wee1 antibody  (Cell Signaling Technology Inc)

Journal: MedComm

Article Title: Dual Targeting of DNA and EGFR by ZYH005 Induces DNA Damage and Mitotic Catastrophe in Glioblastoma

doi: 10.1002/mco2.70717

Figure Lengend Snippet: Z5 induces G2/M arrest and mitotic catastrophe in GBM cells. (A) Cell cycle distribution in GBM cell lines was analyzed by flow cytometry after 24 h of Z5 treatment. n = 3. (B) Representative flow cytometric analysis of PHH3+ and DNA contents > 4n cells. (C) Cell cycle profiles of NC and si‐EGFR GBM cells were assessed by flow cytometry after 24 h Z5 treatment. (D) Expression of WEE1, p‐WEE1, PHH3, CDC2, and p‐CDC2 was analyzed by immunoblotting after 48 h treatment with increasing concentrations of Z5. (E) IF staining was performed with anti‐tubulin (red) and anti‐PHH3 (green) antibodies in U87‐MG and U251‐MG cells treated with DMSO or Z5.

Article Snippet: WEE1 antibody (Cell Signaling Technology, 1:200 dilution) and EGFR antibody (Proteintech, 1:100 dilution) were used to detect the protein.

Techniques: Flow Cytometry, Expressing, Western Blot, Staining

Journal: MedComm

Article Title: Dual Targeting of DNA and EGFR by ZYH005 Induces DNA Damage and Mitotic Catastrophe in Glioblastoma

doi: 10.1002/mco2.70717

Figure Lengend Snippet: Z5 disrupts the nuclear EGFR–WEE1 axis. (A) Correlation between EGFR and WEE1 expression in GBM patients. (B) SPR analysis of the interaction between the kinase domains of EGFR and WEE1. (C) Co‐IP was performed to assess EGFR–WEE1 interaction in U87‐MG cells after 15 min Z5 treatment. (D) Western blot analysis of WEE1, p‐WEE1, p‐CDC2, EGFR, and p‐EGFR in HEK293T cells transfected with empty vector, WT‐EGFR, or E762V‐EGFR. (E) Subcellular localization of EGFR and WEE1 in the cytoplasm and nucleus was analyzed in U87‐MG cells after 15 min Z5 treatment. (F‐G) IF staining of EGFR and WEE1 in NC and siKPNA2‐1400 U87‐MG cells. (H) Growth curves of NC and siKPNA2‐1400 U87 cells after 48 h Z5 treatment. n = 3. (I) Antiproliferative effect of Z5 was assessed by EdU staining in NC and siKPNA2‐1400 U87‐MG cells. (J) Cell cycle distribution was analyzed by flow cytometry in NC and siKPNA2‐1400 U87‐MG cells after 24 h Z5 treatment.

Article Snippet: WEE1 antibody (Cell Signaling Technology, 1:200 dilution) and EGFR antibody (Proteintech, 1:100 dilution) were used to detect the protein.

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot, Transfection, Plasmid Preparation, Staining, Flow Cytometry

Journal: MedComm

Article Title: Dual Targeting of DNA and EGFR by ZYH005 Induces DNA Damage and Mitotic Catastrophe in Glioblastoma

doi: 10.1002/mco2.70717

Figure Lengend Snippet: Z5 suppresses GSCs tumorigenesis and inhibits EGFR‐related signaling in vivo. (A) Representative tumor sphere formation assay in T3359 cells treated with different concentrations of Z5 for 48 h. Scale bar = 400 µm. (B) Quantitative analysis of tumor sphere fragmentation from (A). The data are presented as the mean ± SD, n = 3, compared with the “0” group. (C) Expression of γ‐H2AX, WEE1, p‐WEE1, PHH3, CDC2, p‐CDC2, EGFR, p‐EGFR, ERK, p‐ERK, mTOR, and p‐mTOR in T3359 cells with DMSO or Z5 treatment. (D) H&E staining of intracranial xenografts to assess tumor formation. (E) Survival analysis of mice orthotopically implanted with GSCs. The data are presented as the mean ± SD, n = 4, compared with the NT group. (F) Immunohistochemical analysis of EGFR and WEE1 expression in orthotopic tumor tissues. (G) Quantification of EGFR and WEE1 levels by average optical density from five fields per mouse. The data are presented as the mean ± SD, n = 15, compared with the NT group. (H) Schematic diagram illustrating the mechanism of Z5.

Article Snippet: WEE1 antibody (Cell Signaling Technology, 1:200 dilution) and EGFR antibody (Proteintech, 1:100 dilution) were used to detect the protein.

