Journal: bioRxiv

Article Title: Discovery of Small Molecules and a Druggable Groove That Regulate DNA Binding and Release of the AP1 Transcription Factor ΔFOSB

doi: 10.1101/2025.10.21.683675

Figure Lengend Snippet: A) Cell viability of JPC0661 comm and HTS10307 assessed in the range of 0-20 μM after 24 h (top) and 72 h (bottom) in neuronal progenitor cells (NPC) using the CellTiter-Glo viability assay. Cell viability measures are normalized to the negative control containing no compound but 0.6% DMSO. Dotted lines indicate 100% cell viability. The mean of n replicates is shown for 24 h (n=3) and 72 h (n=2) with error bars indicating the SEM. B) Stability of JPC0661 comm and HTS10307 in mouse liver microsomes. C) and D) Effects of JPC0661 comm (0–100 μM; C ) and JPC0661 (0–100 μM; D ) on AP1-driven luciferase activity in AP1-luc HEK293 cells. The dose-dependent activation of the AP1-driven luciferase reporter was measured based on changes in the luciferase signal and expressed as relative fluorescence units (RFU). Each compound was tested twice (n = 4 wells per experiment; total n = 6–8 wells for JPC0661 comm and n = 7–8 wells for JPC0661 per concentration), with results normalized to the luciferase signals of blank wells from corresponding experiments (n = 8 wells). Nonlinear regression using a three-parameter model was applied to fit the luciferase signal and calculate IC 50 values, which are reported with a 95% confidence interval (CI), and data points are presented as the mean ± SEM. E) Effect of serially diluted JPC0661 (0.003-100 μM) on the viability of AP1-luc HEK293 cells using the Celltiter-Go viability assay (2 h). Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 7 wells). Data points are presented as the mean ± SEM, with a total n = 10–12 wells per concentration. F) Effect of JPC0661 (at doses 12.5, 25, and 50 μM) on the viability of Neuro 2A cells (72 h) using the Celltiter-Go viability assay to assess potential toxicity in mouse neuronal cells. Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 8 wells). Data points are presented as the mean ± SEM, with a total n = 12 wells per concentration.

Article Snippet: The AP1-luciferase reporter human embryonic kidney (HEK293) cell line was obtained from BPS Bioscience (USA) and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Viability Assay, Negative Control, Luciferase, Activity Assay, Activation Assay, Fluorescence, Concentration Assay, Control

Journal: bioRxiv

Article Title: Discovery of Small Molecules and a Druggable Groove That Regulate DNA Binding and Release of the AP1 Transcription Factor ΔFOSB

doi: 10.1101/2025.10.21.683675

Figure Lengend Snippet: A) A panel of JPC0661 analogs designed to probe the role of the sulfonic acid group and the amino-pyrazolone group. B) Table summarizing the JPC0661 analogs tested in AP1-reporter assays using AP1-luc HEK293 cells, yielding cell-based IC50 values and FP-DRC assays. Lower IC 50 values are marked with more plus signs (+) and indicate higher activity. The plots from the FP-DRC and cell-based DRC assays for these compounds are shown in Supplementary Fig. S1 . C) ΔFOSB/JUND bZIP incubated with compounds (0.5 mM) with 100 μM diamide (‘ox’, oxidized) or without diamide (‘red’, reduced) and assessed by SDS-PAGE (with or without reducing agent in the loading buffer). D) ΔFOSB/JUND bZIP protein incubated with compounds (0.5 mM) with no diamide (‘red) or protein alone (no compound) incubated with diamide (‘ox’) as a control and then assessed by SDS-PAGE (with or without reducing agent in the loading buffer). In C) and D) ‘cntrl’ denotes the ΔFOSB/JUND bZIP protein in absence of compound.

