (E) Read density profiles from experiments performed as in D showing readthrough from the gene with the biggest difference in readthrough between siWDR82 and scr, visualized in IGV ( VDAC2 : chr10:75 229 310). (F) Left: Readthrough analysis from chromatin RNA sequencing as in D for all expressed genes (defined as described in materials and methods). Middle: Readthrough analysis for expressed genes with detectable readthrough (log2 ratio > –5). Right: number of expressed genes with log2 ratio >–2. (G) Quantifications of pRNAPII S2 levels on chromatin from flow cytometry analysis, as in . Median levels in segments (shown in ) were divided by pRNAPII S2 levels in the internal barcoding control cells. Thereafter, median levels in each segment of the respective siRNA condition (scr or siCDC73) were divided by the median levels in segment 1 (G1). n = 6. (H) As in G, but for scr, siWDR82, and siWDR82 + siCDC73. n = 3. (I) As in G, but for scr, siPNUTS, and siPNUTS + siCDC73. n = 3. Error bars: SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (two tailed Student’s t-test). Statistical comparisons in G-I: scr versus siCDC73/siWDR82/siPNUTS; siWDR82 + siCDC73 versus siWDR82; siPNUTS + siCDC73 versus siPNUTS. Results in D-F are from a representative experiment.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775, Selleck Biochem) and the CPSF73 inhibitor JTE-607 (MedChemExpress) were used at indicated concentrations, the CDK9 inhibitor 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) (Sigma Aldrich) was used at 100 μM, the CDK7-inhibitor THZ1 (ApexBio) at 1 μM, the XPB inhibitor Triptolide (TPL) (Sigma Aldrich) at 1 μM, and the CHK1 inhibitor AZD7762 (Selleckchem) at 300 nM.

Techniques: Inhibition, Western Blot, Transfection, Flow Cytometry, RNA Sequencing, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Transcription termination counteracts DNA damage after WEE1 inhibition

doi: 10.1093/nar/gkaf1487

Figure Lengend Snippet: Transcription termination suppresses DNA damage in S-phase after WEE1 inhibition. (A) Flow cytometry analysis of cells transfected with scr and siRNA against DDX5 (siDDX5) and XRN2 (siXRN2) with and without 1 µM adavosertib (AZD1775) for 2 h, at 48 h after siRNA transfection. (B) As in A, but cells were transfected with siRNA against CPSF73 (siCPSF73). (C) As in A, but cells were transfected with scr, siWDR82, and siCPSF73 with and without siRNA against CDC73 (siCDC73). Of note is that cells transfected with siCDC73 in the absence of siWDR82 and siCPSF73 were transfected with only siCDC73 (not in combination with scr). (D) Bar chart showing quantifications of median γH2AX levels in S-phase from experiments as in A–C. Error bars represent SEM from ≥3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed Student’s t-test, except one-sample t-test for comparisons to the normalized sample). (E) Western blot showing knockdown levels 48 h after siRNA transfection using the different siRNAs in A–D. (F) As in E. Error bars: SEM of ≥3 independent experiments;* P < 0.05; ** P < 0.01; and *** P < 0.001 (two-tailed Student’s t-test).

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775, Selleck Biochem) and the CPSF73 inhibitor JTE-607 (MedChemExpress) were used at indicated concentrations, the CDK9 inhibitor 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) (Sigma Aldrich) was used at 100 μM, the CDK7-inhibitor THZ1 (ApexBio) at 1 μM, the XPB inhibitor Triptolide (TPL) (Sigma Aldrich) at 1 μM, and the CHK1 inhibitor AZD7762 (Selleckchem) at 300 nM.

Techniques: Inhibition, Flow Cytometry, Transfection, Two Tailed Test, Western Blot, Knockdown

Journal: Nucleic Acids Research

Article Title: Transcription termination counteracts DNA damage after WEE1 inhibition

doi: 10.1093/nar/gkaf1487

Figure Lengend Snippet: Combined targeting of transcription termination with WEE1 synergistically reduces cell survival in prostate cancer cells. (A) Kaplan-Meier plot showing BCR-free survival of prostate cancer patients vs time after prostatectomy. Patients were grouped into two by the median CPSF73 expression based on Illumina Bead Array gene expression data. n = 93. (B) CPSF73 levels versus ISUP grade group (left) and pathological tumor stage (right) in prostate cancer patients. n = 94. (C) Flow cytometry analysis of DU145 cells (left) and PC3 cells (right) without (-) or with JTE-607 (1 µM) and adavosertib (AZD1775) (500 nM) for 24 h. Regions indicate percentage cells with high γH2AX levels. (D) Quantifications from experiments, as in C, showing average % cells with high γH2AX as determined by the regions shown in C. Results are shown relative to the percentage of cells with high γH2AX after treatment with JTE-607 and AZD1775. n = 3. (E) Average viability relative to DMSO control from Cell Titer Glow assays of DU145 cells (left) and PC3 cells (right) treated with the indicated concentrations of JTE-607 and AZD1775 for 5 days. n = 3. (F) Average synergy score (excess over BLISS) calculated from viability data from E. (G) Clonogenic survival of DU145 cells (left) and PC3 cells (right) after treatment with the indicated concentrations of JTE-607 and adavosertib. Data represent the mean of three independent experiments, each performed in triplicate. (H) Working model. See main text for details. Error bars represent SEM of ≥three independent experiments. * P < 0.05; ** P < 0.01; and *** P < 0.001. Statistical testing of synergy in F and G is shown in and .

