sc 5285  (Santa Cruz Biotechnology)

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: Scientific Reports

Article Title: Glutamine modulates cellular size by regulating Wee1 expression

doi: 10.1038/s41598-025-17687-7

Figure Lengend Snippet: Glutamine depletion leads to reduced Wee1. (A) Wee1 protein levels for MDA-MB-231 cells grown with or without glutamine for the indicated time period, analyzed by western blot. GAPDH was used as loading control. The image on the right shows the quantification of the gel. (B) A gel image of semiquantitative RT-PCR of Wee1 gene expression in MDA-MB-231 cells, as described in (A). We used the levels of B-Actin and GAPDH as loading controls. The image on the right shows the quantification of the gel. (C) We analyzed the stability of Wee1 protein levels after treatment with cycloheximide (CHX) for different time periods in MDA-MB-231 cells grown with or without glutamine, using western blot analysis. GAPDH was used as loading control. The image on the right shows the quantification of the gel. (D) Western blot analysis was used to analyze the stability of Wee1 protein levels after treatment with cycloheximide (CHX) for different time periods in HeLa cells grown with or without glutamine, as indicated. GAPDH was used as loading control. The image on the right shows the quantification of the gel. Error bars, SD ( n = 3). A student t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For primary antibodies, we used Wee1 (1:1000; Santa Cruz #5285) and GAPDH (1:1000; Santa Cruz #32233).

Techniques: Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: Scientific Reports

Article Title: Glutamine modulates cellular size by regulating Wee1 expression

doi: 10.1038/s41598-025-17687-7

Figure Lengend Snippet: Wee1 regulates cellular size. (A) HeLa cells were transfected with either Wee1 eSiRNA or control eSiRNA (eSiLuciferase). We analyzed Wee1 levels using a western blot. GAPDH was used as loading control. (B) Microscopy images illustrate the cellular morphology of the cells described in (A). (C) We measured the cellular size (length) of the cells described in (A) using software (Mosaic 2.4, Zeiss) in a Zeiss primvert-inverted cell culture microscope, and indicated the results with a box plot in the lower panel. (D) We transfected either Wee1 eSiRNA or control eSiRNA (eSiLuciferase) into MDA-MB-231 cells. We analyzed Wee1 levels using a western blot. GAPDH was used as loading control. (E) Microscopy images showing the cellular morphology of the cells described in (D). (F) The length of each cell was measured using software (Mosaic 2.4, Zeiss) in a Zeiss primvert-inverted cell culture microscope for the cells shown in (D), as shown by the box plot (lower panel). Error bars, SD ( n = 100). A student t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For primary antibodies, we used Wee1 (1:1000; Santa Cruz #5285) and GAPDH (1:1000; Santa Cruz #32233).

Techniques: Transfection, esiRNA, Control, Western Blot, Microscopy, Software, Cell Culture

Journal: Scientific Reports

Article Title: Glutamine modulates cellular size by regulating Wee1 expression

doi: 10.1038/s41598-025-17687-7

Figure Lengend Snippet: Glutamine supplementation in glutamine-deprived medium increases cellular size and Wee1 expression. (A) Microscopy images showing the morphology of MDA-MB-231 cells grown in glutamine-free medium and in medium supplemented with varying concentrations of glutamine (1.25 mM, 2.5 mM, and 5 mM). (B) Cellular area was measured using Zeiss Mosaic 2.4 software with a Zeiss Primovert inverted cell culture microscope for the cells shown in (A). The results are presented as a box plot. Error bars represent standard deviation (SD); n = 100. (C) Western blot showing Wee1 protein levels in MDA-MB-231 cells grown in glutamine-free medium and in medium supplemented with different concentrations of glutamine. Tubulin was used as a loading control. (D) Densitometric analysis showing the quantification of Wee1/tubulin levels for the samples described in (C). Error bars represent SD; n = 3. A student t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For primary antibodies, we used Wee1 (1:1000; Santa Cruz #5285) and GAPDH (1:1000; Santa Cruz #32233).

Techniques: Expressing, Microscopy, Software, Cell Culture, Standard Deviation, Western Blot, Control

Journal: Scientific Reports

Article Title: Glutamine modulates cellular size by regulating Wee1 expression

doi: 10.1038/s41598-025-17687-7

Figure Lengend Snippet: Glutamine supplementation in glutamine-deprived medium increases cellular size and Wee1 expression in HeLa cells. (A) Microscopy images showing the morphology of HeLa cells cultured in glutamine-free medium and in medium supplemented with different concentrations of glutamine (1.25 mM, 2.5 mM, and 5 mM). (B) Cellular area was measured using Zeiss Mosaic 2.4 software with a Zeiss Primovert inverted cell culture microscope for the cells shown in (A). Results are presented as a box plot. Error bars represent standard deviation (SD); n = 100. (C) Western blot analysis of Wee1 protein levels in HeLa cells cultured in glutamine-free medium and in medium supplemented with increasing concentrations of glutamine. Tubulin was used as a loading control. (D) Quantification of Wee1/tubulin levels from the western blot shown in (C). Error bars represent SD; n = 3. A Student’s t -test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For primary antibodies, we used Wee1 (1:1000; Santa Cruz #5285) and GAPDH (1:1000; Santa Cruz #32233).

Techniques: Expressing, Microscopy, Cell Culture, Software, Standard Deviation, Western Blot, Control

Journal: Scientific Reports

Article Title: Glutamine modulates cellular size by regulating Wee1 expression

doi: 10.1038/s41598-025-17687-7

Figure Lengend Snippet: Wee1 is a critical component in glutamine-mediated cell size regulation. (A,B) Western blot analysis of Wee1 protein levels in HeLa ( A ) and MDA-MB-231 ( B ) cells treated with eSiRNA against Wee1 or Luciferase (control), cultured in glutamine-free medium. Additionally, HeLa cells treated with eSiRNA against Wee1 were cultured in medium supplemented with 2.5 mM glutamine. GAPDH was used as a loading control. Error bars represent SD; n = 3. (C,D) Quantification of Wee1/GAPDH levels from the western blots shown in ( A ). (E,F) Cellular area was measured using Zeiss Mosaic 2.4 software and a Zeiss Primovert inverted cell culture microscope for the cells described in ( A & B ). Results are presented as box plots. Error bars represent standard deviation (SD); n = 100. A Student’s t -test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For primary antibodies, we used Wee1 (1:1000; Santa Cruz #5285) and GAPDH (1:1000; Santa Cruz #32233).

Techniques: Western Blot, esiRNA, Luciferase, Control, Cell Culture, Software, Microscopy, Standard Deviation

Journal: Scientific Reports

Article Title: Glutamine modulates cellular size by regulating Wee1 expression

doi: 10.1038/s41598-025-17687-7

Figure Lengend Snippet: Schematic summarizing the role of glutamine in regulating cellular size. Glutamine regulates c-MYC expression, which in turn controls Wee1 expression. Previous studies have shown that c-MYC binds to the promoter of Wee1, thereby regulating its transcription. Glutamine depletion leads to reduced c-MYC mRNA levels, decreased Wee1 mRNA and protein levels, and ultimately a reduction in cellular size.

Article Snippet: For primary antibodies, we used Wee1 (1:1000; Santa Cruz #5285) and GAPDH (1:1000; Santa Cruz #32233).

Techniques: Expressing