Article Snippet: The AP1-luciferase reporter human embryonic kidney (HEK293) cell line was obtained from BPS Bioscience (USA) and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Activity Assay, Incubation, SDS Page, Control

Journal: iScience

Article Title: Dishevelled localization and function are differentially regulated by structurally distinct sterols

doi: 10.1016/j.isci.2025.112704

Figure Lengend Snippet: DVL2 subcellular localization is shifted in response to sterol change (A) Schematic illustrating the final steps of post-squalene cholesterol synthesis. Small molecule inhibitors are indicated in red, target proteins in blue. (B) Representative western blot of total DVL2 expression following inhibitor treatments. (C) Total cellular DVL2 normalized to GAPDH. N = 3 independent experiments. Data represent mean ± SEM. One-way ANOVA (F (4, 10) = 1.260 p = 0.347555) with Dunnett’s test. (D) Representative western blots of subcellular DVL2 following inhibitor treatments. (E) Subcellular DVL2 normalized to total actin. N = 3–6 independent experiments. Data represent mean ± SEM. One-way ANOVA with Dunnett’s test. (F) Confocal imaging of HEK293T-DVL2-mEGFP-KI cells following respective treatments. (G) Nuclear GFP/unit nuclear area. N = 48–55 individual cells. Data represent mean ± SEM. One-way ANOVA (F (3, 218) = 274.3, p < 0.000001) with Dunnett’s test. (H) Nuclear GFP normalized to total GFP/field. N = 3 independent experiments. One-way ANOVA (F (3, 8) = 62.68, p < 0.0001) with Dunnett’s test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 for all statistical tests. Scale bar = 25 μm.

Article Snippet: HEK293T cells were transduced with lentiviral particles carrying a firefly luciferase reporter under the control of TCF (T cell factor)/LEF (lymphoid enhancer factor)-responsive element located upstream of the minimal TATA promoter (BPS Biosciences, 79787).

Techniques: Western Blot, Expressing, Imaging

Journal: iScience

Article Title: Dishevelled localization and function are differentially regulated by structurally distinct sterols

doi: 10.1016/j.isci.2025.112704

Figure Lengend Snippet: Nuclear DVL2 transport is FoxK2 dependent and sensitive to PDZ-domain inhibition (A) Co-IP between DVL2 and FoxK2 under sterol-impacted conditions. (B) Nuclear DVL2 binding to FoxK2. N = 3 independent experiments. Data represent mean ± SEM. One-way ANOVA with Dunnett’s test; F (2, 6) = 20.58, p = 0.0021 (nucleus); F (2, 6) – 11.22, p = 0.0094 (cytosol). (C) Model for NSC668036, FoxK2, and cholesterol binding to DVL2-PDZ. Predicted NSC668036 binding residues are green, cholesterol residues are purple, FoxK2 residues are cyan. (D) HEK293T-DVL2-mEGFP-KI cells following sterol treatments. (E) Nuclear GFP/unit nuclear area. N > 50 cells from 2 independent experiments. Data represent mean ± SEM. One-way ANOVA (F (4, 438) = 168.5, p < 0.0001) with Tukey’s test. (F) Nuclear GFP normalized to total GFP/field. N = 4 fields from 2 independent experiments. Data represent mean ± SEM. One-way ANOVA (F (4, 15) = 15.74, p < 0.0001) with Tukey’s test. (G) Co-IP between DVL2 and FoxK2. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 for all statistical tests. Scale bar = 25 μm. HC = heavy chain of IgG.

Article Snippet: HEK293T cells were transduced with lentiviral particles carrying a firefly luciferase reporter under the control of TCF (T cell factor)/LEF (lymphoid enhancer factor)-responsive element located upstream of the minimal TATA promoter (BPS Biosciences, 79787).

Techniques: Inhibition, Co-Immunoprecipitation Assay, Binding Assay

Journal: iScience

Article Title: Dishevelled localization and function are differentially regulated by structurally distinct sterols

doi: 10.1016/j.isci.2025.112704

Figure Lengend Snippet: Loss of sterol homeostasis alters the DVL2 protein-protein interaction network (A) Venn diagram of DVL2-interacting proteins in HEK293T cells under sterol-specific conditions. (B) Representative key DVL2 interactions lost within LPDS treatment. (C) DVL2-Tp53 interaction network in FBS, LPDS, and AY9944 treatment. (D) Co-IP between endogenous DVL2 and p53 in HEK293T. (E) β-catenin and Axin1/2 interaction networks with DVL2 under sterol-specific conditions. (F) β-catenin and DVL2 interaction within the nucleus. (G) Co-IP of endogenous DVL2 and Axin2 in HEK293T cells. HC = heavy chain.

Article Snippet: HEK293T cells were transduced with lentiviral particles carrying a firefly luciferase reporter under the control of TCF (T cell factor)/LEF (lymphoid enhancer factor)-responsive element located upstream of the minimal TATA promoter (BPS Biosciences, 79787).

Techniques: Co-Immunoprecipitation Assay