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775, Selleck Biochem) and the CPSF73 inhibitor JTE-607 (MedChemExpress) were used at indicated concentrations, the CDK9 inhibitor 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) (Sigma Aldrich) was used at 100 μM, the CDK7-inhibitor THZ1 (ApexBio) at 1 μM, the XPB inhibitor Triptolide (TPL) (Sigma Aldrich) at 1 μM, and the CHK1 inhibitor AZD7762 (Selleckchem) at 300 nM.

Techniques: Expressing, Gene Expression, Flow Cytometry, Control

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A and B , Dose-response curves for determining the sensitivity of various human cancer cell lines to either adavosertib ( A ) or VIC-1911 ( B ) with multiple dose range (adavosertib: 0.0625-10 μmol/L; VIC-1911: 0.0313-2.5 μmol/L). Cell viability was determined after 4-day exposure to drugs by CellTiter-Glo assay. Cell viability was normalized to control cells. Data are shown as mean ± SEM. C and D , Representative images of cooperative correlation matrix (right) and Loewe plots (left) of HNSCC CAL27 cells ( C ) and NSCLC NCI-H520 cells ( D ) in combinational treatment VIC-1911/adavosertib. E , Status of TP53 mutation, Loewe synergy scores (LSS) and maximum synergistic area scores (LMSAS) of human cancer cell lines are shown for evaluating synergy of combinational VIC-1911 and adavosertib treatment using SynergyFinder. and synergy score >10 and red area indicate synergism. F and G , Dose-response curves of NHTBE ( F ) and BJ1 ( G ) cells treated with control, adavosertib, VIC-1911 or combination from 4-day CellTiter-Glo assay. Cell viability was normalized to control cells. Shown are the means of technical triplicates from one experiment and the data as shown mean ± SEM are representative of three independent experiments with consistent results.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Glo Assay, Control, Mutagenesis

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A , Suppressive effect on clonogenic survival of VIC-1911/adavosertib combination in HNSCC (FaDu and CAL27), NSCLC (NCI-H520 and NCI-H358) and ovarian cancer (A2780) cells. Cells were treated with control, adavosertib (all other cell lines 250 nmol/L; CAL27 500 nmol/L), VIC-1911 (all other cell lines 125 nmol/L; CAL27 250 nmol/L) or combination for 4 days and cultured without drugs without drugs up to 9-12 days. Clonogenic survival was determined via crystal violet staining. Quantification of cell density from crystal violet staining is shown after normalized to control. B , Anchorage independent cell survival of HNSCC and lung cancer cells in response to VIC-1911/adavosertib combination. Cells were treated with the indicated drugs in low density of soft agarose cell culture setting for 4 days and cultured without drugs up to 25-30 days. A colony was defined as containing more than 10 cells, as indicated by >50 pixels in image J. Representative images are viable colony in soft agar. Scale bar = 100 μm. C and D , FaDu, CAL27, NCI-H520 and NCI-H358 cells were cultured with the conditioned media for oncosphere formation assay after 7 to 9 days of 4-day treatment with the indicated drugs as single or combination. Representative image of oncospheres ( C ) and quantification of number of oncospheres ( D ) are shown. Scale bar = 100 μm. Each dot shows a biological replicate (three/condition) and data are shown as mean ± SEM. Statistical significance was determined by Student t test. * P < 0.05; ** P <0.01; *** P < 0.001. E and F , Synergistic antitumor effect of late generation of WEE1 inhibitor azenosertib and VIC-1911 combination in HNSCC FaDu ( E ) and CAL27 ( F ) cells. Azenosertib doses are the same as adaovsertib. Shown are the means of technical triplicates from one experiment and the data as shown mean ± SEM are representative of triplicated and three independent experiments with consistent results.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Control, Cell Culture, Staining, Tube Formation Assay

