Journal: Cell Death Discovery

Article Title: WWP1 gain-of-function drives developmental anoikis through TGFβ pathway during neurodevelopment

doi: 10.1038/s41420-026-02977-4

Figure Lengend Snippet: A Scheme of WWP1 variant-expressing cell line establishment and downstream analysis. The image is created with Biorender.com. B Representative images of dead cell population in WWP1 variants-expressing HeLa cell lines treated with gefitinib. Red, propidium iodide (PI). Scale bar, 200 μm. C Quantification of the PI + counts in HeLa cell lines treated with gefitinib. n = 3 for each condition. Two-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01. D Functional GO enrichment analysis of upregulated and downregulated DEGs between WWP1 E798V and control cell lines. The bar color indicates the cluster categories of the GO term. BP, biological process; CC, cellular component. E Differential PROGENy pathway activity scores of WWP1 E798V relative to control. The pathway with the lowest value, TGFβ, is indicated by a deep blue color. F Gene set enrichment plots for TGFβ signaling and epithelial-mesenchymal transition. NES, normalized enrichment score. G Western blot analysis of phospho-SMAD2 and SMAD2 in WWP1 variant-expressing HeLa cell lines treated with vehicle or hTGFβ1 (10 ng/mL). ACTIN is used as the loading control. H Quantification of normalized pSMAD2 to SMAD2 protein expression ratio in WWP1 variant-expressing HeLa cell lines. n = 5. Kruskal-Wallis test with Dunn’s post hoc test. * p < 0.05; ns not significant. Bar graphs indicate mean ± SEM.

Article Snippet: Non-cytotoxic dosing conditions were determined by the cell viability using Propidium Iodide/RNase staining solution (Cell Signaling Technology, #4087).

Techniques: Variant Assay, Expressing, Functional Assay, Control, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: PTEN-loss confers dependence on the guanylate synthesis enzyme IMPDH in T-cell acute lymphoblastic leukemia

doi: 10.64898/2025.12.22.696045

Figure Lengend Snippet: ( A, B) Indicated human T-ALL cells were treated for 72 hrs with indicated concentrations of the FDA approved IMPDH inhibitor mycophenolic acid. (A) Viable cells were measured using the Cell Titer Glo Cell Viability Assay (Promega), graphed as percent of vehicle-treated cells. (B) Mycophenolic acid IC 50 values, calculated from the experiment in (A). (C, D) T-ALL cells from (A) grown in complete media or serum starved for 18 hours followed by immunoblotting for indicated proteins. (E) Indicated T-ALL cells treated for 72 hrs with vehicle or mycophenolic acid (MPA, 2.5 µM). Cell death was quantified by Annexin V/ Propidium Iodide (AV/PI) staining and graphed as percent of the total cell population. (F) Immunoblots from indicated T-ALL cells treated with vehicle or MPA (1 µM, 48 hrs). (G) Indicated cells treated with indicated doses of MPA, with or without exogenous guanosine (25 µM). Viable cells were measured as in (A) and graphed as percent of vehicle-treated cells. Graphical data are represented as mean ± SEM. **p < 0.0001 by two-way ANOVA. (See also Figure S1).

Article Snippet: Cells were then stained with propidium iodide-RNase solution (CST #4087) for 1 hour at room temperature.

Techniques: Viability Assay, Western Blot, Staining

Journal: bioRxiv

Article Title: PTEN-loss confers dependence on the guanylate synthesis enzyme IMPDH in T-cell acute lymphoblastic leukemia

