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. 2022 May 13;25(6):104404. doi: 10.1016/j.isci.2022.104404

Figure 3.

Figure 3

Activation of IFN-I response by ALS SOD1-mutants is dependent on the cGAS-STING signaling pathway

(A) Knockout efficiency of Ddx58 (RIG-I), Ifih1 (MDA5), Mavs, or Ticam-1 (TRIF) gene in C20 cells examined via western blotting.

(B) WT, DDX58, IFIH1, or MAVS-CRISPR/Cas9-deficient C20 cells either transfected with 0.01 μg Flag-SOD1 WT, G85R, G93A, or MAVS plasmids for 36 h, or 0.5 μg poly(I:C) for 8 h. Ifnb1 mRNA measured using RT-qPCR.

(C) Similar conditions as in panel B performed using WT or MAVS-CRISPR/Cas9-deficient HASTR/ci35 cells, except human MAVS plasmid was replaced with human IRF3 5D plasmid. Ifnb1 mRNA assessed via RT-qPCR.

(D–F) WT, TICAM, cGAS or STING-CRISPR/Cas9-deficient C20 cells either transfected with 0.01 μg Flag-SOD1 WT, G85R, G93A, or MAVS plasmids for 36 h, treated with LPS (100 ng/mL) for 4 h or transfected with HT-DNA (5.0 μg/mL) for 8 h. Ifnb1 mRNA measured via RT-qPCR. The knockout efficiency of cGAS, or STING assessed via western blot and functional assays.

(G) WT C20 cells either treated with RU.521 (5.0 μg/mL) or H.151 (0.5 μg/mL) for 24 h. Cells were then transfected with 0.01 μg of Flag-SOD1 WT, G85R, G93A, or MAVS plasmids, or HT-DNA (1.0 μg/mL). Ifnb1 mRNA was measured via RT-qPCR.

(H) Schematic diagram of Flag-tagged cGAS-WT and mutants (location indicated by asterisk), STING-WT and deletion mutants.

(I) HEK293T cells stably expressing low amounts of Flag-STING-WT clones were first generated. The clones were further co-reconstituted with low expression of either Flag-cGAS-WT or point mutated plasmids. After selection, the double-positive clones cells were then transfected with 0.01 μg of Flag-SOD1 WT, G85R, or G93A plasmids. Ifnb1 mRNA was assessed via RT-qPCR. Protein expression for cGAS-WT and mutants from the 1.0 μg transfected samples were examined. Expression efficiency for each plasmid was evaluated via western blotting.

(J) Similar conditions as in I were used but replaced with Flag-cGAS-WT plasmid co-transfected with either STING-WT or deletion mutant (Δ368). Ifnb1 mRNA was measured using RT-qPCR.

(K) Protein lysates collected from C20 cells expressing the indicated plasmids were subjected to immunoblotting with the indicated antibodies.

(L) Immunofluorescence staining of endogenous cGAS in primary astrocytes cells derived from day-10 WT, or G85R transgenic mice.

(M) Both supernatant and lysates from C20 cells overexpressed with the indicated plasmids were collected and subjected to ELISA analysis of cGAMPs.

(N) Sera was prepared from whole blood derived from SOD1 WT or G85R mice, followed by ELISA analysis of cGAMPs (day-10). All RT-qPCR of RNA samples were normalized to β-actin. Data represent the mean of three independent experiments (mean ± SEM). ∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001, ∗∗∗∗p< 0.0001; (B–G, M, and N: unpaired Student’s t test). See also Figure S4.