Fig 2: Morphometric alterations caused by BMAA in daughter cells.The effects on neuronal development were examined in daughter cells of NSC treated with 50 or 100 µM BMAA. Representative images of cells immunostained with anti-ß III-tubulin (green) and DAPI (blue) (a, f). Morphometric analysis was conducted using an ImageXpress Micro XLS Widefield HCA System (Molecular Devices, Sunnyvale CA, USA), where images were automatically captured and analyzed with the SoftMax Pro Software. Neurite length (b, g), processes per cell (c, i), branches per cell (d, h), and cell body area (e, j) were determined. Values represent mean ± SD from three independent experiments, each with six replicates. Statistically significant differences from control are indicated as follow: ***p < 0.001; **p < 0.01 and *p < 0.05 (one-way ANOVA followed by Tukey–kramer test). Scale bar = 50 µm

Fig 3: BMAA causes morphometric alterations in neurons derived from hippocampal neural stem cells but not in primary neurons.The effects on BMAA treatment with 50 µM to 3 mM BMAA was investigated directly after 24 h exposure of primary neurons, while the neural stem cells were allowed to differentiate for 7 days after the exposure. Representative images of cells immunostained with anti-ß III-tubulin (green), anti-MAP2 (red), and DAPI (blue) are shown (Primary neurons, A; Exposed neural stem cells, B). Morphometric analysis was conducted using an ImageXpress Micro XLS Widefield HCA System (Molecular Devices, Sunnyvale CA, USA), where images were automatically captured and analyzed with the MetaXpress Software. Neurite length (C, D), the number of processes per cell (E, F), the number of branches per cell (G, H), and the cell body area (I, J) were determined. Values represent mean ± SD from three independent experiments, each with five to six replicates. Statistically significant differences from control are indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001 (one-way ANOVA followed by Tukey–Kramer test). Scale bar = 50 µm.

Fig 4: The effects of selected A. planci fraction on the level of LDL-C uptake by the HepG2 cell line at 3 different time points: 12, 24, and 36 h, respectively. Cells were either untreated or treated with 3.13, 6.25, or 12.5 µg/mL of the selected fraction for 12, 24, and 36 h. Images were taken using a high-content screening platform (ImageXpress® Micro, Molecular Devices) (A), and analyzed using ImageJ 1.52a; Java 1.8.0_191 (B). Berberine sulfate (BS)-treated cells were used as the positive control. ‘*’ denotes statistically significance (p < 0.05) as compared to untreated control (DMSO).

Fig 5: General outline of the study. First: A high-content screening (HCS) assay based on H1 hESCs and NTera-2 cells was developed, to explore pluripotency and differentiation. Several morphometrical features, as well as intensity-related measurements (from OCT4 and Cyclin B1 staining), were obtained from nuclei and cytoplasm compartments. Second: The HCS assay was used in a miR functional screen in 96-well plates to investigate the effects of 31 miRs. Following transfection and culture, images were acquired with an ImageXpress micro XLS HCS system (Molecular Devices). Third: Following image and data analysis (with CellProfiler and KNIME softwares, respectively), the multiparametric phenotypic profile describing the functional effect of each miR was submitted to hierarchical clustering (using Cluster 3.0 and Java Treeview), allowing the identification of miRs with similar functional effects (pro-pluripotency or pro-differentiation). Fourth: TargetScan was used to identify predicted targets shared by miRs in the same cluster (i.e., inducing similar phenotypes). Signaling pathways and biological processes enriched for these shared targets were identified using the DAVID tool