Figure S1.

Elevated NF-κB and Type I IFN Signaling Because of TDP-43 In Vitro, Related to Figure 1

(A) Doxycycline (Dox inducible wild-type (WT) or ALS mutant (Q331K) TDP-43 was stably transduced into the mouse neuronal cell line NSC-34. 72 hr after TDP-43 induction, RNA was collected for qPCR of Ifnb1, Ifna6, Ifit1 and Tnf or (B) cells were lysed for western blot of p-TBK1, p-IRF3, p-p65, TDP-43 and actin as control. Blots are representative of three independent experiments. (C) IFNβ ELISA was performed on the supernatant from cells in (A). (D) Representative western blot of MAVS, PKR, cGAS, STING, FLAG, TDP-43 and Actin from cells in Figure. 1A. (E) IFNβ ELISA was performed on the supernatant from MEFs after 72hrs induction of WT and Q331K TDP-43. (F) cGAMP ELISA was performed on the lysates of human THP-1 cells overexpressing TDP-43 (WT or Q331K) after 72hrs induction. (G) Images of healthy control and TDP-43-ALS patient iPSC during differentiation into premature MNX1⁺ motor neurons (day 18) and further into mature MNX1⁺/ChAT⁺ motor neurons (day 28). (red - MNX1 or ChAT, green – β3-tubulin and blue - DAPI). (scale: 40 μm). (H) MNX1 and CHAT expression, measured by qPCR in undifferentiated (day 0) and differentiated iPSC-derived MNs (day 28). (I) Representative western blot of p-TBK1, total TBK1, TDP-43, TFAM and Actin for cells in (G) (lysed in 1% NP-40). Data are mean ± SEM from 3-4 independent experiments. P values were calculated using one-way or two-way ANOVA. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.