Additional file 1 of Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells
Creators: name: Limanaqi, Fiona
name: Zecchini, Silvia
name: Saulle, Irma
name: Strizzi, Sergio
name: Vanetti, Claudia
name: Garziano, Micaela
name: Cappelletti, Gioia
name: Parolin, Debora
name: Caccia, Sonia
name: Trabattoni, Daria
name: Fenizia, Claudio
name: Clerici, Mario
name: Biasin, Mara
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Figure S1. A. Trypan Blue Exclusion assay in A549-hACE2 cells. Transfection with either NT-siRNA or SNCA-siRNA does not significantly affect cell viability compared with Mock- or SARS-CoV-2-infected CTR cells. Again, transfection with SNCA-siRNA does not significantly affect cell viability compared with NT-siRNA in either Mock- or SARS-CoV-2-infected cells. Results are expressed as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed by applying Two-way ANOVA. ns = non-significant. B and C. MTT assay rules out any cytotoxic effects of exogenous α-syn, IFN-β, or their combination, in A549-hACE2 (B) and CaLu-3 cells (C). Results are expressed as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed through Wilcoxon matched-pairs signed rank test (to test for differences between MOCK and VIRUS groups) and the Kruskal–Wallis test (to test for differences among various treatments within the same group). ns = non-significant. Figure S2. Thioflavin-S-α-syn co-staining rules out an amyloid nature of permeabilization-resistant α-syn species induced by IFN-β. Representative immunofluorescence images for α-syn protein combined with Thioflavin-S (Th-S) staining in Mock- and SARS-CoV-2-Infected CaLu-3 epithelial lung cells 48 h post-infection (MOI 0.05), in the absence or presence of exogenous α-syn monomers, or IFN-β. Cells were fixed for 15 min in Formaldehyde solution, followed by 15 min permeabilization with 0.3% Triton X-100. Bars correspond to 20 μm. Figure S3. A. HUVECs immunofluorescence negative controls. Negative control was performed in Mock cells by omitting primary antibodies. Some non-specific background signal for the secondary antibody Goat anti-mouse Alexa Fluor 488 (ab150113) was observed in HUVECs, likely due to their inherent fluorescence in the green spectrum. B. Trypan Blue Exclusion Assay in HUVECs. Exogenous α-syn, or IFN-β did not produce cytotoxicity in HUVECs. Results are shown as mean ± SEM from n = 3 independent experiments. Data were analyzed by applying Two-Way ANOVA. ns = non-significant. Figure S4. Representative Western Blot at low exposure showing distribution of exogenously added α-syn, as well as SARS-CoV-2 N protein immunostaining in A549-hACE2 cells at 48 h post infection, in the absence and presence of IFN-β. Endogenous α-syn was not detected at short exposure. Exogenous α-syn addition enhanced SARS-CoV-2 N protein immunostaining compared with untreated infected cells, which disappeared following combination with IFN-β. Figure S5. In the absence of productive SARS-CoV-2 infection, exogenous α-syn administration fails to decrease α-syn multimers and multimer:monomer ratio. A. Western blot for α-syn detection in HUVECs 78 h post- Mock and SARS-CoV-2 infection in the absence and presence of IFN-β or exogenous α-syn monomers. Blots of the experimental groups without exogenous α-syn are only representative, while groups treated with exogenous α-syn were quantified and graphed (B and C). Normalization was performed against total protein (Loading control). Results show raw normalized values presented as mean ± SEM from n = 2 independent experiments. Multimer:monomer α-syn ratio is expressed as absolute value calculated as “multimers/monomers”. Data were analyzed by applying Two-Way ANOVA (for total, multimer, monomer α-syn quantification) or One Way ANOVA (for multimer:monomer ratio). ns = non-significant. Table S1. Sequences of the primers employed in the study. FW forward; RV reverse. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), synuclein alpha (SNCA), SARS-CoV-2 Nucleocapsid 2 (SARS-CoV-2 N2); Interferon beta (IFNB), tumor necrosis factor alpha (TNFA), 2'-5'-oligoadenylate synthetase 1 (OAS1), retinoic acid-inducible gene I (RIG-I), Myxovirus Resistance Protein 1 (MX1), Myxovirus Resistance Protein 2 (MX2), Toll-like receptor 8 (TLR8), Toll-like receptor 9 (TLR9).