pcmv6 xl4 wee1  (OriGene)

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. IHC staining for WEE1 in Human EAC tissue sections depicting strong cytosolic localization B. Quantification of the Cytoplasmic and Nuclear Signals from A using Cell Profiler C. Cell fractionation of FLO1 and OE33 EAC cell lines followed by western blot for WEE1, p84 (Nuclear marker) and ὰ-Tubulin (Cytoplasmic marker) D- Co-Immunofluorescence of WEE1 (green) and C-MYC (Red) along with DAPI nuclear stain (Blue) in EAC cell lines FLO1 and OE33, captured at 40X Magnification.E.MYC mRNA expression in WEE1 high Vs WEE1 low EAC tissue samples derived from TCGA and 4 different GEO datasets. F. Gene Set Enrichment Analysis (GSEA) of MYC target genes in WEE1 high Vs WEE1 low EAC samples G. WEE1 mRNA expression in non-cancerous normal esophagus (Normal) and Esophageal cancer (Tumor) tissue samples, analyzed by TNM plot.com . *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. H. MYC mRNA expression in non-cancerous normal esophagus (Normal) and Esophageal cancer (Tumor) tissue samples, analyzed by TNM plot.com . I. Co-Immunofluorescence of WEE1 (green) and C-MYC (Red) along with DAPI nuclear stain (blue) in normal esophagus and gastroesophageal adenocarcinoma tissue sections in TMA, captured at 20x magnification. J- Quantification of fluorescence intensity from I K – Correlation analysis between WEE1 and MYC signal intensity

Article Snippet: For WEE1 over expression, pCMV6-XL4-WEE1 (Origene, sc117999, Rockville, MD) at a concentration of 0.05 μg and 0.1 μg was transfected using Polyjet transfection reagent in a 6-well plate as described above.

Techniques: Immunohistochemistry, Cell Fractionation, Western Blot, Marker, Immunofluorescence, Staining, Expressing, Derivative Assay, Fluorescence

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. Western blot analysis of WEE1, P-CDC2 (Y15), CDC2, C-MYC, and β-ACTIN in OE33, OE19, and SK-GT4 cells treated with Control siRNA and WEE1 siRNA for 48 hours. B.Cell cycle analysis of the OE19 cell line treated with Control siRNA and WEE1 siRNA for 48 hours. C.Quantification of cell population in various phases of the cell cycle from B. D.Luciferase reporter assay to determine MYC transcriptional activity in Control siRNA and WEE1 siRNA treated OE33, OE19, and SK-GT4 cells, previously transfected with control plasmid/ C-MYC overexpressing plasmid *P<0.05, **P<0.01, *** P<0.001. E. qRT-PCR analysis of MYC target genes – ABCC1, MNT & CDK4 mRNA normalized to HPRT1 gene in Control siRNA and WEE1 siRNA treated OE33 and OE19 cells *P<0.05, **P<0.01, ***P<0.001. F. Co-Immunofluorescence staining of WEE1 (green), C-MYC (Red), along with DAPI nuclear stain (blue) in SK-GT4 and OE33 cell lines transfected with Control siRNA and WEE1 siRNA, captured at 20X magnification. G. Downregulation of C-MYC target genes in WEE1 siRNA-treated OE33 cells vs. control siRNA-treated cells, from RNA sequencing analysis.

Article Snippet: For WEE1 over expression, pCMV6-XL4-WEE1 (Origene, sc117999, Rockville, MD) at a concentration of 0.05 μg and 0.1 μg was transfected using Polyjet transfection reagent in a 6-well plate as described above.

