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: Cell Reports

Article Title: The insertion of an ATTTC repeat in an Alu element hyperactivates a neurodevelopmental enhancer in spinocerebellar ataxia type 37

doi: 10.1016/j.celrep.2026.117146

Figure Lengend Snippet: The DRL shows neurodevelopmental enhancer activity (A) Schematic representation of the DRL/FragT sequence in DAB1 -antisense strand. The zoom-in box (pale brown box) shows the (AAAAT) n (blue box) in the middle A-rich region of the AluJb element with left (LM) and right (RM) monomers, close to the CpG island (yellow box) and the TFBS (green box), flanked by CTCF-binding sites. The ENCODE-annotated regulatory EH38E1350977 element is represented in pink. Lines underneath represent the sequences cloned for the zebrafish transgenic lines generation, across and within the DRL sequence. (B) Stereomicroscopic images of EGFP expression in the eye and whole brain, at 24 hpf, in transgenic lines with FragT14-AS and Frag3-AS sequences cloned upstream of a promoter-less EGFP; no expression is seen in control embryos; images were processed with the Fiji software. (C) Left: representative confocal images confirming FragT14-AS and Frag3-AS expression in the eye, forebrain, midbrain, and hindbrain in these zebrafish transgenic lines at 24 hpf; maximum intensity z-projections of 14 planes; scale bars: 100 μm. Right: zoom-in images for the same zebrafish lines; orange and yellow delimitations represent eye and midbrain-hindbrain boundary close-ups, respectively; scale bars: 100 μm. (D) Left: schematic representation of the DRL sequences cloned upstream of promoter-less firefly luciferase ( luc+ ) for reporter assays in human HEK293T cells. Right: graphic representation of luciferase activity (fold change) for each sequence, as luc+/NanoLuc (Nluc) expression ratios in HEK293T cells, compared to the negative control (data are from three biological replicates, shown as the mean ± SD; one-way ANOVA test with Dunnett’s post hoc test; ∗∗∗∗ p < 0.0001 and ∗∗∗ p < 0.001). FB, forebrain; MB, midbrain; HD, hindbrain; OV, optic vesicle; MA, mutant allele; scale bars: 100 μm. Asterisks represent autofluorescence regions. See also .

Article Snippet: As positive control, total protein extracts were extracted from HEK293T cells transfected with pCMV6-XL4-DAB1 mammalian expression vector (#SC113027, Origene) and 1 μg was loaded on SDS-PAGE gel.

Techniques: Activity Assay, Sequencing, Binding Assay, Clone Assay, Transgenic Assay, Expressing, Control, Software, Luciferase, Negative Control, Mutagenesis

Journal: Cell Reports

Article Title: The insertion of an ATTTC repeat in an Alu element hyperactivates a neurodevelopmental enhancer in spinocerebellar ataxia type 37

doi: 10.1016/j.celrep.2026.117146

Figure Lengend Snippet: DRL interaction with neural regulatory elements and silencer activity outside of the DAB1 expression domain (A) Schematics of interactions between the DRL sequence and the zebrafish midbrain-specific Z48 enhancer at 48 hpf. Top: FragT14-AS, Frag1-AS, and Frag3-AS sequences cloned upstream of a promoter-less EGFP and Z48 with schematics of their driven EGFP expression to zebrafish somites and upon Z48 interaction only to the whole midbrain. Bottom left: representative stereomicroscopic images of different EGFP expression patterns in the midbrain from FragT14-AS, Frag1-AS, and Frag3-AS interactions with the Z48 enhancer in embryos. Bottom right: graphic representation of the percentage of embryos expressing EGFP in midbrain and/or somites (shown as the mean ± SD of three replicates; EGFP-positive embryos, FragT14-AS = 238, Frag1-AS = 195, and Frag3-AS = 148, ꭕ 2 , ∗∗∗∗ p < 0.0001). (B) Left: schematic representation of the DRL sequences cloned upstream the TK promoter in the control of luciferase expression for silencer assay in HEK293T cells; right: luciferase activity in HEK293T cells, measured by the fold change in luciferase/NanoLuc (luc2/Nluc) expression ratios compared to the negative control (shown as the mean ± SD of three biological replicates; one-way ANOVA test with Dunnett’s post hoc test; ∗∗∗∗ p < 0.0001 and ∗∗∗ p < 0.001). (C) Silencer activity of the DRL in vivo . Top: schematics of the putative silencer placed between the Z48 enhancer and the cardiac actin promoter in the reporter vector. Intermediate: graphical representation showing EGFP fluorescence intensity signal in midbrain and somites triggered by (left) FragT14-S and FragT-S-MA and (right) Frag1-S and Frag3-S sequences at 24 hpf; negative control, empty vector (data are represented as the mean ± SD; n = 60 zebrafish embryos/condition from three replicates, Kruskal-Wallis test followed by Dunn’s post hoc test; ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, and ∗ p < 0.05). Bottom: representative stereomicroscopic images of EGFP expression in zebrafish midbrain and somites driven by FragT14-S, FragT-S-MA, Frag1-S, and Frag3-S putative silencer elements; scale bars: 200 μm; arrows indicate EGFP expression in midbrain. MA, mutant allele. Asterisks show the area of autofluorescence. See also .

Article Snippet: As positive control, total protein extracts were extracted from HEK293T cells transfected with pCMV6-XL4-DAB1 mammalian expression vector (#SC113027, Origene) and 1 μg was loaded on SDS-PAGE gel.

Techniques: Activity Assay, Expressing, Sequencing, Clone Assay, Control, Luciferase, Negative Control, In Vivo, Plasmid Preparation, Fluorescence, Mutagenesis