Journal: Molecular and Cellular Biology

Article Title: DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries

doi: 10.1128/MCB.00285-18

Figure Lengend Snippet: DDX3 regulates the expression of PACT, STAT1, GNB2, Rac1, TAK1, and p38 MAPK proteins in THP-1 and HeLa cells. THP-1 and HeLa cells were transduced with either the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing the indicated shRNAs. After 24 h, puromycin was added to the culture medium for selection. The cells were harvested for analysis at 3 days posttransduction. Immunoblotting was performed using antibodies against DDX3, α-tubulin, PACT, STAT1, GNB2, Rac1, TAK1, and p38 MAPK. The detection of α-tubulin served as a loading control.

Article Snippet: The p38α-expressing vector pMT3 p38 was a gift from John Kyriakis (Addgene plasmid 12658).

Techniques: Expressing, Transduction, Plasmid Preparation, Selection, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries

doi: 10.1128/MCB.00285-18

Figure Lengend Snippet: Polysome profile analysis of STAT1, GNB2, Rac1, TAK1, and p38 MAPK translation in the DDX3 knockdown and mock-treated HEK293 cells. HEK293 cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing DDX3 shRNA (shDDX3-2). The cells were harvested for analysis at 3 days posttransduction. The cytoplasmic extracts of the cells were loaded on a linear 15% to 40% sucrose gradient and subjected to ultracentrifugation. (A) After sucrose gradient centrifugation, the polysome profiles were plotted using A254 values. (B) After sucrose gradient centrifugation, the mRNA/ribosome complexes were separated into 12 fractions. Total RNA was extracted from each fraction for analysis. The purified RNA was resolved by 1% denaturing agarose gel electrophoresis. 18S and 28S rRNAs were visualized using ethidium bromide for staining. (C) Total RNA was extracted from each fraction for analysis. The amount of mRNA was detected by quantitative real-time RT-PCR with specific primers for STAT1, GNB2, Rac1, TAK1, MAPK14 (p38α), MAPK11 (p38β), and MAPK12 (p38γ) mRNAs. The sum of a specific mRNA from the total fractions (fractions 1 to 11) was assumed to be 1 (or 100%), and the relative mRNA level in each fraction was expressed as a percentage of the amount of total specific mRNA. (D) The translational efficiencies of STAT1, GNB2, Rac1, TAK1, MAPK14 (p38α), MAPK11 (p38β), MAPK12 (p38γ), and GAPDH mRNAs in the mock-treated (pLKO.1) and DDX3 knockdown (shDDX3-2) HEK293 cells were calculated and are represented as percentages. The detection of GAPDH mRNA served as a negative control. The bar graph shows the changes in translational efficiencies as means ± SDs from three independent experiments.*, P < 0.05; **, P < 0.01.

Article Snippet: The p38α-expressing vector pMT3 p38 was a gift from John Kyriakis (Addgene plasmid 12658).

Techniques: Knockdown, Transduction, Plasmid Preparation, Expressing, shRNA, Gradient Centrifugation, Purification, Agarose Gel Electrophoresis, Staining, Quantitative RT-PCR, Negative Control

Journal: Molecular and Cellular Biology

Article Title: DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries

doi: 10.1128/MCB.00285-18

Figure Lengend Snippet: DDX3 controls the translation of PACT, STAT1, GNB2, Rac1, TAK1, and p38 MAPK mRNAs that contain complex 5′ UTRs. (A) The 5′ UTR sequences of PACT, STAT1, GNB2, Rac1, TAK1, and p38 MAPK (α, β, and γ) mRNAs obtained from the NCBI Reference Sequence (RefSeq) database were analyzed. The length, GC content, and minimum free energy of the 5′ UTR sequences are indicated. (B) Secondary structures within the 5′ UTRs were predicted using the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). The nucleotides are colored according to their probabilities in the structure. (C) HEK293 cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing DDX3 shRNA (shDDX3-2). After 48 h, the HEK293 cells were cotransfected with firefly luciferase (Fluc) reporters containing the 5′ UTRs of PACT, STAT1, GNB2, Rac1, TAK1, p38β MAPK, and β-actin mRNAs and the control pRL-SV40 vector encoding the Renilla luciferase (Rluc). The cells were lysed for analysis at 24 h posttransfection. For each transfectant, the Fluc activity was normalized to that of the Rluc control. The bar graph shows the relative Fluc/Rluc activities in DDX3 knockdown cells compared with those in the mock-treated controls. Data are shown as means ± SDs from three independent experiments. **, P < 0.01; ***, P < 0.001.

Article Snippet: The p38α-expressing vector pMT3 p38 was a gift from John Kyriakis (Addgene plasmid 12658).

Techniques: Sequencing, Transduction, Plasmid Preparation, Expressing, shRNA, Luciferase, Control, Transfection, Activity Assay, Knockdown

Journal: Molecular and Cellular Biology

Article Title: DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries

doi: 10.1128/MCB.00285-18

Figure Lengend Snippet: Expression of proinflammatory cytokines is impaired in DDX3 knockdown THP-1 cells. (A) The locations of various cytokine capture antibodies on the human cytokine array (R&D Systems) are indicated. (B) THP-1 cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing DDX3 shRNA (shDDX3-2). At 3 days posttransduction, the cells were stimulated with 80 ng/ml of LPS or 1 μg/ml of poly(I·C) for 6 h to mimic bacterial or viral infection, respectively. After 6 h of treatment, cell culture supernatants were collected and analyzed according to the manufacturer’s instructions. (C) The bar graph shows the relative protein level of LPS-induced cytokines in DDX3 knockdown THP-1 cells compared to those in the mock-treated controls (top). The relative mRNA levels of the cytokines normalized to GAPDH were analyzed using quantitative real-time RT-PCR (bottom). (D) The bar graph shows the relative protein level of poly(I·C)-induced cytokines in DDX3 knockdown THP-1 cells compared to mock-treated controls (top). The relative mRNA level of cytokines normalized to GAPDH was analyzed using quantitative real-time RT-PCR (bottom). Data are shown as means ± SDs from two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) The experiment was essentially analogous to that for panel B, except that cells were transiently transfected with the mock or p38α-expressing vector for 24 h. At 3 days posttransduction, total RNA was extracted for analysis after 6 h of LPS stimulation. The relative mRNA level of CCL2 normalized to GAPDH was analyzed using quantitative real-time RT-PCR. Data are shown as means ± SDs from three independent experiments. *, P < 0.05; ***, P < 0.001.

Article Snippet: The p38α-expressing vector pMT3 p38 was a gift from John Kyriakis (Addgene plasmid 12658).

Techniques: Expressing, Knockdown, Transduction, Plasmid Preparation, shRNA, Infection, Cell Culture, Quantitative RT-PCR, Transfection

Journal: Molecular and Cellular Biology

Article Title: DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries

doi: 10.1128/MCB.00285-18

Figure Lengend Snippet: Model of DDX3-mediated translational control in virus- and bacterium-induced inflammation and phagocytosis. In epithelial and phagocytic cells, DDX3 has functions in the signaling pathways of proinflammatory cytokines and IFN-β through translational control of PACT, TAK1, and p38 MAPK. In phagocytes, DDX3 also participates in phagocyte migration and phagocytosis through translational control of STAT1, GNB2, and Rac1.

Article Snippet: The p38α-expressing vector pMT3 p38 was a gift from John Kyriakis (Addgene plasmid 12658).

Techniques: Control, Virus, Protein-Protein interactions, Migration