Journal: Nature

Article Title: DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

doi: 10.1038/s41586-020-2041-2

Figure Lengend Snippet: a, b, Immunofluorescence staining of endogenous KU86 (a) and DNA-PKcs (b) in U2OS cells. DDX21 RNA helicase is used as a positive control for nucleoli. The CSK buffer contains Triton X-100 for pre-extraction before fixation (see Methods). When indicated, the cells were treated with 50 nM ActD for 1 h before pre-extraction, fixation and staining. c, Localization of ectopically expressed GFP-tagged KU70 in mouse ES cells. a–c, n = 3 biologically independent experiments. d, U3 ChIRP-qRT–PCR analysis from HeLa cells. Enrichment levels, relative to input samples, of the U3, 7SK, 18S, and RMRP RNAs were assessed from experimental (−RNase A) or control (+RNase A) ChIRP samples. Data are from two independent biological replicates. e, DNA-PK was also recovered from U3 ChIRP-MS in IMR90 cells. Peptide spectral match (PSM) counts for control (RNase A) and experimental (U3) samples are shown. n = 2 biological replicates.

Article Snippet: Fixed cells were then incubated with primary antibodies in 3% BSA for 1 h at 25 °C, including mouse anti-human KU86 (ThermoFisher, MA5–12933, 1:100), rabbit anti-human DDX21 (Novus, NB100–1718, 1:500) or anti-DNA-PKcs (ThermoFisher, Ab-4(cocktail)), followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rabbit, and cyanine3-conjugated anti-mouse, Invitrogen, 1:500) for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Positive Control, Extraction, Quantitative RT-PCR, Control

Journal: FEBS letters

Article Title: Identification of nucleolar effects in JNK-deficient cells.

doi: 10.1016/j.febslet.2008.08.004

Figure Lengend Snippet: Fig. 1. JNK promotes nucleolar localization of DDX21. (A) Localization of endogenous DDX21 in wild-type, JNK1 /, JNK2 / and JNK1,2 / MEFs as examined by wide-field fluorescence microscopy. (B) A graph showing the ratio of DDX21 staining in nucleoplasm/nucleolus depicted as mean ± S.D. of 50 cells/group. (C) Localization of endogenous DDX21 in HT1080 cells 1 h after 20 lM PD98059, SB302850 and SP600125 treatment. (D) A graph showing the ratio of DDX21 staining in nucleoplasm/nucleolus depicted as mean ± S.D. of 50 cells/group. (E) Localization of endogenous DDX21 in non-treated or SP600125 treated JNK1,2 / MEFs as examined by wide-field fluorescence microscopy. (F) A graph showing the ratio of DDX21 staining in nucleoplasm/nucleolus depicted as mean ± S.D. of 50 cells/group. (A, C and E) Shown are images representing the dominant phenotype observed in the cell population. Each experiment was repeated two to three times with similar results.

Article Snippet: Primary antibodies used were rabbit monoclonal c-Jun (Signal Transduction), mouse monoclonal HA (Santa Cruz Biotechnology) and human polyclonal DDX21 [16].

Techniques: Microscopy, Staining

Journal: FEBS letters

Article Title: Identification of nucleolar effects in JNK-deficient cells.

doi: 10.1016/j.febslet.2008.08.004

Figure Lengend Snippet: Fig. 2. JNK inhibits DDX21 nucleolar mobility. (A) Exogenously expressed EGFP-DDX21 proteins in untreated or SP600125 treated (20 lM, 1 h) HT1080 cells was subjected to photobleach by a 488 nm laserline in indicated nucleoli (white arrowheads). FITC (515 nm) shows recovery of the photobleached EGFP-DDX21 proteins. (B) Quantitation of the nucleolar recovery rate of EGFP-DDX21 in untreated or SP600125 treated cells as measured by relative fluorescence intensity passing through the 515 nm filter.

Article Snippet: Primary antibodies used were rabbit monoclonal c-Jun (Signal Transduction), mouse monoclonal HA (Santa Cruz Biotechnology) and human polyclonal DDX21 [16].

Techniques: Quantitation Assay

Journal: FEBS letters

Article Title: Identification of nucleolar effects in JNK-deficient cells.

doi: 10.1016/j.febslet.2008.08.004

Figure Lengend Snippet: Fig. 3. c-Jun and DDX21 levels are reduced in JNK1,2 / cells. (A) The protein levels of actin, c-Jun, DDX21 and JNK1 in MEF WT and JNK1,2 / whole cell extract was examined by Western blot analysis of the respective proteins. Quantification of the DDX21 protein expression levels is also shown. Quantitation has been done by MCID imaging analysis software and correlated to actin expression from the same sample. (B) The protein levels of actin, c-Jun, DDX21 and phospho-c-Jun in untreated and SP600125 treated (20 lM, 24 h) HT1080 cells whole cell extract was examined by Western blot analysis of the respective proteins. (A and B) Quantitation of the DDX21 protein expression levels correlated to actin expression from the same sample.

Article Snippet: Primary antibodies used were rabbit monoclonal c-Jun (Signal Transduction), mouse monoclonal HA (Santa Cruz Biotechnology) and human polyclonal DDX21 [16].

Techniques: Western Blot, Expressing, Quantitation Assay, Imaging, Software

Journal: FEBS letters

Article Title: Identification of nucleolar effects in JNK-deficient cells.

doi: 10.1016/j.febslet.2008.08.004

Figure Lengend Snippet: Fig. 4. JNK affects DDX21 phosphorylation status. (A) DDX21 phosphorylation status was examined by 32P in vivo labelling and DDX21 immunoprecipitation in non-treated and SP600125 (20 lM, 1 h) treated HT1080 cells as well as in c-Jun / and JNK1,2 / MEF cells. The upper panel shows an autoradiogram of DDX21 and the lower panel a DDX21 Western blot. Quantitation of DDX21 phosphorylation levels correlated to immunoprecipitated DDX21 protein levels from the same sample. (B) Localization of EGFP-DDX21 wt, a putative EGFP-DDX21 JNK binding mutant as examined by wide-field fluorescence microscopy. (C) Wide-field images of EGFP-DDX21 wt, EGFP-DDX21 S71A, EGFP-DDX21 S89A, EGFP-DDX21 S121A, EGFP-DDX21 S171A. (D) Wild-type (c-Jun-WT), JNK-binding (c-JunD31–57) and phosphorylation (c-JunAla) deficient HA- tagged c-Jun rescues nucleolar localization of DDX21 in Jun / MEFs. Transfected HA-positive cells are indicated by white arrowheads. (A–D) Shown are images representing the dominant phenotype observed in the cell population. Each experiment was repeated two to three times with similar results.

Article Snippet: Primary antibodies used were rabbit monoclonal c-Jun (Signal Transduction), mouse monoclonal HA (Santa Cruz Biotechnology) and human polyclonal DDX21 [16].

Techniques: Phospho-proteomics, In Vivo, Immunoprecipitation, Western Blot, Quantitation Assay, Binding Assay, Mutagenesis, Microscopy, Transfection