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. 2022 Apr 8;119(15):e2118819119. doi: 10.1073/pnas.2118819119

Fig. 1.

Fig. 1.

αSyn-PFFs cause glial genotoxicity and cGAS/STING activation in vitro. (A) Representative Western blot analysis of cytoplasmic (Triton X-100 soluble) cell fractions isolated from primary cerebrocortical cultures of mouse neurons or mixed cultures of microglia and astrocytes (MA) treated with PBS vehicle or α-synuclein preformed fibrils (αSyn-PFF) for 24 h. (B) Representative Western blot analysis of the DNA double-strand break marker γH2A.X in MA glial cultures treated as in (A). (C) Representative Western blot of pTBK1 in noncytoplasmic fractions (Triton X-100 insoluble) of MA cultures treated for 24 h with PBS vehicle or αSyn-PFF ± STING inhibitor H-151 (5 µM). (D, E) Quantifications of actin-normalized γH2A.X band intensity from B (n = 3) or pTBK1 band intensity from C (n = 5). P value from two-tailed t test (t = 6.13, df = 4) in D and from one-way ANOVA with Tukey post hoc comparisons in E (PBS vs. PFF: q = 11.32; PFF vs. PFF+H151: q = 6.42; df = 12). (F) Enzyme-linked immunosorbent assay (ELISA) for the cGAS activation product and STING ligand 2′3′-cyclic-GMP-AMP (cGAMP) in MA glial cultures treated with PBS vehicle or αSyn-PFF for 24 h (n = 6). P value from two-tailed t test (t = 5.43, df = 10).