Journal: Cell Reports

Article Title: MRNIP/C5orf45 Interacts with the MRN Complex and Contributes to the DNA Damage Response

doi: 10.1016/j.celrep.2016.07.087

Figure Lengend Snippet: MRNIP Promotes ATM-CHK2 Signaling and Resistance to IR (A) HCT116 cells were transfected with a non-targeting control siRNA or two individual siRNAs targeting MRNIP. After 72 hr, cells were exposed to IR (3 Gy) for the indicated times before lysis and western blotting using the indicated antibodies. Quantification of these data reveals an ∼30%–70% reduction in ATM substrate phosphorylation at 1 hr post-IR in MRNIP-depleted cells compared to cells transfected with control siRNA. (B) Assessment of IR-induced G2-M checkpoint in cells transfected with either control non-targeting or MRNIP-targeting siRNA. Shown is the fold increase in mitotic cells in irradiated (2 Gy) MRNIP siRNA-treated cells compared to control siRNA-treated cells. Data shown represent the mean from three experimental repeats with their respective SEMs ( ∗ p ≤ 0.05 compared to control siRNA-transfected cells). (C) IR-induced RAD51 foci in U2OS cells transfected with either control siRNA or two individual siRNAs targeting MRNIP. After 72 hr post-transfection, cells were exposed to IR (3 Gy) fixed after the indicated times post-IR exposure and stained with a RAD51 antibody. Cells were counterstained with DAPI, and cells with greater than five foci were scored as positive. Data shown represent the mean from three experimental repeats with their respective SEMs ( ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01 compared to control siRNA-transfected cells). (D) HR frequency in HEK293 cells transfected with control siRNA or two individual siRNAs targeting MRNIP. After 48 hr, cells were transfected with either I-SceI or DR-GFP constructs alone as negative controls or both I-SceI and DR-GFP constructs. After a further 24 hr, cells were trypsinized and the GFP-positive fraction was assessed directly by flow cytometry. Data shown represent the mean from three experimental repeats with their respective SEMs ( ∗∗ p ≤ 0.01 compared to control siRNA-transfected cells). (E) Cells were transfected as in (A), irradiated, and fixed after the indicated times before staining for γH2AX. Cells were counterstained with DAPI, and the number of cells with greater than five foci were scored as above. Data shown represent the mean from three experimental repeats with their respective SEMs ( ∗ p ≤ 0.05 compared to control siRNA-transfected cells). (F) Cells were transfected and irradiated as in (A), and the percentage of cells with associated micronuclei was determined. Data shown represent the mean from three experimental repeats with their respective SEMs ( ∗ p ≤ 0.05 compared to control siRNA-transfected cells). (G) Cells were transfected in 96-well plates as in (A) and exposed to IR (0, 1, or 2.5 Gy). After 5 days, survival was determined by MTT assay across three independent experiments. Errors shown are SEMs. Comparable clonogenic survival curves are shown in Figure S1 F.

Article Snippet: The antibodies used in this study are as follows: Abcam: C5orf45 (ab150917), pATM Ser1981 (ab36810), total ATM (2C1; ab78), pDNA-PKcs Thr2609, GFP (ab290), phospho-histone H3 Ser10 (ab14955), b-tubulin (ab7792), and b-actin (ab8224); Cell Signaling Technologies: gH2AX Ser139 (no. 2577), phospho-CHK2 (Ser68; no. 2661), Chk2 (no. 2662), total H3 (no. 4499); GeneTex: RAD50 (13B3); Bethyl: phospho-KAP1 (Ser824; A300-767), KAP1 (A300-274A), NBS1 (A300-187A); and R&D Systems: MRE1111.

