Fig. 11.

TNFα contributed to Hippo/Yap pathway activation and FD-induced hepatomegaly. The tissue homogenate prepared from larvae at 11 dpf (a) and the used FD medium after cultivating Huh7 cells for 8 days (b) were quantified for TNFα protein level with ELISA. Huh7 cells cultivated in regular α-MEM with/without the exogenously added TNFα protein for 48 h were examined for cell size with flow cytometry (c) and analyzed for YAP expression and phosphorylation (d). The liver sizes of the larvae incubated in embryo water containing TNFα inhibitor LEN (38.5 µM) or CAY10500 (1 µM) starting from 7 dpf were imaged and quantified at 11 dpf under a fluorescence dissecting microscope (e). f The livers isolated from the larvae of 11 dpf were dispersed and the hepatocytes were examined for cell size with FACS. Shown here are the averaged results of at least four independent trials with each sample prepared from 20 to 30 larvae. g LEN (TNFα inhibitor, 4 µM) was added to FD cultured medium for Huh7 cells 5-day post-seeding. Cell sizes were measured on 8 dps with flow cytometry and calculated for the percentage of rescuing efficiency. h The expression of mmp13a and adam17b in the liver isolated from FD larvae at 11 dpf were quantified with real-time PCR. i The liver size of larvae exposed to MMP13 inhibitor was quantified at 11 dpf. The continuous presence of MMP13 inhibitor in embryos water upon FD induction significantly alleviated liver hypertrophy. All the data presented above are the averages of at least three independent trials. The cell size obtained with flow cytometry (FSC) was reported in arbitrary units (a.u.). Statistical results are represented in the mean ± SEM. CTL control (cells or larvae without FD), FD folate deficiency, LEN lenalidomide, MMP13-I MMP13 inhibitor