Fig 2: DDX3 regulates translation of candidate target genes involved in oogenesis in 293T cells.293T cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing shDDX3-1. Cells were harvested for analysis at day 3 post-transduction. A Western blot analysis was performed using antibodies against DDX3 and α-tubulin proteins. Detection of α-tubulin served as a loading control. B Cytoplasmic extracts were loaded on a linear 15-40% sucrose gradient ultracentrifugation. Total RNA was extracted from each fraction for analysis. Polysome profile analysis and quantitative real-time RT-PCR were performed to assess translational efficiency of APC1, CCNE1, P38γ, B56β, PP2Aβ, PKACα, FBXW11, RAC1, P38β, GNAS, and GAPDH mRNAs in DDX3 knockdown 293T cells (shDDX3-1) compared to control cells (pLKO.1). Detection of GAPDH mRNA served as a negative control. The bar graph shows the changes in translational efficiency as mean and standard deviation from three independent experiments. Statistical significance was determined using the Student’s t-test (ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001).

Fig 3: The APC substrates SKP2 and Cyclin B exhibit a phosphorylation-dependent mechanism for the interaction with core APC during cell cycle progression.a Western blot of HeLa cells synchronized by double thymidine block using phospho-specific antibodies. b Western blot of immunoprecipitates from HeLa cells synchronized by double thymidine block and collected at serial times for immunoprecipitation using SKP2 (upper panel), Cyclin B (middle panel), or ID2 (lower panel) antibodies. E47 and HEB are controls for the immunoprecipitation of endogenous ID2. c Loss of CDH1-mediated regulation of SKP2-S64D stability. Western blot of HeLa cells expressing FLAG-SKP2 WT, S64A, or S64D in the presence or absence of CDH1. d Loss of CDH1-mediated regulation of Cyclin B-S126D stability. Western blot of HeLa cells expressing FLAG-Cyclin B WT, S126A, or S126D in the presence or absence of CDH1. e In vivo ubiquitylation of HeLa cells co-expressing FLAG-SKP2 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lyasates immunoprecipitated with FLAG antibody. WCL, whole cellular lysates. f In vivo ubiquitylation of HeLa cells co-expressing FLAG-Cyclin B1 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lysates immunoprecipitated with FLAG antibody. WCL whole cellular lysates. g Loss of interaction between the SKP2-S64D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-SKP2 WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). h Loss of interaction between the Cyclin B-S126D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-Cyclin B WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). Total and phosphorylated proteins are from independent gels; APC1, APC3, CDH1 are from the same gel in each panel; E47, HEB, pHH3, SKP2 and Cyclin B1 are from independent gels; HA and FLAG/Vinculin in panel e are from different gels; Loading controls are from the same gel as FLAG or HEB in panel a. Molecular weight markers are indicated in kDa. Experiments were repeated two times with similar results.

Fig 4: DDX3 regulates the protein expression of candidate target genes involved in oogenesis in HeLa cells.HeLa cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing DDX3 shRNAs (shDDX3-1 and shDDX3-2). Cells were harvested for analysis at day 3 post-transduction. A Western blot analysis was performed using antibodies against DDX3, APC1, cyclin E1, p38 MAPK, B56β, PP2Aβ, PKACα, FBXW11, Rac1, GNAS, and α-tubulin proteins. Detection of α-tubulin served as a loading control. B The expression of APC1, CCNE1, P38γ, B56β, PP2Aβ, PKACα, FBXW11, RAC1, P38β, GNAS, DDX3, and GAPDH mRNAs were detected by quantitative real-time RT-PCR. The bar graph shows the relative mRNA levels normalized to GAPDH as mean and standard deviation from three independent experiments. Statistical significance was tested by one-way ANOVA (ns not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig 5: The N-terminal region of ID2 including Ser-5 is necessary for the interaction between ID2 and core APC subunits.a GST-ID2 WT and deletion mutants of the N- and C-terminus were incubated with lysates from HeLa cells. Bound proteins were analyzed by western blot. Lower panel, Coomassie staining of GST-ID2 proteins used in the GST pull-down. b GST pull-down using GST-ID2 WT and N-terminus deletion mutants and HeLa cell lysates. Bound proteins were analyzed by western blot. Lower panel, Coomassie staining of GST-ID2 proteins used in the GST pull-down. c GST pull-down using GST-ID2 proteins and U-2 OS cell lysates followed by western blot. Arrowhead, specific band; asterisk, non-specific band. Lower panel, Coomassie staining of GST-ID2 proteins used in the GST pull-down. d In vivo interaction between ID family members expressed in HeLa cells and core APC subunits, CDH1 coactivator, and bHLH transcription factors (E47 or HEB). ID interacting proteins were detected by FLAG immunoprecipitation followed by western blot for endogenous proteins as indicated. e Interaction between ID2 phosphorylation mutants and D-Box mutant (DBM) expressed in HeLa cells and core APC subunits or CDH1 coactivator. ID2 interacting proteins were detected by FLAG immunoprecipitation followed by western blot for endogenous proteins as indicated. f FLAG-ID2-S5 phospho-mutants expressed in HeLa cells were immunoprecipitated and probed by western blot for the association with endogenous E47. g HeLa cells transduced with non-targeting or CDH1 siRNA were transfected with FLAG-ID2 WT or ID2-S5 phospho-mutants. Cellular lysates were used in FLAG immunoprecipitation followed by western blot for core APC subunits and CDH1 (left panel). Right panel, whole cellular lysates. Molecular weight markers are indicated in kDa. Coomassie staining in a–c was performed on SDS-gels loaded with the same GST-fusion protein amounts used in the binding reactions. APC proteins and CDH1 are from the same blots in each panel; E47 and HEB in panel d are from two independent gels; FLAG is from an independent gel; Loading controls are from the same gel as HEB in d, FLAG in e, E47 in f, APC1/APC3/CDH1 in g. Experiments were repeated two times with similar results.