Journal: Nucleic acids research
Article Title: KRCC1, a modulator of the DNA damage response.
doi: 10.1093/nar/gkac890
Figure Lengend Snippet: Figure 1. KRCC1 promotes CHK1 activation and efficient checkpoint. (A, B) OV90 and U2OS cells were transfected with control siRNA (siCTL) or siRNA targeting KRCC1 (siKRCC1) for 72 h. Cells were stained with DAPI and anti-RPA2 antibody and visualized by fluorescence microscopy (scale bar, 20 m). Percentage of cells with >10 foci is quantitated. The data are from manual scoring of ∼200 cells per condition and from three experiments ± SDs. (C) Lysates from above transfected siCTL and siKRCC1 OV90 and U2OS cells were analyzed by immunoblotting for CHK1-mediated DDR markers. (D) Immunoblotting for CHK1-mediated DDR markers after 72 h KRCC1 silencing in the presence or absence of CPT (1 M for 1 h). (E) KRCC1 interaction with CHK1 and 14-3-3 was evaluated using co-immunoprecipitation in EV or HA-tagged KRCC1 (HA-KRCC1) overexpressed cells treated with or without CPT (3 M, 2 h). (F) CHK1 interaction with KRCC1 was evaluated using co-immunoprecipitation in EV or Halo-tagged KRCC1 (Halo-KRCC1) overexpressed cells treated with CPT (3 M, 2 h) in the presence or absence of ATR inhibitor (ATRi, 5 M, 4 h) and quantification of immunoprecipitated Halo-KRCC1 by densitometry analysis using NIH ImageJ and normalized to their respective CHK1 levels and compared to CPT treatment only, which was set to 1. Experiments were repeated three times. Data represent mean ± SD. Marker (M) and IgG lanes are shown. (G) OV90 cells transfected with EV or HA-KRCC1 were treated with CPT (1 M for 1 h) and released for up to 180 min. Cells were collected at the indicated time points and processed for immunoblotting. The black right-pointing triangle indicates KRCC1. Short exposure (SE) and long exposure (LE) blots for KRCC1 are shown. The right panel depicts quantification of pCHK1-S296 by densitometry analysis using NIH ImageJ, normalized to their respective CHK1 levels and compared to the no treatment control group (NT), which was set to 1. (H) Proposed model of CHK1 activation. Following DNA damage and ATR-mediated phosphorylation of CHK1 at S345, CHK1 associates directly or indirectly with KRCC1 and 14-3-3. We posit that this association induces a conformational change in CHK1 to favor autophosphorylation at S296 and enhance kinase activity toward CDC25A.
Article Snippet: The following primary antibodies were used: pCHK1-S345 (2348), pCHK1S296 (2349), H2AX (2577), CHK1 (2360), HA-tag (3724), pH3-S10 (3377) and pan 14-3-3 (8312) from Cell Signaling Technology (Danvers, MA, USA); KRCC1 (16916-1- AP) from Proteintech (Rosemont, IL, USA); CDC25A (sc7389) and CDC7 (sc-56275) from Santa Cruz Biotechnol- ogy (Dallas, TX, USA); pRPA-S33 (A300-246A), AND1 (A301-141A), PSF3 (A304-124A), CHK1 (A300-298A) and pMCM2-S40/41 (A300-788A) from Bethyl Laboratories (Montgomery, TX, USA); DBF4 (ab124707) and pCyclinB1-S126 (ab55184) from Abcam; anti-Halo-tag (G921A) from Promega; RPA32 (MABE285) from EMD Millipore; RAD51 (NB100-148) from Novus Biological; 53BP1 (88439) from Cell Signaling Technology; and - tubulin and -actin from Sigma–Aldrich.
Techniques: Activation Assay, Transfection, Control, Staining, Fluorescence, Microscopy, Western Blot, Immunoprecipitation, Marker, Phospho-proteomics, Activity Assay
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![Identification of genes differentially regulated by LPS in M-MØ and GM-MØ. a Experimental design. b Normalized fluorescence intensity of the indicated cytokine mRNA in untreated and LPS-treated M-MØ and GM-MØ. c Scatter plot of microarray results, showing the LPS-induced gene expression changes in M-MØ (log2 M-MØ + LPS/M-MØ, adj p < 0.05, x-axis) plotted against the difference in the LPS-induced gene expression changes in M-MØ and GM-MØ (log2FC [M-MØ + LPS/M-MØ] − log2FC [GM-MØ + LPS/GM-MØ]) (y-axis). The relative position of some informative genes is indicated. d GSEA on the ranked list of genes obtained from the comparison of the transcriptome of M-MØ + LPS versus GM-MØ + LPS using Gene set 1 genes. The identity of the genes within the leading edge is shown. e Identification of the gene sets including genes with the highest differential LPS responsiveness in M-MØ (Gene set 1) and GM-MØ (Gene set 2). The number of genes in each gene set and associated gene ontology terms (Enrichr) are indicated. f Expression of selected members of Gene set 1 in untreated and LPS-treated M-MØ and GM-MØ (left panel), untreated and HMGB1-treated M-MØ and GM-MØ (middle panel), and untreated and PAM3CSK4-treated M-MØ (right panel), as determined by qRT-PCR on 4 independent samples. Results are indicated as the mRNA levels of each gene in activated cells relative to the level of the same mRNA in untreated cells (n = 3–4; *, p < 0.05; **, p < 0.005; ***, p < 0.0005). g SOCS2 protein levels in GM-MØ and M-MØ stimulated with LPS for the indicated times, as determined by Western blot. Shown is 1 representative experiment (n = 2). hCCL19 mRNA and <t>CCL19</t> protein levels in untreated (−) and LPS-treated M-MØ and GM-MØ. Shown are the means and SD of 5 independent experiments (n = 5; *, p < 0.05). i Expression of the indicated Gene set 1 genes in M-MØ stimulated with LPS (4 h) in the presence of U0126, BIRB0796, or BIRB0796 and U0126. Results indicate the expression of each gene relative to its expression in LPS-stimulated M-MØ. Shown are the means and SD of 3–4 independent experiments (n = 3–4; *, p < 0.05; **, p < 0.01; ***, p < 0.005). LPS, lipopolysaccharide; GSEA, gene set enrichment analysis; M-MØ, monocyte-derived human macrophages.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9444/pmc09149444/pmc09149444__jin-0014-0243-g02.jpg)