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A-C , Fork progression speed in FaDu and NCI-H358 cells treated with control (Ctrl), adavosertib (Ada; 250 nmol/L), VIC-1911 (VIC; 125 nmol/L) or combination. A , The experimental design is shown. Cells were pre-treated with the indicated drugs for 24 hours and followed by sequential incubation with CIdU and IdU for 20 minutes each. B, Representative images of fibers (left) and quantification of fork progression as measured by fiber assays of FaDu cells (right). C, Quantification of fork progression as measured by fiber assays of NCI-H358 cells. Fibers (n=45-62 for FaDu; n=161-190 for NCI-H358) were examined. The box plots (as for those in all other figures) show median values (central lines) and all shown points as Min to Max. kb, kilobases. Scale bar = 10 μm. D-F , Synergistic increases of replication stress marker pRPA2 (S33)-positive foci in FaDu or NCI-H358 cells treated with VIC-1911/adavosertib. Cells were treated with control, adavosertib, VIC-1911 or combination for 24 hours and followed by immunofluorescence staining with pRPA2 (S33) and γH2A.X (S139) antibodies. Representative images of FaDu cells ( D ) and quantification of pRPA2 (S33)-positive foci in a cell of FaDu ( E ) and NCI-H358 ( F ). n ≥ 35 cells were examined. G , Transcription-replication conflicts induced by VIC-1911/adavosertib combination in FaDu cells. Cells treated with the indicated drugs as single or combination for 24 were assessed PLA intensity derived from interaction between RNA polymerase II and PCNA replisome. The negative control (Neg.Ctrl) shows primary antibodies only in cells treated with the combination. Representative images of PLA under different conditions, Scale bar = 10 μm (Top). Quantification of the PLA signals (Bottom). Each dot representative the mean PLA signal of all cells in a field as determined by area of PLA signal in total number of DAPI-positive nuclei, compared with the solvent control (Ctrl). n = 14 fields for the Neg.Ctrl, Ctrl, Ada and VIC; n = 21 fields for the Combo, and the data as shown mean ± SEM are representative of three independent experiments with consistent results. Statistical significance was determined by unpaired two-tailed t -test comparing two conditions (VIC-1911 vs Combination; **** P < 0.0001). H , Heatmaps showing relative expression changes of DNA replication regulators genes under treatment with control, adavosertib, VIC-1911 or combination at different time points following by thymidine synchronization in FaDu cells. Only significantly changed genes based on RNA-seq analysis are shown. Each column indicates a biological replicate (n=3/condition). I , Pathway enhichment was dertermined by GSEA. J, Immunoblot analysis of the indicated proteins in FaDu and CAL27 cells upon the treatments at 15 minutes after CDK1 inhibitor-mediated synchronization. Cells were synchronized with Ro-3306 (9 μmol/L for 20 hours) and followed by release along with treatment of adavosertib and/or VIC-1911 versus control. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. K , Immunoblot analysis of the indicated proteins in Ro-3306-mediated synchronized FaDu cells upon the treatments with VIC-1911 (125 nmol/L) and/or AZD6783 (1 μmol/L) for 15 minutes.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Control, Incubation, Marker, Immunofluorescence, Staining, Derivative Assay, Negative Control, Solvent, Two Tailed Test, Expressing, RNA Sequencing, Western Blot, SDS Page