Techniques: In Vivo, Tube Formation Assay, Expressing, Staining, Immunohistochemical staining

Journal: iScience

Article Title: Preclinical drug screen identifies WEE1 inhibitor and vinca alkaloid as a combination treatment concept for Li-Fraumeni syndrome medulloblastoma

doi: 10.1016/j.isci.2025.114564

Figure Lengend Snippet: Drug combination screening and drug hits validation in in vitro TP53 mut brain cancer cell models (A) Average synergy scores estimated for combinations of cell cycle checkpoint kinase inhibitors and micronucleus-associated compounds across TP53 mut brain cancer cell lines; black points indicate the average value across all calculated synergy scores (ATM/ATRi, ATM/ATR inhibitors; CHK1/2i, CHEK1/CHEK2 inhibitors; WEE1i, WEE1 inhibitor; VBL, vinblastine; VCR, vincristine) (see also ). (B) Model representing mechanism of action of adavosertib in TP53 wt and TP53 mut cancer cells exposed to DNA damaging agents (Created with BioRender.com ). (C) Adavosertib (adav) decreases CDK1 phosphorylation levels in SJ-GBM2 and UW228-2 cell lines; Student’s t test was used for statistical analysis, data are represented as mean ± SEM (Δ t , exposure time; ∗ p < 0.05; ∗∗ p < 0.01). (D) WEE1 knockdown (KD) and vincristine (VCR) effect on metabolic activity of in vitro TP53 mut brain cancer cell lines: data are represented as mean ± SEM (siContr, control siRNA; siWEE1, WEE1 siRNA; NS, not significant; ∗ p < 0.05; ∗∗ p < 0.01) (see also C). (E) WEE1 KD and VCR effect on in vitro caspase-3 activity in TP53 mut brain cancer cell lines: data are represented as mean ± SEM (NS, not significant; ∗ p < 0.05; ∗∗ p < 0.01) (see also C). (F) Dose-response curves of WEE1 inhibitors (adavosertib, Debio 0123, and ZN-c3) in in vitro TP53 mut brain cancer cell lines: data are represented as mean ± SEM. (G) Synergy scores of vincristine and WEE1 inhibitors combination treatment in in vitro TP53 mut brain cancer cell lines: data are represented as mean.

Article Snippet: WEE1 knockdown at protein level was confirmed by Western blot (α-WEE1 antibody [B-11], cat.no. sc-5285, Santa Cruz Biotechnology).

Techniques: Biomarker Discovery, In Vitro, Phospho-proteomics, Knockdown, Activity Assay, Control

Journal: iScience

Article Title: Preclinical drug screen identifies WEE1 inhibitor and vinca alkaloid as a combination treatment concept for Li-Fraumeni syndrome medulloblastoma

doi: 10.1016/j.isci.2025.114564

Figure Lengend Snippet: WEE1 as a therapeutic target in pediatric brain tumor entities (A) WEE1 mRNA expression in MB tumors by methylation group (MB dataset: Northcott [ n = 491], cerebellum dataset: Roth [ n = 9] ). (B) WEE1 mRNA expression in MB subgroups (Cavalli [ n = 763] ). (C) Adavosertib sensitivity score in pediatric brain tumors (Petralia [ n = 218] ). (D) Adavosertib sensitivity score in MB groups (Cavalli [ n = 763] ). (E) Adavosertib sensitivity score in MB tumors by methylation group (Cavalli [ n = 763] ) (see also B). Data are represented as mean ± SEM (Student’s t test significance levels: NS, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001).

Article Snippet: WEE1 knockdown at protein level was confirmed by Western blot (α-WEE1 antibody [B-11], cat.no. sc-5285, Santa Cruz Biotechnology).

Techniques: Expressing, Methylation

Journal: iScience

Article Title: Preclinical drug screen identifies WEE1 inhibitor and vinca alkaloid as a combination treatment concept for Li-Fraumeni syndrome medulloblastoma

doi: 10.1016/j.isci.2025.114564

Figure Lengend Snippet: In vivo validation of adavosertib and vincristine (VCR) in LFS SHH-MB models (A) Survival of mice injected with BT084 patient-derived xenograft (PDX) model during treatment with adavosertib and VCR; log rank test was used for statistical analysis. (B) Tumor growth dynamics of BT084 PDX model during treatment with adavosertib and vincristine (VCR): data are represented as mean ± SEM. (C) Phospho (p)-CDK1 in LFS SHH-MB PDX cells (HS231222 and LFS primary) following in vivo treatment with adavosertib and VCR: numbers below the blot represent normalized fold change relative to non-treated control. (D) Tumor growth dynamics of LFS MB PDX models expressing WEE1 shRNA (shWEE1) and control shRNA (shSCRAMBLE). (E) Survival of mice injected with LFS MB PDX models expressing shWEE1 and shSCRAMBLE; log rank test was used for statistical analysis.