Techniques: Knockdown, Western Blot, Control, Cell Cycle Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence, Staining, RNA Sequencing

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. Western blot analysis of WEE1, P-CDC2 (Y15), CDC2, C-MYC and β-ACTIN in FLO1, OE33, SK-GT4 and OE19 cells untreated/ treated with MK-1775 for 24 hours. B. Cell cycle analysis of the OE19 cell line, untreated / treated with MK-1775 for 24 hours. C. Quantification of cell population in various phases of the cell cycle from B. D. Luciferase reporter assay to determine MYC transcriptional activity in untreated/ MK-1775 treated OE33, OE19, and SK-GT4 cells, previously transfected with control plasmid/ C-MYC overexpressing plasmid *P<0.05, **P<0.01, *** P<0.001. Lower panel – western blot analysis of P-CDC2 Y15, C-MYC & β-ACTIN in the cell lysates from D. E. qRT-PCR analysis of MYC target genes – ABCC1, MNT & CDK4 mRNA normalized to HPRT1 gene in OE33 and OE19 cell lines untreated / treated with MK-1775 for 24 hours *P<0.05, **P<0.01, ***P<0.001. F. Co-Immunofluorescence staining of P-CDC2 (Y15) (green), C-MYC (Red), along with DAPI nuclear stain (blue) in FLO1 and OE33 cell lines, untreated / treated with MK-1775 for 24 hours, captured at 20x Magnification.

Article Snippet: For WEE1 over expression, pCMV6-XL4-WEE1 (Origene, sc117999, Rockville, MD) at a concentration of 0.05 μg and 0.1 μg was transfected using Polyjet transfection reagent in a 6-well plate as described above.

Techniques: Inhibition, Activity Assay, Western Blot, Cell Cycle Assay, Luciferase, Reporter Assay, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. Western Blot analysis of WEE1, P-CDC2 (Y15), CDC2, C-MYC, and β-ACTIN in OE19, OE33, and SK-GT4 cells treated with MG 132 or MK-1775 alone or in combination. B. Western Blot analysis of WEE1, P-CDC2 (Y15), CDC2, C-MYC, and β-ACTIN in OE19 and OE33 cells treated with siRNA or MG 132 alone or in combination. C. Western blot analysis of WEE1, C-MYC, and β-ACTIN in Cycloheximide-treated (0 min, 10 min, 30 min, & 60 min) OE19, OE33 & SK-GT4 cell lines. These cells were previously untreated (control), or MK-1775 treated for 24 hours. D, E & F. Half-life determination of C-MYC in OE19, OE33, and SK-GT4 cell lines through linear regression analysis. The signal intensity of the C-MYC bands was normalized to the respective β-ACTIN bands and used for quantification.

Article Snippet: For WEE1 over expression, pCMV6-XL4-WEE1 (Origene, sc117999, Rockville, MD) at a concentration of 0.05 μg and 0.1 μg was transfected using Polyjet transfection reagent in a 6-well plate as described above.

Techniques: Inhibition, Knockdown, Western Blot, Control

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. Western Blot analysis of P-GSK3β (S9), GSK3β, P-C-MYC T58, C-MYC & β-ACTIN in untreated control as well as MK-1775 (0.5μM & 1μM) treated OE33, SK-GT4 & OE19 cell lines. B. Western Blot analysis of P-GSK3β (S9), GSK3β, P-C-MYC T58, C-MYC & β-ACTIN in control siRNA and WEE1 siRNA treated OE33, SK-GT4 & OE19 cell lines. C. PLA to visualize C-MYC and GSK3β interaction (red spots) in untreated Control & MK-1775 treated OE19, OE33, and SK-GT4 cell lines. Captured at 40X magnification. D. Western blot analysis of WEE1, P-CDC2 Y15, C-MYC, P-GSK3β (S9), GSK3β, and β-ACTIN in empty vector as well as WEE1-CDS vector transfected OE33, SK-GT4, and OE19 cell lines. E. Western blot analysis of WEE1, P-CDC2 Y15, CDC2, C-MYC, and β-ACTIN in empty vector, WEE1-CDS-WT, and WEE1-CDS-K328A kinase dead mutant vector transfected SK-GT4 cells.

Article Snippet: For WEE1 over expression, pCMV6-XL4-WEE1 (Origene, sc117999, Rockville, MD) at a concentration of 0.05 μg and 0.1 μg was transfected using Polyjet transfection reagent in a 6-well plate as described above.