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A-C , FaDu and NCI-H358 cells were treated with control, adavosertib (250 nmol/L), VIC-1911 (125 nmol/L) or combination for 24 hours, and followed by immunofluorescent staining with anti-tubulin (Green) and anti-phospho-Histone H3 (pHH3; S10; Red). Nucleus was stained with TOPRO3. A , Representative images of mitotic cells were captured by confocal microscopy. Scale bar = 10 μm. B , Percentage of dual pHH3 and TOPRO3-positive FaDu and NCI-H358 cells. Fields were randomly captured and counted more than 300 TOPRO3-positive cells. C , Percentage of normal or abnormal mitotic cells in pHH3-positive FaDu and NCI-H358 cells. D , Representative time-lapse live cell images of synchronized FaDu cells. EGFP-αTubulin and mCherry-Histone 2B stably expressing FaDu cells were synchronized by double thymidine block and followed by treatment with control, VIC-1911, adaovosertib or combination over 24 hours. Images were captured every 10 minutes interval and time indicates hours after release from double thymidine blocking with drug treatment. E , HNSCC (FaDu and CAL27) and lung cancer (NCI-H1437 and NCI-H358) cells were treated with the indicated single drug or combination for 48 hours and followed by staining with Annexin V and PI for apoptosis induction by flow cytometry analyses. Percentage of apoptotic cells in either Annexin V-positive or Annexin V/PI-positive cells. Each dot shows replicate from three independent experiments with similar results and data are shown as mean ± SEM. Statistical significance was determined by Student t test. * P < 0.05; ** P <0.01; *** P < 0.001. F , The indicated cell lines were exposed to the drugs singly or in combination for 48 hours. Cell lysates were subjected to SDS-PAGE and immuno-blotting with antibodies against cleaved PARP and cleaved Caspase-3 for apoptosis induction. G , Representative image of cleaved Caspase-3 induction in protein array for apoptosis panel of FaDu cells at 48 hours after exposure to the indicated drugs as single or combination. Enlarged signal are inserted. H , Pathway enhichment was dertermined by GSEA using RNA-Seq profile derived from FaDu cells treated with control, adavosertib, VIC-1911 or combination for 24 hours.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Control, Staining, Confocal Microscopy, Stable Transfection, Expressing, Blocking Assay, Flow Cytometry, SDS Page, Protein Array, RNA Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A , FaDu and CAL27 cells were synchronized with Ro-3306 for 20 hours and released with complete media (supplement with 10% FBS) including the indicated drugs for 15 minutes. Cell lysates were subjected to SDS-PAGE and immuno-blotting. B , FaDu cells were synchronized with double thymidine block and followed by release with 10% FBS-containing media including control, VIC-1911 (125 nmol/L), adavosertib (250 nmol/L) or combination for the indicated time points. Cell lysates were subjected to SDS-PAGE and immuno-blotting. C , The indicated cell lines were cultured for 24 hours and cell lysates were subjected to SDS-PAGE and immuno-blotting for the indicated proteins. D , TP53- isogenic PCI-13 cells stably expressing control, TP53 WT , G245D , C238F , or R273H mutations were cultured in the complete media for 24 hours and RNA expression of the indicated genes were determined by qRT-PCR. mRNA expression of the genes was normalized with its expression in TP53 WT PCI-13 cells. E , Bliss synergy plotting for evaluating synergy score of combinational VIC-1911 and adavosertib treatment for 4 days. TP53-isogenic PCI-13 cells were treated with the indicated doses of adavosertib or VIC-1911 as single or combination as well as control for 4 days and cell viability was assessed by CellTiter-Glo assay. Loewe plotting were generated by conversion to bliss plots using cooperative correlation. Red area and Bliss MSA score indicate synergy; antagonism (< - 10), additive (-10 to 10) or synergism (> 10). F , Cell cycle distribution in synchronized PCI-13 TP53 isogeneic cells upon exposure to combinational treatment VIC-1911 (125 nmol/L)/adavosertib (250 nmol/L). Double-thymidine block-mediated synchronized PCI-13 cells were treated with control, adavosrtib, VIC-1911 or combination for 24 hours, and followed by flow cytometry of PI-stained cells G, PCI-13 cells were treated with control, VIC-1911 (125 nmol/L), adavosertib (250 nmol/L) or combination for 24 hours and cell lysates were subjected to SDS-PAGE and immuno-blotting.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: SDS Page, Blocking Assay, Control, Cell Culture, Stable Transfection, Expressing, RNA Expression, Quantitative RT-PCR, Glo Assay, Generated, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A-D , Mice bearing FaDu ( A ), CAL27 ( B ), NCI-H358 ( C ) or A549 ( D ) subcutaneous xenografts (FaDu n = 4; CAL27 n = 8; NCI-H358 n = 6; A549 n =5 per group) were treated daily (6 days on-1 day off) with vehicle, adavosertib 120 mg kg -1 , VIC-1911 30 mg kg -1 , or the combination for 3 weeks when tumor reached ∼200 mm 3 . Tumor growth curves were determined by measuring tumor volumes other day. B , CAL27 xenografts were treated as described above and tumor growth was further monitored after treatment duration was completed. Data are shown as mean ± SEM. *, P < 0.05. F-H . Mice bearing HNSCC PDX PDXN04 xenografts (n = 5-6) were treated as described above. F , Relative changes in tumor volume of HNSCC PDX model PDX04 during treatment with the indicated inhibitors. Relative tumor volume was calculated normalizing the measured tumor volume to the tumor volume on baseline (Day 1). G , Waterfall plot showing tumor volume changes after 10-day treatment. H , Kaplan-Meier survival curve of PDXN04 xenografted mice treated as indicated. Data are shown as mean ± SEM. ***, P < 0.001. I-K , Mice bearing LUAD PDX PDXPRH xenografts (n = 3-4) were treated as described above. I , Relative changes in tumor volume of HNSCC PDX model PDX04 during treatment with the indicated inhibitors calculated as described above. J , Waterfall plot showing tumor volume changes after 12-day treatment. K , Kaplan-Meier survival curve of PDXPRH xenografted mice treated as indicated. Data are shown as mean ± SEM. *, P < 0.05. L and M , Representative images of IHC staining for Ki-67 (L) and cleaved caspase-3 in tumor samples at the endpoint. Data are quantified in the graphs. Scale bars = 100 μm. Inset, high-power field. One-way ANOVA multiple comparison with Tukey’s correction were used to analyze the data. ****, P < 0.0001.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Immunohistochemistry, Comparison

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A and B , Dose-response curves for determining the sensitivity of various human cancer cell lines to either adavosertib ( A ) or VIC-1911 ( B ) with multiple dose range (adavosertib: 0.0625-10 μmol/L; VIC-1911: 0.0313-2.5 μmol/L). Cell viability was determined after 4-day exposure to drugs by CellTiter-Glo assay. Cell viability was normalized to control cells. Data are shown as mean ± SEM. C and D , Representative images of cooperative correlation matrix (right) and Loewe plots (left) of HNSCC CAL27 cells ( C ) and NSCLC NCI-H520 cells ( D ) in combinational treatment VIC-1911/adavosertib. E , Status of TP53 mutation, Loewe synergy scores (LSS) and maximum synergistic area scores (LMSAS) of human cancer cell lines are shown for evaluating synergy of combinational VIC-1911 and adavosertib treatment using SynergyFinder. and synergy score >10 and red area indicate synergism. F and G , Dose-response curves of NHTBE ( F ) and BJ1 ( G ) cells treated with control, adavosertib, VIC-1911 or combination from 4-day CellTiter-Glo assay. Cell viability was normalized to control cells. Shown are the means of technical triplicates from one experiment and the data as shown mean ± SEM are representative of three independent experiments with consistent results.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Glo Assay, Control, Mutagenesis

