wee1  (MedChemExpress)

Journal: bioRxiv

Article Title: WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2

doi: 10.1101/2025.03.12.642825

Figure Lengend Snippet: a Western blot of RPE TP53 -/- cells treated for 24 hour treatments with increasing concentrations of AZD1775, with and without 1 μM GCN2iB. b,c Resazurin-based cell viability assay of RPE-1 TP53 -/- dCas9-KRAB cells expressing sgRNAs targeting GCN2, GCN1, or the AAVS1 locus. Cells were treated with varying concentrations of AZD1775 for 72 hours in a 96-well plate format (biological n=4). Graphs are depicted with means ± SD. Validations of the CRISPRi knockdown of GCN2 and GCN1 are shown in Supplementary Fig.9. d Representative immunofluorescence images of a panel of cell lines (RPE-1, U2OS, HAP1, SAOS2, MCF10A, HELA and MEF cell lines) probed for nuclear ATF4 treated with either DMSO, AZD1775 (650 nM for all cell lines except HAP1, which were treated at 300 nM), or AZD1775 in combination with 1 μM GCN2iB followed by row Z-score heatmap normalised per cell line summarising the immunofluorescence nuclear ATF4 intensity across different cell lines (biological n=4). e Ribosome profiling showing the log fold change of RPE-1 TP53 -/- cells treated with 650 nM AZD1775 vs DMSO for 10 hours (biological n=3). f Summary of flow cytometry based AHA experiment on the RPE-1 TP53 -/- cell line. An example of the flow cytometry gating strategy is available in Supplementary Fig.7a. g Bar chart of flow cytometry results showing the median fluorescence intensity of clickable AHA. RPE- 1 TP53 -/- cells (6 well plate format) were treated with 1 μg/mL cycloheximide, 350 nM AZD1775, 1.5 μM Debio0123 and 1 μM GCN2iB alone or in combination. A cell population not treated with AHA served as an unstained negative control. Median fluorescence intensity results were normalised to the untreated control (UT) (biological n=3). Bar charts are depicted with means ± SD, points represent each biological replicate. Statistical analyses were performed by one-way ANOVA with multiple comparisons, comparing to untreated condition, ns= not significant, ** p LJ

Article Snippet: GCN2iB and WEE1-IN-4 was obtained from Medchemexpress.

Techniques: Western Blot, Viability Assay, Expressing, Knockdown, Immunofluorescence, Flow Cytometry, Fluorescence, Negative Control, Control

Journal: bioRxiv

Article Title: WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2

doi: 10.1101/2025.03.12.642825

Figure Lengend Snippet: a Schematic showing flow cytometry based CRISPRi two-colour growth competition assays in RPE-1 TP53 -/- dCas9-KRAB cells. Cells were transduced with either sgLacZ-mCherry virus or sgGOI (gene of interest)-GFP. The mCherry and GFP expressing cell populations were mixed at a 50:50 ratio and treated with DMSO, AZD1775 (150 nM, 250 nM or 300 nM), 100 nM ISRIB or 1 μM GCN2iB, alone or in combination, for 9 days. Cells were assessed by flow cytometry, passaged and treated with fresh DMSO or drug every 3 days. The flow cytometry gating strategy is available in Supplementary Fig.7b. b Example of a normalised GFP/mCherry fitness graph. Values above 1 indicate the cell population expressing sgGOI causes increased relative cell fitness compared to cells expressing sgLacZ control, whereas values below 1 indicate decreased fitness. This is followed by an area under the curve that is generated from the GFP/mCherry fitness graph. c, d, e Bar charts quantifying the area under the curve of the normalised GFP/mCherry fitness graphs for different sgGOI-GFP populations vs sgLacZ-mCherry controls. Data were normalised to the respective DMSO-treated conditions. Values >1 indicate increased relative fitness compared to DMSO. Values <1 indicate decreased relative fitness compared to DMSO. Gray bars represent DMSO only, blue bars represent AZD1775 treatment only, blue/navy striped bars represent AZD1775 + 100 nM ISRIB, blue/pink striped bars represent AZD1775 + 1 μM GCN2iB. AZD1775 concentrations used: 150 nM for GSPT1, ALKBH8, and PKMYT1 CRISPRi; 250 nM for RRM2 and FZR1 CRISPRi; 300 nM for DUT CRISPRi. Original GFP/mCherry fitness graphs for all conditions (including sgAAVS1-GFP vs sgLacZ-mCherry controls) as well as ISRIB and GCN2iB only treatment controls are provided in Supplementary Fig.10-13, with CRISPRi knockdown validations in Supplementary Fig.9. Bar charts are depicted with means ± SD, points represent each biological replicate (biological n=3). Statistical analyses were performed by one-way ANOVA with multiple comparisons, comparing to DMSO treatment, ns= not significant, * p LJ

Article Snippet: GCN2iB and WEE1-IN-4 was obtained from Medchemexpress.

Techniques: Flow Cytometry, Transduction, Virus, Expressing, Control, Generated, Knockdown, Viability Assay

Journal: bioRxiv

Article Title: WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2

doi: 10.1101/2025.03.12.642825

Figure Lengend Snippet: a Western blot showing the time course of WEE1 degradation in the RPE TP53 -/- cell line following treatment with 1 μM HRZ-057-1 or HRZ-1-098-1. b A schematic of the experiment in (c) . c Western blot of the RPE TP53 -/- cell line pre-treated with DMSO or 1 μM WEE1 molecular glues (HRZ-057-1 and HRZ-1-098-1) for 1 hour (total treatment duration: 7 hours), followed by DMSO or 650 nM AZD1775 for an additional 6 hours. d A schematic of the in vitro FLAG-tagged GCN2 experiment e Western blot of an in vitro experiment probing the total and phosphorylated GCN2 in the presence of DMSO, Neratinib, WEE1i (AZD1775 and Debio0123) and PKMYT1i (RP-6306). f Immunofluorescence analysis of nuclear ATF4 and γH2AX in the RPE TP53 -/- cell line treated with 650 nM AZD1775 across multiple timepoints (biological n=3). Box plots show median and quartile ranges. g Representative images showing ATF4 and γH2AX intensity in untreated (UT) cells and those treated with 650 nM AZD1775 alone or in combination with 1 μM GCN2iB for 24 hours in the RPE TP53 -/- cell line.

Article Snippet: GCN2iB and WEE1-IN-4 was obtained from Medchemexpress.

Techniques: Western Blot, In Vitro, Immunofluorescence