Fig 1: DDX21 relocates from the nucleolus to the cytoplasm induced by PCV4 Cap overexpression. (A) Immunofluorescence analysis of DDX21 localization during PCV4 Cap expression. PK-15 cells were transfected 4.0 µg of pcDNA3.0-PCV4-Cap plasmid. The cells were fixed at 24, 48, and 72 h post-transfection (hpt) and incubated with the antibodies corresponding PCV4 Cap, and DDX21 followed by the fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (green) and Alexa Fluor-546 conjugated donkey anti-rabbit IgG (red) secondary antibodies. Nuclei were stained with DAPI (blue) and then observed under a confocal microscope. Scale bar, 10µm. (B) The proportion of co-localization of PCV4 Cap and DDX21 proteins was analyzed using ImageJ software at 24, 48, and 72 hpt. (C) The three-layer dimensional confocal stack analysis was performed to verify the co-localization of PCV4 Cap and DDX21 proteins at 48 and 72 hpt. (D) The cell nuclear and cytoplasmic fractions were extracted after PK-15 cells transfected 4.0 µg of pcDNA3.0-PCV4-Cap plasmid. At 48 and 72 hpt, the protein samples were prepared and analyzed by western blotting using antibodies against PCV4 Cap and DDX21. Histone H3 and ß-tubulin served as fractionation quality controls. (E) The DDX21 protein band intensity was analyzed using ImageJ software at 48, 72 hpt. Data are presented as means ± SD of three independent experiments. *p < 0.05.
Fig 2: 763GSRSNRFQNK772 of DDX21 is crucial for binding to PCV4 Cap. (A) Schematic representation of the NTD, Helicase D, and CTD of DDX21 and their truncation mutants used in this study. (B–D) The DDX21-CTD-(582–784 aa) interacted with PCV4 Cap. HEK293T cells were co-transfected with expression plasmids GFP- DDX21-WT or its serial GFP-DDX21 truncated mutants M1 to M6, together with Flag-PCV4-Cap or Flag-gst-PCV4-Cap plasmid. The cell lysate extracts were immunoprecipitated or GST pulled-down followed by western blotting using the indicated antibodies. (E,F) Identification the critical amino acids of DDX21-CTD essential for interaction with PCV4 Cap. HEK293T cells were co-transfected with DDX21 or DDX21 truncated mutants M7 to M9, along with Flag-PCV4-Cap or Flag-gst-PCV4-Cap plasmid, and the cell lysate extracts were immunoprecipitated or GST pulled-down followed by western blotting using the indicated antibodies.
Fig 3: Both the NTD and CTD of RNase H1 contribute to the binding with NAT10 and DDX21. (A) The schematics of different RNase H1 domains constructed on the BioID2-HA vector. (B) The different RNase H1 domains fused with BioID2-HA and Flag-tagged NAT10 were expressed in 293CT cells for 48hrs. “-”means mock transfection control. The expression of these tagged proteins in the cell lysates was confirmed by Western analyses (B, left panel). Immunoprecipitation using anti-HA antibody was performed. Co-precipitated NAT10 was detected by Western analyses using anti-Flag antibody, and different H1 domains on beads were detected using anti-HA antibody (B, right panel). (C) HA-tagged RNase H1 domains and Flag-tagged DDX21 were co-expressed in 293CT cells. Anti-Flag antibody-conjugated beads were used to immunoprecipitate the tagged DDX21-associated RNase H1 protein domains. The expression of tagged proteins (left panel) and co-isolated proteins (right panel) was determined by Western analyses. Arrows indicate the expressed proteins at expected sizes.
Fig 4: The C-terminal domains of purified DDX21 and NAT10 can bind RNase H1. (A) The schematic domain structure of DDX21. The RNase H1 -domain (601–783) is highlighted with a red line. (B) Western analyses of RNase H1 protein co-isolated with different GST-tagged DDX21 domains. About 5 μg GST-tagged different DDX21 protein domains were immobilized on the glutathione resin, incubated with 40 μM or without toxic PS-ASOs, then washed off the extra unbounded ASOs, and finally incubated with 5 μg purified MBP-RNase H1. After washing, resin-retained proteins were co-isolated and analyzed by Western analyses. A stained image of the same Western gel before transfer to membrane is presented in the lower panel and served as a control for loading. The GST-tagged DDX21 domains are indicated with arrows. (C) The domain structure of NAT10. The C-terminus (541–1025) associated with purified RNase H1 is highlighted by a red line. (D) Western analyses of RNase H1 protein co-isolated with different MBP-tagged NAT10 domains. About 5 μg of different MBP-tagged NAT10 domains coated on amylose resin was first incubated without or with 40 μM toxic PS-ASOs (558807), and then incubated with 5 μg GST-fused full-length RNase H1. Co-isolated proteins were detected by Western analysis. A stained image (Blazin’ Bright™ Luminescent UV Protein Gel Stain, P-825-1; Gold Biotechnology, Inc.) of the same Western gel before transfer to the membrane is presented in the lower panel and served as a control for loading. The MBP-tagged NAT10 domains are indicated with arrows.
Fig 5: Reduction of NAT10 and DDX21 increases cytotoxicities induced by toxic PS-ASOs. (A) Live cell imaging of cells transfected with a safe PS-MOE-ASO (446654) for different times. Nucleolar localization of PS-ASOs is marked with arrows. Scale bars, 20 μm. (B) Live cell imaging of cells transfected with a toxic PS-MOE-ASOs (828939) for 3 h. (C) Immunofluorescence staining of P54nrb proteins in different siRNA-treated cells, followed by transfection of toxic PS-ASOs (558807). Scale bars, 20 μm. (D) Quantification of P54nrb and PS-ASO signals in the nucleolar area, as exemplified in (C). The ratio between nucleolar P54nrb and PS-ASO signal intensity was calculated and plotted. Error bars represent standard deviations from 30 cells. P values were calculated based on t-test using prism. ***P < 0.001. (E, F) qRT-PCR quantification of P21 mRNA levels in different siRNA-treated HeLa cells, followed by transfection of different concentrations of a nontoxic PS-ASO (25691, E) or toxic PS-ASO (558807, F). Error bars represent standard deviations from four repeated experiments.
Supplier Page from Abcam for Anti-DDX21 antibody [EPR14495]