Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: H2O2-induced different cell viability in M17, PC12 and MN9D cells as measured by MTT assay (A & B) and trypan blue dye exclusion assay (C). Each bar from Figs. represents data obtained from 5 separate experiments (N = 5 of independent cell culture preparations, which is the same in the following legends). *p < 0.05, **p < 0.01, ***p < 0.001, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: MTT Assay, Exclusion Assay, Cell Culture, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on DDRs in M17 and MN9D cells as demonstrated by measurements of γH2AX (A) and p53 (B). Cells were exposed to 100–400 μM H2O2 for 3 h. Top panels: autoradiograph obtained by western blotting. Button panel: the quantitative analysis of band densities in western blotting. Each bar from Figs. represents data obtained from 5–7 separate experiments (N = 5–7). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Autoradiography, Western Blot, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: H2O2 treatment induces single-strand DNA breaks as determined by the Comet assay in M17 and MN9D cells. Cells were exposed to different concentrations of H2O2 for 3 h. The cells were processed for comet assays run under alkaline conditions to identify DNA SSBs. Tail moment (B), tail length (C) and % DNA in the tail (D) were analyzed to evaluate DNA damage. Each bar from Figs. represents data obtained from 5 separate experiments (N = 5). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Single Cell Gel Electrophoresis, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on ROS production in M17 and MN9D cells. Cells were exposed to different concentrations of for 3 h. ROS was measured by the DCFH2-DA assay. Each bar from Figs. represents data obtained from 6 separate experiments (N = 6). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on Ctr1 (A) and OGG1 (B) protein levels in M17 and MN9D cells. Each bar represents data obtained from 5 separate experiments (N = 5) in (A), and 4 separate experiments (N = 4) in (B). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on Cav1.2 (A) and Cav1.3 (B) protein levels in M17 and MN9D cells as determined by western blotting. Cells were exposed to different concentrations of H2O2 for 3 h. Each bar in Figs. represents data obtained from 6 to 7 separate experiments (N = 6–7). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Western Blot, Control

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 2 Synergistic toxicity evoked by combined treatment with the CHK1 inhibitor PF477736 (PF) and the RAD51 inhibitor B02 in J82CisPt. (A) The viability of J82CisPt cells was analyzed after treatment with differently combined low and moderate toxic doses of CHK1i PF477736 and RAD51i B02 as indicated. Cell viability was measured after a 72 h treatment period using the AlamarBlue Assay. Based on the data obtained from three independent experiments each performed in quadruplicate, the combination indices (CIs) were calculated using CompuSyn software (CI < 0.9 indicating synergistic effects, CI ≈1 additive effects and CI > 1.2 antagonistic effects). (B) Protein expression and activation of different apoptosis-related factors were examined via Western Blot analyses using protein extracts of J82CisPt

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Alamar Blue Assay, Software, Expressing, Activation Assay, Western Blot

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 3 S-phase arrest in J82CisPt cells following treatment with B02 and PF477736. (A, B) Inhibitors (10 μM B02 ± 1 μM PF477736) were added 24 h after seeding and cell cycle distribution was analyzed after a treatment period of 24 h (A) and 72 h (B) employing propidium iodide staining and flow cytometric analysis. Data are presented as mean + SD from n = 3 independent experiments. (C) The EdU incorporation of J82CisPt cells was analyzed after treatment with 10 μM B02 or/and 1 μM PF477736. EdU incorporation was analyzed after 24 h treatment period with an EdU pulse of 2 h. The graph shows the mean + SD of n = 3 independent experiments (1000–2000 nuclei analyzed per sample). The scale bars in the representative pictures correspond to 50 μm. ***p ≤.001; **p ≤.01; *p ≤.05; significant compared to control (*), to B02 mono- treatment (#) and to PF477736 mono-treatment (+). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Staining, Control

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 4 Hampered replication occurring after combined treatment of J82CisPt with PF477736 and B02. (A) 24 h after seeding, J82CisPt cells were treated with either 10 μM B02, 1 μM PF477736 or both substances for 6 h. After the treatment, cells were incubated for 20 min with CldU, followed by 20 min incubation with IdU. The BrdU analogs were labeled by immunofluorescence, staining was analyzed microscopically and fiber lengths were measured using ImageJ. Data presented are from two independent experiments, whereby 200 fibers were measured for each sample. Each dot represents one analyzed fiber and the black lines show the mean ± SEM. The mean value is also given above the graphs. Upper panel, nascent DNA elongation, graphically displayed as IdU track lengths of bi-colored DNA fibers. Middle panel, table summarizing the evaluation of proportions of origins and terminations in the total fiber population (ns, not significant). Lower panel, as measure of DNA replication fork stalling fork asymmetry was determined from three-colored replication origins as the ratio of the longer red IdU fiber track length versus the shorter red IdU fiber track length departing from the same green CldU track. (B) Formation of RPA foci in the nuclei of J82CisPt cells was analyzed after 6 and 24 h treatment with 10 μM B02 or/and 1 μM PF477736 via immunocytochemical staining. Data are shown with each dot representing one analyzed nucleus and the black lines showing the mean ± SEM from three independent experiments, where in each case 50 nuclei were counted. The scale bars in the representative pictures correspond to 10 μm. ***p ≤.001; **p ≤.01; *p ≤.05; significant compared to control (*), to B02 mono-treatment (#) and to PF477736 mono-treatment (+). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Incubation, Labeling, Immunofluorescence, Staining, Control

