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  • 99
    Millipore ku 55933 ku
    IKKβ inhibitor is equally potent as DNA-PK and ATM inhibitors in inhibition of the repair of IR-induced DSBs. (A) and (B) MCF-7 cells were incubated with vehicle (0.1% DMSO) or 5 µM BMS-345541 (BMS), NU-7026 (NU) or <t>KU-55933</t> (KU) for 1 h before exposure to 2 Gy IR. DSBs were analyzed by γH2AX and 53BP1 immunofluorescent staining before IR (CTL) or at various time points after IR. The average numbers of γH2AX and 53BP1 foci/cell from three independent experiments are presented as mean ± SE. *** p
    Ku 55933 Ku, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Selleck Chemicals ku 0063794 ku
    Catalytic inhibitor of mTOR increases eIF2alpha phosphorylation: a . Cells were treated with the noted concentrations of <t>Ku-0063794</t> for 48 hours, then harvested and processed for Western blot analysis of eIF2α phosphorylation. b . Ku-0063794 induced changes in eIF2α (e), p-eIF2α (p) and the ratio p-eIF2α/eIF2α (p/e) represent the analysis of 3 determinations for 100 and 1000 nM and of 2 determinations for 300 nM. *Denotes significant change relative to control - p
    Ku 0063794 Ku, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam ku 55933
    SIRT 7 modulates levels of H3K18Ac at  DSB H3K18Ac  IF  in  WT  and  SirT7 −/−  fibroblasts. Cells were pulsed with EdU (green), stained with antibody against H3K18Ac (magenta), and then counterstained with  DAPI  (blue). (A) Representative  IF  images of late S‐phase nuclei (EdU positive; scale bar 5 μm). (B) Quantitation of (A) throughout the cell cycle (mean ± SEM of  >  30 cells per cell cycle, time point, and genotype; 3 mice per genotype). Same as in (B), in S‐phase cells at the indicated times after  IR  (1 Gy) (mean ±  SEM  of  >  30 cells per cell cycle, time point, and genotype; 3 mice per genotype). Laser‐induced  DNA  damage in  HT 1080 cells expressing  HA ‐tagged  SirT7  stained with antibodies against γH2 AX  (red) and  HA  (green), then counterstained with  DAPI  (blue) 30 min postdamage (scale bar 3 μm). Ch IP  assays of  SIRT 7 enrichment at the indicated loci as described in Fig   5 H (mean ±  SEM ; one of two independent experiments is shown). Recruitment kinetics of  GFP ‐tagged  SirT7  after laser‐induced microirradiation. (Top) Representative cell at the indicated times after induction of  DNA  damage (white circle; scale bar 2 μm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5 μM  KU ‐55933  ATM  inhibitor ( ATM i ), 10 μM olaparib  PARP  inhibitor ( PARP i ), or  DMSO .  KU ‐55933 and olaparib were added 30 min and 1 h, respectively, prior to  DNA  damage (mean ±  SEM ; sample size:  SIRT 7‐ GFP ,  n  = 34;  SIRT 7‐GFP + ATM i ,  n  = 36;  SIRT 7‐GFP + PARP i  = 18). (Bottom) Same as (middle) except quantitation of  SIRT 7‐ GFP  relative intensity within the nucleolus. Data were acquired at 5‐s intervals over a span of five min. Ch IP  assays of H3K18Ac enrichment at the indicated loci as described in Fig   5 H. H3K18Ac enrichment adjacent to the induced  DSB  in control and  SIRT 7‐depleted cells (bottom left), or in cells overexpressing  SIRT 7 in the presence or absence of  PARP  inhibitors ( PARP i ) (bottom right) (mean ±  SEM ; one of two independent experiments is shown). Data information: * P 
    Ku 55933, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Tocris ku 55933
    Relative modification efficiency in D1 cells transfected with SDF-PCR-WT and treated with α-Amanitin, 1,5-Isoquinolinediol and <t>KU-55933.</t> Transfected samples were analyzed three days after transfection (3 days; black columns) or in parallel treated 24 hours after transfection with 0.5 µM of 5-Aza-dC for 48 hours (1 day+2 days 5-Aza-dC; white columns). No statistically significant differences were observed at 3 days (black bars) respect to untreated cells (SDF-PCR-WT). Demethylating effect of 5-Aza-dC increased eGFP detection in all samples (white columns) in a statistically significant manner (Δ p = 0.003; +p = 0.01; • p = 0.0007). 5-Aza-dC addition also disclosed the effect of 1,5-Isoquinolinediol on SDF-mediated correction in a statically significant manner respect either to cells not treated with 5-Aza-dC (**p = 0.0002) and to the cells transfected with SDF-PCR-WT in which no drug was added (*p = 0.003). Dashed lines refers to modification efficiency observed in cells without addition of any drug but treated by 5-Aza-dC. Results are from mean values of three independent experiments and are reported as relative modification efficiencies in respect to control without drugs (SDF-PCR-WT).
    Ku 55933, supplied by Tocris, used in various techniques. Bioz Stars score: 98/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Tocris ku 0063794
    mTORC1 represses PIP 3 production and controls the mTORC2 requirement for activation of AKT (a) Immunoblot analysis of PTEN expression in CTLs cultured ± 48 h rapamycin. (b) HPLC-MS based analysis of PtdIns(3,4,5)P 3 levels in control IL-2/IL-12 maintained CTLs (DM) and CTLs treated with PI(3)K p110δ inhibitor IC-87114 (IC, 1 h) and rapamycin for the indicated times. (c) Immunoblot analysis of Akt T308 and S473 phosphorylation levels in CTLs ± 48 h rapamycin. (d, e) The data show immunoblots of Akt T308 phosphorylation in control and mTORC1 inhibited CTLs treated with (d) IC-87114 or (e) AKTi1/2. (f) Immunoblot analysis of PTEN expression in CTLs cultured ± 48 h rapamycin or <t>KU-0063794.</t> (g) HPLC-MS based analysis of PtdIns(3,4,5)P 3 levels in control CTLs and CTLs treated with KU-0063794 for indicated durations. (h) Immunoblot analysis of Akt T308, Akt S473, FOXO1/3A T24/T32 phosphorylation and phosphorylation of the mTORC1 substrates S6K T389 and 4EBP1 S65 in CTLs treated with KU-0063794 for the indicated times. (i) Scatter plot depicting correlation of mean (n=3) fold changes in transcript expression from Affymetrix microarray analysis of control CTLs vs CTLs treated with rapamycin (x-axis) or KU-0063794 (y-axis). Immunoblots are representative of at least three biological replicates. (b, g) Individual data points and means are shown. P -values are determined by one-way ANOVA (Holm-Sidak) vs. DMSO (DM) as control. * P
    Ku 0063794, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore ku 60019
    Q6 induced G2/M arrest and apoptosis is ATM/Chk2 dependent in hypoxia. A. HepG2 and Bel-7402 cells, treated with Q6 (5 μM) in the presence or absence of caffeine (2 mM) for 24 h under hypoxia (1% O 2 ), were collected and prepared for cytometric analysis of cell cycle distribution. B C. HepG2 cells treated with Q6 (10 μM) in the presence or absence of caffeine (2 mM) for 24 h under hypoxia (1% O 2 ). Detection of apoptosis by flow cytometry (B) and caspase cascade by Western blot (C) were then performed. D E. HepG2 cells treated with Q6 in the presence or absence of ATM specific inhibitor <t>KU-60019</t> (3 μM) under hypoxia (1% O 2 ), and subjected to sub-G1 analyses (D) and Western blot analyses (E), respectively. F. Western blot was used to assess the role of ATM during apoptosis induced by Q6 in hypoxia. HepG2 cells were treated with ATM RNAi or vector RNAi in the presence or absence of Q6 (5 μM) under hypoxia (1% O 2 ) condition.
    Ku 60019, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Selleck Chemicals ku 60019
    Role of CK2 kinase in XPC phosphorylation and CPD repair. ( A ) Immunoblot analysis of XPC phosphorylated at S94 (p-XPC S94), total XPC and GAPDH 30 min after UVB exposure (20 mJ/cm 2 ) in XPC Null cells expressing pLenti-XPC WT or mutant S94A. ( B ) HaCaT cells were treated with vehicle, rapamycin (25 nM), CX-4945 (5 μM), <t>KU-60019</t> (1 μM), LY294002 (10 μM), NU7441 (1 μM), TCS JNK 60 (10 μM), SB203580 (10 μM), PD98059 (20 μM) or Y27632 (10 μM) for 1 h. The levels of XPC phosphorylated at S94 (p-XPC S94), total XPC and GAPDH were analyzed by immunoblot assay. ( C ) Immunoblot analysis of XPC phosphorylated at S94 (p-XPC S94), total XPC, CK2A1, CK2A2 and GAPDH 30 min after UVB exposure (20 mJ/cm 2 ) in HaCaT cells transfected with siRNA targeting CK2A1 and CK2A2 (siCK2) or non-targeting control siRNA (siNC). ( D ) Slot blot analysis of the levels of CPD at the indicated times post-UVB (20 mJ/cm 2 ) in HaCaT cells transfected with siRNA targeting CK2A1 and CK2A2 (siCK2) or non-targeting control siRNA (siNC). Methylene blue staining was used for loading control. ( E ) Quantification of percentage (%) of CPD repair from (D). ** P
    Ku 60019, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 97/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris ku 60019
    Role of CK2 kinase in XPC phosphorylation and CPD repair. ( A ) Immunoblot analysis of XPC phosphorylated at S94 (p-XPC S94), total XPC and GAPDH 30 min after UVB exposure (20 mJ/cm 2 ) in XPC Null cells expressing pLenti-XPC WT or mutant S94A. ( B ) HaCaT cells were treated with vehicle, rapamycin (25 nM), CX-4945 (5 μM), <t>KU-60019</t> (1 μM), LY294002 (10 μM), NU7441 (1 μM), TCS JNK 60 (10 μM), SB203580 (10 μM), PD98059 (20 μM) or Y27632 (10 μM) for 1 h. The levels of XPC phosphorylated at S94 (p-XPC S94), total XPC and GAPDH were analyzed by immunoblot assay. ( C ) Immunoblot analysis of XPC phosphorylated at S94 (p-XPC S94), total XPC, CK2A1, CK2A2 and GAPDH 30 min after UVB exposure (20 mJ/cm 2 ) in HaCaT cells transfected with siRNA targeting CK2A1 and CK2A2 (siCK2) or non-targeting control siRNA (siNC). ( D ) Slot blot analysis of the levels of CPD at the indicated times post-UVB (20 mJ/cm 2 ) in HaCaT cells transfected with siRNA targeting CK2A1 and CK2A2 (siCK2) or non-targeting control siRNA (siNC). Methylene blue staining was used for loading control. ( E ) Quantification of percentage (%) of CPD repair from (D). ** P
    Ku 60019, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore ku 63794
    Inhibition of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling induction in TR146 cells infected with Candida albicans. A , Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on cytokine production after 24 hours (multiplicity of infection [MOI] = 0.01) shown as percentage of the dimethyl sulfoxide (DMSO) vehicle control. B , Effect of inhibition of PI3K/Akt (wortmannin [1 µM] and LY294002 [50 µM]) or p38 (SB203580 [10 µM]) signaling on cell damage (lactate dehydrogenase [LDH] release) after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). C , Effect of inhibition of mTOR activity with 10 μM <t>Ku-63794</t> on cell damage (LDH release) after 24 hours (MOI] = 0.01) shown as percentage of DMSO vehicle control. D , Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cytokine production after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). E , Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of IκBα 2 hours postinfection (MOI = 10). F , Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of MKP1 and c-Jun and on the production of c-Fos 2 hours postinfection (MOI = 10). Candida albicans was added as 100% yeast, which switched to hyphal growth by 2 hours postinfection. Data are the mean ( A – D ) or representative ( E and F ) of at least 3 independent experiments. * P
    Ku 63794, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam ku 60019
    ATM-depleted fibroblasts progressively accumulate ROS and oxidative protein damage. ( A ) Representative Western blot analysis on ATM-depleted fibroblasts showing upregulation of proteins involved in the antioxidant response. TIG1 cells were transfected with either a control siRNA (siCtrl) or an ATM-targeting siRNA (siATM#1, siATM #2). Actin was used as loading control. Densitometric quantification of the indicated proteins is reported at the bottom of the gel ( N = 2). ( B ) Western blot analysis on a representative time course depletion of ATM. TIG1 fibroblasts were treated as indicated and ATM expression was monitored in nuclear cell extracts. Lamin A/C was used as loading control. ( C ) Quantification of ROS content using flow-cytometry in siATM-treated fibroblasts. TIG1 fibroblasts were treated as indicated. H 2 O 2 (25 μM, 30 min) was used as a positive control for induction of ROS. ATM inhibitors (Ku-55933 – ATMi #1 and <t>Ku-60019</t> – ATMi #2, both 10 μM) were provided fresh every 24 h for a total of 72 h ( N = 3). ( D ) Quantification of mitochondrial membrane potential (ΔΨm) in siATM-treated fibroblasts using flow-cytometry. Cells were treated as in panel C. Staurosporine (2 μM, 2 h) was used as a positive control for mitochondrial membrane depolarisation ( N = 3). ( E ) Detection of protein carbonylation in nuclear extracts obtained from fibroblasts depleted of ATM for the indicated time. Carbonylated proteins were detected by derivatisation with dinitrophenylhydrazone (DNP) followed by Western blot using an anti-DNP antibody. Equal amounts of cell extract were loaded in each lane. H 2 O 2 (1 mM, 30 minutes) was used as a positive control for induction of protein carbonylation. ( F ) Quantification of protein carbonylation in ATM-depleted fibroblasts; the histogram displays densitometric data from the analysis reported in panel E ( N = 6). Results are expressed as mean ± SD from the indicated number ( N ) of independent experiments: * P
    Ku 60019, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Selleck Chemicals ku 57788
    Lack of functional DNA-PKcs reduces chemotherapy-induced mutations ( a ) DNA-PKcs protein expression in DNA-PKcs expressing (control) and DNA-PKcs knockout cells was assessed by immunoblot. Probing for GAPDH was used as a loading control. ( b ) Cells were treated with indicated doses of drugs or left untreated (UT) for 24 hours, harvested and clonogenicity assays were performed to determine the proportion of cells maintaining clonogenic competency. ( c ) Surviving cells were grown in 6-TG to select for the emergence of any HPRT mutants. ( d ) TK6 cells were pre-treated with 10 μM <t>KU-57788</t> (DNA-PKi) or DMSO for 1 hour, then cultured in media containing 10 nM doxorubicin (Dox), 1 μM cisplatin (Cis), 0.1 nM vincristine (Vin), 10 μM GDC-0152 (GDC) or untreated for 24 hours. Cells were harvested and clonogenicity or ( e ) HPRT assays were performed on surviving cells. Error bars represent mean ± SEM from at least three independent experiments. Two-way ANOVA analyses with Bonferroni post-tests were used to estimate the probability that random chance accounted for the differences observed in drug responses between control cells and either each DNA-PKcs null line or both KO lines (denoted by the square brackets) ( b , c ), or DMSO-treated versus inhibitor-treated cells ( d , e ). P values
    Ku 57788, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 88/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Selleck Chemicals ku 0060648
    HDR stimulation by NU7441 and <t>KU-0060648</t> is comparable to other HDR enhancing tools. a Western blot showing knock down efficiency obtained with the siRNAs targeting either Ku70 or Ku80. b Quantitation of genome editing events from cells transfected with pQCiG-TLR and ΔeGFP donor in the presence of 2 μM NU7441, 250 nM KU-0060648, siRNAs targeting Ku70, Ku80, DNA-PKcs or DNA ligase IV, 1 μM Scr7, or a combination of Scr7 and 2 μM NU7441 or 250 nM KU-0060648. The HDR and NHEJ values are relative to those obtained with Cas9, sgRNA , and ΔeGFP donor in the presence of vehicle (DMSO). Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant. c Western blot showing expression of Adenovirus 5 proteins E1B55K and E4orf6 following transfection into the 293/TLR line. d Quantitation of genome editing efficiencies was as in B except that adenovirus 5 proteins E1B55K and E4orf6 expression vectors were co-transfected with pQCiG-TLR and ΔeGFP donor plasmids and cultured in presence or absence of 2 μM NU7441 or 250 nM KU-0060648. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant
    Ku 0060648, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Selleck Chemicals nu7441 ku 57788
    HDR stimulation by NU7441 and <t>KU-0060648</t> is comparable to other HDR enhancing tools. a Western blot showing knock down efficiency obtained with the siRNAs targeting either Ku70 or Ku80. b Quantitation of genome editing events from cells transfected with pQCiG-TLR and ΔeGFP donor in the presence of 2 μM NU7441, 250 nM KU-0060648, siRNAs targeting Ku70, Ku80, DNA-PKcs or DNA ligase IV, 1 μM Scr7, or a combination of Scr7 and 2 μM NU7441 or 250 nM KU-0060648. The HDR and NHEJ values are relative to those obtained with Cas9, sgRNA , and ΔeGFP donor in the presence of vehicle (DMSO). Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant. c Western blot showing expression of Adenovirus 5 proteins E1B55K and E4orf6 following transfection into the 293/TLR line. d Quantitation of genome editing efficiencies was as in B except that adenovirus 5 proteins E1B55K and E4orf6 expression vectors were co-transfected with pQCiG-TLR and ΔeGFP donor plasmids and cultured in presence or absence of 2 μM NU7441 or 250 nM KU-0060648. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant
    Nu7441 Ku 57788, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ku 0060648
    Comparison of PK levels of <t>KU-0060648</t> and DNA-PK activity within ex vivo SW620 tumour samples following i.v dosing
    Ku 0060648, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Tocris atmi ku 55933
    XAB2 prevents Ku retention on seDSBs induced by temozolomide. a , Quantification of the average number of Ku80 foci detected by immunofluorescence microscopy in control and XAB2-depleted U87 cells exposed to 15 μM TMZ (or DMSO) for 2 h and allowed to recover for 48 h in the absence or presence of the <t>ATMi</t> KU-55933. b , Representative immunofluorescence images used for the quantification of the number of colocalizing Ku80 (red)-RPA32 (cyan) foci following exposure of control and XAB2-depleted U87 cells to TMZ and recovery for 48 h. Examples of colocalized foci are highlighted by yellow squares, with one representative example (indicated by an arrow) shown in the close up section (scale bar = 0.1 μm). c - d , Percentage of colocalized Ku80-RPA32 foci per cell following exposure to TMZ as in c , quantified based on foci examination in individual cells, as presented in a violin plot ( d ). e - g , Same as b - d for the analysis of Ku80 (red) and RAD51 (cyan) foci colocalization. Scale bar = 5µm. The images are representative of 3 independent biological repeats. Bars represent mean ± s.e.m. Significant differences between specified comparisons were assessed by a t-test (unpaired, 2-tails) and are highlighted by stars (*P
    Atmi Ku 55933, supplied by Tocris, used in various techniques. Bioz Stars score: 89/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    IKKβ inhibitor is equally potent as DNA-PK and ATM inhibitors in inhibition of the repair of IR-induced DSBs. (A) and (B) MCF-7 cells were incubated with vehicle (0.1% DMSO) or 5 µM BMS-345541 (BMS), NU-7026 (NU) or KU-55933 (KU) for 1 h before exposure to 2 Gy IR. DSBs were analyzed by γH2AX and 53BP1 immunofluorescent staining before IR (CTL) or at various time points after IR. The average numbers of γH2AX and 53BP1 foci/cell from three independent experiments are presented as mean ± SE. *** p