doi: 10.64898/2025.12.22.696045

Figure Lengend Snippet: (A) Immunoblots from parental Jurkat cells and Jurkat cells with doxycycline-inducible PTEN-addback, treated with mycophenolic acid (MPA, 1 µM, 48 hrs) in the presence or absence of doxycycline (Dox, 1 µg/mL), following 24 hr doxycycline pre-treatment. (B-D) Ba/F3 cells with CRISPR control (sgCtl) or Pten deletion (sgPTEN) were (B) treated with indicated doses of the IMPDH inhibitor mizoribine for 72 hours. Viable cells were measured using the Cell Titer Glo Cell Viability Assay and graphed as percent of vehicle-treated cells. (C) Immunoblots on indicated proteins treated with vehicle or mizoribine (5 µM, 48 hrs). (D) Ba/F3 cells treated with vehicle or mizoribine (5 µM, 72 hrs). Cell death was quantified by Annexin V/ Propidium Iodide (AV/PI) staining and graphed as percent of the total cell population. (E, F) Immunoblots from the indicated PTEN-deficient T-ALL cells treated for 48 hrs with MPA (1 µM) with or without the mTORC1 inhibitor RMC-5552 (1 nM) or the AKT inhibitor MK-2206 (2 µM). (G, H) Indicated cells treated for 72 hrs with MPA (2.5 µM) with or without RMC-5552 (1 nM) or MK-2206 (2 µM). Cell death was quantified and graphed as in (D) . Graphical data are represented as mean ± SEM. **p < 0.0001 by two-way ANOVA. (See also Figure S2).

Article Snippet: Cells were then stained with propidium iodide-RNase solution (CST #4087) for 1 hour at room temperature.

Techniques: Western Blot, CRISPR, Control, Viability Assay, Staining

Journal: bioRxiv

Article Title: PTEN-loss confers dependence on the guanylate synthesis enzyme IMPDH in T-cell acute lymphoblastic leukemia

doi: 10.64898/2025.12.22.696045

Figure Lengend Snippet: (A) Immunoblots from indicated cells treated with mycophenolic acid (MPA, 1 µM) for indicated times. (B) Indicated cells were treated with MPA (1 µM, 24 hrs). Cell cycle distribution was quantified by flow cytometry for propidium iodide staining. (C) Indicated cells were treated with MPA (1 µM, 16 hrs) followed by flow cytometry for phosho-H2AX S139 immunostaining, graphed as percent of positive cells. (D, E) Immunoblots on indicated cells treated for 24 hrs with MPA (1 µM) with or without RMC-5552 (1 nM) or MK-2206 (2 µM). (F, G) RNA synthesis measured in indicated cells using the RNA Synthesis Assay kit (Abcam). Cells were treated for 3 hrs with vehicle or actinomycin D (Act. D) and then labeled for 1 hr with 5-ethynyl-uridine (EU), followed by quantification by flow cytometry. (H) Relative abundance of the indicated metabolites, measured by LC-MS/MS, in cells treated with MPA (1 µM, 6 hours), and graphed normalized to vehicle-treated cells. Graphical data are represented as mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005 by unpaired Welch’s t-test. (See also Figure S3).

Article Snippet: Cells were then stained with propidium iodide-RNase solution (CST #4087) for 1 hour at room temperature.

Techniques: Western Blot, Flow Cytometry, Staining, Immunostaining, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: PTEN-loss confers dependence on the guanylate synthesis enzyme IMPDH in T-cell acute lymphoblastic leukemia

doi: 10.64898/2025.12.22.696045

Figure Lengend Snippet: (A) Indicated cells were treated for 72 hrs with indicated concentrations of mycophenolic acid. Viable cells were measured using the Cell Titer Glo Cell Viability Assay (Promega) and are graphed as percent of vehicle-treated cells. (B) Immunoblots of indicated cells grown in complete media or serum starved for 18 hours. (C) Indicated cells treated for 72 hrs with MPA (2.5 µM) with or without RMC-5552 (1 nM). Cell death was quantified by Annexin V/ Propidium Iodide (AV/PI) staining, and graphed as percent of the total cell population. (D) Indicated cells were treated with MPA (1 µM, 16 hrs) followed by flow cytometry for phosho-H2AX S139 immunostaining, graphed as percent of positive cells. (E) Immunoblots from indicated cells treated with MPA (1 µM) for indicated times. (F, G) Immunoblots from indicated cells treated for 24 hrs with MPA (1 µM) with or without RMC-5552 (1 nM), rapamycin (20 nM), MK-2206 (2 µM) or SCH-772984 (1 µM). Graphical data are represented as mean ± SEM. (See also Figure S5).

Article Snippet: Cells were then stained with propidium iodide-RNase solution (CST #4087) for 1 hour at room temperature.

Techniques: Viability Assay, Western Blot, Staining, Flow Cytometry, Immunostaining