Fig 2: DDX39B regulates NF-?B via MAVS and TRIF. a Luciferase assay using the -1C reporter in primary mouse embryonic fibroblasts (MEFs) isolated from wild-type, MAVS-/-, and Tmem173-/- mice following infection with sh-Ddx39b (Ddx) or non-targeting control. Data show mean relative value, ± SEM of two independent experiments. Inset: IB with anti-DDX39B antibody. b Luciferase assay performed as in a using wild-type, Myd88-/-, and Trif-/- MEFs. Data show mean relative value, ± SEM of two independent experiments. c IB in the indicated MEFs infected with sh-Ddx39b or non-targeting control using anti-phospho-p65 antibody. d Luciferase assay in U87 control or DDX39B CRISPR cells. Data show mean relative value, ± SEM of two independent experiments. e IB in U87 control and DDX39B CRISPR clones transfected with si-MAVS or si-control probed with the indicated antibodies. *P < 0.05, **P < 0.01 (two-tailed t test)

Fig 3: DDX39B inhibits NF-?B activity. a Luciferase assay using a reporter bearing -1C ?B-sites in A172 cells expressing two independent sh-DDX39B constructs or a control vector. Data show mean value normalized to EV, ± SEM from two independent experiments. b Luciferase assay in U87 cells transfected with empty vector (EV) or DDX39B. Data show mean value normalized to EV, ± SEM from two independent experiments. c Immunoblot (IB) in A172 GBM cells expressing two sh-DDX39B constructs or sh-control. IB was performed with anti-phospho-I?Ba, anti-I?Ba, or anti-GAPDH as loading control. d IB in GBM34 and GBM44 GSCs probed with anti-DDX39B. e Immunoblot in GBM44 GSCs expressing shRNA constructs as in c. IB performed as in c. f Representative immunofluorescence (IF) analysis of endogenous p65 in A172 cells expressing sh-DDX39B. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar, 10 µm. g IB in GBM44 GSCs from e expressing shRNA constructs probed with anti-phospho-p65 and anti-p65 antibodies. h IB in GBM34 GSCs stably expressing empty vector (EV) or S-tagged DDX39B with anti-phospho-p65 and anti-p65. *P < 0.05, **P < 0.01 (two-tailed t test)

Fig 4: DDX39B stability is regulated by SUMO-dependent ubiquitination. a IB in U87 cells stably expressing S-tagged wild-type DDX39B (WT) or E55A-DDX39B (E55A) in the presence of cycloheximide (CHX). Cells were harvested at the indicated time after CHX treatment and IB performed with anti-S-tag or anti-GAPDH antibody. b IB in 293T cells transfected with S-tagged DDX39B (WT or the indicated mutant) and Myc-ubiquitin (Myc-Ub). IP performed with S-agarose and IB with anti-Myc or anti-S-tag. c IB in U87 cells transfected with si-PIASx or si-control. IP was performed with anti-DDX39B and IB with anti-ubiquitin. Input was probed with the indicated antibodies. d IB in 293T cells transfected with empty vector or S-tag DDX39B, Myc-Ub, and Flag-PIAS1 or FLAG-PIASx-β. IP with S-agarose was followed by IB with anti-Myc. Input was probed with the indicated antibodies. e IB in U87 cells expressing S-tagged wild-type DDX39B (WT) in the presence of cycloheximide (CHX) and either vehicle or MG132 (10 μM). Cells were harvested at the indicated time after start of CHX treatment, and IB performed with anti-S-tag or anti-GAPDH antibody. f IB in 293T cells transfected with S-tag DDX39B and treated with vehicle or TMZ (100 μM, 12 h). IP was performed with S-agarose and IB with anti-ubiquitin. Input was probed with the indicated antibodies. g IB in U87 cells treated with vehicle or TMZ (100 μM) for the indicated time in the presence of CHX with anti-DDX39B

Fig 5: ECD interaction with DDX39A is required for mRNA export. RNA FISH analysis was performed on Ecdfl/fl MEFs overexpressing either vector, ECD, DDX39A, or ECD-P617G after treatment with GFP (control) or Cre-GFP adenovirus to delete ECD. (A) WB shows Cre-mediated knockdown of ECD. Exogenous ECD indicated by red marks. ß-Actin was used as a loading control. (B) Cells were fixed and hybridized with 12 µM oligo(dT) 22-nt Quasar 570 probe and then imaged at ×63 magnification. Bars, 10 µm. (C) Quantifications of nucleus-to-cytoplasmic signal ratios were conducted using ImageJ software in at least 75 cells, and graphs were plotted by normalizing to the control cells. Significance of the ratios was calculated using Student’s two-tailed t test between groups as indicated, error bars represent standard errors from the means. *, P = 0.05; **, P = 0.01; ***, P = 0.001.