    Journal: PLoS ONE

    Article Title: IKK? Regulates the Repair of DNA Double-Strand Breaks Induced by Ionizing Radiation in MCF-7 Breast Cancer Cells

    doi: 10.1371/journal.pone.0018447

    Figure Lengend Snippet: IKKβ inhibitor is equally potent as DNA-PK and ATM inhibitors in inhibition of the repair of IR-induced DSBs. (A) and (B) MCF-7 cells were incubated with vehicle (0.1% DMSO) or 5 µM BMS-345541 (BMS), NU-7026 (NU) or KU-55933 (KU) for 1 h before exposure to 2 Gy IR. DSBs were analyzed by γH2AX and 53BP1 immunofluorescent staining before IR (CTL) or at various time points after IR. The average numbers of γH2AX and 53BP1 foci/cell from three independent experiments are presented as mean ± SE. *** p

    Article Snippet: KU-55933 (KU), BMS-345541 (BMS), Hoechst-33342 (Hoe) and Propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Inhibition, Incubation, Staining, CTL Assay

    miR‐106a and  LITAF  regulate  ATM  expression to confer radioresistance. (A)  qRT ‐ PCR  for  ATM mRNA  expression (normalized to  GAPDH ) in  PC 3 and  DU 145 control(m)/miR‐106a(106a) mimic and control(si)/ LITAF  si RNA  cells. (B) Representative western blot for phospho‐ ATM  ( pATM ), total  ATM , and β‐actin (loading control) in  PC 3 control/miR‐106a mimic and control/ LITAF  si RNA  cells at 0 Gy and 30 min after 6 Gy irradiation ( IR ). (C)  ATM  promoter luciferase in  PC 3 and  DU 145 control/miR‐106a mimic cells. (D) Representative western blot for  pATM , total  ATM , and β‐actin from  PC 3 cells were treated with  DMSO  only (vehicle), 1, 2, 5, and 10 μ m  of  KU ‐55933 2 hr before exposure to 6 Gy  IR , and baseline at 0 Gy with  DMSO  only. (E)  SA ‐β‐galactosidase assay was performed with  DU 145 control/miR‐106a cells 7d after 6 Gy  IR . Cells were treated with  DMSO  and 2 μ m  KU ‐55933. Representative images show  DU 145 control/miR‐106a cells after 6 Gy stained for  SA ‐β‐galactosidase with  DMSO  or  KU ‐55933 (scale bar = 200 μm). (F) Clonogenic survival assays were performed with  DU 145 cells transfected with control and miR‐106a mimic.  PC 3 and  DU 145 cells were treated with  DMSO  or 2 μ m  KU ‐55933 ( KU )  ATM  inhibitor 2 h prior to 6 Gy or 8 Gy radiation, respectively. Mean,  SEM , and statistical significance are denoted; *,  P 

