Fig 2: Selective miRNAs are regulated by DDX3 and PACT in HEK293T cells. (A) HEK293T cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing the indicated shRNAs. Cells were harvested for analysis 3 days after transduction. Western blot analysis was performed with antibodies against DDX3, α‐tubulin and PACT (n = 3). The detection of α‐tubulin was used as a loading control. (B) HEK293T cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing the indicated shRNAs. Total RNA was extracted using the TRIzol reagent 3 days after transduction. miRNAs were analyzed by qRT‐PCR. Bar graphs show the relative expression levels of miRNAs normalized to U6 snRNA. Data are shown as the mean ± SD from three independent experiments. (C) HEK293T cells were transiently transfected with the pcDNA3.1 vector or the pcDNA3.1 vector expressing FLAG‐tagged DDX3, DDX3 mutant (S382L) or PACT. Cells were harvested for analysis 2 days after transfection. Western blot analysis was performed with antibodies against DDX3, α‐tubulin and PACT (n = 3). The detection of α‐tubulin was used as a loading control. (D) HEK293T cells were transiently transfected with the pcDNA3.1 vector or the pcDNA3.1 vector expressing FLAG‐tagged DDX3, DDX3 mutant (S382L) or PACT. Total RNA was extracted using the TRIzol reagent 2 days after transfection. miRNAs were analyzed by qRT‐PCR. Bar graphs show the relative expression levels of miRNAs normalized to U6 snRNA. Data are shown as the mean ± SD from three independent experiments. Statistical analyses were carried out using Student's t test (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001).

Fig 3: Selective miRNAs are regulated by DDX3 and PACT in HCT116 and HeLa cells. (A) HCT116 and HeLa cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing the indicated shRNAs. Cells were harvested for analysis 3 days after transduction. Western blot analysis was performed with antibodies against DDX3, α‐tubulin and PACT (n = 3). The detection of α‐tubulin was used as a loading control. (B) HCT116 and HeLa cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing the indicated shRNAs. After 24 h, 2 μg·mL−1 puromycin was added to the medium for selecting infected cells. Total RNA was extracted using the TRIzol reagent 3 days after transduction. miRNAs were analyzed by qRT‐PCR. Bar graphs show the relative expression levels of miRNAs normalized to U6 snRNA. Data are shown as the mean ± SD from three independent experiments. Statistical analyses were carried out using Student's t test (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001).

Fig 4: PACT protein expression profiling at the interphase and mitosis of dividing cells. Immunofluorescence images of LX-2, HepG2, and HCT116 cells, investigating PACT expression and distribution in both interphase and mitotic cells. PACT-immunodetection pattern is found diffused in the cytoplasm, whereas it maintains an intense signal during mitosis (yellow arrows) in all the examined cell lines, regardless of the malignant phenotype (LX-2 versus HepG2) or the absence of the wild-type p53 protein (HCT116-p53+/+ versus HCT116-p53−/−). Green color: PACT; Red color: α-Tubulin; Blue color: Nucleus (DAPI). Scale bars: 10 µm.

Fig 5: DDX3 and PACT are required for shRNA‐induced RNAi. (A) Schematic representation of the strategy for measuring the gene‐silencing activity of shFluc. (B) HEK293T, HCT116 and HeLa cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing shDDX3 or shPACT. After 24 h, cells were transiently transfected with the pLKO.1 vector or the pLKO.1 vector expressing shFluc together with the firefly luciferase (Fluc) and Renilla luciferase (Rluc) reporter constructs. Cells were lysed for dual‐luciferase reporter assay 3 days after transduction. For each transfectant, the Fluc activity was normalized to that of the Rluc control. Bar graphs show the relative Fluc/Rluc ratio in DDX3‐ or PACT‐depleted cells compared to mock‐treated (pLKO.1) cells. Data are shown as the mean ± SD from three independent experiments. Statistical analyses were carried out using Student's t test (**P < 0.01; ***P < 0.001).