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A , Suppressive effect on clonogenic survival of VIC-1911/adavosertib combination in HNSCC (FaDu and CAL27), NSCLC (NCI-H520 and NCI-H358) and ovarian cancer (A2780) cells. Cells were treated with control, adavosertib (all other cell lines 250 nmol/L; CAL27 500 nmol/L), VIC-1911 (all other cell lines 125 nmol/L; CAL27 250 nmol/L) or combination for 4 days and cultured without drugs without drugs up to 9-12 days. Clonogenic survival was determined via crystal violet staining. Quantification of cell density from crystal violet staining is shown after normalized to control. B , Anchorage independent cell survival of HNSCC and lung cancer cells in response to VIC-1911/adavosertib combination. Cells were treated with the indicated drugs in low density of soft agarose cell culture setting for 4 days and cultured without drugs up to 25-30 days. A colony was defined as containing more than 10 cells, as indicated by >50 pixels in image J. Representative images are viable colony in soft agar. Scale bar = 100 μm. C and D , FaDu, CAL27, NCI-H520 and NCI-H358 cells were cultured with the conditioned media for oncosphere formation assay after 7 to 9 days of 4-day treatment with the indicated drugs as single or combination. Representative image of oncospheres ( C ) and quantification of number of oncospheres ( D ) are shown. Scale bar = 100 μm. Each dot shows a biological replicate (three/condition) and data are shown as mean ± SEM. Statistical significance was determined by Student t test. * P < 0.05; ** P <0.01; *** P < 0.001. E and F , Synergistic antitumor effect of late generation of WEE1 inhibitor azenosertib and VIC-1911 combination in HNSCC FaDu ( E ) and CAL27 ( F ) cells. Azenosertib doses are the same as adaovsertib. Shown are the means of technical triplicates from one experiment and the data as shown mean ± SEM are representative of triplicated and three independent experiments with consistent results.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Control, Cell Culture, Staining, Tube Formation Assay

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A-C , Fork progression speed in FaDu and NCI-H358 cells treated with control (Ctrl), adavosertib (Ada; 250 nmol/L), VIC-1911 (VIC; 125 nmol/L) or combination. A , The experimental design is shown. Cells were pre-treated with the indicated drugs for 24 hours and followed by sequential incubation with CIdU and IdU for 20 minutes each. B, Representative images of fibers (left) and quantification of fork progression as measured by fiber assays of FaDu cells (right). C, Quantification of fork progression as measured by fiber assays of NCI-H358 cells. Fibers (n=45-62 for FaDu; n=161-190 for NCI-H358) were examined. The box plots (as for those in all other figures) show median values (central lines) and all shown points as Min to Max. kb, kilobases. Scale bar = 10 μm. D-F , Synergistic increases of replication stress marker pRPA2 (S33)-positive foci in FaDu or NCI-H358 cells treated with VIC-1911/adavosertib. Cells were treated with control, adavosertib, VIC-1911 or combination for 24 hours and followed by immunofluorescence staining with pRPA2 (S33) and γH2A.X (S139) antibodies. Representative images of FaDu cells ( D ) and quantification of pRPA2 (S33)-positive foci in a cell of FaDu ( E ) and NCI-H358 ( F ). n ≥ 35 cells were examined. G , Transcription-replication conflicts induced by VIC-1911/adavosertib combination in FaDu cells. Cells treated with the indicated drugs as single or combination for 24 were assessed PLA intensity derived from interaction between RNA polymerase II and PCNA replisome. The negative control (Neg.Ctrl) shows primary antibodies only in cells treated with the combination. Representative images of PLA under different conditions, Scale bar = 10 μm (Top). Quantification of the PLA signals (Bottom). Each dot representative the mean PLA signal of all cells in a field as determined by area of PLA signal in total number of DAPI-positive nuclei, compared with the solvent control (Ctrl). n = 14 fields for the Neg.Ctrl, Ctrl, Ada and VIC; n = 21 fields for the Combo, and the data as shown mean ± SEM are representative of three independent experiments with consistent results. Statistical significance was determined by unpaired two-tailed t -test comparing two conditions (VIC-1911 vs Combination; **** P < 0.0001). H , Heatmaps showing relative expression changes of DNA replication regulators genes under treatment with control, adavosertib, VIC-1911 or combination at different time points following by thymidine synchronization in FaDu cells. Only significantly changed genes based on RNA-seq analysis are shown. Each column indicates a biological replicate (n=3/condition). I , Pathway enhichment was dertermined by GSEA. J, Immunoblot analysis of the indicated proteins in FaDu and CAL27 cells upon the treatments at 15 minutes after CDK1 inhibitor-mediated synchronization. Cells were synchronized with Ro-3306 (9 μmol/L for 20 hours) and followed by release along with treatment of adavosertib and/or VIC-1911 versus control. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. K , Immunoblot analysis of the indicated proteins in Ro-3306-mediated synchronized FaDu cells upon the treatments with VIC-1911 (125 nmol/L) and/or AZD6783 (1 μmol/L) for 15 minutes.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Control, Incubation, Marker, Immunofluorescence, Staining, Derivative Assay, Negative Control, Solvent, Two Tailed Test, Expressing, RNA Sequencing, Western Blot, SDS Page