    Journal: Molecular Oncology

    Article Title: mi RNA‐106a and prostate cancer radioresistance: a novel role for LITAF in ATM regulation

    doi: 10.1002/1878-0261.12328

    Figure Lengend Snippet: miR‐106a and LITAF regulate ATM expression to confer radioresistance. (A) qRT ‐ PCR for ATM mRNA expression (normalized to GAPDH ) in PC 3 and DU 145 control(m)/miR‐106a(106a) mimic and control(si)/ LITAF si RNA cells. (B) Representative western blot for phospho‐ ATM ( pATM ), total ATM , and β‐actin (loading control) in PC 3 control/miR‐106a mimic and control/ LITAF si RNA cells at 0 Gy and 30 min after 6 Gy irradiation ( IR ). (C) ATM promoter luciferase in PC 3 and DU 145 control/miR‐106a mimic cells. (D) Representative western blot for pATM , total ATM , and β‐actin from PC 3 cells were treated with DMSO only (vehicle), 1, 2, 5, and 10 μ m of KU ‐55933 2 hr before exposure to 6 Gy IR , and baseline at 0 Gy with DMSO only. (E) SA ‐β‐galactosidase assay was performed with DU 145 control/miR‐106a cells 7d after 6 Gy IR . Cells were treated with DMSO and 2 μ m KU ‐55933. Representative images show DU 145 control/miR‐106a cells after 6 Gy stained for SA ‐β‐galactosidase with DMSO or KU ‐55933 (scale bar = 200 μm). (F) Clonogenic survival assays were performed with DU 145 cells transfected with control and miR‐106a mimic. PC 3 and DU 145 cells were treated with DMSO or 2 μ m KU ‐55933 ( KU ) ATM inhibitor 2 h prior to 6 Gy or 8 Gy radiation, respectively. Mean, SEM , and statistical significance are denoted; *, P 

    Article Snippet: 2.12 KU‐55933 KU‐55933 (Cat#: SML1109; Sigma‐Aldrich, Oakville, ON, Canada) was dissolved in DMSO, and cells were treated with vehicle (DMSO) or KU‐55933.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Irradiation, Luciferase, Staining, Transfection

    Proposed model by which miR‐106a confers radiation resistance. miR‐106a upregulates  ATM  expression through  LITAF  knockdown, to inhibit senescence and confer radioresistance in prostate cancer.  KU ‐55933 has been identified as a therapeutic intervention for miR‐106a‐induced radioresistance.

    Journal: Molecular Oncology

    Article Title: mi RNA‐106a and prostate cancer radioresistance: a novel role for LITAF in ATM regulation

    doi: 10.1002/1878-0261.12328

    Figure Lengend Snippet: Proposed model by which miR‐106a confers radiation resistance. miR‐106a upregulates ATM expression through LITAF knockdown, to inhibit senescence and confer radioresistance in prostate cancer. KU ‐55933 has been identified as a therapeutic intervention for miR‐106a‐induced radioresistance.

    Article Snippet: 2.12 KU‐55933 KU‐55933 (Cat#: SML1109; Sigma‐Aldrich, Oakville, ON, Canada) was dissolved in DMSO, and cells were treated with vehicle (DMSO) or KU‐55933.

    Techniques: Expressing

    ATM kinase inhibitor (KU-55933), phosphoinositide 3 kinase inhibitor (wortmannin) and ATM/ATM-Rad3-related inhibitor (caffeine) prevent MG132-induced upregulation of MICB. A549 cells were treated with KU-55933 (10 µM) for 9 h or wortmannin (6 µM) or caffeine (10 mM) for 10 h, or MG132 for 8 h, or with KU-55933 for 9 h or wortmannin or caffeine for 10 h, with the addition of MG132 during the final 8 h. The expression of MICB was measured and was normalized to that of control DMSO-treated cells (Con). (A) RNA levels and (B) flow cytometry. Multiple comparisons were performed with one-way analysis of variance. *P

    Journal: Molecular Medicine Reports

    Article Title: MG132 selectively upregulates MICB through the DNA damage response pathway in A549 cells

    doi: 10.3892/mmr.2018.9676

    Figure Lengend Snippet: ATM kinase inhibitor (KU-55933), phosphoinositide 3 kinase inhibitor (wortmannin) and ATM/ATM-Rad3-related inhibitor (caffeine) prevent MG132-induced upregulation of MICB. A549 cells were treated with KU-55933 (10 µM) for 9 h or wortmannin (6 µM) or caffeine (10 mM) for 10 h, or MG132 for 8 h, or with KU-55933 for 9 h or wortmannin or caffeine for 10 h, with the addition of MG132 during the final 8 h. The expression of MICB was measured and was normalized to that of control DMSO-treated cells (Con). (A) RNA levels and (B) flow cytometry. Multiple comparisons were performed with one-way analysis of variance. *P

    Article Snippet: MG132, KU-55933, caffeine and wortmannin were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

    Techniques: Expressing, Flow Cytometry, Cytometry

    Catalytic inhibitor of mTOR increases eIF2alpha phosphorylation: a . Cells were treated with the noted concentrations of Ku-0063794 for 48 hours, then harvested and processed for Western blot analysis of eIF2α phosphorylation. b . Ku-0063794 induced changes in eIF2α (e), p-eIF2α (p) and the ratio p-eIF2α/eIF2α (p/e) represent the analysis of 3 determinations for 100 and 1000 nM and of 2 determinations for 300 nM. *Denotes significant change relative to control - p

    Journal: PLoS ONE

    Article Title: Eukaryotic Initiation Factor 2? - a Downstream Effector of Mammalian Target of Rapamycin - Modulates DNA Repair and Cancer Response to Treatment

    doi: 10.1371/journal.pone.0077260

    Figure Lengend Snippet: Catalytic inhibitor of mTOR increases eIF2alpha phosphorylation: a . Cells were treated with the noted concentrations of Ku-0063794 for 48 hours, then harvested and processed for Western blot analysis of eIF2α phosphorylation. b . Ku-0063794 induced changes in eIF2α (e), p-eIF2α (p) and the ratio p-eIF2α/eIF2α (p/e) represent the analysis of 3 determinations for 100 and 1000 nM and of 2 determinations for 300 nM. *Denotes significant change relative to control - p

    Article Snippet: It remains to be determined if the phenomenon we demonstrated in our study e.g. increased eIF2α phosphorylation following inhibition of mTOR by rapalogues or by Ku-0063794, results from inactivation of mTORC1, mTORC2 – Akt pathway, or whether additional mTOR mediated pathways are involved.

    Techniques: Western Blot

    SIRT 7 modulates levels of H3K18Ac at  DSB H3K18Ac  IF  in  WT  and  SirT7 −/−  fibroblasts. Cells were pulsed with EdU (green), stained with antibody against H3K18Ac (magenta), and then counterstained with  DAPI  (blue). (A) Representative  IF  images of late S‐phase nuclei (EdU positive; scale bar 5 μm). (B) Quantitation of (A) throughout the cell cycle (mean ± SEM of  >  30 cells per cell cycle, time point, and genotype; 3 mice per genotype). Same as in (B), in S‐phase cells at the indicated times after  IR  (1 Gy) (mean ±  SEM  of  >  30 cells per cell cycle, time point, and genotype; 3 mice per genotype). Laser‐induced  DNA  damage in  HT 1080 cells expressing  HA ‐tagged  SirT7  stained with antibodies against γH2 AX  (red) and  HA  (green), then counterstained with  DAPI  (blue) 30 min postdamage (scale bar 3 μm). Ch IP  assays of  SIRT 7 enrichment at the indicated loci as described in Fig   5 H (mean ±  SEM ; one of two independent experiments is shown). Recruitment kinetics of  GFP ‐tagged  SirT7  after laser‐induced microirradiation. (Top) Representative cell at the indicated times after induction of  DNA  damage (white circle; scale bar 2 μm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5 μM  KU ‐55933  ATM  inhibitor ( ATM i ), 10 μM olaparib  PARP  inhibitor ( PARP i ), or  DMSO .  KU ‐55933 and olaparib were added 30 min and 1 h, respectively, prior to  DNA  damage (mean ±  SEM ; sample size:  SIRT 7‐ GFP ,  n  = 34;  SIRT 7‐GFP + ATM i ,  n  = 36;  SIRT 7‐GFP + PARP i  = 18). (Bottom) Same as (middle) except quantitation of  SIRT 7‐ GFP  relative intensity within the nucleolus. Data were acquired at 5‐s intervals over a span of five min. Ch IP  assays of H3K18Ac enrichment at the indicated loci as described in Fig   5 H. H3K18Ac enrichment adjacent to the induced  DSB  in control and  SIRT 7‐depleted cells (bottom left), or in cells overexpressing  SIRT 7 in the presence or absence of  PARP  inhibitors ( PARP i ) (bottom right) (mean ±  SEM ; one of two independent experiments is shown). Data information: * P 

    Journal: The EMBO Journal

    Article Title: SIRT7 promotes genome integrity and modulates non‐homologous end joining DNA repair

    doi: 10.15252/embj.201593499

    Figure Lengend Snippet: SIRT 7 modulates levels of H3K18Ac at DSB H3K18Ac IF in WT and SirT7 −/− fibroblasts. Cells were pulsed with EdU (green), stained with antibody against H3K18Ac (magenta), and then counterstained with DAPI (blue). (A) Representative IF images of late S‐phase nuclei (EdU positive; scale bar 5 μm). (B) Quantitation of (A) throughout the cell cycle (mean ± SEM of > 30 cells per cell cycle, time point, and genotype; 3 mice per genotype). Same as in (B), in S‐phase cells at the indicated times after IR (1 Gy) (mean ±  SEM of >  30 cells per cell cycle, time point, and genotype; 3 mice per genotype). Laser‐induced DNA damage in HT 1080 cells expressing HA ‐tagged SirT7 stained with antibodies against γH2 AX (red) and HA (green), then counterstained with DAPI (blue) 30 min postdamage (scale bar 3 μm). Ch IP assays of SIRT 7 enrichment at the indicated loci as described in Fig  5 H (mean ±  SEM ; one of two independent experiments is shown). Recruitment kinetics of GFP ‐tagged SirT7 after laser‐induced microirradiation. (Top) Representative cell at the indicated times after induction of DNA damage (white circle; scale bar 2 μm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5 μM KU ‐55933 ATM inhibitor ( ATM i ), 10 μM olaparib PARP inhibitor ( PARP i ), or DMSO . KU ‐55933 and olaparib were added 30 min and 1 h, respectively, prior to DNA damage (mean ±  SEM ; sample size: SIRT 7‐ GFP , n  = 34; SIRT 7‐GFP + ATM i , n  = 36; SIRT 7‐GFP + PARP i  = 18). (Bottom) Same as (middle) except quantitation of SIRT 7‐ GFP relative intensity within the nucleolus. Data were acquired at 5‐s intervals over a span of five min. Ch IP assays of H3K18Ac enrichment at the indicated loci as described in Fig  5 H. H3K18Ac enrichment adjacent to the induced DSB in control and SIRT 7‐depleted cells (bottom left), or in cells overexpressing SIRT 7 in the presence or absence of PARP inhibitors ( PARP i ) (bottom right) (mean ±  SEM ; one of two independent experiments is shown). Data information: * P 

    Article Snippet: Pharmacologic inhibition of ATM was performed using the ATM‐specific inhibitor, KU‐55933 (Abcam), at the dose of 5 μM 30 min prior to DNA damage induction.

