Figure 2. GACC shows modest differential sensitivity in vitro and reduces tumor burden in zebrafish with low overt hepatotoxicity readouts. (A–D) CCK-8 viability assays of PLC/PRF/5 (A); Hep3B (B); HepG2 (C) and THLE-2 (D) cells treated with GACC at the indicated concentration for 72 h. Viability is normalized to vehicle control (0.1% DMSO, set to 100%). Data are shown as mean ± SEM from n ≥ 3 independent biological replicates, each measured in triplicate technical wells. IC50 values were estimated by 4-parameter logistic regression. (E) Zebrafish embryotoxicity dose–response following immersion exposure to GACC beginning at 6 hpf with daily medium renewal for 5 days. Survival/morphology endpoints were used to estimate IC50. Data represent mean ± SEM from 3 biological replicates with 10 embryos per concentration per replicate (total embryos indicated in the panel). Vehicle control contained 0.1% DMSO. (F) Zebrafish xenograft assay. DiI-labeled Hep3B cells were microinjected into the yolk sac of 2 dpf embryos, followed by immersion treatment starting at 1 dpi with vehicle (0.1% DMSO), GACC (50 µM), or sorafenib (10 µM) for 48 h. Representative fluorescence images (left) and quantification of tumor burden (right) expressed as integrated fluorescence density normalized to vehicle. Data are mean ± SEM; n = 10 embryos per group pooled from 3 independent experiments. Scale bar: top-left and bottom-left 500 µm, all others 250 µm as indicated. (G,H) Larval hepatotoxicity readouts using Tg(fabp10a:EGFP-mCherry) larvae exposed to compounds by immersion from 2 to 5 dpf. (G) Quantification of liver area and (H) mean fluorescence intensity (MFI) (normalized to body length as described in Methods). Data are mean ± SEM; n = 10 larvae per group from >4 independent experiments. Statistical tests: one-way ANOVA with Tukey’s post hoc test. *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, not significant.