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A-C , FaDu and NCI-H358 cells were treated with control, adavosertib (250 nmol/L), VIC-1911 (125 nmol/L) or combination for 24 hours, and followed by immunofluorescent staining with anti-tubulin (Green) and anti-phospho-Histone H3 (pHH3; S10; Red). Nucleus was stained with TOPRO3. A , Representative images of mitotic cells were captured by confocal microscopy. Scale bar = 10 μm. B , Percentage of dual pHH3 and TOPRO3-positive FaDu and NCI-H358 cells. Fields were randomly captured and counted more than 300 TOPRO3-positive cells. C , Percentage of normal or abnormal mitotic cells in pHH3-positive FaDu and NCI-H358 cells. D , Representative time-lapse live cell images of synchronized FaDu cells. EGFP-αTubulin and mCherry-Histone 2B stably expressing FaDu cells were synchronized by double thymidine block and followed by treatment with control, VIC-1911, adaovosertib or combination over 24 hours. Images were captured every 10 minutes interval and time indicates hours after release from double thymidine blocking with drug treatment. E , HNSCC (FaDu and CAL27) and lung cancer (NCI-H1437 and NCI-H358) cells were treated with the indicated single drug or combination for 48 hours and followed by staining with Annexin V and PI for apoptosis induction by flow cytometry analyses. Percentage of apoptotic cells in either Annexin V-positive or Annexin V/PI-positive cells. Each dot shows replicate from three independent experiments with similar results and data are shown as mean ± SEM. Statistical significance was determined by Student t test. * P < 0.05; ** P <0.01; *** P < 0.001. F , The indicated cell lines were exposed to the drugs singly or in combination for 48 hours. Cell lysates were subjected to SDS-PAGE and immuno-blotting with antibodies against cleaved PARP and cleaved Caspase-3 for apoptosis induction. G , Representative image of cleaved Caspase-3 induction in protein array for apoptosis panel of FaDu cells at 48 hours after exposure to the indicated drugs as single or combination. Enlarged signal are inserted. H , Pathway enhichment was dertermined by GSEA using RNA-Seq profile derived from FaDu cells treated with control, adavosertib, VIC-1911 or combination for 24 hours.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Control, Staining, Confocal Microscopy, Stable Transfection, Expressing, Blocking Assay, Flow Cytometry, SDS Page, Protein Array, RNA Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A , FaDu and CAL27 cells were synchronized with Ro-3306 for 20 hours and released with complete media (supplement with 10% FBS) including the indicated drugs for 15 minutes. Cell lysates were subjected to SDS-PAGE and immuno-blotting. B , FaDu cells were synchronized with double thymidine block and followed by release with 10% FBS-containing media including control, VIC-1911 (125 nmol/L), adavosertib (250 nmol/L) or combination for the indicated time points. Cell lysates were subjected to SDS-PAGE and immuno-blotting. C , The indicated cell lines were cultured for 24 hours and cell lysates were subjected to SDS-PAGE and immuno-blotting for the indicated proteins. D , TP53- isogenic PCI-13 cells stably expressing control, TP53 WT , G245D , C238F , or R273H mutations were cultured in the complete media for 24 hours and RNA expression of the indicated genes were determined by qRT-PCR. mRNA expression of the genes was normalized with its expression in TP53 WT PCI-13 cells. E , Bliss synergy plotting for evaluating synergy score of combinational VIC-1911 and adavosertib treatment for 4 days. TP53-isogenic PCI-13 cells were treated with the indicated doses of adavosertib or VIC-1911 as single or combination as well as control for 4 days and cell viability was assessed by CellTiter-Glo assay. Loewe plotting were generated by conversion to bliss plots using cooperative correlation. Red area and Bliss MSA score indicate synergy; antagonism (< - 10), additive (-10 to 10) or synergism (> 10). F , Cell cycle distribution in synchronized PCI-13 TP53 isogeneic cells upon exposure to combinational treatment VIC-1911 (125 nmol/L)/adavosertib (250 nmol/L). Double-thymidine block-mediated synchronized PCI-13 cells were treated with control, adavosrtib, VIC-1911 or combination for 24 hours, and followed by flow cytometry of PI-stained cells G, PCI-13 cells were treated with control, VIC-1911 (125 nmol/L), adavosertib (250 nmol/L) or combination for 24 hours and cell lysates were subjected to SDS-PAGE and immuno-blotting.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: SDS Page, Blocking Assay, Control, Cell Culture, Stable Transfection, Expressing, RNA Expression, Quantitative RT-PCR, Glo Assay, Generated, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: AURKA inhibition amplifies DNA replication stress to foster WEE1 kinase dependency and synergistic antitumor effects with WEE1 inhibition in cancers