    Techniques: Staining, Quantitation Assay, Mouse Assay, Expressing

    SIRT 7 protects cells from endogenous and induced  DNA  damage Quantitation of neutral comet assays, using passage 3  WT  and  SirT7 −/−  primary  MEF s, showing (left) the amount (%) of  DNA  in the tail and (right) the tail moment (see   Fig S3A  for representative images). Western blot showing  ATM  and  KAP 1 phosphorylation and total protein levels in  WT  and  SirT7 −/−  primary fibroblasts after  IR  (8 Gy).  ATM  inhibitor ( ATM i )  KU ‐55933 was added 30 min prior to irradiation where indicated. One representative blot from four independent experiments is shown. IF  analysis of  WT  and  SirT7 −/−  primary fibroblasts showing γH2 AX  dynamics after  DNA  damage induction. Cells were untreated (No IR ) or treated with 1 Gy of X‐rays ( IR ) and fixed at the indicated times postinsult. Cells were pulsed with EdU (green) 30 min prior to fixation, then stained for γH2 AX  (red), and counterstained with  DAPI  (blue) ( n >  30 cells per group/mice; 3 mice per genotype). (C) Representative images of untreated S‐phase (No IR , EdU positive)  WT  and  SirT7 −/−  nuclei (scale bar 5 μm). (D) Quantitation of the number of γH2 AX  foci per nucleus at the indicated time points post‐ IR  (1 Gy) and indicated cell cycle phases (mean ± SEM from three independent experiments). (E) Quantitation of experiment described in (C) showing mean number of γH2 AX  foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after  IR  in G2 (mean ±  SEM  from three independent experiments). Total γH2 AX  foci and γH2 AX  foci overlapping with or at the periphery of heterochromatic regions were enumerated. Nuclei and pericentric heterochromatin (chromocenters) were segmented by  DAPI  staining. Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number. (F) Representative images of  WT  and  SirT7 −/−  primary fibroblasts in G2‐phase showing γH2 AX  (red) and  DAPI  (blue) at the indicated period of time after  IR . (Right) 3D rendering of the  IF  segmentation depicting nuclei (pale blue), chromocenters (darker blue), and γH2 AX  (yellow denotes foci associated with pericentric heterochromatin, otherwise foci are red). Scale bar 2 μm. FACS  quantitation of  WT  and  SirT7 −/− MEF  cells in S‐phase after insult with 10  mM  hydroxyurea ( HU ) for 24 h. Cells were fixed and stained with 7 AAD  and cell cycle was monitored by  FACS  (mean ±  SEM ; five samples per genotype). DNA  fiber labeling analysis was used to assess  DNA  replication fork progression in passage 3 and passage 6 primary  WT  and  SirT7 −/− MEF s. (H) Representative images from cells labeled for 20 min with IdU (green) followed by 20 min of CldU (red). (I) Quantitation of fork velocity (fiber length/labeling time; mean ±  SEM ; three samples per genotype per condition). Scale bar 10 μm. Data information: * P 

    Journal: The EMBO Journal

    Article Title: SIRT7 promotes genome integrity and modulates non‐homologous end joining DNA repair

    doi: 10.15252/embj.201593499

    Figure Lengend Snippet: SIRT 7 protects cells from endogenous and induced DNA damage Quantitation of neutral comet assays, using passage 3 WT and SirT7 −/− primary MEF s, showing (left) the amount (%) of DNA in the tail and (right) the tail moment (see Fig S3A for representative images). Western blot showing ATM and KAP 1 phosphorylation and total protein levels in WT and SirT7 −/− primary fibroblasts after IR (8 Gy). ATM inhibitor ( ATM i ) KU ‐55933 was added 30 min prior to irradiation where indicated. One representative blot from four independent experiments is shown. IF analysis of WT and SirT7 −/− primary fibroblasts showing γH2 AX dynamics after DNA damage induction. Cells were untreated (No IR ) or treated with 1 Gy of X‐rays ( IR ) and fixed at the indicated times postinsult. Cells were pulsed with EdU (green) 30 min prior to fixation, then stained for γH2 AX (red), and counterstained with DAPI (blue) ( n >  30 cells per group/mice; 3 mice per genotype). (C) Representative images of untreated S‐phase (No IR , EdU positive) WT and SirT7 −/− nuclei (scale bar 5 μm). (D) Quantitation of the number of γH2 AX foci per nucleus at the indicated time points post‐ IR (1 Gy) and indicated cell cycle phases (mean ± SEM from three independent experiments). (E) Quantitation of experiment described in (C) showing mean number of γH2 AX foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after IR in G2 (mean ±  SEM from three independent experiments). Total γH2 AX foci and γH2 AX foci overlapping with or at the periphery of heterochromatic regions were enumerated. Nuclei and pericentric heterochromatin (chromocenters) were segmented by DAPI staining. Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number. (F) Representative images of WT and SirT7 −/− primary fibroblasts in G2‐phase showing γH2 AX (red) and DAPI (blue) at the indicated period of time after IR . (Right) 3D rendering of the IF segmentation depicting nuclei (pale blue), chromocenters (darker blue), and γH2 AX (yellow denotes foci associated with pericentric heterochromatin, otherwise foci are red). Scale bar 2 μm. FACS quantitation of WT and SirT7 −/− MEF cells in S‐phase after insult with 10 mM hydroxyurea ( HU ) for 24 h. Cells were fixed and stained with 7 AAD and cell cycle was monitored by FACS (mean ±  SEM ; five samples per genotype). DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7 −/− MEF s. (H) Representative images from cells labeled for 20 min with IdU (green) followed by 20 min of CldU (red). (I) Quantitation of fork velocity (fiber length/labeling time; mean ±  SEM ; three samples per genotype per condition). Scale bar 10 μm. Data information: * P 

    Article Snippet: Pharmacologic inhibition of ATM was performed using the ATM‐specific inhibitor, KU‐55933 (Abcam), at the dose of 5 μM 30 min prior to DNA damage induction.

    Techniques: Quantitation Assay, Western Blot, Irradiation, Staining, Mouse Assay, FACS, Labeling

    Relative modification efficiency in D1 cells transfected with SDF-PCR-WT and treated with α-Amanitin, 1,5-Isoquinolinediol and KU-55933. Transfected samples were analyzed three days after transfection (3 days; black columns) or in parallel treated 24 hours after transfection with 0.5 µM of 5-Aza-dC for 48 hours (1 day+2 days 5-Aza-dC; white columns). No statistically significant differences were observed at 3 days (black bars) respect to untreated cells (SDF-PCR-WT). Demethylating effect of 5-Aza-dC increased eGFP detection in all samples (white columns) in a statistically significant manner (Δ p = 0.003; +p = 0.01; • p = 0.0007). 5-Aza-dC addition also disclosed the effect of 1,5-Isoquinolinediol on SDF-mediated correction in a statically significant manner respect either to cells not treated with 5-Aza-dC (**p = 0.0002) and to the cells transfected with SDF-PCR-WT in which no drug was added (*p = 0.003). Dashed lines refers to modification efficiency observed in cells without addition of any drug but treated by 5-Aza-dC. Results are from mean values of three independent experiments and are reported as relative modification efficiencies in respect to control without drugs (SDF-PCR-WT).

    Journal: PLoS ONE

    Article Title: Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System

    doi: 10.1371/journal.pone.0030851

    Figure Lengend Snippet: Relative modification efficiency in D1 cells transfected with SDF-PCR-WT and treated with α-Amanitin, 1,5-Isoquinolinediol and KU-55933. Transfected samples were analyzed three days after transfection (3 days; black columns) or in parallel treated 24 hours after transfection with 0.5 µM of 5-Aza-dC for 48 hours (1 day+2 days 5-Aza-dC; white columns). No statistically significant differences were observed at 3 days (black bars) respect to untreated cells (SDF-PCR-WT). Demethylating effect of 5-Aza-dC increased eGFP detection in all samples (white columns) in a statistically significant manner (Δ p = 0.003; +p = 0.01; • p = 0.0007). 5-Aza-dC addition also disclosed the effect of 1,5-Isoquinolinediol on SDF-mediated correction in a statically significant manner respect either to cells not treated with 5-Aza-dC (**p = 0.0002) and to the cells transfected with SDF-PCR-WT in which no drug was added (*p = 0.003). Dashed lines refers to modification efficiency observed in cells without addition of any drug but treated by 5-Aza-dC. Results are from mean values of three independent experiments and are reported as relative modification efficiencies in respect to control without drugs (SDF-PCR-WT).

    Article Snippet: KU-55933 (Tocris, Bristol, U.K.), a potent, selective and competitive ATM kinase inhibitor, was used at 10 µM one hour prior transfection.

    Techniques: Modification, Transfection, Polymerase Chain Reaction

    mTORC1 represses PIP 3 production and controls the mTORC2 requirement for activation of AKT (a) Immunoblot analysis of PTEN expression in CTLs cultured ± 48 h rapamycin. (b) HPLC-MS based analysis of PtdIns(3,4,5)P 3 levels in control IL-2/IL-12 maintained CTLs (DM) and CTLs treated with PI(3)K p110δ inhibitor IC-87114 (IC, 1 h) and rapamycin for the indicated times. (c) Immunoblot analysis of Akt T308 and S473 phosphorylation levels in CTLs ± 48 h rapamycin. (d, e) The data show immunoblots of Akt T308 phosphorylation in control and mTORC1 inhibited CTLs treated with (d) IC-87114 or (e) AKTi1/2. (f) Immunoblot analysis of PTEN expression in CTLs cultured ± 48 h rapamycin or KU-0063794. (g) HPLC-MS based analysis of PtdIns(3,4,5)P 3 levels in control CTLs and CTLs treated with KU-0063794 for indicated durations. (h) Immunoblot analysis of Akt T308, Akt S473, FOXO1/3A T24/T32 phosphorylation and phosphorylation of the mTORC1 substrates S6K T389 and 4EBP1 S65 in CTLs treated with KU-0063794 for the indicated times. (i) Scatter plot depicting correlation of mean (n=3) fold changes in transcript expression from Affymetrix microarray analysis of control CTLs vs CTLs treated with rapamycin (x-axis) or KU-0063794 (y-axis). Immunoblots are representative of at least three biological replicates. (b, g) Individual data points and means are shown. P -values are determined by one-way ANOVA (Holm-Sidak) vs. DMSO (DM) as control. * P

    Journal: Nature immunology

    Article Title: The cytotoxic T cell proteome and its shaping by mammalian Target of Rapamycin

    doi: 10.1038/ni.3314

    Figure Lengend Snippet: mTORC1 represses PIP 3 production and controls the mTORC2 requirement for activation of AKT (a) Immunoblot analysis of PTEN expression in CTLs cultured ± 48 h rapamycin. (b) HPLC-MS based analysis of PtdIns(3,4,5)P 3 levels in control IL-2/IL-12 maintained CTLs (DM) and CTLs treated with PI(3)K p110δ inhibitor IC-87114 (IC, 1 h) and rapamycin for the indicated times. (c) Immunoblot analysis of Akt T308 and S473 phosphorylation levels in CTLs ± 48 h rapamycin. (d, e) The data show immunoblots of Akt T308 phosphorylation in control and mTORC1 inhibited CTLs treated with (d) IC-87114 or (e) AKTi1/2. (f) Immunoblot analysis of PTEN expression in CTLs cultured ± 48 h rapamycin or KU-0063794. (g) HPLC-MS based analysis of PtdIns(3,4,5)P 3 levels in control CTLs and CTLs treated with KU-0063794 for indicated durations. (h) Immunoblot analysis of Akt T308, Akt S473, FOXO1/3A T24/T32 phosphorylation and phosphorylation of the mTORC1 substrates S6K T389 and 4EBP1 S65 in CTLs treated with KU-0063794 for the indicated times. (i) Scatter plot depicting correlation of mean (n=3) fold changes in transcript expression from Affymetrix microarray analysis of control CTLs vs CTLs treated with rapamycin (x-axis) or KU-0063794 (y-axis). Immunoblots are representative of at least three biological replicates. (b, g) Individual data points and means are shown. P -values are determined by one-way ANOVA (Holm-Sidak) vs. DMSO (DM) as control. * P

    Article Snippet: In brief, 50 × 106 CTLs grown in ‘light’ SILAC media and treated with either rapamycin or KU-0063794 were mixed with 50 × 106 CTLs grown in ‘heavy’ SILAC treated with DMSO and washed twice with ice cold HBSS.

