E1383

NSC-34 cells were cultured in DMEM media (Gibco) on 13 mm coverslips (Menzel-Glasser) in 24 well plates and transiently transfected with EGFP-tagged TDP-43 using Lipofectamine 2000 (Invitrogen). To induce DNA damage, cells were treated with 13.5 μM etoposide (E1383, Sigma) or DMSO as a control. After 48 h, cells were fixed with 4% PFA and mounted with Dako fluorescence mounting media. Images were taken with an epi-fluorescent microscope (Zeiss). For identification of SGs containing TDP-43, immunocytochemistry using a mouse anti-HuR (Invitrogen) antibody was performed. Primary cortical neurons were cultured in B27 neurobasal medium supplemented with 1% glutamate and 1% penicillin/streptomycin. Primary neurons were transfected with EGFP-tagged TDP-43 with Lipofectamine 2000 at 3 or 5 DIV. After 24 h neurons were treated with 13.5 μM (E1383, Sigma) or DMSO as a control for 1 h and fixed with 4% paraformaldehyde. Slides were mounted with Dako fluorescence mounting media. Images were taken with an epi-fluorescent microscope (Zeiss).

Antibodies to P-H2AX (Cell Signaling, 80312S), TOP2β (Abcam, ab72334), CTCF (Abcam, ab128873), ER-α (Cell Signaling, 8644S), and Alexa Fluor Plus 488 (Invitrogen, A32723) were used. Two siRNA were used to deplete TOP2A and TOP2B separately, and NC siRNA was used for negative control. Three shRNA were used to knockdown ESR1, ESR2 and NRF1 separately, and NC shRNA was used for negative control. The siRNA sequences and shRNA sequences can be found in the supplementary materials [see Additional file 1]. All siRNA transfections were performed using Lipofectamine 2000™ (Thermo Fisher, 11668019) according to the manufacturer’s protocol and 20 nM siRNA. All lentiviral vectors expressing the scramble shRNA transfections were performed using polybrene (Sigma-Aldrich, TR-1003). Etoposide (Sigma-Aldrich, E1383) and E2 (Sigma-Aldrich, E8875) was dissolved in DMSO (Sigma-Aldrich, D2650). All primers and primer sequences can be found in the supplementary materials [see Additional file 1].

Generally, 2 × 10⁵ cells were seeded per well of a 12-well plate and infected the next day at the indicated multiplicity of infection (MOI). Cells from separate wells were trypsinized and counted to determine cell number. Virus inoculum was prepared in PBS supplemented with 0.3% BSA, 1 mM Ca²⁺/Mg²⁺, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated with the inoculum for 1 h, washed 3 times with PBS, and then overlaid with DMEM supplemented with 0.1% FBS, 0.3% BSA, 20 mM Hepes, 100 units/mL penicillin, and 100 μg/mL streptomycin. Where indicated, IFNα2a (Roferon-A; Roche), etoposide (Sigma-Aldrich; E1383), KU55933 (Sigma-Aldrich; SML1109), MG132 (Sigma-Aldrich; M7449), lactacystin (Enzo Life Sciences; BML-PI104-0200), ruxolitinib (Santa Cruz; sc-364729), or BX-795 (Sigma-Aldrich; SML0694) was added to the medium at the indicated concentration. Virus titers in the supernatants at the indicated time points were determined by standard plaque assay on MDCK cells. All infections with IBV were performed at 33 °C, while 37 °C was used for all other conditions.