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 5 S-phase-dependent formation of DNA damage and activation of DDR- and DNA repair-related mechanisms following treatment of J82CisPt with PF477736 and B02. (A) Protein expression and activation of different replication stress- and DDR-related factors was examined via Western Blot analyses with protein extracts of J82CisPt cells treated for 6 h or 24 h with 10 μM B02, 1 μM PF477736 or both substances. (B) Formation of DNA strand breaks was analyzed via alkaline Comet Assay after 24 h mono- and combination-treatment with 10 μM B02 and 1 μM PF477736. Tail intensity (% DNA in tail) is displayed as dot for every analyzed cell and the mean ± SEM calculated from n = 3; N = 50. ***p ≤.001; **p ≤.01; *p ≤.05; significant compared to control (*), to B02 mono-treatment (#) and to PF477736 mono-treatment (+). (C) To analyze in which cell cycle phase the damage predominantly occurs, a double staining with γH2AX antibody and propidium iodide was applied and examined by flow cytometry after a treatment period of 6 and 24 h in J82CisPt. Displayed representative images of the flow cytometrical analyses were generated using FlowJo software. (D) J82CisPt cells were co-treated with 10 μM B02 + 1 μM PF477736 for 24 h, afterwards immunocytochemical co-staining of γH2AX and RPA was performed to analyze the correlation of both markers. For γH2AX, the mean fluorescence intensity of the nuclei was measured and the number of RPA foci per nucleus were counted. Data are shown with each dot representing one analyzed nucleus and the black lines showing the mean ± SEM from two independent experiments, where in each case 50 nuclei were measured. The scale bar in the representative picture corresponds to 20 μm. ***p ≤.001; significant compared to nuclei with <10 RPA foci. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Activation Assay, Expressing, Western Blot, Alkaline Single Cell Gel Electrophoresis, Control, Double Staining, Flow Cytometry, Generated, Software, Staining, Fluorescence

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 6 Combining B02 or PF477736 with other CHK1- or RAD51-inhibitors, respectively, likewise induces S-phase arrest, replication stress, and DNA damage in J82CisPt. J82CisPt cells were co-treated with 10 μM B02 + 1 μM LY2603618 (LY) (A) or 1 μM PF477736 (PF) + 30 μM RI(dl)2 (RI2) (B). Following 24 h treatment, propidium iodide-based cell cycle analysis was performed by flow cytometry with emphasis on the proportion of cells in S-phase. A total of 10,000 counts were measured for quantification. Induction of γH2AX and pRPA32 (S4, S8) was examined via Western Blot analyses with protein extracts of J82CisPt cells treated for 6 or 24 h with the corresponding combination or mono- treatments. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Cell Cycle Assay, Flow Cytometry, Western Blot

Journal: Cell

Article Title: TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

doi: 10.1016/j.cell.2012.06.039

Figure Lengend Snippet: Figure 6. Deregulation of TRIP12 and UBR5 Alters the Dynamics of DNA Repair (A) U-2-OS cells were transfected with the indi- cated siRNA for 72 hr and irradiated. WCE were analyzed by immunoblotting. (B) U-2-OS cells were irradiated (2 Gy), and 1 hr later, they were immunostained with an antibody to RPA (the FITC channel); nuclear DNA was coun- terstained by DAPI. Cell-cycle distribution was determined for each individual cell by quantifying the total DAPI and chromatin-bound RPA intensity per nucleus. (C) U-2-OS cells were transfected with the indi- cated siRNAs, irradiated, immunostained with antibodies to RPA and RAD51, sorted according to cell cycle as in (B), and subjected to an automated analysis of RAD51 focus formation. At least 500 cells were analyzed for each condition; represen- tative images are shown. (D) U-2-OS cells were transfected with siRNAs for 72 hr and irradiated. Where indicated, cells were treated for 2 hr with ATM or DNA-PK inhibitors prior to irradiation. WCE were analyzed by immuno- blotting (left). In parallel, cells were treated with the indicated siRNAs and inhibitors as indicated, irra- diated (2 Gy), and after 4 hr, were subjected to an automated single cell analysis for the number of MDC1 nuclear foci (right). (E) U-2-OS cells were treated with the indicated siRNAs and inhibitors, irradiated, and analyzed as in (D). Scale bar, 10 mm. See also Figure S7.

