Fig 2: Biological activity of BA103 in xenograft models of GBM. (A–D) Mice were inoculated s.c. with U87 cells, randomly distributed in two groups (CTR and BA103), and treated according to the protocol shown in panel (A). Tumor growth was monitored by measuring tumor volume (B), and at the end point, tumors were weighed, and the resulting mean values for each group are reported in the histogram (C). Recovered tumor masses were processed for analysis of DDX3X expression by immunohistochemistry, and representative images acquired at 400× magnification are shown in panel (D). (E) Survival curve from in vivo orthotopic model of GBM. Mice were divided in two groups, control (CTR, n = 5) receiving vehicle, and inhibitor-treated (n = 8) receiving an i.p. injection of 50 mg/kg BA103. Mice were monitored for two months after U87 cells inoculation.

Fig 3: DDX3X is not required for stress-dependent inhibition of necroptosis.Primary BMDMs were stimulated as indicated. (A) Primary BMDMs derived from control (Ddx3xfl/fl) or myeloid-specific Lyz2creDdx3xfl/fl mice were treated as indicated, and representative images (6 h) were obtained from automated IncuCyte analysis where necroptotic cells were quantified by uptake of membrane-impermeant Sytox Green (green, with red analysis mask outline). (B) Necroptosis was quantified by automated analysis of Sytox Green–positive nuclei. (C) Immunoblots were performed from BMDM lysates at the indicated time-points to confirm DDX3X expression was reduced and assess necroptosis signaling (via phosphorylation of MLKL [p-MLKL]) in unstressed or thapsigargin-stressed BMDMs treated with zVAD + TNF. (D) Primary BMDMs derived from control (Ddx3xfl/fl) or myeloid-specific Lyz2creDdx3xfl/fl mice were treated as indicated, and necroptosis was quantified by automated analysis of Sytox Green–positive nuclei. Ddx3xfl/fl and Lyz2creDdx3xfl/fl quantifications overlap upon zVAD + Ars + TNF treatment. (E) Representative images (6 h) and IncuCyte quantification of NLRP3-dependent pyroptosis (via LPS + nigericin treatment) in BMDMs derived from the indicated genotypes. Significance was determined (B, D, E) by two-way ANOVA followed by (B, D) Dunnett’s or (E) Sidak’s multiple comparisons test, ****P < 0.0001. Data are generated from three images per replicate well (n = 3) and are representative of at least three independent biological replicate experiments. Scale bar (black) indicates 50 μm. Data are presented as mean ± SEM.

Fig 4: DDX3X is a central regulator of the NLRP3 inflammasome, stress granules, and type I IFN responses during IAV infection.A, confocal microscopy imaging of Ddx3xfl/fl and Ddx3xfl/flLysMCre bone marrow–derived macrophages (BMDMs) infected with influenza A virus (IAV; MOI 5) or IAV–ΔNS1 (MOI 5), stained for G3BP1 and DDX3X to visualize stress granules and DAPI to visualize nuclei. The scale bars represent 10 μm (whole image) and 5 μm (magnified image). Representative images (n = 2). B, quantification of the number of cells with stress granules from confocal microscopy images. Each circle represents the percentage of cells in a field of view with stress granules. ∗∗∗p = 0.0004 (unpaired two-sided t test). Data are the mean ± SEM. (n = 2 independent experiments). C, immunoblot analysis of caspase-1 (CASP1) cleavage (pro-CASP1 (p45) and cleaved CASP1 (p20)) in Ddx3xfl/fl and Ddx3xfl/flLysMCre BMDMs infected with IAV–PR8 (MOI 20) and ELISA measurement of IL-1β and IL-18 in Ddx3xfl/fl and Ddx3xfl/flLysMCre BMDMs infected with IAV–PR8 (MOI 20). ∗∗p = 0.0054, ∗p = 0.0436 (unpaired two-sided t test; n ≥ 2). Data are the mean ± SEM. D, ELISA measurement of IFN-β in Ddx3xfl/fl and Ddx3xfl/flLysMCre BMDMs infected with IAV and IAV–ΔNS1 (MOI 5). ∗p = 0.0263, ∗∗p = 0.0054 (unpaired two-sided t test; n > 3). Data are the mean ± SEM. ΔNS1, NS1 deletion mutant; DDX3X, DEAD-box helicase 3 X-linked; G3BP1, GTPase-activating protein-binding protein 1.

Fig 5: DDX3X interacts with Drosha/DGCR8 complex and affects the pri-miRNA and mature miRNA expression levels.(A) The interactions between DDX3X and Drosha complex tested by immunoprecipitation (IP) experiment. The uncropped full-length blots were presented in Figure S3. (B) The interactions between DDX3X and Dicer tested by immunoprecipitation (IP) experiment. The uncropped full-length blots were presented in Figure S3. (C) Relative pri-miRNA expression levels of DDX3X-dependent miRNAs (miR-1, miR-141, miR-145, miR-19b, miR-20a and miR-34a) in DDX3X KD cell lines compared with negative control (NC) cell lines. The DDX3X-independent miRNA (miR-21) was used as an internal control. The asterisk(s) (*,**) indicate a significant difference (p < 0.05, p < 0.01) respectively. (D) Relative mature miRNA expression levels of DDX3X-dependent miRNAs in DDX3X KD cell lines compared with NC cell lines. The DDX3X-independent miRNA (miR-21) was used as an internal control. The asterisks (**) indicate a significant difference (p < 0.01). (E) Relative pri-miRNA expression levels of DDX3X-dependent miRNAs in DDX3X OE cell lines compared with NC cell lines. The DDX3X-independent miRNA (miR-21) was used as an internal control. The asterisk(s) (*,**) indicate a significant difference (p < 0.05, p < 0.01) respectively. (F) Relative mature miRNA expression levels of DDX3X-dependent miRNAs in DDX3X OE cell lines compared with NC cell lines. The DDX3X-independent miRNA (miR-21) was used as an internal control. The asterisk(s) (*,**) indicate a significant difference (p < 0.05, p < 0.01) respectively.