    Techniques: Activation Assay, Expressing, Cell Culture, High Performance Liquid Chromatography, Mass Spectrometry, Western Blot, Microarray

    The mTORC1 regulated CTL proteome (a) Immunoblot analysis of mTORC1/2 substrates in P14 TCR transgenic CTLs cultured with IL-2/IL-12 ± 48 h treatment with either rapamycin or KU-0063794. (b) Protein synthesis was examined by monitoring 3 H-Met incorporation into nascent proteins in CTLs cultured in IL-2/IL-12 and treated with rapamycin for the indicated time. (c) Cellular protein content of CTLs ± 48 h rapamycin. (d, e,) Volcano plots showing fold changes in proteins vs. log-transformed P -values from mass spectrometry analysis of CTLs ± 48 h rapamycin. (d) Total proteins. Known rapamycin sensitive proteins perforin and L-selectin are highlighted. (e) CTL effector molecules. (f) IFN-γ secretion by CTLs ± 48 h rapamycin measured by ELISA. (h) Immunoblot analysis of T-bet in CTLs ± 48 h rapamycin. (h, i) Validation of up-regulated proteins: (h) ELISA of shed CD62L in cell supernatants prepared from CTLs ± 48 h rapamycin. (i) Immunoblot analysis of IRS2 in CTLs ± 48 h rapamycin. (a, g, i): representive immunoblots of at least three biological replicates. (b, c, f, h): individual data points and means are shown. P -values shown determined by (b): one-way ANOVA (Holm-Sidak) vs. DMSO as control on non-normalized data; (c, f, h): two-tailed Student’s t-test. Data based on three (b, f, h) or four (c) biological replicates. * P

    Journal: Nature immunology

    Article Title: The cytotoxic T cell proteome and its shaping by mammalian Target of Rapamycin

    doi: 10.1038/ni.3314

    Figure Lengend Snippet: The mTORC1 regulated CTL proteome (a) Immunoblot analysis of mTORC1/2 substrates in P14 TCR transgenic CTLs cultured with IL-2/IL-12 ± 48 h treatment with either rapamycin or KU-0063794. (b) Protein synthesis was examined by monitoring 3 H-Met incorporation into nascent proteins in CTLs cultured in IL-2/IL-12 and treated with rapamycin for the indicated time. (c) Cellular protein content of CTLs ± 48 h rapamycin. (d, e,) Volcano plots showing fold changes in proteins vs. log-transformed P -values from mass spectrometry analysis of CTLs ± 48 h rapamycin. (d) Total proteins. Known rapamycin sensitive proteins perforin and L-selectin are highlighted. (e) CTL effector molecules. (f) IFN-γ secretion by CTLs ± 48 h rapamycin measured by ELISA. (h) Immunoblot analysis of T-bet in CTLs ± 48 h rapamycin. (h, i) Validation of up-regulated proteins: (h) ELISA of shed CD62L in cell supernatants prepared from CTLs ± 48 h rapamycin. (i) Immunoblot analysis of IRS2 in CTLs ± 48 h rapamycin. (a, g, i): representive immunoblots of at least three biological replicates. (b, c, f, h): individual data points and means are shown. P -values shown determined by (b): one-way ANOVA (Holm-Sidak) vs. DMSO as control on non-normalized data; (c, f, h): two-tailed Student’s t-test. Data based on three (b, f, h) or four (c) biological replicates. * P

    Article Snippet: In brief, 50 × 106 CTLs grown in ‘light’ SILAC media and treated with either rapamycin or KU-0063794 were mixed with 50 × 106 CTLs grown in ‘heavy’ SILAC treated with DMSO and washed twice with ice cold HBSS.

    Techniques: CTL Assay, Transgenic Assay, Cell Culture, Transformation Assay, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Western Blot, Two Tailed Test

    Antiproliferative effects of ku-0063794 on EGFR TKI-sensitive and -resistant NSCLC cells. (A) Cells were treated for 72 hr with ku-0063794 at the IC 50 concentrations, and cell lysates were immunoblotted to detect the indicated proteins. The experiment, repeated three times, yielded similar results. (B) The ratios of mTOR signaling pathway associated phosphorylated proteins over total proteins in the basal state and after treatment with ku-0063794 at the IC 50 concentrations in all four cell lines.

    Journal: PLoS ONE

    Article Title: Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

    doi: 10.1371/journal.pone.0069104

    Figure Lengend Snippet: Antiproliferative effects of ku-0063794 on EGFR TKI-sensitive and -resistant NSCLC cells. (A) Cells were treated for 72 hr with ku-0063794 at the IC 50 concentrations, and cell lysates were immunoblotted to detect the indicated proteins. The experiment, repeated three times, yielded similar results. (B) The ratios of mTOR signaling pathway associated phosphorylated proteins over total proteins in the basal state and after treatment with ku-0063794 at the IC 50 concentrations in all four cell lines.

    Article Snippet: Antibodies and Regents Ku-0063794 (TOCRIS, Minneapolis, MN, USA) and pure gefitinib, kindly provided by AstraZeneca, were prepared in DMSO and diluted with culture medium before use. mTOR (#2983), p-mTOR (#2971), Rictor (#2114 and #5379), Raptor (#2280 and #5382), Akt (#9272), p-Akt ser473 (#4060), p-p70S6K (#9208), p-4EBP1 (#9459), FOXO1 (#2880) and p-FOXO1 (#9461) antibodies for western blot and immunoprecipitation were all purchased from CST (Beverly, MA, USA). p70S6K (#ab47504) and p-p70S6K (#ab60947) antibodies for western blot and immunohistochemistry were purchased from Abcam (Cambridge, MA, USA).

    Techniques:

    Ku-0063794 inhibited cell proliferation and induced G1 cell cycle arrest in EGFR mutant NSCLC cells. (A) Inhibition efficiency of ku-0063794 in the four NSCLC cell lines. (B) Ku-0063794 induced cell cycle arrest in both EGFR TKI-sensitive and -resistant cells. The upper panel represents basal cell cycle distributions of the PC9, PC9GR, H1650, and H1975 cell lines, respectively, and the lower panel represents the cell cycle distributions of PC9, PC9GR, H1650, and H1975 cells after treatment with the IC 50 concentrations of ku-0063794 for 72 hr.

    Journal: PLoS ONE

    Article Title: Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

    doi: 10.1371/journal.pone.0069104

    Figure Lengend Snippet: Ku-0063794 inhibited cell proliferation and induced G1 cell cycle arrest in EGFR mutant NSCLC cells. (A) Inhibition efficiency of ku-0063794 in the four NSCLC cell lines. (B) Ku-0063794 induced cell cycle arrest in both EGFR TKI-sensitive and -resistant cells. The upper panel represents basal cell cycle distributions of the PC9, PC9GR, H1650, and H1975 cell lines, respectively, and the lower panel represents the cell cycle distributions of PC9, PC9GR, H1650, and H1975 cells after treatment with the IC 50 concentrations of ku-0063794 for 72 hr.

    Article Snippet: Antibodies and Regents Ku-0063794 (TOCRIS, Minneapolis, MN, USA) and pure gefitinib, kindly provided by AstraZeneca, were prepared in DMSO and diluted with culture medium before use. mTOR (#2983), p-mTOR (#2971), Rictor (#2114 and #5379), Raptor (#2280 and #5382), Akt (#9272), p-Akt ser473 (#4060), p-p70S6K (#9208), p-4EBP1 (#9459), FOXO1 (#2880) and p-FOXO1 (#9461) antibodies for western blot and immunoprecipitation were all purchased from CST (Beverly, MA, USA). p70S6K (#ab47504) and p-p70S6K (#ab60947) antibodies for western blot and immunohistochemistry were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Mutagenesis, Inhibition

    Q6 induced G2/M arrest and apoptosis is ATM/Chk2 dependent in hypoxia. A. HepG2 and Bel-7402 cells, treated with Q6 (5 μM) in the presence or absence of caffeine (2 mM) for 24 h under hypoxia (1% O 2 ), were collected and prepared for cytometric analysis of cell cycle distribution. B C. HepG2 cells treated with Q6 (10 μM) in the presence or absence of caffeine (2 mM) for 24 h under hypoxia (1% O 2 ). Detection of apoptosis by flow cytometry (B) and caspase cascade by Western blot (C) were then performed. D E. HepG2 cells treated with Q6 in the presence or absence of ATM specific inhibitor KU-60019 (3 μM) under hypoxia (1% O 2 ), and subjected to sub-G1 analyses (D) and Western blot analyses (E), respectively. F. Western blot was used to assess the role of ATM during apoptosis induced by Q6 in hypoxia. HepG2 cells were treated with ATM RNAi or vector RNAi in the presence or absence of Q6 (5 μM) under hypoxia (1% O 2 ) condition.

    Journal: PLoS ONE

    Article Title: Hypoxia-Targeted Drug Q6 Induces G2-M Arrest and Apoptosis via Poisoning Topoisomerase II under Hypoxia

    doi: 10.1371/journal.pone.0144506

    Figure Lengend Snippet: Q6 induced G2/M arrest and apoptosis is ATM/Chk2 dependent in hypoxia. A. HepG2 and Bel-7402 cells, treated with Q6 (5 μM) in the presence or absence of caffeine (2 mM) for 24 h under hypoxia (1% O 2 ), were collected and prepared for cytometric analysis of cell cycle distribution. B C. HepG2 cells treated with Q6 (10 μM) in the presence or absence of caffeine (2 mM) for 24 h under hypoxia (1% O 2 ). Detection of apoptosis by flow cytometry (B) and caspase cascade by Western blot (C) were then performed. D E. HepG2 cells treated with Q6 in the presence or absence of ATM specific inhibitor KU-60019 (3 μM) under hypoxia (1% O 2 ), and subjected to sub-G1 analyses (D) and Western blot analyses (E), respectively. F. Western blot was used to assess the role of ATM during apoptosis induced by Q6 in hypoxia. HepG2 cells were treated with ATM RNAi or vector RNAi in the presence or absence of Q6 (5 μM) under hypoxia (1% O 2 ) condition.

    Article Snippet: TPZ (tirapazamine) was purchased from Topharman Shanghai Co. Ltd.. Etoposide (VP16), KU-60019 and caffeine were all purchased from Sigma (St. Louis, MO).

