Figure S2 . (D) Subcellular fractionations of TC71 cells demonstrate the presence of DDX3, RAD51, RECQL1, RPA32, and XRCC2 in both cytoplasmic and nuclear compartments. Results are representative of three independent experiments. See also
. CL, whole-cell lysate. (E) Immunofluorescent images of TC71 EWS cells 6 h after treatment with 5 μM RK-33 and 2 Gy IR. Green = γ-H2A.X staining of double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm. " width="100%" height="100%">
Journal: iScience
Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma
doi: 10.1016/j.isci.2024.108925
Figure Lengend Snippet: DDX3 interacts with components of the homologous DDR pathway (A) Western blots of DR-U2OS and EJ5-U2OS cell lysates following siRNA knockdown of DDX3. (B) DR-U2OS, EJ5-U2OS, and SSA-U2OS cell lines were treated with either scramble or siDDX3 and then transfected with the I Sce I-pCAGGS vector or an empty vector to induce DNA damage. Effective DDR was visualized by induction of GFP expression and quantified using flow cytometry. Results represent three independent experiments per cell line. Data represent frequency of DNA recombination events ±SEM. ∗∗∗p < 0.001 determined by multiple unpaired t tests followed by Šídák’s multiple comparisons test. I, induction of DNA damage with I Sce I-pCAGGS; UI, non-induction with empty pCAGGS vector; HR, homologous recombination; NHEJ, non-homologous end-joining; SSA, single-strand annealing. (C) Immunoprecipitation (IP) of TC71 EWS cell lysates using anti-DDX3 antibodies conjugated to magnetic beads. Western blots demonstrate co-immunoprecipitation of various DDR proteins with endogenous DDX3. Iso IgG, immunoprecipitation of TC71 cell lysates using control isotype antibodies. Results are representative of three independent experiments. See also Figure S2 . (D) Subcellular fractionations of TC71 cells demonstrate the presence of DDX3, RAD51, RECQL1, RPA32, and XRCC2 in both cytoplasmic and nuclear compartments. Results are representative of three independent experiments. See also Figure S2 . CL, whole-cell lysate. (E) Immunofluorescent images of TC71 EWS cells 6 h after treatment with 5 μM RK-33 and 2 Gy IR. Green = γ-H2A.X staining of double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm.
Article Snippet: Mouse IgG1 Isotype Control (clone MOPC-21) , Thermo Fisher Scientific , Cat#MA1-10407; RRID: AB_2536775.
Techniques: Western Blot, Knockdown, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Homologous Recombination, Non-Homologous End Joining, Immunoprecipitation, Magnetic Beads, Control, Staining
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Figure S2 . (D) Subcellular fractionations of TC71 cells demonstrate the presence of DDX3, RAD51, RECQL1, RPA32, and XRCC2 in both cytoplasmic and nuclear compartments. Results are representative of three independent experiments. See also 