Article Snippet: WEE1 knockdown at protein level was confirmed by Western blot (α-WEE1 antibody [B-11], cat.no. sc-5285, Santa Cruz Biotechnology).

Techniques: In Vivo, Biomarker Discovery, Injection, Derivative Assay, Control, Expressing, shRNA

Journal: Neuro-Oncology

Article Title: Transcriptomics-guided high-throughput drug screening identifies potent therapies for P53 pathway altered DIPG/DMG

doi: 10.1093/neuonc/noaf216

Figure Lengend Snippet: Transcriptomic Screening Identifies ATR Pathway Inhibitor AZ20 as a Promising Candidate to Overcome SN-38 Resistance in TP53 -Mutant DIPG. ( A ) KEGG analysis of 190313 cells treated with SN-38 (10 nM, 72 h) revealed upregulation of cell cycle and DNA replication pathways (* P < .05). (B) HALLMARK pathway analysis identified enrichment of E2F target genes (* P < .05). (C) Correlation analysis showed strong associations between E2F (E2F1, E2F2, E2F7, E2F8) and DNA damage repair genes (ATR, CHK1, PARP1, etc.) (* P < .05). (D) Western blot analysis showed significant upregulation of ATR, CHK1, PARP1, and WEE1 protein expression in TP53 -mutant DIPG cell lines (190313, 190326, 150728) treated with 10 nM SN-38 for 72 h, compared to untreated controls. (E-G) PARP1 inhibitor Olaparib had minimal effect on TP53-mutant DIPG (IC50 > 10 μM) and showed no synergy with SN-38 (NS). (H) The CHK1 inhibitor (SCH900776) exhibited potent cytotoxic effects on TP53 -mutant DIPG cells (190326), with an IC50 of ∼100 nM and minimal toxicity to PPCs. (I-J) Co-treatment with SCH900776 (100 nM) and SN-38 (10 nM) demonstrated significant synergy, reducing cell viability (* P < .05). (K) Screening of 23 ATR pathway inhibitors identified AZ20 as the most potent (>70% viability reduction at 1 μM). Heatmap includes TP53-KD and PPM1D-KD isogenic lines. (L) AZ20 exhibited strong activity in TP53-mutant DIPG (IC50 ∼200 nM) and limited toxicity to PPCs (IC50 > 1 μM). Viability assessed by CellTiter-Glo (mean ± SD, n = 3).

Article Snippet: P53 (DO-1, Cat# 18032S), WIP1 (E2X1I, Cat# 94886S), and GAPDH (D4C6R, Cat# 97166S) antibodies were obtained from Cell Signaling Technology (CST); BCL2 (Cat# 68103-1-Ig), BAX (Cat# 60267-1-Ig), Vinculin (Cat# 66305-1-Ig), and PARP1 (Cat# 66520-1-Ig) antibodies were purchased from ProteinTech; CHK1 (Cat# BM3968), WEE1 (Cat# A01319-2), and ATR (Cat# A00262-3) antibodies were sourced from Boster.

Techniques: Mutagenesis, Western Blot, Expressing, Activity Assay

Journal: Neuro-Oncology

Article Title: Transcriptomics-guided high-throughput drug screening identifies potent therapies for P53 pathway altered DIPG/DMG