Article Snippet: Rabbit polyclonal antibodies included the following: RNF168 (Stewart et al., 2009; provided by Daniel Durocher), RNF8 (rabbit antiserum, provided by Xiaochun Yu), 53BP1 (sc-22760, Santa Cruz), MDC1 (ab11171, Abcam), TRIP12 (A301-814A, Bethyl), UBR5/EDD (A300-573A, Bethyl), PARP1 (sc-7150, Santa Cruz), RPA32 phospho Ser33 (A300-246A, Bethyl), RPA32-phospho S4/S8 (A300245A, Bethyl), CHK1 (sc-8404, Santa Cruz), CHK1 phospho S345 (#2341, Cell Signaling), pCHK1-S317 (#2344, Cell Signaling), KAP1 (A300-274A, Bethyl), KAP1 phospho S824 (ab70369, Abcam), Herc2 (Bekker-Jensen et al., 2010), Smc1 (ab9262, Abcam), Rap80 (A300-763A, Bethyl), RAD51 (sc-8394, Santa Cruz), H2AX (NB100-638 Novus Biologicals).

Techniques: Transfection, Irradiation, Western Blot, Single-cell Analysis

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: TRIM33 interacts with ALC1 upon induction of DNA damage. A, HEK293 cells expressing either vector or FLAG-ALC1 were treated with either vehicle or bleomycin, subjected to immunoprecipitation using FLAG beads, and then processed by LC-MS/MS. The relative peptide counts for each condition are shown for ALC1, PARP1, APLF, histone H2B, Ku70, and TRIM33. As shown, TRIM33 peptides were identified only in the bleomycin-treated samples. B, endogenous interaction of TRIM33 and ALC1 upon hydroxyurea (HU) and bleomycin treatment. DNase-treated nuclear extracts from untreated (Un) cells or cells treated with HU (3 mm, 3 h) or bleomycin (300uM, 1 h) were immunoprecipitated with anti-TRIM33 antibody. Immunoprecipitates were processed for Western blotting (WB) using antibodies to TRIM33 and ALC1 (top two panels, IP). Aliquots of nuclear extract were also directly processed for Western blotting with these antibodies (bottom two panels, Inputs).

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: Localization of TRIM33 to DNA breaks is dependent upon PARP and ALC1. A, PAR (top two panels) and TRIM33 (bottom two panels) were localized by IF in untreated (Un) and in cells pretreated with 1 μm PARPi (Pi) ABT-888 for 1 h. B, quantitation of PAR and TRIM33 colocalization with γH2AX at sites of laser scissors. *, p < 0.005. C, Parp1+/+ or Parp1−/− mouse embryonic fibroblasts were treated with laser scissors, and γH2AX and TRIM33 were localized by IF. Images are shown at identical magnification. D, quantitation of PAR and TRIM33 colocalization with γH2AX at sites of laser scissors. *, p < 0.005. E, APLF, WtALC1, C1 (ALC1 macro domain only), and TRIM33 proteins were dot-blotted onto a nitrocellulose membrane and incubated with 32P-labeled PAR. F, TRIM33 localization to DNA breaks is ALC1-dependent. U2OS cells stably expressing control sh, ALC1sh, or cells expressing ALC1 sh were reconstituted with WT ALC1, KR (kinase dead) and DA (PAR binding mutant) were analyzed. All cells were subjected to UV laser scissor-induced DNA breaks. After 10 min, cell were fixed, and IF was performed using antibodies to γH2AX and TRIM33. G, Western blot analyses showing levels of ALC1 and TRIM33 in U2OS cells expressing control and ALC1 shRNA and different constructs of ALC1 in ALC1sh cells. H, quantitation of relative intensity of TRIM33 at sites of DNA damage. Each data point is mean ± S.D. of at least 20 cells. *, p < 0.005.

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Quantitation Assay, Incubation, Labeling, Stable Transfection, Expressing, Binding Assay, Mutagenesis, Western Blot, shRNA, Construct

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: TRIM33 dynamically interacts with ALC1 in a PARP-dependent manner. A, HeLa cells were untreated (Un) or treated with IR or UV light, with or without PARP inhibitor (PARPi), and DNase-treated nuclear extracts were prepared at 5 and 10 min. TRIM33 IP was performed, followed by Western blotting (WB) using the indicated antibodies. IP, immunoprecipitation; No Ab, no antibody. B, quantitation of ALC1 interaction with TRIM33. The plot shows the ratio of the signal of ALC1 coimmunoprecipitation to ALC1 input. C, The FLAG WtALC1, ALC1K77R, and ALC1D723A mutants were expressed in 293 cells and subjected to UV irradiation. Protein extracts were prepared after 5 min and immunoprecipitated with anti-FLAG antibodies, followed by Western blotting using antibody against TRIM33 and FLAG. D, PAR-bound ALC1 does not bind TRIM33 in vitro. WtALC1, KR (ATPase dead) and DA (PAR binding mutant) ALC1 mutants and TRIM33 proteins were dot-blotted onto a nitrocellulose membrane and incubated with PAR, washed, and then incubated with purified TRIM33. Membranes were then processed for Western blot analysis with antibodies to PAR (top panel) or antibody to TRIM33 (bottom panel).

