Journal: Oncology reports

Article Title: Novel SAHA‑bendamustine hybrid NL‑101 in combination with daunorubicin synergistically suppresses acute myeloid leukemia.

doi: 10.3892/or.2020.7591

Figure Lengend Snippet: Figure 3. NL‑101 in combination with DNR promotes DNA damage‑related signaling. MV4‑11 cells were treated with 300 nM NL‑101, 8 nM DNR and the combination; HL‑60 cells were treated with 500 nM NL‑101, 16 nM DNR and the combination for 48 h. Western blot analysis was conducted for γ‑H2AX, ATR, p‑ATR, ATM, p‑ATM, CHK1, p‑CHK1, CHK2 and p‑CHK2 protein levels. DNR, daunorubicin; p, phosphorylated.

Article Snippet: Antibodies against caspase-3 (cat. no. 9662), caspase-7 (cat. no. 9492), PARP (cat. no. 9532), BAD (cat. no. 9239), BIM (cat. no. 2933), cyclin B1 (cat. no. 4135), cell division cycle protein 2 (CDC2; cat. no. 9116), γ-H2AX (Ser139; cat. no. 9718), phosphorylated (p)-ATR (Thr1981; cat. no. 30632), p-ATM (Ser1981; cat. no. 5883), p-CHK1 (Ser345; cat. no. 2348), p-CHK2 (Thr68; cat. no. 2197), GAPDH (cat. no. 5174) and β-actin (cat. no. 4970) were purchased from Cell Signaling Technology, Inc. ATR (cat. no. 19787-1-AP), ATM (cat. no. 27156-1-AP), CHK1 (cat. no. 10362-1-AP) and CHK2 (cat. no. 13954-1-AP) antibodies were purchased from ProteinTech Group, Inc. Anti-human (h) CD45‐fluorescein isothiocyanate (FITC) (cat. no. ab134199) was obtained from Abcam.

Techniques: Western Blot

Journal: Endocrinology

Article Title: The role of exchange protein directly activated by cyclic AMP 2-mediated calreticulin expression in the decidualization of human endometrial stromal cells.

doi: 10.1210/en.2013-1478

Figure Lengend Snippet: Figure 4. Knock-down of EPAC2 or CRT in ESCs is associated with senescence. ESCs were treated for 24 hours with nontargeting control (Cont), EPAC2, or CRT siRNA and then cultured for 72 hours. A, B, ESCs were stained with SA--Gal (A). Scale bars, 100 m. The graph shows the relative levels of SA--Gal-positive cells. The data from three independent experiments are presented as ratios and show the mean SEM (B). *, P .01 vs Cont. C, D, Cell lysates were subjected to immunoblot analysis with anti-p53, p21, CRT, or EPAC2 antibody. The same blot was stripped and reprobed with anti-GAPDH antibody as a loading control (C). The graphs show the relative levels of p53 or p21 normalized to GAPDH levels (D). The data (mean SEM) from three independent experiments are presented as ratios. *, P .01 vs Cont.

Article Snippet: Antibodies against CRT, p53 (clone 1C12), p21 (clone DCS60), EPAC1 (clone 5D3), and EPAC2 (clone 5B1) were purchased from Cell Signaling Technology.

Techniques: Knockdown, Control, Cell Culture, Staining, Western Blot

Journal: Biological & pharmaceutical bulletin

Article Title: Glionitrin A, a new diketopiperazine disulfide, activates ATM-ATR-Chk1/2 via 53BP1 phosphorylation in DU145 cells and shows antitumor effect in xenograft model.

doi: 10.1248/bpb.b13-00719

Figure Lengend Snippet: Fig. 2. Glionitrin A Elevates H2A.X Phosphorylation by Responding to DNA Damage Resulting from the Activation of ATM/ATR-Chk1/2 in a 53BP1-Dependent Manner

Article Snippet: Primary antibodies against phospho-H2A.X (Ser139), phospho-53BP1 (S11778), 53BP1, phospho-ATM (Ser1981), phospho-ATR (Ser428), ATR, phospho-Chk1 (Ser317), phospho-Chk1 (Ser345), Chk1, phospho-Chk2 (Thr68), phosphoChk2 (Thr387), Chk2, cdc25A, phosphor-cdc2 (Tyr15), cdc2, cleaved caspase-8, Bid, Bax, cleaved caspase-9, cleaved caspase-3, poly(ADP-ribose) polymerase (PARP), apoptosisinducing factor (AIF), endonuclease G (EndoG), histone deacetylase 1 (HDAC1), cytochrome c oxidase subunit IV (COX IV) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.).