doi: 10.1093/neuonc/noaf216

Figure Lengend Snippet: Synergistic anti-tumor effects of AZ20 and SN-38 in TP53 -mutant DIPG cells through inhibition of ATR pathway signaling and induction of apoptosis. (A) Twenty-one ATR pathway inhibitors were screened in combination with SN-38 (1 μM each) in TP53-mutant DIPG cells. Viability was measured by CellTiter-Glo ( n = 3) analyzed by a two-tailed unpaired t -test. (B-D) Synergy analysis using the BLISS model confirmed a robust synergistic interaction between SN-38 and AZ20 in 190326 cells (D). In contrast, this synergistic effect was not observed in TP53 wild-type DIPG cells (150714, DIPG17) (B and C). (E-G) Cell viability was measured after 24, 48, and 72 h of treatment with DMSO, SN-38 (10 nM), AZ20 (10 nM), or both in 190326, 150714, and DIPG17 cells. Combination significantly reduced viability in 190326 (**** P < .0001). (H) Western blot analysis of protein expression in 190326 cells following 72 h of treatment with DMSO (vehicle control), SN-38 (10 nM), AZ20 (10 nM), or their combination. SN-38 monotherapy activated ATR and its downstream targets, CHK1 and WEE1, while combination treatment with SN-38 and AZ20 suppressed ATR activation and downregulated CHK1 and WEE1 expression. The combination treatment also induced apoptosis, as evidenced by increased levels of cleaved PARP1. (I) Chou-Talalay-based combination index (CI) heatmap for SN-38 and AZ20 in TP53-mutant DIPG cell line 190326. Combination index values were calculated from a 72-h viability assay using fixed-ratio matrix combinations of SN-38 and AZ20. CI < 1 indicates synergy, CI = 1 indicates additivity, and CI > 1 indicates antagonism. (J) 190326 cells transfected with siATR and treated with SN-38 or AZ20 showed reduced viability in SN-38 + siATR and AZ20 + SN-38 + siATR groups (**** P < .0001).

Article Snippet: P53 (DO-1, Cat# 18032S), WIP1 (E2X1I, Cat# 94886S), and GAPDH (D4C6R, Cat# 97166S) antibodies were obtained from Cell Signaling Technology (CST); BCL2 (Cat# 68103-1-Ig), BAX (Cat# 60267-1-Ig), Vinculin (Cat# 66305-1-Ig), and PARP1 (Cat# 66520-1-Ig) antibodies were purchased from ProteinTech; CHK1 (Cat# BM3968), WEE1 (Cat# A01319-2), and ATR (Cat# A00262-3) antibodies were sourced from Boster.

Techniques: Mutagenesis, Inhibition, Two Tailed Test, Western Blot, Expressing, Control, Activation Assay, Viability Assay, Transfection

Journal: Nature Communications

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1038/s41467-025-66514-0

Figure Lengend Snippet: a Relative ATF4-mScarlet MFI in RPE1 ATF4-mScarlet reporter cells after treatment with 125, 250, 500, or 1000 nM of AZD1775, RP-6306, VE-822 or AZD7762. Data represent mean ± SD (DMSO: n = 4; other conditions: n = 3). Statistical analysis was performed using a one-way ANOVA with Dunnett’s multiple comparisons with p ≤ 0.05 considered significant. b Immunoblot of RPE1 TP53 KO cells treated with siRNA targeting WEE1 for 72 h and AZD1775 (250 nM) for 2.5 or 5 h. Representative blot of n = 3 experiments. WEE1 and GCN2 engagement as measured by NanoBRET kinase target engagement assay. HEK293 cells were transfected with WEE1-NanoLuc ( c ) and NanoLuc-GCN2 ( d ) and incubated with K-10 tracer (0.5 μM), and indicated doses of CC1, GCN2iB, AZD1775, Debio 0123, ZNL-02-096, RP-6306 or neratinib for 2 h prior to substrate addition and BRET signal detection. Data represent mean ± SD (n = 3). Representative histograms ( e ) and quantification ( f ) of ATF4-mScarlet flow cytometry measurements in RPE1 TP53 KO ATF4-mScarlet reporter cells treated with siRNAs targeting GCN1 and AZD1775 or neratinib (1 µM) for 5 h. Data represent mean ± SD (n = 3), statistical analysis: two-way ANOVA with Tukey’s multiple comparisons test, with p ≤ 0.05 considered significant. g Model of WEE1 inhibitor mode of action and consequences. All replicates are biological replicates unless indicated otherwise. Source data are provided as a file.

Article Snippet: Immunodetection was performed using antibodies directed against puromycin (MABE343, Merck, 1:10000), GCN2 (3302, Cell Signaling, 1:1000), pGCN2 (ab75836, Abcam, 1:500), ATF4 (11815, Cell Signaling, 1:1000), Vinculin (ab129002, Abcam, 1:2000), HSP90 (sc-13119, Santa Cruz, 1:5000), pCDK1/2/3/5 (ab133463, Abcam, 1:1000), eIF2A (ab169528, Abcam, 1:1000), WEE1 (4936S, Cell signaling, 1:1000), rabbit anti-cyclin B1 (Santa Cruz, sc-752, 1:1000), and GCN1L1 (A301-843A, Bethyl laboratories, 1:1000) followed by staining with secondary antibodies Goat Anti-Rabbit Immunoglobulins/HRP (Dako, P0448) or Rabbit Anti-Mouse Immunoglobulins/HRP (Dako, P0260).

Techniques: Western Blot, Drug discovery, Transfection, Incubation, Flow Cytometry