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Western Blot, Immunoprecipitation, Quantitation Assay, Irradiation, In Vitro, Binding Assay, Mutagenesis, Incubation, Purification

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: TRIM33 knockdown induces delayed dissociation of ALC1 but does not affect PAR dissociation from sites of DNA damage. A, HeLa cells expressing either control shRNA, TRIM33 shRNA, TRIM33shRNA + WtTRIM33, or the TRIM33shRNA + TRIM33CA mutant were subjected to UV laser scissors and stained for γH2AX (green) and ALC1 (red) at the time points indicated. B, Western blot analysis showing levels of TRIM33 in cells treated with control shRNA (lane 1), TRIM33 shRNA (lane 2), TRIM33sh RNA + WtTRIM33 (lane 3), and TRIM33sh RNA + TRIM33CA (lane 4). C, quantification of ALC1 localization to DNA damage in TRIM33 knockdown, WtTRIM33, or TRIM33CA reconstituted cells. Each data point is mean ± S.D. of at least 20 cells. D, TRIM33 knockdown does not affect ALC1 protein levels. Shown is a Western blot analysis showing levels of ALC1, TRIM33, and tubulin in control sh (1), TRIM33sh and TRIM33sh (2) cells reconstituted with either TRIM33Wt (3) or TRIM33CA (4). Protein extracts were collected from untreated or UV-treated cells, as indicated, and probed by Western blot analysis using the antibodies indicated. E, HeLa cells expressing either control shRNA or TRIM33 sh2 RNA were treated with laser scissors and stained by IF for γH2AX (green) and PAR (red). F, quantification of PAR intensity was performed 5 and 45 min after induction of DNA damage. Data are plotted as the mean ± S.D. of at least 20 cells. *, p < 0.005 for control versus TRIM33 shRNA-treated cells. G, Western blot analysis showing TRIM33 knockdown. HeLa cells transfected with control or TRIM33 shRNA and proteins levels were detected by Western blot analysis by probing with antibodies against TRIM33 and tubulin.

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Expressing, shRNA, Mutagenesis, Staining, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: TRIM33 rescues ALC1 overexpression sensitivity to DNA-damaging agents. A, ALC1 overexpression results in delayed dissociation of ALC1 from DNA damage. FLP-In ALC1 and FLP-In ALC1 + WtTRIM33 cells were exposed to UV laser scissor-induced DNA damage, and cells were fixed at the indicated time points. Cells were stained by IF for γH2AX (green) and ALC1 (red). B, Western blot analysis showing levels of TRIM33 and ALC1 in ALC1-overexpressing cells. C, quantification of ALC1 intensity was performed at the indicated time points after induction of DNA damage. Data are plotted as the mean ± S.D. of at least 20 cells. D, effect of TRIM33 on sensitivity of ALC1-overexpressing cells to bleomycin. U2OS cells stably expressing either empty vector (FLP-In-FLAG), WtALC1 (FLP-In-ALC1), WtALC1 (FLP-In-ALC1) + WtTRIM33, and WtALC1 (FLP-In-ALC1) + TRIM33CA were treated with increasing concentrations of bleomycin for 3 days. Relative cell counts at day 3 were measured by MTS assay and were plotted normalized to untreated cells. Data are mean ± S.D. of three experiments. E, induction of DNA breaks in cells (n > 200 cells) of indicated genotypes after treatment with increasing amounts of phleomycin were measured by alkaline comet assay. Data are plotted as the mean ± S.D. of at least three experiments.

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Over Expression, Staining, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, MTS Assay, Alkaline Single Cell Gel Electrophoresis

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Hepatic MTH1+CD3+ T cells correlated with disease activity in AIH. (A) Representative confocal staining of CD3 (red), MTH1 (green), and DAPI (for nuclei in blue) (magnification ×400) in the liver of patients with AIH. Scale bar, 20 μm. (B) Number of MTH1+CD3+ T cells in portal areas was positively correlated with degree of hepatic inflammation but showed no clear link with fibrosis stages in AIH. (C) Number of MTH1+CD3+ T cells in portal areas had a significant positive correlation with levels of serum ALT and AST in patients with AIH. (D) Number of MTH1+CD3+ T cells in portal areas was positively correlated with levels of serum ALP and GGT. Bars reflect the mean ± SEM. Abbreviations: DAPI, 4 0, 6‐diamidino‐2‐phenylindole; hpf, high‐power field.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: Activity Assay, Staining

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib significantly inhibited T‐cell proliferation in human T cells in vitro . Isolated human CD3+ T cells were cultured with/without anti‐CD3/CD28 beads for 72 hours. Representative western blot analyses of MTH1 72 hours after anti‐CD3/CD28 beads stimulation. (B,C) Isolated T cells were activated with anti‐CD3/CD28 beads with or without 2 μM karonudib for 72 hours. The percentage of CD25+ and CD69+ T cells was determined on day 3 by flow cytometry. (D) Analysis of T‐cell number treated with karonudib for 72 hours after anti‐CD3/CD28 beads stimulation. (E) Statistical analysis of the T‐cell proliferation assay treated with/without 2 μM karonudib for 72 hours from HCs and patients with AIH who were treatment naive. (F) Representative western blot analyses of P53, P21, P27, CDK2, and cyclin E 72 hours after anti‐CD3/CD28 beads stimulation. The GAPDH blot was used as a loading control. Data are from one experimental representative of at least three independent experiments and represent triplicate wells. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Cell Culture, Western Blot, Flow Cytometry, Proliferation Assay, Control

