Additional file 2 of Assessment of developmental neurotoxicity induced by chemical mixtures using an adverse outcome pathway concept
Creators: name: Pistollato, Francesca
name: Gyves, Emilio Mendoza De
name: Carpi, Donatella
name: Bopp, Stephanie K.
name: Nunes, Carolina
name: Worth, Andrew
name: Bal-Price, Anna
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified

description: Additional file 2: Figure S1. Effects elicited by Bisphenol A (BPA); quantitative evaluation of immunocytochemistry using HCI (Cellomics platform). IMR90-derived NSCs were differentiated for 7 DIV and treated for either 3 or 14 days with three different concentrations of BPA (0.29 μM, IC20/100, white bars; 12.74 μM, IC5, grey bars; 28.96 μM, IC20, black bars) in comparison to solvent control (0.1% DMSO, Ctr) at the respective time point. Analysis of BPA effects on: (A) total (i.e., cell body and neurites) and in neurites only expression of SYP (pre-synaptic) and PSD95 (post-synaptic proteins (MAP2 staining was used as a marker of neurites), and the number of overlapping SYP/PSD95 spots (synapses) in the neurites; (B) neurite length and branch points per neurite; (C) total BDNF protein levels and BDNF expression ratio, comparing neurite to cell body. Data are represented as mean ± S.E.M. of 3–4 biological replicates. Figure S2. Effects elicited by Chlorpyrifos (CPF); quantitative evaluation of immunocytochemistry using HCI (Cellomics platform). IMR90-derived NSCs were differentiated for 7 DIV and treated for either 3 or 14 days with three different concentrations of CPF (0.37 μM, IC20/100, white bars; 21.01 μM, IC5, grey bars; 37.10 μM, IC20, black bars) in comparison to solvent control (0.1% DMSO, Ctr) at the respective time point. Analysis of CPF effects on: (A) total (i.e., cell body and neurites) and in neurites only expression of SYP (pre-synaptic) and PSD95 (post-synaptic proteins (MAP2 staining was used as a marker of neurites), and the number of overlapping SYP/PSD95 spots (synapses) in the neurites; (B) neurite length and branch points per neurite; (C) total BDNF protein levels and BDNF expression ratio, comparing neurite to cell body. Data are represented as mean ± S.E.M. of 3–4 biological replicates. Figure S3. Effects elicited by Lead(II) chloride (Lead); quantitative evaluation of immunocytochemistry using HCI (Cellomics platform). IMR90-derived NSCs were differentiated for 7 DIV and treated for either 3 or 14 days with three different concentrations of Lead (0.0073 μM, IC20/100, white bars; 0.17 μM, IC5, grey bars; 0.73 μM, IC20, black bars) in comparison to solvent control (0.1% DMSO, Ctr) at the respective time point. Analysis of Lead effects on: (A) total (i.e., cell body and neurites) and in neurites only expression of SYP (pre-synaptic) and PSD95 (post-synaptic proteins (MAP2 staining was used as a marker of neurites), and the number of overlapping SYP/PSD95 spots (synapses) in the neurites; (B) neurite length and branch points per neurite; (C) total BDNF protein levels and BDNF expression ratio, comparing neurite to cell body. Data are represented as mean ± S.E.M. of 3–4 biological replicates. Figure S4. Effects elicited by Methylmercury(II) chloride (Methyl-Hg); quantitative evaluation of immunocytochemistry using HCI (Cellomics platform). IMR90-derived NSCs were differentiated for 7 DIV and treated for either 3 or 14 days with three different concentrations of Methyl-Hg (0.0013 μM, IC20/100, white bars; 0.05 μM, IC5, grey bars; 0.13 μM, IC20, black bars) in comparison to solvent control (0.1% DMSO, Ctr) at the respective time point. Analysis of Methyl-Hg effects on: (A) total (i.e., cell body and neurites) and in neurites only expression of SYP (pre-synaptic) and PSD95 (post-synaptic proteins (MAP2 staining was used as a marker of neurites), and the number of overlapping SYP/PSD95 spots (synapses) in the neurites; (B) neurite length and branch points per neurite; (C) total BDNF protein levels and BDNF expression ratio, comparing neurite to cell body. Data are represented as mean ± S.E.M. of 3–4 biological replicates. Figure S5. Effects elicited by PCB138; quantitative evaluation of immunocytochemistry using HCI (Cellomics platform). IMR90-derived NSCs were differentiated for 7 DIV and treated for either 3 or 14 days with three different concentrations of PCB138 (0.0593 μM, IC20/100, white bars; 3.53 μM, IC5, grey bars; 5.93 μM, IC20, black bars) in comparison to solvent control (0.1% DMSO, Ctr) at the respective time point. Analysis of PCB138 effects on: (A) total (i.e., cell body and neurites) and in neurites only expression of SYP (pre-synaptic) and PSD95 (post-synaptic proteins (MAP2 staining was used as a marker of neurites), and the number of overlapping SYP/PSD95 spots (synapses) in the neurites; (B) neurite length and branch points per neurite; (C) total BDNF protein levels and BDNF expression ratio, comparing neurite to cell body. Data are represented as mean ± S.E.M. of 3–4 biological replicates. Figure S6. Effects elicited by Valproic acid (VA); quantitative evaluation of immunocytochemistry using HCI (Cellomics platform). IMR90-derived NSCs were differentiated for 7 DIV and treated for either 3 or 14 days with three different concentrations of VA (2.1 μM, IC20/100, white bars; 70 μM, IC5, grey bars; 210 μM, IC20, black bars) in comparison to solvent control (0.1% DMSO, Ctr) at the respective time point. Analysis of VA effects on: (A) total (i.e., cell body and neurites) and in neurites only expression of SYP (pre-synaptic) and PSD95 (post-synaptic proteins (MAP2 staining was used as a marker of neurites), and the number of overlapping SYP/PSD95 spots (synapses) in the neurites; (B) neurite length and branch points per neurite; (C) total BDNF protein levels and BDNF expression ratio, comparing neurite to cell body. Data are represented as mean ± S.E.M. of 3–4 biological replicates.