Techniques: Transfection, Control, Lysis, Western Blot, Phospho-proteomics, Irradiation, Staining, Construct, Flow Cytometry, MTT Assay

Journal: Molecular Cancer

Article Title: Survivin safeguards chromosome numbers and protects from aneuploidy independently from p53

doi: 10.1186/1476-4598-13-107

Figure Lengend Snippet: Activation of ATM and CHK2 at DNA lesions in SBC-2 cells with knockdown of Survivin. a: Representative immunofluorescence analysis of SBC-2 cells, with knockdown of Survivin and stained for activated ATM (ATM S1981) and ɣH2AX. Note double positive ɣH2AX, ATM S1981 loci indicative for DDR occurring in polyploid nuclei (arrowheads). Inlet showing magnification of indicated multinucleated cell with colocalized ɣH2AX and ATM S1981. b: Representative image depicting ɣH2AX, ATM S1981 staining in shLuc-transduced controls. Magnification bars: 10 μm. c: Representative image of SBC-2 cells after knockdown of Survivin showing the appearance of activated CHK2 (CHK2 T68) in ɣH2AX-positive DNA lesions. d: ɣH2AX and CHK2 T68 do not appear in shLuc-transduced SBC-2 cells. Magnification bars: 10 μm.

Article Snippet: The membranes were probed with primary antibodies in blocking solution over night at 4°C including rabbit polyclonal anti-Survivin (R&D Systems), polyclonal mouse anti-p21 waf/cip (R&D Systems), monoclonal rabbit anti-p21 waf/cip (Cell Signaling), polyclonal goat anti-p53 (R&D Systems), monoclonal rabbit anti-p53 S15 (Abcam), polyclonal anti-p53 S20, polyclonal anti-p53 S37 (Cell Signaling), monoclonal mouse anti-α-Tubulin and monoclonal anti-Actin (Sigma), monoclonal rabbit anti-ATM S1981 (Epitomics), polyclonal rabbit anti-ATM (Merck), monoclonal rabbit anti-Caspase 3 (Cell Signaling), monoclonal rabbit anti-CHK2 T68 (Cell Signaling), polyclonal rabbit anti-Cyclin D1 (Santa Cruz), polyclonal rabbit anti-Cyclin E (Santa Cruz) and monoclonal mouse anti-γH2AX (Millipore).

Techniques: Activation Assay, Knockdown, Immunofluorescence, Staining

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 5 Impaired DNA damage repair in p21-deficient cells after low-dose doxorubicin treatment. A Western blot analysis of phosphorylated ATM (p-ATM), p-Chk1, Chk1, p-Chk2, and Chk2 in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for various time points. β-actin was used as a loading control. B Fluorescence microscopy of Lamin B1 (red) and γ-H2AX foci (green) in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 48 h. Scale Bar = 20 μm. C Fluorescence microscopy showing nuclei (blue) and γ-H2AX foci (green) in HCT116 WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 24 h and then released into fresh media for various time points. Scale bar = 20 μm. Quantification of nuclei with >10 γ-H2AX foci (n = 20 cells per group, repeated 4 times). Data are mean ± SD ****p < 0.0001.

Article Snippet: The following antibodies were used: β-actin (sc-69879), p53 (sc-126), Lamin B1(sc-365214), αtubulin (sc-5286), Noxa (sc-515840, and MKLP1 (sc-136473) (Santa Cruz Biotechnologies, Dallas, TX, USA); p21 (ab109520), phospho-ATM (S1981) (ab81292), and phospho-DNA PKcs (S2056) (ab18192)(Abcam, Cambridge, MA, USA); phospho-Chk1 (Ser345)(# 2348), Chk1 (#2360), phospho-Chk2 (Thr68)(# 2197), Chk2 (#6334), cleaved caspase-3 (#9664), phospho-Histone H2A.X (Ser139)(# 9718), Mcl-1 (#39224), and Aurora B (#3094) (Cell signaling technology, Danvers, MA, USA); Mre11(GTX70212) and α-tubulin (GTX112141) (Genetex, San Antonio, Texas, USA); DNA-PKcs (19983-1-AP) (Proteintech, Rosemont, IL, USA); HRP-conjugated rabbit IgG and mouse IgG (Jackson Immunoresearch, West Grove, PA, USA).

Techniques: Western Blot, Control, Fluorescence, Microscopy