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib rendered activated T cells more susceptible to DNA damage in vitro . (A) Representative fields corresponding to each treatment were photographed. Isolated human T cells were activated with/without anti‐CD3/CD28 beads in the presence of DMSO or 2 μM karonudib for 72 hours. The alkaline comet assay was conducted and nucleoids were visualized by epifluorescence microscopy using a fluorescein isothiocyanate filter. (B) Quantification of comet tail moment. Values represent mean ± SEM from three independent experiments (100 comets per experiment). (C) Levels of cleaved‐PARP and phosphorylated histone H2AX (γ‐H2AX) were determined by immunoblot analysis. The GAPDH blot was used as a loading control. (D,E) Intracellular flow cytometry assessment of annexin V and propidium iodide expression in anti‐CD3/CD28 beads‐stimulated CD4+ and CD8+ T lymphocytes treated with 2 μM karonudib or DMSO after 72 hours. Data are from one experimental representative of at least three independent experiments. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Alkaline Single Cell Gel Electrophoresis, Epifluorescence Microscopy, Western Blot, Control, Flow Cytometry, Expressing

Journal: Cell Death and Differentiation

Article Title: Glycoprotein PTGDS promotes tumorigenesis of diffuse large B-cell lymphoma by MYH9-mediated regulation of Wnt–β-catenin–STAT3 signaling

doi: 10.1038/s41418-021-00880-2

Figure Lengend Snippet: A The expression level of PTGDS mRNA was reduced by adriamycin (ADR) and bendamustine (BEN). B and C The treatment of AT56 (50 μM) enhanced the cytotoxicity of ADR and BEN in LY1 and LY3 cells. D and E AT56 sensitized DLBCL cells to ADR and BEN in cell apoptosis. F Representative images and quantification of the tail of DLBCL cells treated with AT65 (75 and 125 µM) in the comet assay. Bar = 40 μm. G and H Western blotting and immunofluorescent images indicated that AT56 (75 μM) increased the expression of p-H2AX in DLBCL cells. Bar = 40 μm. Data are shown as the mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Adriamycin, bendamustine, and WP1066 were purchased from Selleck Chemicals (TX, USA).

Techniques: Expressing, Single Cell Gel Electrophoresis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: Impact of the modulation of TYRO3 expression on the radiosensitivity of bladder cancer cell lines. ( a – c ) Representative clonogenic survival curves of RT112 and 5637 cell lines ( upper panel). 48 h after transfection with siLUC (control, red), siTYRO3#4 (blue), siTYRO3#801 (green) or diluted concentration of siRNA#1 (black) cells were exposed to increased doses of gamma-rays. In each case, downregulation was confirmed by western blot at 48 h after transfection. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( d ) Representative survival curves of UM-UC-3 control (empty plasmid, red) and TYRO3 over-expressed cell lines (TYRO3 encoding- plasmid, purple) ( upper panel). The upregulation was confirmed by western blot. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( e ) Phosphorylated TYRO3 shown in western blot ( lower panel) after immunoprecipitation of the cell extracts with phospho-Tyrosine antibodies and its relative expression in 5637 cells quantified using Image-J software and normalized to that of total TYRO3 ( upper panel). Data represents mean ± SD of 3 independent experiments. Unpaired t-test analysis: * p < 0.05; ** p < 0.005, ns: not significant.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Expressing, Transfection, Control, Concentration Assay, Western Blot, Plasmid Preparation, Immunoprecipitation, Software

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on Ionizing Radiation-Induced Foci and DNA damage. γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 downregulated RT112 ( a ) or 5637 ( c ) cells (scale bar 5 microns); Quantification of cells containing more than 10 γH2AX foci at 24 h after 2 Gy of irradiation in the downregulated RT112 ( b ) and 5637 ( d ) cell lines; ( e ) γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 overexpressing UM-UC-3 cell line (Scale bar 5 microns); ( f ) Quantification of cells containing more than 10 γH2AX foci at 30 min and 24 h after 2 Gy of irradiation in TYRO3 overexpressing cell lines. The data shown above is from three different experiments and error bars represent the SD. Unpaired t -test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0005, ns: non-significant. Representative images of the alkaline Comet assay performed on TYRO3 downregulated RT112 ( g ) and 5637 ( h ) cells and the resulting Olive tail moments analysis in TYRO3 downregulated RT112 ( i ) and 5637 ( j ) cells irradiated at 6 Gy. The data shown here is from three independent experiments analyzing 200 nucleus per condition per experiment, horizontal bars represent the median values. Kruskal-Wallis nonparametric tests with Multiple comparisons were used: * p < 0.05; ** p < 0.005, ns: non-significant; ( k ) western blot of DNA damage repair proteins; TYRO3 was downregulated, irradiated at 6 Gy and the lysates were prepared and analyzed 0.5–24 h after.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Alkaline Single Cell Gel Electrophoresis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on DNA damage response pathways. ( a ) Volcano plot showing the fold change (Log2 Ratio) versus negative log of the p-value of differentially expressed genes after Nanostring analysis between BCa (RT112 and 5637) 6 Gy irradiated cells versus BCa 6 Gy irradiated cells after complete TYRO3 knock-down (siTYRO3#4 and siTYRO3#801). Significant: p -value < 0.05 ( b ) Log2 ratio of the significantly expressed genes ( p < 0.05) after Nanostring analysis between the two groups. The name of the up- or downregulated genes are listed on the left. On the right are the corresponding Nanostring gene annotations. ( c ) Protein-protein association network of the up and downregulated genes assessed using the STRING database.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 downregulation affects cell cycle following irradiation. Analysis of the cell cycle distribution 24 h after 6 Gy irradiation on TYRO3-downregulated RT112 ( a ) and 5637 ( b ) cells. Comparison of percentage (%) of cells in G2/M in RT112 ( c ) and 5637 ( d ) cells. Data shown is from three different experiments and error bars represent the SD. Unpaired t-test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005, ns: non-significant. ( e ) western blot of cell cycle proteins 30 min and up to 24 h after irradiation.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Comparison, Western Blot