Techniques: Inhibition, Knockdown, Phospho-proteomics, Western Blot, Control, Plasmid Preparation, Transfection, Mutagenesis

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. Co-Immunofluorescence staining of MRP1 (Green), MYC (Red), along with DAPI nuclear stain (blue) in Normal non-cancerous esophagus (NE) and Gastroesophageal Junction adenocarcinoma TMA. Captured at 20X Magnification. B. Relative quantification of MRP1 fluorescence intensity and MYC fluorescence intensity in Normal esophagus (NE) and EAC tissue sections. C. Co-relation analysis between MRP1 fluorescence intensity and MYC fluorescence intensity from B. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. D. Western blot analysis of C-MYC, MRP1, and β-ACTIN in untreated control and MK-1775-treated OE33, FLO1, and SK-GT4 cell lines. E & F. Rhodamine 123 intracellular fluorescence intensity during influx (red) and after 4 hours of efflux (blue) in control (E) & MK-1775 treated OE33 cells (F) G. Percentage of intracellular Rhodamine 123 retained after efflux in Control and MK-1775 treated OE33 cells * P <0.05, **P<0.01, ***P<0.001. H & I. Rhodamine 123 intracellular fluorescence intensity during influx (red) and after 4 hours of efflux (blue) in control (E) & MK-1775 treated SK-GT4 cells (F) J. Percentage of intracellular Rhodamine 123 retained after efflux in Control and MK-1775 treated SK-GT4 cells *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: For WEE1 over expression, pCMV6-XL4-WEE1 (Origene, sc117999, Rockville, MD) at a concentration of 0.05 μg and 0.1 μg was transfected using Polyjet transfection reagent in a 6-well plate as described above.

Techniques: Inhibition, Immunofluorescence, Staining, Quantitative Proteomics, Fluorescence, Western Blot, Control

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. 21 positive hits (red), which synergized with MK-1775 in O19 cells during the first round of screening using an 892 FDA-approved drug screening library. B. Panobinostat (red arrow) was found to synergize with MK-1775 in FLO1 (blue) and SK-GT4 (red) cell lines during the second round of screening. C. Synergy Finder analysis results in FLO1, OE33, OE19, and SK-GT4 cell lines treated with MK-1775 and Panobinostat. *P<0.05, **P<0.01, ***P<0.001. D. Flow cytometric analysis of Annexin V & SYTOX red dual staining in OE33 cells treated with MK-1775/Panobinostat alone or a combination. E. Percentage of apoptotic cells from Fig D *P<0.05, **P<0.01, ***P<0.001. F. Western blot analysis of PARP, Cl-PARP, Caspase 3, Cl-Caspase 3, and β-ACTIN in OE33 cells treated with MK-1775/ Panobinostat alone or a combination. G. Flow cytometric analysis of Annexin V & SYTOX red dual staining in OE19 cells treated with MK-1775/Panobinostat alone or a combination. H. Percentage of apoptotic cells from Fig G *P<0.05, **P<0.01, ***P<0.001. I. Western blot analysis of PARP, Cl-PARP, Caspase 3, Cl-Caspase 3, and β-ACTIN in OE19 cells treated with MK-1775/ Panobinostat alone or a combination.

Article Snippet: For WEE1 over expression, pCMV6-XL4-WEE1 (Origene, sc117999, Rockville, MD) at a concentration of 0.05 μg and 0.1 μg was transfected using Polyjet transfection reagent in a 6-well plate as described above.

Techniques: Drug discovery, Staining, Western Blot

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. Human EAC PDX-derived Organoids treated with MK-1775 / Panobinostat alone or in combination. B. Quantification of organoid diameter from A. C. Tumor growth curves in 4 experimental groups (Untreated control, MK-1775, Panobinostat, combination of MK-1775 & Panobinostat) at the end of the experiment. D. Western blot analysis of PARP, Cl-PARP, Caspase 3, CL-Caspase3, and β-ACTIN in Mouse Xenografts. E. Immunofluorescence staining for P-CDC2 Y15 (green), C-MYC (red), MRP1 (green), and Ki67 (red) in Mouse EAC PDXs, captured at 20X Magnification. F. Quantification data from E. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: For WEE1 over expression, pCMV6-XL4-WEE1 (Origene, sc117999, Rockville, MD) at a concentration of 0.05 μg and 0.1 μg was transfected using Polyjet transfection reagent in a 6-well plate as described above.

Techniques: In Vivo, Derivative Assay, Control, Western Blot, Immunofluorescence, 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 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