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: DDX3 is associated with AGO 2 and Dicer in HEK293T cells. (A) HEK293T cells (5 × 106) were transiently transfected with the pEGFP‐C1 vector (GFP) or the pEGFP‐C1 vector expressing GFP‐DDX3. After 24 h, cells were harvested for immunoprecipitation with an anti‐GFP antibody. Co‐precipitated proteins were resolved on 10% SDS/PAGE, followed by western blot analysis with anti‐AGO2 and anti‐GFP antibodies (n = 3). Ig H represents the immunoglobulin heavy chain. (B) HEK293T cells (1 × 107) were harvested for immunoprecipitation with anti‐AGO2 or mouse IgG control antibody. Co‐precipitated proteins were resolved on 8% SDS/PAGE, followed by western blot analysis with anti‐DDX3 and anti‐AGO2 antibodies (n = 2). Ig H represents the immunoglobulin heavy chain. (C) HEK293T cells (5 × 106) were transiently transfected with the pcDNA3.1 vector or the pcDNA3.1 vector expressing FLAG‐tagged DDX3 or PACT. After 24 h, cells were harvested for immunoprecipitation with an anti‐FLAG antibody. Co‐precipitated proteins were resolved on 8% SDS/PAGE, followed by western blot analysis with anti‐Dicer and anti‐FLAG antibodies (n = 2).

Fig 4: DDX3 and PACT are vital for cell proliferation and transformation. (A) HEK293T and HCT116 cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing shDDX3 or shPACT. After 2 days, infected cells were seeded at 3 × 103 cells per well in a 96‐well plate. Cell proliferation was assessed by the MTS reagent for 3 days. After incubation with the MTS reagent for 1 h, the absorbance is measured at 490 nm using a plate reader. Data are shown as the mean ± SD from three independent experiments. (B) HEK293T cells were transiently transfected with the pcDNA3.1 vector or the pcDNA3.1 vector expressing FLAG‐tagged DDX3 or PACT. After 24 h, transfected cells were seeded at 3 × 103 cells per well in a 96‐well plate. Cell proliferation was assessed by the MTS reagent for 4 days. After incubation with the MTS reagent for 1 h, the absorbance is measured at 490 nm using a plate reader. Data are shown as the mean ± SD from three independent experiments. (C) HEK293 cells were stably transfected with the pcDNA3.1 vector (Mock) or the pcDNA3.1 vector expressing FLAG‐tagged DDX3 (DDX3 overexpression). After selection with G418 for 2 weeks, stable cells were cultured in 6‐cm dishes. Images were acquired with an AxioObserver A1 equipped with a AxioCam MRm camera (n = 3, representative images shown) (Zeiss, Oberkochen, Germany).

Fig 5: DDX3 specifically regulates the expression of PACT in human cells and C. elegans. (A) HEK293T, HeLa and HCT116 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, PACT, TRBP and α‐tubulin (n = 3). The detection of α‐tubulin was used as a loading control. (B) HEK293T cells were treated with dimethyl sulfoxide or RK‐33 for 24 h. Cells were harvested for western blot analysis with antibodies against DDX3, PACT, TRBP and α‐tubulin (n = 2). The detection of α‐tubulin was used as a loading control. (C) A stable HEK293 cell line that overexpresses DDX3 was established. Total cell extracts prepared from DDX3‐overexpressing HEK293 cells were subjected to western blot analysis with antibodies against DDX3, PACT, TRBP and α‐tubulin (n = 2). The detection of α‐tubulin was used as a loading control. (D) Transgenic C. elegans C860 were fed with the various RNAi bacteria for gene silencing. Total worm extracts were prepared from RDE‐4‐GFP‐expressing C. elegans and subjected to western blot analysis with antibodies against VBH‐1, GFP and β‐actin (n = 2). The detection of β‐actin was used as a loading control.