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet:

Article Snippet: Antibody , Anti- PERK (Rabbit monoclonal) , Cell Signaling Technology , 3192 , WB (1:1000).

Techniques: Cloning, CRISPR, Knock-Out, Expressing, Recombinant, Plasmid Preparation, Transfection, Sequencing, Software, Diffusion-based Assay, Single Particle

Journal: Cell death and differentiation

Article Title: Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast.

doi: 10.1038/cdd.2009.1

Figure Lengend Snippet: Figure 1 Cell membrane integrity of BeWo cells was monitored with anti-human b-catenin immunofluorescence. (a) Immunofluorescent image of BeWo cells undergoing low spontaneous level of fusion (* indicates 3 or more DAPI-stained nuclei within an intact membrane was considered as fusion event). (b) Forskolin-induced excessive cell– cell fusion resulted in membrane dissolution and loss of distinct membrane staining. (c) Silenced BeWo cells and (d) silenced BeWo cells treated with forskolin show no cell– cell fusion shown by intact membrane staining. Scale bar ¼ 100 mm. (e) Relative GCM1 mRNA expression (n ¼ 6) and (f) representative western blot analysis of GCM1 protein expression (n ¼ 6) in BeWo cells stably expressing siRNA to GCM1 in cells treated with 25 mM of forskolin and in cells stably expressing siRNA þ forskolin compared with non-silencing (sense control) siRNA-transfected control BeWo cells. Forskolin treatment of BeWo cells significantly upregulated GCM1 RNA and protein expression. GCM1 silencing eliminated this effect. Bar graph showing the mean±s.e. (n ¼ 6). NS, non-silenced control; F, forskolin (25 mM). (g) The two-color fusion assay shows a significant decrease of de novo fusion in the silenced BeWo group with forskolin treatment compared with that in control and forskolin-treated non-silenced BeWo cells. Difference between the non-forskolin-treated sense control group and stably expressing GCM1 siRNA group is not significant because of the low level of basal fusion (n ¼ 6). (h) A proliferation assay of control BeWo cells, overexpressing GCM1 (25 mM forskolin treatment) and BeWo cells stably expressing siRNA to GCM1 over 3 days in culture. On the third day of culture, cells overexpressing GCM1 show significant reduction in the number of cells, whereas BeWo cells stably expressing GCM1; siRNA show significant increase in cell numbers as compared with control cells (n ¼ 3). Mock controls showed no difference to non-silenced controls in all experiments (data not shown). *Po0.005, **Po0.001

Article Snippet: Membranes were blocked with 5% skimmed milk in 0.1% (v/v) Tween Tris-buffer saline (TBST) for 2 h at room temperature and incubated with anti-GCM1 antibody (1:3000) at 41C overnight (Aviva System Biology, San Diego, CA, USA).

Techniques: Membrane, Staining, Dissolution, Expressing, Western Blot, Stable Transfection, Control, Transfection, Single Vesicle Fusion Assay, Proliferation Assay

Journal: Cell death and differentiation

Article Title: Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast.

doi: 10.1038/cdd.2009.1

Figure Lengend Snippet: Figure 3 GCM1 antisense oligonucleotides and siRNA treatment after trypsinization of SCT result in an accumulation of 4.9±1.5 layers of CT cells (semi-thin section, a) that proliferate (Ki-67 immunopositive in brown, d) but do not regenerate a new layer of SCT. (b) Treatment of non-denuded first trimester explants with GCM1 antisense oligonucleotide or siRNA results in the accumulation of 3.8±0.4 layers of cytotrophoblast cells underneath a necrotic SCT layer (note the dissolution of syncytial nuclei, r). (e) Accumulating V-CT exhibited a high level of proliferation (Ki-67 positive). (c) Forskolin treatment results in the accumulation of multilayer of syncytial nuclei, but reduced the numbers of V-CT cells (0.9±0.3 layers) and (f) the rate of Ki-67 immunopositivity in the trophoblast layer. Scale bars ¼ 1000 mm (a–c), 50 mm (d–f). (g) Western blot analysis of GCM1 protein levels in siRNA or antisense oligonucleotide-treated explants. (h) Forskolin treatment of first trimester floating villous explants regulates GCM1 protein levels in time-dependent manner. White bars ¼ controls, black bars ¼ forskolin-treated explants. Significant difference from control explants is indicated by *Po0.05

Article Snippet: Membranes were blocked with 5% skimmed milk in 0.1% (v/v) Tween Tris-buffer saline (TBST) for 2 h at room temperature and incubated with anti-GCM1 antibody (1:3000) at 41C overnight (Aviva System Biology, San Diego, CA, USA).

