(A) Representation of the zebrafish Nile red fat metabolism assay. Strong fluorescence signal is present in zebrafish larvae from the solvent control around the yolk sac and stomach/intestine. Compounds 1 and 2 decreased the Nile red staining, in contrast to chlorophyll a and b. (B) Quantification of lipid-reducing activity in the zebrafish Nile red fat metabolism assay after exposure over 48 h. Solvent control was 0.1% dimethyl sulfoxide (DMSO) and positive control was 50 µM resveratrol (REV). Values are expressed as mean fluorescence intensity (MFI) relative to the DMSO group and are derived from six to eight individual larvae per treatment group. The data are represented as box-whisker plots from the fifth to 95th percentiles. Asterisks highlight significant altered fluorescence intensities that indicate changes of neutral lipid level (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05).

Quantification of lipid content (Nile red) and viability (calcein AM) in differentiated 3T3-L1 spheroids after exposure to 1 and 2 over 48 h. (A) Results of quantification of fluorescence by CellProfiler software (mean ± SD). (B) Representative images from fluorescence microscopy. Statistical differences to the solvent control were analyzed by one-way ANOVA, followed by a Dunnett’s multiple comparison post-test (*** p <0.001, ** p < 0.01, * p < 0.05). (C) Quantification of free glycerol on the medium where 3T3-L1 organoids were exposed to 1 and 2 over 48 h. Data represent means ± SD. No significant alterations on free glycerol content in the medium were observed. Kolmogorov–Smirnov test was used to test normality of the data, followed by a Dunnett’s multiple comparison post-test (*** p <0.001, ** p < 0.01, * p < 0.05).