Techniques: Phospho-proteomics, Activation Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Effect of circadian clock mutations on DNA damage response in mammalian cells.

doi: 10.4161/cc.21771

Figure Lengend Snippet: Figure 8. PER1 expression neither sensitizes human lung cancer cells to apoptosis nor affects Chk2 phosphorylation following ionizing radiation (IR). siRNA transfected NCI-H460 human lung cancer cells were irradiated with 0, 10 or 30 Gy of IR and allowed to grow for 48 h, protein lysates were prepared for immunoblot analysis. (A) Protein levels were analyzed by immunoblotting 48 h after irradiation with IR with the indicated antibodies from NCI-H460 cells transfected with either negative control (non-target, NT) or Per1 siRNAs. (B) Quantitative analysis of the immunoblots. (C) Protein levels were analyzed by immunoblotting 48 h after irradiation with IR with the indicated antibodies from NCI-H460 cells transfected with either empty vector (pCDNA3) or a mPer1-overexpressing vector for 72 h. (D) Quantitative analysis of the immunoblots. Averages and standard deviations are from at least three independent experiments. T-PARP and C-PARP stand for total PARP and cleaved PARP, respectively.

Article Snippet: Protein lysates from whole-cell lysates were prepared as described previously,42 and the protein expression levels were determined by immunoblot assay.42 The following antibodies were used to detect the respective proteins: XPA, Cyclophilin-B, p53, Chk1, Chk2 and actin (Santa Cruz Biotechnology); cleaved PARP, cleaved caspase-3, P-chk1, P-chk2 and GAPDH (Cell Signaling Technology); Cry2, Per2, Clock and Bmal1 (Bethyl Labs); Per1 (Kerafast) and Cry1.38 IR dose (0, 2, 2.5, 4, 5, 6, 7.5, 8 and 10 Gy).

Techniques: Expressing, Phospho-proteomics, Transfection, Irradiation, Western Blot, Negative Control, Plasmid Preparation

Journal: Oncoimmunology

Article Title: Uric acid accumulation in DNA-damaged tumor cells induces NKG2D ligand expression and antitumor immunity by activating TGF-β-activated kinase 1

doi: 10.1080/2162402X.2021.2016159

Figure Lengend Snippet: ATM and Chk silencing blocks genotoxic drug-induced ERK phosphorylation, uric acid production, and MICA/B expression . HeLa cells were transfected with scrambled control siRNA, ATM siRNA, Chk1 siRNA, or Chk2 siRNA. After incubation for 24 hr, the cells were left untreated or treated with 5-FU (10 μM) or gemcitabine (2 μM) for another 24 hr. Cell lysates were prepared and analyzed for TAK1 and ERK phosphorylation by Western blot (a) and uric acid levels by using a uric acid assay kit (b). Relative phosphorylation levels were analyzed by quantifying the density of phosphorylated TAK1 and ERK bands normalized by the density of their corresponding total protein bands with NIH Image-J software and presented as bar graphs. Data are the mean ± SD of three experiments. **p < .01, compared to the untreated control. ## p < .01; ns, not significant, compared to the corresponding control siRNA-transfected controls. Single-cell suspensions were prepared and analyzed for the levels of MICA/B expression (c) by flow cytometry. Green line, isotype control; Red line, MICA/B. The data represent one of three independent experiments with similar results.

Article Snippet: TAK1 siRNA (6317S), ATM siRNA (6328S), Chk1 siRNA (6241S), and Chk2 siRNA (6276S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Phospho-proteomics, Expressing, Transfection, Control, Incubation, Western Blot, Uric Acid Assay, Software, Flow Cytometry

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