    Techniques: Flow Cytometry, Cytometry, Western Blot, Plasmid Preparation

    Role of CK2 kinase in XPC phosphorylation and CPD repair. ( A ) Immunoblot analysis of XPC phosphorylated at S94 (p-XPC S94), total XPC and GAPDH 30 min after UVB exposure (20 mJ/cm 2 ) in XPC Null cells expressing pLenti-XPC WT or mutant S94A. ( B ) HaCaT cells were treated with vehicle, rapamycin (25 nM), CX-4945 (5 μM), KU-60019 (1 μM), LY294002 (10 μM), NU7441 (1 μM), TCS JNK 60 (10 μM), SB203580 (10 μM), PD98059 (20 μM) or Y27632 (10 μM) for 1 h. The levels of XPC phosphorylated at S94 (p-XPC S94), total XPC and GAPDH were analyzed by immunoblot assay. ( C ) Immunoblot analysis of XPC phosphorylated at S94 (p-XPC S94), total XPC, CK2A1, CK2A2 and GAPDH 30 min after UVB exposure (20 mJ/cm 2 ) in HaCaT cells transfected with siRNA targeting CK2A1 and CK2A2 (siCK2) or non-targeting control siRNA (siNC). ( D ) Slot blot analysis of the levels of CPD at the indicated times post-UVB (20 mJ/cm 2 ) in HaCaT cells transfected with siRNA targeting CK2A1 and CK2A2 (siCK2) or non-targeting control siRNA (siNC). Methylene blue staining was used for loading control. ( E ) Quantification of percentage (%) of CPD repair from (D). ** P

    Journal: Nucleic Acids Research

    Article Title: Phosphorylation of xeroderma pigmentosum group C regulates ultraviolet-induced DNA damage repair

    doi: 10.1093/nar/gky239

    Figure Lengend Snippet: Role of CK2 kinase in XPC phosphorylation and CPD repair. ( A ) Immunoblot analysis of XPC phosphorylated at S94 (p-XPC S94), total XPC and GAPDH 30 min after UVB exposure (20 mJ/cm 2 ) in XPC Null cells expressing pLenti-XPC WT or mutant S94A. ( B ) HaCaT cells were treated with vehicle, rapamycin (25 nM), CX-4945 (5 μM), KU-60019 (1 μM), LY294002 (10 μM), NU7441 (1 μM), TCS JNK 60 (10 μM), SB203580 (10 μM), PD98059 (20 μM) or Y27632 (10 μM) for 1 h. The levels of XPC phosphorylated at S94 (p-XPC S94), total XPC and GAPDH were analyzed by immunoblot assay. ( C ) Immunoblot analysis of XPC phosphorylated at S94 (p-XPC S94), total XPC, CK2A1, CK2A2 and GAPDH 30 min after UVB exposure (20 mJ/cm 2 ) in HaCaT cells transfected with siRNA targeting CK2A1 and CK2A2 (siCK2) or non-targeting control siRNA (siNC). ( D ) Slot blot analysis of the levels of CPD at the indicated times post-UVB (20 mJ/cm 2 ) in HaCaT cells transfected with siRNA targeting CK2A1 and CK2A2 (siCK2) or non-targeting control siRNA (siNC). Methylene blue staining was used for loading control. ( E ) Quantification of percentage (%) of CPD repair from (D). ** P

    Article Snippet: Where indicated, cells were treated with the inhibitors rapamycin (25 nM, LC Laboratories, R-5000), CX-4945 (5 μM, Selleck Chemicals, S2248), KU-60019 (1 μM, Selleck Chemicals, S1570), LY294002 (10 μM, Promega, V1201), NU7441 (1 μM, Selleck Chemicals, S2638), TCS JNK 60 (10 μM, Torcis, 3222), SB203580 (10 μM, Promega, V1161), PD98059 (20 μM, Promega, V1191), Y27632 (10 μM, Torcis, 1254) or with the vehicle (DMSO, Sigma-Aldrich).

    Techniques: Expressing, Mutagenesis, Transfection, Dot Blot, Staining

    Inhibition of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling induction in TR146 cells infected with Candida albicans. A , Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on cytokine production after 24 hours (multiplicity of infection [MOI] = 0.01) shown as percentage of the dimethyl sulfoxide (DMSO) vehicle control. B , Effect of inhibition of PI3K/Akt (wortmannin [1 µM] and LY294002 [50 µM]) or p38 (SB203580 [10 µM]) signaling on cell damage (lactate dehydrogenase [LDH] release) after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). C , Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cell damage (LDH release) after 24 hours (MOI] = 0.01) shown as percentage of DMSO vehicle control. D , Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cytokine production after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). E , Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of IκBα 2 hours postinfection (MOI = 10). F , Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of MKP1 and c-Jun and on the production of c-Fos 2 hours postinfection (MOI = 10). Candida albicans was added as 100% yeast, which switched to hyphal growth by 2 hours postinfection. Data are the mean ( A – D ) or representative ( E and F ) of at least 3 independent experiments. * P

    Journal: The Journal of Infectious Diseases

    Article Title: Protection Against Epithelial Damage During Candida albicans Infection Is Mediated by PI3K/Akt and Mammalian Target of Rapamycin Signaling

    doi: 10.1093/infdis/jit824

    Figure Lengend Snippet: Inhibition of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling induction in TR146 cells infected with Candida albicans. A , Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on cytokine production after 24 hours (multiplicity of infection [MOI] = 0.01) shown as percentage of the dimethyl sulfoxide (DMSO) vehicle control. B , Effect of inhibition of PI3K/Akt (wortmannin [1 µM] and LY294002 [50 µM]) or p38 (SB203580 [10 µM]) signaling on cell damage (lactate dehydrogenase [LDH] release) after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). C , Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cell damage (LDH release) after 24 hours (MOI] = 0.01) shown as percentage of DMSO vehicle control. D , Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cytokine production after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). E , Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of IκBα 2 hours postinfection (MOI = 10). F , Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of MKP1 and c-Jun and on the production of c-Fos 2 hours postinfection (MOI = 10). Candida albicans was added as 100% yeast, which switched to hyphal growth by 2 hours postinfection. Data are the mean ( A – D ) or representative ( E and F ) of at least 3 independent experiments. * P

    Article Snippet: Wortmannin, LY294002, Ku-63794, and SB203580 were from Calbiochem.

    Techniques: Inhibition, Infection, Activity Assay

    ATM-depleted fibroblasts progressively accumulate ROS and oxidative protein damage. ( A ) Representative Western blot analysis on ATM-depleted fibroblasts showing upregulation of proteins involved in the antioxidant response. TIG1 cells were transfected with either a control siRNA (siCtrl) or an ATM-targeting siRNA (siATM#1, siATM #2). Actin was used as loading control. Densitometric quantification of the indicated proteins is reported at the bottom of the gel ( N = 2). ( B ) Western blot analysis on a representative time course depletion of ATM. TIG1 fibroblasts were treated as indicated and ATM expression was monitored in nuclear cell extracts. Lamin A/C was used as loading control. ( C ) Quantification of ROS content using flow-cytometry in siATM-treated fibroblasts. TIG1 fibroblasts were treated as indicated. H 2 O 2 (25 μM, 30 min) was used as a positive control for induction of ROS. ATM inhibitors (Ku-55933 – ATMi #1 and Ku-60019 – ATMi #2, both 10 μM) were provided fresh every 24 h for a total of 72 h ( N = 3). ( D ) Quantification of mitochondrial membrane potential (ΔΨm) in siATM-treated fibroblasts using flow-cytometry. Cells were treated as in panel C. Staurosporine (2 μM, 2 h) was used as a positive control for mitochondrial membrane depolarisation ( N = 3). ( E ) Detection of protein carbonylation in nuclear extracts obtained from fibroblasts depleted of ATM for the indicated time. Carbonylated proteins were detected by derivatisation with dinitrophenylhydrazone (DNP) followed by Western blot using an anti-DNP antibody. Equal amounts of cell extract were loaded in each lane. H 2 O 2 (1 mM, 30 minutes) was used as a positive control for induction of protein carbonylation. ( F ) Quantification of protein carbonylation in ATM-depleted fibroblasts; the histogram displays densitometric data from the analysis reported in panel E ( N = 6). Results are expressed as mean ± SD from the indicated number ( N ) of independent experiments: * P

    Journal: Nucleic Acids Research

    Article Title: Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells

    doi: 10.1093/nar/gkx635

    Figure Lengend Snippet: ATM-depleted fibroblasts progressively accumulate ROS and oxidative protein damage. ( A ) Representative Western blot analysis on ATM-depleted fibroblasts showing upregulation of proteins involved in the antioxidant response. TIG1 cells were transfected with either a control siRNA (siCtrl) or an ATM-targeting siRNA (siATM#1, siATM #2). Actin was used as loading control. Densitometric quantification of the indicated proteins is reported at the bottom of the gel ( N = 2). ( B ) Western blot analysis on a representative time course depletion of ATM. TIG1 fibroblasts were treated as indicated and ATM expression was monitored in nuclear cell extracts. Lamin A/C was used as loading control. ( C ) Quantification of ROS content using flow-cytometry in siATM-treated fibroblasts. TIG1 fibroblasts were treated as indicated. H 2 O 2 (25 μM, 30 min) was used as a positive control for induction of ROS. ATM inhibitors (Ku-55933 – ATMi #1 and Ku-60019 – ATMi #2, both 10 μM) were provided fresh every 24 h for a total of 72 h ( N = 3). ( D ) Quantification of mitochondrial membrane potential (ΔΨm) in siATM-treated fibroblasts using flow-cytometry. Cells were treated as in panel C. Staurosporine (2 μM, 2 h) was used as a positive control for mitochondrial membrane depolarisation ( N = 3). ( E ) Detection of protein carbonylation in nuclear extracts obtained from fibroblasts depleted of ATM for the indicated time. Carbonylated proteins were detected by derivatisation with dinitrophenylhydrazone (DNP) followed by Western blot using an anti-DNP antibody. Equal amounts of cell extract were loaded in each lane. H 2 O 2 (1 mM, 30 minutes) was used as a positive control for induction of protein carbonylation. ( F ) Quantification of protein carbonylation in ATM-depleted fibroblasts; the histogram displays densitometric data from the analysis reported in panel E ( N = 6). Results are expressed as mean ± SD from the indicated number ( N ) of independent experiments: * P

    Article Snippet: The ATM kinase inhibitors Ku-55933 and Ku-60019 were from Abcam and R & D Systems, respectively. siRNA transfections were carried out as previously described ( ).