doi: 10.1101/2025.05.28.656693

Figure Lengend Snippet: A-D , Mice bearing FaDu ( A ), CAL27 ( B ), NCI-H358 ( C ) or A549 ( D ) subcutaneous xenografts (FaDu n = 4; CAL27 n = 8; NCI-H358 n = 6; A549 n =5 per group) were treated daily (6 days on-1 day off) with vehicle, adavosertib 120 mg kg -1 , VIC-1911 30 mg kg -1 , or the combination for 3 weeks when tumor reached ∼200 mm 3 . Tumor growth curves were determined by measuring tumor volumes other day. B , CAL27 xenografts were treated as described above and tumor growth was further monitored after treatment duration was completed. Data are shown as mean ± SEM. *, P < 0.05. F-H . Mice bearing HNSCC PDX PDXN04 xenografts (n = 5-6) were treated as described above. F , Relative changes in tumor volume of HNSCC PDX model PDX04 during treatment with the indicated inhibitors. Relative tumor volume was calculated normalizing the measured tumor volume to the tumor volume on baseline (Day 1). G , Waterfall plot showing tumor volume changes after 10-day treatment. H , Kaplan-Meier survival curve of PDXN04 xenografted mice treated as indicated. Data are shown as mean ± SEM. ***, P < 0.001. I-K , Mice bearing LUAD PDX PDXPRH xenografts (n = 3-4) were treated as described above. I , Relative changes in tumor volume of HNSCC PDX model PDX04 during treatment with the indicated inhibitors calculated as described above. J , Waterfall plot showing tumor volume changes after 12-day treatment. K , Kaplan-Meier survival curve of PDXPRH xenografted mice treated as indicated. Data are shown as mean ± SEM. *, P < 0.05. L and M , Representative images of IHC staining for Ki-67 (L) and cleaved caspase-3 in tumor samples at the endpoint. Data are quantified in the graphs. Scale bars = 100 μm. Inset, high-power field. One-way ANOVA multiple comparison with Tukey’s correction were used to analyze the data. ****, P < 0.0001.

Article Snippet: The WEE1 inhibitors adavosertib (AZD1775; HY-10993) and azenosertib (Zn-c3; E1000) were purchased from MedChemExpress and Selleck Chemicals, respectively.

Techniques: Immunohistochemistry, Comparison

Journal: Cancer biology & therapy

Article Title: The polymeric fluoropyrimidine CF10 overcomes limitations of 5-FU in pancreatic ductal adenocarcinoma cells through increased replication stress.

doi: 10.1080/15384047.2024.2421584

Figure Lengend Snippet: Figure 1. (a) Structure of CF10 with PEG6 (blue) and AraC (yellow) modifications. (b) Differential metabolism of CF10 and 5-FU resulting in increased DNA damage with CF10 treatment and vulnerability to inhibitors of DNA repair. (c) Activation of the intra-S-phase checkpoint thru the ATR/Chk1 pathway and phosphorylation of Wee1. Our studies showed that inhibition of ATR or Wee1 enhances cytotoxicity of CF10, but not 5-FU, to PDAC cells. The combination of CF10+ATRi/Wee1 inhibition may overcome 5-FU resistance for improved treatment of PDAC.

Article Snippet: The Wee1 inhibitor AZD1775 (adavosertib) and the ATR inhibitor AZD6738 were obtained from Selleck Chemicals.

Techniques: Activation Assay, Phospho-proteomics, Inhibition

Journal: Cancer biology & therapy

Article Title: The polymeric fluoropyrimidine CF10 overcomes limitations of 5-FU in pancreatic ductal adenocarcinoma cells through increased replication stress.

doi: 10.1080/15384047.2024.2421584

Figure Lengend Snippet: Figure 4. Viability effect of Wee1i or ATRi with single agent compounds in four PDAC cell lines. (a) The Wee1 inhibitor AZD1775 (adavosertib, green) showed increased single agent efficacy as compared to 5-FU (magenta), while treatment with both drugs were not additive (black). (b) ATRi (blue) showed increased single agent efficacy as compared to 5-FU (magenta), while treatment with both drugs did not show an additive effect (black).

Article Snippet: The Wee1 inhibitor AZD1775 (adavosertib) and the ATR inhibitor AZD6738 were obtained from Selleck Chemicals.

Techniques:

Journal: Cancer biology & therapy

Article Title: The polymeric fluoropyrimidine CF10 overcomes limitations of 5-FU in pancreatic ductal adenocarcinoma cells through increased replication stress.

doi: 10.1080/15384047.2024.2421584

Figure Lengend Snippet: Figure 6. CF10, but not 5-FU, is enhanced by Wee1i and ATRi in four PDAC cell lines. (a, b) the Wee1i (green) and the ATRi (blue) showed enhanced cytotoxicity in all tested cell lines when administered concurrently to CF10 (black, single agent orange). (c, d) Conversely, enhanced effect is not seen in three out of four cell lines, and only modestly in the CFPAC1 cell line, when the Wee1i (green) and ATRi (blue) were administered either concurrently (black) to 5-FU (single agent, magenta).

Article Snippet: The Wee1 inhibitor AZD1775 (adavosertib) and the ATR inhibitor AZD6738 were obtained from Selleck Chemicals.