Techniques: Dissolution, Western Blot, Control

Journal: Cell death and differentiation

Article Title: Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast.

doi: 10.1038/cdd.2009.1

Figure Lengend Snippet: Figure 4 Histological section of (a) control, (b) GCM1-silenced, and (c) forskolin-treated floating villous placental explants stained for BrdU (blue nuclei). The index of proliferation of villous trophoblast cells in explants cultured with (d) GCM1 siRNA, (e) the GCM1 antisense oligonucleotide and (f) forskolin. Scale bars ¼ 40 mm (a, b); 50 mm (c). n ¼ 9; *Po0.05

Article Snippet: Membranes were blocked with 5% skimmed milk in 0.1% (v/v) Tween Tris-buffer saline (TBST) for 2 h at room temperature and incubated with anti-GCM1 antibody (1:3000) at 41C overnight (Aviva System Biology, San Diego, CA, USA).

Techniques: Control, Staining, Cell Culture

Journal: Cell death and differentiation

Article Title: Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast.

doi: 10.1038/cdd.2009.1

Figure Lengend Snippet: Figure 5 Dissecting light microscope image of (a) sense oligonucleotide-treated first trimester explant cultured on Matrigel. Typical organized extravillous trophoblast outgrowth formation; eight outgrowth fingers are numbered. (b) H&E staining of control explants. (c) The outgrowths are a1-Integrin-positive as well as (d) GCM1- immunopositive. (e) GCM1 antisense oligonucleotide-treated explants do not form extravillous outgrowths, (f) cells do not invade Matrigel (H&E staining). (g) Cells are negative for the invasive marker a1-Integrin. (h) Immunofluorescent staining to GCM1 protein. Scale bars ¼ 100 mm (a, e); 50 mm (b, f); 25 mm (c, d, g, h)

Article Snippet: Membranes were blocked with 5% skimmed milk in 0.1% (v/v) Tween Tris-buffer saline (TBST) for 2 h at room temperature and incubated with anti-GCM1 antibody (1:3000) at 41C overnight (Aviva System Biology, San Diego, CA, USA).

Techniques: Light Microscopy, Cell Culture, Staining, Control, Marker

Journal: Nature neuroscience

Article Title: Aberrant Topoisomerase-1-DNA Lesions are Pathogenic in Neurodegenerative Genome Instability Syndromes

doi: 10.1038/nn.3715

Figure Lengend Snippet: a. The repair kinetics of quiescent astrocytes following treatment with the Topoisomerase-1 poison camptothecin (CPT) at the indicated time points is shown. Although Top1-induced DNA breaks are repaired more slowly in Atm −/− astrocytes, deficiency in the related kinase, DNA-dependent protein kinase, catalytic subunit ( Prkdc −/− ) results in comparable DNA single-strand break repair rates as wild-type (Ctrl) astrocytes. Inset panel shows quiescent GFAP-positive Atm −/− astrocytes. b. Inhibiting Atm kinase activity fails to recapitulate the repair defect observed in Atm −/− cells after CPT. Western blot analysis (inset) of irradiated wild type (Ctrl) astrocytes co-treated with 10 μM ATM inhibitor KU55933 (ATMi) and radiation confirms Atm inhibition by defective DNA damage-induced Chk2 modification (black arrows) and p53 phosphorylation in Atm −/− cerebella and astrocytes. NBS1 was used as a loading control. Comet analysis (bar graph) indicates Atm −/− astrocytes (red arrow) accumulate significantly more CPT-induced DNA damage than ATMi-treated ctrl astrocytes (yellow arrow), indicating ATM kinase-independent repair of Top1-DNA lesions. Full-length Western blots are presented in . c. ICE analysis of quiescent primary murine astrocytes following treatment with DNA damaging agents can result in accumulation of Top1cc. Treatment conditions were; 14 μm CPT for 60 mins at 37°C; 20Gy IR followed by 60 mins recovery at 37°C; 150 μm H 2 O 2 for 5 mins at 4°C followed by 60 mins recovery at 37°C; 0.20 mg/ml MMS for 10 mins at 37°C. Top1cc were identified by blotting genomic DNA with anti-Top1 and gDNA levels were assessed by re-probing with 32 P-labelled mouse ES cell genomic DNA ( 32 P-DNA). d. Alkaline comet analysis of quiescent Atm −/− astrocytes show defective DNA single-strand break repair after treatments listed for ‘c’. For each in vitro comet assay, 100 cells/comet corresponding to each genotype and treatment were analyzed and experiments were performed in triplicate (total of n=300 cells/genotype/treatment). e. In vivo comet analysis comparing relative DNA strand break repair rates amongst ctrl, Atm −/− and Tdp1 −/− cerebellar granule cell neurons following ionizing radiation (15 Gy) and a 30 min recovery. Bar graphs represent mean comet tail moments from experiments that were repeated in duplicate (2 mice/treatment) with cells isolated from each cerebella also measured in duplicate (total n=400 independent comet tail moments measured per line/treatment); error bars represent standard error of means (S.E.M.). Scatterplots indicate representative cellular comet tail moments from each corresponding cell/treatment type. For all graphs */** denotes p-values < 0.0001.

