Additional file 3 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 3: Figure S7. Single chemicals and mixtures effects on synaptogenesis. HiPSC-derived NSCs were differentiated for 7 DIV, and then treated for either 3 days (A-C) or 14 days (D-F) with single chemicals (BPA, CPF, Lead, Methyl-Hg, PCB138 and VA) or three types of mixtures: (i) a mixture with the 3 similar MoA chemicals (‘3-Sim’), (ii) a mixture with the 3 dissimilar MoA chemicals (‘3-Diss’), and (iii) a mixture with all 6 chemicals (‘All’). Graphs show: (A, D) total PSD95 protein levels; (B, E) total SYP protein levels, and (C, F) the number or overlapping SYP and PSD95 spots at the levels of nerites (i.e., SYP/PSD95 co-localization, synapses). All samples were normalized to solvent control (0.1% DMSO, Ctr) at the respective time point. LOAEC-syn (red curves) and their serial dilutions (respectively black (1:2) and blue (1:4) curves) were tested as indicated in Table 3 (reporting concentrations tested for each individual chemical). Data are represented as mean ± S.E.M. of 3–4 biological replicates. Figure S8. Single chemicals and mixtures effects on neurite outgrowth. HiPSC-derived NSCs were differentiated for 7 DIV, and then treated for either 3 days (A-D) or 14 days (E-G) with single chemicals (BPA, CPF, Lead, Methyl-Hg, PCB138 and VA) or three types of mixtures: (i) a mixture with the 3 similar MoA chemicals (‘3-Sim’), (ii) a mixture with the 3 dissimilar MoA chemicals (‘3-Diss’), and (iii) a mixture with all 6 chemicals (‘All’). Graphs show: (A, E) neurite length, (B, F) the number of branch points per neurite, (C, G) the number of neurites per neuron, and (D, H) the percentage of β-III-Tubulin+ cells. All samples were normalized to solvent control (0.1% DMSO, Ctr) at the respective time point. LOAEC-neu (red curves) and their serial dilutions (respectively black (1:2), blue (1:4) and light blue (1:8) curves) were tested as indicated in Table 3 (reporting concentrations tested for each individual chemical). Data are represented as mean ± S.E.M. of 3–4 biological replicates. Figure S9. Effects of chlorpyrifos (CPF) and valproic acid (VA) withdrawal from mixtures on neurite outgrowth. HiPSC-derived NSCs were differentiated for 7 DIV, and then treated for 14 days with: (i) the ‘3-Sim’ mixture (BPA, Lead and CPF) and the same mixture without CPF, (ii) the ‘3-Dissim’ mixture (Methyl-Hg, PCB138 and VA) and the same mixture without VA, (iii) the ‘All’ mixture (BPA, CPF, Lead, Methyl-Hg, PCB138, and VA) and the same mixture excluding CPF and VA. (A) Representative pictures at 10x magnification (β-III-Tubulin, in red). (B-E) Graphs show neurite outgrowth after 14 day treatment with LOAEC/2-neu (black bars), and LOAEC/4-neu concentrations (blue bars). Graphs show total live (non-pyknotic) cell numbers (B, based on DAPI staining), neurite length (C), the number of branch points per neurite (D), and the number of neurites per neuron (E) analysed upon treatments with LOAEC/2-neu and LOAEC/4-neu concentrations as indicated in Table 3 (reporting concentrations tested for each individual chemical). Values were normalised to the corresponding complete mixtures, and data are represented as mean ± S.E.M. of 3 biological replicates. Statistical significance was assessed by unpaired t-test, comparing each differential mixture vs the original mixture (e.g., ‘3-Sim’ vs ‘3-Sim (−CPF)’). p