Fig 2: eIF3e’s mechanism of regulation involves eIF3d and DDX3X. (A) Western blot analysis of the indicated eIF3 subunits in U251 cells treated with control (Scr) or eIF3e (3e) siRNAs (i). eIF3d protein levels in (i) were normalized to ß-actin protein levels and plotted relatively to the Scr condition (ii). Data are presented as mean values ± SEM of nine independent experiments; *P < 0.05 (Wilcoxon test). (B) Venn diagram showing unique or common 5'UTR CLIP-derived binding sites between DDX3X (32) and eIF3d (12). (C) Immunoprecipitation of U251 total extracts, followed by western blot analysis and probing with the indicated antibodies. Shown is a representative result from two independent experiments. (D) RNA chromatography using the 5'UTR of the CDC45 and GADD45a RNAs either capped or not and total cell extracts from U251 cells treated with control (Scr) or eIF3e (3e) siRNAs, followed by western blot analysis. Shown is a representative result from two independent experiments. (E) Quantitative RT-qPCR analysis from pooled NP, LP and HP fractions, using specific primers for indicated mRNAs extracted from U251 cells treated with control (Scr) or DDX3X siRNAs. Data are plotted relatively to input mRNA amounts and are presented as mean values ± SEM of three independent experiments; NS: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005 (paired t-test). (F) Western blot analysis of the indicated proteins in U251 cells treated with control (Scr) or DDX3X siRNAs. Shown is a representative image of three independent experiments. (G) Ratio of Renilla/Firefly luciferase activities (Rluc/Fluc) determined using U251 cells treated with control (siScr) or DDX3X siRNAs, followed by co-transfection with Rluc and Fluc mRNA reporters. Data are presented as mean values ± SEM of three independent experiments; NS: non-significant, **P < 0.005 (paired t-test).

Fig 3: Verification of Ring1b-interacting proteins.A, B Ring1b interacts with DDX3X and DDX5, or with Snail1 and Twist2. The 293 T cells were transfected with plasmids as indicated for 24 h. Proteins were captured by IP and analyzed by western blot. C Heatmap of interaction between Ring1b-associated proteins. D Schematic depiction of Ring1b complexes. E–G Ring1b complexes verified in breast cell lines. Lysates from 10 A, 10A-Ring1b, MCF-7, and 231 cells were analyzed by IP followed by western blot. H Expression of endogenous Ring1b-associated proteins in breast cell lines. Total protein extractions from cells were analyzed by western blot.

Fig 4: DDX3 activates AREG mRNA translation(A) Schematic diagram showing humanized Renilla luciferase (hRL) reporters containing either or both of the 5′ and -3′ UTRs of AREG.(B) In vivo translation assay using the indicated hRL reporters in siC- or siD#1-transfected SAS cells. The activity of human firefly luciferase (hFL) encoded by the same reporters was used as an internal control. Bar graph shows relative hRL/hFL activity (mean ± SD); ∗p < 0.01.(C) Schematic diagram showing DDX3 in which two RecA-like domains are labeled in blue and violet and 11 conserved motifs (Q, I-VI), and the two mutations are indicated. An in vivo translation assay was performed using the indicated hRL reporters in endogenous DDX3-depleted (by siD#1) and exogenous DDX3 (wild-type or mutants)-overexpressing SAS cells. Bar graph shows relative hRL/hFL activity.(D) SAS cells were transfected with siD#1 and the indicated DDX3 expression vectors. The lysates were analyzed by immunoblotting using antibodies as indicated.(E) DDX3 modulates the translation of AREG mRNA via both its 5′ and -3′ UTR.

Fig 5: The DDX3-AREG axis promotes angiogenesis and macrophage recruitment and suppresses cytotoxic immune cells in syngeneic tumors(A) Experimental procedure for using a syngeneic graft model to evaluate the effect of DDX3 knockdown and rAREGm injection on tumor growth.(B) Immunoblotting for DDX3 and GAPDH in the tumor extracts collected at 7 and 14 days after transplantation.(C) Average size of the tumors from control (siC) or DDX3 knockdown (siD#1) MOC-L1 or the latter supplemented with rAREG (siD#1 + rAREGm). Bar graph shows average weight (mean ± SEM) of tumors as in (B). p < 0.01. Eight tumors from each frank of four mice in each group were analyzed.(D) Representative images of each type of the tumors as in (C) with similar sizes are shown in the top image. Periodic acid-Schiff (PAS) and immunofluorescence staining of tumor sections. Arrowheads indicate the PAS-positive regions. Bar graphs show PAS intensity and the ratios of F4/80 or granzyme B-positive cells. Scale bars represent 1 mm and 100 µm in PAS and immunofluorescence images, respectively. Error bars represent mean ± SEM; *p < 0.01.(E) A summary of the tumor-promoting effects of the DDX3-AREG axis.