Additional file 1 of Inhibition of microglial β-glucocerebrosidase hampers the microglia-mediated antioxidant and protective response in neurons
Creators: name: Brunialti, Electra
name: Villa, Alessandro
name: Mekhaeil, Marianna
name: Mornata, Federica
name: Vegeto, Elisabetta
name: Maggi, Adriana
name: Di Monte, Donato A.
name: Ciana, Paolo
Publisher: name: figshare
Year: 2021
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Supplemental Figure 1. SK-ARE-luc2 clone selection. Following transfection with the pARE-luc2-ires-tdTomato plasmid, SK-N-BE cells were subject for 4 weeks to a selective pressure with G418 and two representative clones #1.2 and #3 tested for the ability to report NFE2L2 upregulation 2, 6 or 24 hours after the treatment with 80 μM tBHQ or vehicle. Clone #1.2 was selected, amplified and used in the present study. Luciferase enzyme activity expressed as relative luciferase units (RLU) per μg protein, data are mean values ± SEM (n = 3) of a single experiment, which is representative of at least two other independent experiments. *p&lt;0.05 vs vehicle calculated by one-way ANOVA followed by Tukey’s multiple comparison test. Supplemental Figure 2. GCase inhibition in mice and SK-ARE-luc2 cells treated with CBE. (A) Residual activity of GCase in the brain of mice treated with 100 mg/kg CBE for three days. (B) Residual activity of GCase in SK-ARE-luc2 cells treated with 200 μM CBE for 48 hours. Data are mean values of the enzymatic activity quantified as μmol 4-MU generated in 1-hour reaction per μg of proteins expressed as % of the activity detected versus vehicle treated animals (A) or cells (B) ±SD of n=2 in duplicate (in vivo experiments), n=2 in triplicate (cell culture experiments). **p &lt; 0.01, ***p&lt;0.001 versus vehicle calculated by unpaired t-test. Supplemental Figure 3. Different ratio of BV-2:SK-ARE-luc2 cocultures differentially modulate neuronal NFE2L2 activity. Luciferase activity is expressed as RLU is reported on the graph as FC on monoculture; bars are mean values ±SD of n=5 measures in triplicate. ***p&lt;0.001 by one-way ANOVA followed by Dunnet’s multiple comparisons test. Supplemental Figure 4. Nuclear localization of NFE2L2 in neuronal-microglia culture treated with CBE. Representative immunocytochemistry analysis of SK-N-BE and BV-2 cell lines in monoculture and coculture, treated with 200 μM CBE or vehicle for 48 hours; cells were co-stained with anti- NFE2L2 (red) and anti-CD11b antibodies (green), and with DAPI (blue). Supplemental Figure 5. GCase inhibition dampen neuroprotective functions of microglia. Representative single parameter histograms of flow cytometry analyses related to propidium iodide fluorescence: (A) SK-N-BE treated with 0.5 mM MPP+ or vehicle for 24 hours; (B) SK-N-BE cultured alone or in coculture with BV-2 and treated with 0.5 mM MPP+ for 24 hours; (C) cocultures treated with 200 μM CBE for 48 hours or (D) with 200 μM CBE for 48 hours and 0.5 mM MPP+ for 24 hours. Supplemental Figure 6. GCase inhibitions in primary microglia. Representative images of primary microglia marked with GFP showing that the treatment with 200 μM CBE for 48 hours increases a microglia sub-population characterized by a round shaped morphology.