Techniques:

Journal: Cancer biology & therapy

Article Title: The polymeric fluoropyrimidine CF10 overcomes limitations of 5-FU in pancreatic ductal adenocarcinoma cells through increased replication stress.

doi: 10.1080/15384047.2024.2421584

Figure Lengend Snippet: Figure 7. Adding of deoxynucleotides rescues viability in cells treated with CF10 or CF10+Wee1i/ATRi. (a) Four PDAC cell lines were treated with CF10 (orange) or CF10 and 10 µm dNTPs (black). (b) The same cell lines were tested with CF10 + Wee1i (green) or CF10 + Wee1i and 10 µm dNTPs (black). (c) The same cell lines were tested with CF10 + ATRi (blue) or CF10 + ATRi and 10 µm dNTPs (black).

Article Snippet: The Wee1 inhibitor AZD1775 (adavosertib) and the ATR inhibitor AZD6738 were obtained from Selleck Chemicals.

Techniques:

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Primers used in this study.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Sequencing

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Association of WEE1 expression with the clinicopathological features and prognosis of CRC. ( a ) Immunohistochemical staining of WEE1 in surgical CRC specimens (100× magnification). Based on the staining intensity, the cells were classified as strong, moderate, weak, and negative, with strong and moderate being classified as the WEE1-positive group and weak and negative as the WEE1-negative group. ( b ) Immunohistochemical staining of p53 in surgical CRC specimens (100× magnification). Based on the distribution of immunostained positive cells, the cells were classified as diffuse strong, nested, negative, mosaic, sporadic, and diffuse weak, with diffuse strong, nested, and negative being classified as the p53 mutations and mosaic, sporadic, and diffuse weak as p53 wild types. ( c ) Database analysis based on the UCSC cancer genomic browser relationship between WEE1 expression and prognosis (overall survival rate) in CRC (GDC, The Cancer Genome Atlas (TCGA), Colon Adenocarcinoma (COAD) n = 487). ( d ) Association between WEE1 expression and prognosis (overall survival rate) in 152 patients with surgically resected CRC at our hospital. ( e ) Comparison of the overall survival rates of patients ( n = 152) who underwent surgery for CRC classified by WEE1 expression and KRAS mutation status. Log-rank test *: p < 0.05, n.s.: not significant.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison, Mutagenesis

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: WEE1 expression and clinicopathological features.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: WEE1 expression and gene mutation status.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Mutagenesis

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Quantification of WEE1 expression in CRC cell lines and effect of WEE1 inhibitor on cell proliferation. ( a ) Relative expression levels of WEE1 in various CRC cell lines measured using real-time PCR, where its expression level in normal colonic mucosa was set as 1. ( b ) Growth curves of various CRC cell lines after exposure to various concentrations of the WEE1 inhibitor, adavosertib (200, 500, and 1000 nM), and untreated cells (control). The mean values are plotted. One-way ANOVA followed by Dunnett’s post hoc test, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Effects of the WEE1 inhibitor on cell cycle and apoptosis induction in CRC cell lines. ( a ) Changes over time in the percentage of cells in the G1, S, and G2/M phases of the cell cycle as measured by cell cycle analysis in untreated (0 h) and various adavosertib-treated (1000 nM) CRC cell lines (at 4, 12, and 24 h). Comparison of percentage of apoptotic cells before (0 h) and 24 h after addition of adavosertib. *: p < 0.05, n.s.: not significant. ( b ) Changes over time in the percentage of apoptotic, viable, and necrotic cells in various untreated (0 h) and adavosertib-treated colon cancer cell lines. The cell lines were treated with 1000 nM adavosertib, and the analysis was performed 6, 24, and 48 h after treatment. Comparison of percentage of apoptotic cells before (0 h) and 48 h after the addition of adavosertib. *: p < 0.05, n.s.: not significant.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Cell Cycle Assay, Comparison

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Effects of the WEE1 inhibitor, adavosertib, on tumor volume in an orthotopic transplanted mouse model of CRC. ( a ) Orthotopic transplanted mouse model of CRC using MC38: transplant design and treatment schedule. n = 5, control group; n = 4, treatment group. ( b ) Macroscopic images of mouse tumors obtained following euthanasia on day 35 post-transplantation. ( c ) Comparison of tumor volume between control and treatment groups. ( d ) Orthotopic transplanted mouse model of CRC using CT26: transplant design and treatment schedule. n = 6, control group; n = 6, treatment group. ( e ) Macroscopic images of mouse tumors obtained following euthanasia on day 24 post-transplantation. ( f ) Comparison of tumor volume between control and treatment groups. Welch’s t -test, **: p < 0.01, n.s.: not significant.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control, Transplantation Assay, Comparison

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Evaluation of the gene expression profiles in transplanted tumors using RNA sequencing. ( a ) Statistics of differentially expressed genes (DEGs; p < 0.05 and fold change > 2). ( b ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. ( c ) Gene Ontology (GO) enrichment analysis. ( d ) Gene Set Enrichment Analysis (GSEA) in orthotopically transplanted tumors treated with or without WEE1 inhibitor. NES: normalized enrichment score.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, RNA Sequencing Assay