    Techniques: Western Blot, Transfection, Expressing, Flow Cytometry, Cytometry, Positive Control

    Lack of functional DNA-PKcs reduces chemotherapy-induced mutations ( a ) DNA-PKcs protein expression in DNA-PKcs expressing (control) and DNA-PKcs knockout cells was assessed by immunoblot. Probing for GAPDH was used as a loading control. ( b ) Cells were treated with indicated doses of drugs or left untreated (UT) for 24 hours, harvested and clonogenicity assays were performed to determine the proportion of cells maintaining clonogenic competency. ( c ) Surviving cells were grown in 6-TG to select for the emergence of any HPRT mutants. ( d ) TK6 cells were pre-treated with 10 μM KU-57788 (DNA-PKi) or DMSO for 1 hour, then cultured in media containing 10 nM doxorubicin (Dox), 1 μM cisplatin (Cis), 0.1 nM vincristine (Vin), 10 μM GDC-0152 (GDC) or untreated for 24 hours. Cells were harvested and clonogenicity or ( e ) HPRT assays were performed on surviving cells. Error bars represent mean ± SEM from at least three independent experiments. Two-way ANOVA analyses with Bonferroni post-tests were used to estimate the probability that random chance accounted for the differences observed in drug responses between control cells and either each DNA-PKcs null line or both KO lines (denoted by the square brackets) ( b , c ), or DMSO-treated versus inhibitor-treated cells ( d , e ). P values

    Journal: Scientific Reports

    Article Title: Mutagenic assessment of chemotherapy and Smac mimetic drugs in cells with defective DNA damage response pathways

    doi: 10.1038/s41598-018-32517-9

    Figure Lengend Snippet: Lack of functional DNA-PKcs reduces chemotherapy-induced mutations ( a ) DNA-PKcs protein expression in DNA-PKcs expressing (control) and DNA-PKcs knockout cells was assessed by immunoblot. Probing for GAPDH was used as a loading control. ( b ) Cells were treated with indicated doses of drugs or left untreated (UT) for 24 hours, harvested and clonogenicity assays were performed to determine the proportion of cells maintaining clonogenic competency. ( c ) Surviving cells were grown in 6-TG to select for the emergence of any HPRT mutants. ( d ) TK6 cells were pre-treated with 10 μM KU-57788 (DNA-PKi) or DMSO for 1 hour, then cultured in media containing 10 nM doxorubicin (Dox), 1 μM cisplatin (Cis), 0.1 nM vincristine (Vin), 10 μM GDC-0152 (GDC) or untreated for 24 hours. Cells were harvested and clonogenicity or ( e ) HPRT assays were performed on surviving cells. Error bars represent mean ± SEM from at least three independent experiments. Two-way ANOVA analyses with Bonferroni post-tests were used to estimate the probability that random chance accounted for the differences observed in drug responses between control cells and either each DNA-PKcs null line or both KO lines (denoted by the square brackets) ( b , c ), or DMSO-treated versus inhibitor-treated cells ( d , e ). P values

    Article Snippet: Drugs used were doxorubicin (Sigma; St. Louis, MO, USA), cisplatin (Sigma), vincristine (Selleck Chemicals; Houston, TX, USA), GDC-0152 (Selleck Chemicals), KU-60019 (Selleck Chemicals), KU-57788 (Selleck Chemicals), RI-1 (Selleck Chemicals), B02 (Selleck Chemicals) and 6-thioguanine (6-TG) (Sigma).

    Techniques: Functional Assay, Expressing, Knock-Out, Cell Culture

    HDR stimulation by NU7441 and KU-0060648 is comparable to other HDR enhancing tools. a Western blot showing knock down efficiency obtained with the siRNAs targeting either Ku70 or Ku80. b Quantitation of genome editing events from cells transfected with pQCiG-TLR and ΔeGFP donor in the presence of 2 μM NU7441, 250 nM KU-0060648, siRNAs targeting Ku70, Ku80, DNA-PKcs or DNA ligase IV, 1 μM Scr7, or a combination of Scr7 and 2 μM NU7441 or 250 nM KU-0060648. The HDR and NHEJ values are relative to those obtained with Cas9, sgRNA , and ΔeGFP donor in the presence of vehicle (DMSO). Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant. c Western blot showing expression of Adenovirus 5 proteins E1B55K and E4orf6 following transfection into the 293/TLR line. d Quantitation of genome editing efficiencies was as in B except that adenovirus 5 proteins E1B55K and E4orf6 expression vectors were co-transfected with pQCiG-TLR and ΔeGFP donor plasmids and cultured in presence or absence of 2 μM NU7441 or 250 nM KU-0060648. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant

    Journal: Genome Medicine

    Article Title: Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing

    doi: 10.1186/s13073-015-0215-6

    Figure Lengend Snippet: HDR stimulation by NU7441 and KU-0060648 is comparable to other HDR enhancing tools. a Western blot showing knock down efficiency obtained with the siRNAs targeting either Ku70 or Ku80. b Quantitation of genome editing events from cells transfected with pQCiG-TLR and ΔeGFP donor in the presence of 2 μM NU7441, 250 nM KU-0060648, siRNAs targeting Ku70, Ku80, DNA-PKcs or DNA ligase IV, 1 μM Scr7, or a combination of Scr7 and 2 μM NU7441 or 250 nM KU-0060648. The HDR and NHEJ values are relative to those obtained with Cas9, sgRNA , and ΔeGFP donor in the presence of vehicle (DMSO). Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant. c Western blot showing expression of Adenovirus 5 proteins E1B55K and E4orf6 following transfection into the 293/TLR line. d Quantitation of genome editing efficiencies was as in B except that adenovirus 5 proteins E1B55K and E4orf6 expression vectors were co-transfected with pQCiG-TLR and ΔeGFP donor plasmids and cultured in presence or absence of 2 μM NU7441 or 250 nM KU-0060648. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant

    Article Snippet: NU7441 and KU-0060648 were purchased from Selleckchem (Houston, TX, USA).

    Techniques: Western Blot, Quantitation Assay, Transfection, Non-Homologous End Joining, Expressing, Cell Culture

    DNA-PK inhibitors stimulate homology-directed repair at the p53 locus. a Outline of experimental approach. b Position of target sites and partial sequence of oligonucleotides used as repair templates for p53 exons 5 and 7. A schematic representation of the murine p53 locus is shown with the locations of sgp53 -1 and sgp53 -3. The sequence of the donor templates for HDR ( sgp53 -1; 105 nucleotides, sgp53 -3; 106 nucleotides) are indicated with red nucleotides denoting missense mutations and a dash indicating a frameshift mutation leading to a premature stop codon (in bold). c Fold stimulation of HDR at the sgp53 -1 and sgp53 -3 sites by NU7441 (2 μM) or KU-0060648 (250 nM). Results are shown as the fraction of all retrieved mutated sequences that correspond to HDR events in the presence of the DNA-PK inhibitors relative to vehicle. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant

    Journal: Genome Medicine

    Article Title: Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing

    doi: 10.1186/s13073-015-0215-6

    Figure Lengend Snippet: DNA-PK inhibitors stimulate homology-directed repair at the p53 locus. a Outline of experimental approach. b Position of target sites and partial sequence of oligonucleotides used as repair templates for p53 exons 5 and 7. A schematic representation of the murine p53 locus is shown with the locations of sgp53 -1 and sgp53 -3. The sequence of the donor templates for HDR ( sgp53 -1; 105 nucleotides, sgp53 -3; 106 nucleotides) are indicated with red nucleotides denoting missense mutations and a dash indicating a frameshift mutation leading to a premature stop codon (in bold). c Fold stimulation of HDR at the sgp53 -1 and sgp53 -3 sites by NU7441 (2 μM) or KU-0060648 (250 nM). Results are shown as the fraction of all retrieved mutated sequences that correspond to HDR events in the presence of the DNA-PK inhibitors relative to vehicle. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant

    Article Snippet: NU7441 and KU-0060648 were purchased from Selleckchem (Houston, TX, USA).

    Techniques: Sequencing, Mutagenesis

    HDR directed by oligonucleotide donors are stimulated by DNA-PKcs inhibitors. a Quantitation of genome editing events from cells transfected with pQCiG-TLR and either ΔeGFP donor (2.5 kbp homology upstream and 1.5 kbp homology downstream of target site) or oligonucleotides (sense or antisense) (110 nucleotides) spanning the sgGFP target site and exposed to vehicle, 2 μM NU7441, or 250 nM KU-0060648. The HDR and NHEJ values are relative to those obtained with Cas9, sgRNA , and ΔeGFP donor in the presence of vehicle (DMSO). N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant. b Representative examples of FACS plot obtained in ( a ). Note that mCherry + cells are reporting on only one-third of all NHEJ events. c Quantitation of genome editing events from experiments performed as described in (a) except using WT Cas9 or the D10A and H840A nickase variants and the ΔeGFP donor. Values are set relative to editing frequencies observed in 293/TLR cells transfected with WT Cas9 expression vector and propagated in the presence of vehicle. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant

    Journal: Genome Medicine

    Article Title: Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing

    doi: 10.1186/s13073-015-0215-6

    Figure Lengend Snippet: HDR directed by oligonucleotide donors are stimulated by DNA-PKcs inhibitors. a Quantitation of genome editing events from cells transfected with pQCiG-TLR and either ΔeGFP donor (2.5 kbp homology upstream and 1.5 kbp homology downstream of target site) or oligonucleotides (sense or antisense) (110 nucleotides) spanning the sgGFP target site and exposed to vehicle, 2 μM NU7441, or 250 nM KU-0060648. The HDR and NHEJ values are relative to those obtained with Cas9, sgRNA , and ΔeGFP donor in the presence of vehicle (DMSO). N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant. b Representative examples of FACS plot obtained in ( a ). Note that mCherry + cells are reporting on only one-third of all NHEJ events. c Quantitation of genome editing events from experiments performed as described in (a) except using WT Cas9 or the D10A and H840A nickase variants and the ΔeGFP donor. Values are set relative to editing frequencies observed in 293/TLR cells transfected with WT Cas9 expression vector and propagated in the presence of vehicle. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant

    Article Snippet: NU7441 and KU-0060648 were purchased from Selleckchem (Houston, TX, USA).

    Techniques: Quantitation Assay, Transfection, Non-Homologous End Joining, FACS, Expressing, Plasmid Preparation

    DNA-PKcs inhibitors stimulate HDR following Cas9 induction of a DSB. a Schematic outline of the Traffic Light Reporter assay. The eGFP and mCherry open reading frames (ORF) are fused by a ribosome skipping sequence (T2A) where translating ribosomes skip from a Gly to a Pro codon without forming a peptide bond while maintaining the reading frame to produce two distinct polypeptides [ 39 , 40 ]. In the TLR, the eGFP and mCherry ORFs are positioned in different frames and NHEJ repair of a DSB directed to the GFP ORF will, in one out of three events, place ΔeGFP in frame with mCherry - leading to mCherry production. Since the eGFP ORF contains an insertion harboring a premature termination codon (and lacks a start codon), its expression can only be recovered upon HDR-mediated repair following delivery of an appropriate homologous template. The reading frame for each fluorescent protein is indicated in parentheses. b Outline of siRNA and drug treatment protocol. In one set of experiments, the 293/TLR cell line was transfected with the indicated siRNAs ( yellow box ) and 24 h later expression constructs driving synthesis of Cas9 and sgRNAs and an eGFP donor template were introduced into the cells. In a second set of experiments, the 293/TLR cell line was first transfected with expression constructs driving synthesis of Cas9 and sgRNAs and an eGFP donor template. Compounds were added 16 h later ( orange box ). In both cases, cells were allowed to propagate for 5 days before FACS analysis. c Knockdown of DNA-PK, DNA Ligase IV, and PI3K-p110α in 293/TLR cells transfected with the indicated siRNAs. Western blots were probed with antibodies to the indicated proteins. The dashed line separates two different sets of membranes. d Quantitation of genome editing events in 293/TLR cells transfected with the indicated siRNAs. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to scrambled siRNAs) was calculated using the Student’s t -test: * P ≤0.05; ** P ≤0.01; ns, not significant. e DNA-PK inhibitors prevent H2AX phosphorylation upon γ-irradiation. Cells were pre-incubated with the indicated small molecules for 1 h followed by 4 GY of γ-irradiation (IR). Extracts for western blotting were prepared from non-irradiated (IR) cells (lane 1) or IR-exposed cells incubated in the presence of vehicle (lane 2), 2 μM NU77441 (lane 3), and 250 nM KU-0060648 (lane 4). Western blots were probed with antibodies directed to the proteins indicated to the left of the blot. The ratio of p-H2AX and eEF2 band intensities is indicated below the blots. f Flow cytometry analysis of 293/TLR cells transfected with pQCX vectors driving synthesis of Cas9 and sgRNAs targeting Rosa26 or eGFP . When indicated, the GFP repair template, pRRL SFFV d20GFP.T2A.mTagBFP (ΔeGFP Donor) was also included. NU7441 and KU-0060648 were used at a final concentration of 2 μM and 250 nM, respectively. Cells were gated for the live population as determined by PI staining. Transfection efficiencies were greater than 70 %. g Quantitation of genome editing events from cells propagated in the presence of the indicated concentrations of DNA-PKcs inhibitors. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant

    Journal: Genome Medicine

    Article Title: Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing

    doi: 10.1186/s13073-015-0215-6

    Figure Lengend Snippet: DNA-PKcs inhibitors stimulate HDR following Cas9 induction of a DSB. a Schematic outline of the Traffic Light Reporter assay. The eGFP and mCherry open reading frames (ORF) are fused by a ribosome skipping sequence (T2A) where translating ribosomes skip from a Gly to a Pro codon without forming a peptide bond while maintaining the reading frame to produce two distinct polypeptides [ 39 , 40 ]. In the TLR, the eGFP and mCherry ORFs are positioned in different frames and NHEJ repair of a DSB directed to the GFP ORF will, in one out of three events, place ΔeGFP in frame with mCherry - leading to mCherry production. Since the eGFP ORF contains an insertion harboring a premature termination codon (and lacks a start codon), its expression can only be recovered upon HDR-mediated repair following delivery of an appropriate homologous template. The reading frame for each fluorescent protein is indicated in parentheses. b Outline of siRNA and drug treatment protocol. In one set of experiments, the 293/TLR cell line was transfected with the indicated siRNAs ( yellow box ) and 24 h later expression constructs driving synthesis of Cas9 and sgRNAs and an eGFP donor template were introduced into the cells. In a second set of experiments, the 293/TLR cell line was first transfected with expression constructs driving synthesis of Cas9 and sgRNAs and an eGFP donor template. Compounds were added 16 h later ( orange box ). In both cases, cells were allowed to propagate for 5 days before FACS analysis. c Knockdown of DNA-PK, DNA Ligase IV, and PI3K-p110α in 293/TLR cells transfected with the indicated siRNAs. Western blots were probed with antibodies to the indicated proteins. The dashed line separates two different sets of membranes. d Quantitation of genome editing events in 293/TLR cells transfected with the indicated siRNAs. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to scrambled siRNAs) was calculated using the Student’s t -test: * P ≤0.05; ** P ≤0.01; ns, not significant. e DNA-PK inhibitors prevent H2AX phosphorylation upon γ-irradiation. Cells were pre-incubated with the indicated small molecules for 1 h followed by 4 GY of γ-irradiation (IR). Extracts for western blotting were prepared from non-irradiated (IR) cells (lane 1) or IR-exposed cells incubated in the presence of vehicle (lane 2), 2 μM NU77441 (lane 3), and 250 nM KU-0060648 (lane 4). Western blots were probed with antibodies directed to the proteins indicated to the left of the blot. The ratio of p-H2AX and eEF2 band intensities is indicated below the blots. f Flow cytometry analysis of 293/TLR cells transfected with pQCX vectors driving synthesis of Cas9 and sgRNAs targeting Rosa26 or eGFP . When indicated, the GFP repair template, pRRL SFFV d20GFP.T2A.mTagBFP (ΔeGFP Donor) was also included. NU7441 and KU-0060648 were used at a final concentration of 2 μM and 250 nM, respectively. Cells were gated for the live population as determined by PI staining. Transfection efficiencies were greater than 70 %. g Quantitation of genome editing events from cells propagated in the presence of the indicated concentrations of DNA-PKcs inhibitors. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t -test: * p ≤0.05; ** p ≤0.01; ns, not significant

    Article Snippet: NU7441 and KU-0060648 were purchased from Selleckchem (Houston, TX, USA).

    Techniques: Reporter Assay, Sequencing, Non-Homologous End Joining, Expressing, Transfection, Construct, FACS, Western Blot, Quantitation Assay, Irradiation, Incubation, Flow Cytometry, Cytometry, Concentration Assay, Staining

    Comparison of PK levels of KU-0060648 and DNA-PK activity within ex vivo SW620 tumour samples following i.v dosing

    Journal: Molecular cancer therapeutics

    Article Title: Chemosensitisation of cancer cells by KU-0060648; a dual inhibitor of DNA-PK and PI-3K

    doi: 10.1158/1535-7163.MCT-11-0535

    Figure Lengend Snippet: Comparison of PK levels of KU-0060648 and DNA-PK activity within ex vivo SW620 tumour samples following i.v dosing

    Article Snippet: To determine PI-3K activity, cells were exposed to a range of concentrations of KU-0060648 for 1 hr prior to a 30-minute treatment with 50 ng/ml insulin-like growth factor-1 (Calbiochem, Merck Biosciences, Nottingham, UK).

    Techniques: Activity Assay, Ex Vivo

    Chemopotentiation by KU-0060648 in vitro

    Journal: Molecular cancer therapeutics

    Article Title: Chemosensitisation of cancer cells by KU-0060648; a dual inhibitor of DNA-PK and PI-3K

    doi: 10.1158/1535-7163.MCT-11-0535

    Figure Lengend Snippet: Chemopotentiation by KU-0060648 in vitro

    Article Snippet: To determine PI-3K activity, cells were exposed to a range of concentrations of KU-0060648 for 1 hr prior to a 30-minute treatment with 50 ng/ml insulin-like growth factor-1 (Calbiochem, Merck Biosciences, Nottingham, UK).

    Techniques: In Vitro

    Growth inhibition, cytotoxicity and chemosensitisation of KU-0060648 in human colon and breast cancer cell lines

    Journal: Molecular cancer therapeutics

    Article Title: Chemosensitisation of cancer cells by KU-0060648; a dual inhibitor of DNA-PK and PI-3K

    doi: 10.1158/1535-7163.MCT-11-0535

    Figure Lengend Snippet: Growth inhibition, cytotoxicity and chemosensitisation of KU-0060648 in human colon and breast cancer cell lines

    Article Snippet: To determine PI-3K activity, cells were exposed to a range of concentrations of KU-0060648 for 1 hr prior to a 30-minute treatment with 50 ng/ml insulin-like growth factor-1 (Calbiochem, Merck Biosciences, Nottingham, UK).

    Techniques: Inhibition

    Efficacy of etoposide and KU-0060648 in MCF7 and SW620 sub-cutaneous xenografts

    Journal: Molecular cancer therapeutics

    Article Title: Chemosensitisation of cancer cells by KU-0060648; a dual inhibitor of DNA-PK and PI-3K

    doi: 10.1158/1535-7163.MCT-11-0535

    Figure Lengend Snippet: Efficacy of etoposide and KU-0060648 in MCF7 and SW620 sub-cutaneous xenografts

    Article Snippet: To determine PI-3K activity, cells were exposed to a range of concentrations of KU-0060648 for 1 hr prior to a 30-minute treatment with 50 ng/ml insulin-like growth factor-1 (Calbiochem, Merck Biosciences, Nottingham, UK).

    Techniques:

    Determination of the cellular specificity of KU-0060648 for DNA-PK-dependent cell survival following exposure to etoposide or doxorubicin

    Journal: Molecular cancer therapeutics

    Article Title: Chemosensitisation of cancer cells by KU-0060648; a dual inhibitor of DNA-PK and PI-3K

    doi: 10.1158/1535-7163.MCT-11-0535

    Figure Lengend Snippet: Determination of the cellular specificity of KU-0060648 for DNA-PK-dependent cell survival following exposure to etoposide or doxorubicin

    Article Snippet: To determine PI-3K activity, cells were exposed to a range of concentrations of KU-0060648 for 1 hr prior to a 30-minute treatment with 50 ng/ml insulin-like growth factor-1 (Calbiochem, Merck Biosciences, Nottingham, UK).

    Techniques:

    Plasma pharmacokinetics of KU-0060648 following different routes of administration

    Journal: Molecular cancer therapeutics

    Article Title: Chemosensitisation of cancer cells by KU-0060648; a dual inhibitor of DNA-PK and PI-3K

    doi: 10.1158/1535-7163.MCT-11-0535

    Figure Lengend Snippet: Plasma pharmacokinetics of KU-0060648 following different routes of administration

    Article Snippet: To determine PI-3K activity, cells were exposed to a range of concentrations of KU-0060648 for 1 hr prior to a 30-minute treatment with 50 ng/ml insulin-like growth factor-1 (Calbiochem, Merck Biosciences, Nottingham, UK).

    Techniques:

    XAB2 prevents Ku retention on seDSBs induced by temozolomide. a , Quantification of the average number of Ku80 foci detected by immunofluorescence microscopy in control and XAB2-depleted U87 cells exposed to 15 μM TMZ (or DMSO) for 2 h and allowed to recover for 48 h in the absence or presence of the ATMi KU-55933. b , Representative immunofluorescence images used for the quantification of the number of colocalizing Ku80 (red)-RPA32 (cyan) foci following exposure of control and XAB2-depleted U87 cells to TMZ and recovery for 48 h. Examples of colocalized foci are highlighted by yellow squares, with one representative example (indicated by an arrow) shown in the close up section (scale bar = 0.1 μm). c - d , Percentage of colocalized Ku80-RPA32 foci per cell following exposure to TMZ as in c , quantified based on foci examination in individual cells, as presented in a violin plot ( d ). e - g , Same as b - d for the analysis of Ku80 (red) and RAD51 (cyan) foci colocalization. Scale bar = 5µm. The images are representative of 3 independent biological repeats. Bars represent mean ± s.e.m. Significant differences between specified comparisons were assessed by a t-test (unpaired, 2-tails) and are highlighted by stars (*P

    Journal: bioRxiv

    Article Title: XAB2 prevents abortive recombinational repair of replication-associated DNA double-strand breaks and its loss is synthetic lethal with RAD52 inhibition

    doi: 10.1101/2020.04.10.035410

    Figure Lengend Snippet: XAB2 prevents Ku retention on seDSBs induced by temozolomide. a , Quantification of the average number of Ku80 foci detected by immunofluorescence microscopy in control and XAB2-depleted U87 cells exposed to 15 μM TMZ (or DMSO) for 2 h and allowed to recover for 48 h in the absence or presence of the ATMi KU-55933. b , Representative immunofluorescence images used for the quantification of the number of colocalizing Ku80 (red)-RPA32 (cyan) foci following exposure of control and XAB2-depleted U87 cells to TMZ and recovery for 48 h. Examples of colocalized foci are highlighted by yellow squares, with one representative example (indicated by an arrow) shown in the close up section (scale bar = 0.1 μm). c - d , Percentage of colocalized Ku80-RPA32 foci per cell following exposure to TMZ as in c , quantified based on foci examination in individual cells, as presented in a violin plot ( d ). e - g , Same as b - d for the analysis of Ku80 (red) and RAD51 (cyan) foci colocalization. Scale bar = 5µm. The images are representative of 3 independent biological repeats. Bars represent mean ± s.e.m. Significant differences between specified comparisons were assessed by a t-test (unpaired, 2-tails) and are highlighted by stars (*P

    Article Snippet: The ATMi KU-55933 (Tocris Biosciences) was used as described in experiments with CPT [ ]; it was added during the last half of the 48 h recovery period following exposure to TMZ.

    Techniques: Immunofluorescence, Microscopy