Article Snippet: Blots were immunostained with the following antibodies: anti-Chk2 (mouse, 1:1000, Upstate, cat# 05-649), anti-phospho-Chk2 T68 (rabbit, 1:1000, Cell Signaling, cat# 2661), anti-p53 ser15 (rabbit, 1:1000, Cell Signaling, cat# 9284), anti-Nbs1 antibody (rabbit, 1:500, Cell Signaling cat# 3002), anti-ATM (MAT3, mouse, 1:1000, Sigma; D2E2, rabbit, 1:2000, Cell Signaling, cat# 2873), anti-pATM S1981 (10H11.E12, 1:1000, Abcam, cat# ab36810), anti-Tdp1 (mouse, 1:1000, Abnova, cat# H00055775-A01), anti-Topoisomerase 1 (rabbit, 1:1000, Bethyl, cat# 302-590A), anti-KAP1 (rabbit, 1:2000, Abcam, cat# ab10484) and anti-phospho-KAP1 S824 (rabbit, 1:1000, Bethyl, cat# A300-767A), followed by appropriate horseradish peroxidase–conjugated secondary antibodies (1:2,000, GE Healthcare) and detected using ECL or ECL Prime chemiluminescence reagent (GE Healthcare).

Techniques: Activity Assay, Western Blot, Irradiation, Inhibition, Modification, Phospho-proteomics, Control, In Vitro, Single Cell Gel Electrophoresis, In Vivo, Isolation

Journal: Nature neuroscience

Article Title: Aberrant Topoisomerase-1-DNA Lesions are Pathogenic in Neurodegenerative Genome Instability Syndromes

doi: 10.1038/nn.3715

Figure Lengend Snippet: a . Primary Atm −/− astrocytes form few γH2AX foci after CPT treatment (5 μM for 60 min) while abundant γH2AX foci are seen in WT and Tdp1 −/− astrocytes. All genotypes show equivalent levels of γH2AX after IR. Upper panels show immunofluorescence analysis of typical γH2AX foci, which are quantified in the graphs below, as are the foci observed after IR. b. Similar to astrocytes, murine embryonic fibroblasts (MEFs) also exhibit Atm-dependent γH2AX after CPT. 53BP1 foci also show a similar induction after CPT and co-localize with γH2AX foci; these data are quantified in the adjacent graphs. c. In contrast to ATM −/− cells (A-T), the loss of NBS1 does not affect γH2AX foci formation upon CPT treatment. d. While bleomycin treatment (10 μg/ml 30 mins) induces γH2AX foci at similar levels in WT, Atm −/− and Tdp1 −/− cells, pre-treatment with CPT prevents bleomycin-induced γH2AX foci formation in Atm −/− and Atm −/− ;Tdp1 −/− cells. The ATM inhibitor (ATMi) KU55933 prevents CPT-induced γH2AX foci formation indicating that Atm kinase activity is required for H2AX phosphorylation. e. Quantitation of γH2AX foci/cell for the different treatments and genotypes presented in ‘d’ is shown. f. When CPT treated (5μM CPT, 60mins) Atm −/− or Atm −/− ;Tdp1 −/− cells MEFs are subsequently incubated with CPT-free media, DNA damage signaling is activated in a DNA-dependent protein kinase, catalytic subunit (DNA-PKcs/Prkdc)-dependent manner as γH2AX foci fail to form in the presence of the DNA-PKcs inhibitor, NU7441 (2μM). For all foci quantification experiments, 30 cells for each cell line and corresponding treatment were counted and experiments were repeated in quadruplicate (total n=120 independent cells measured per line/treatment). Bar graphs represent mean cellular foci values of all replicates, error bars represent standard error of means (S.E.M.) and p-value is calculated using student’s unpaired t-test. The y-axis on graphs in ‘b’ and ‘f’ is non-linear to indicate increased foci number in specific genotypes after DNA damage.

Article Snippet: Blots were immunostained with the following antibodies: anti-Chk2 (mouse, 1:1000, Upstate, cat# 05-649), anti-phospho-Chk2 T68 (rabbit, 1:1000, Cell Signaling, cat# 2661), anti-p53 ser15 (rabbit, 1:1000, Cell Signaling, cat# 9284), anti-Nbs1 antibody (rabbit, 1:500, Cell Signaling cat# 3002), anti-ATM (MAT3, mouse, 1:1000, Sigma; D2E2, rabbit, 1:2000, Cell Signaling, cat# 2873), anti-pATM S1981 (10H11.E12, 1:1000, Abcam, cat# ab36810), anti-Tdp1 (mouse, 1:1000, Abnova, cat# H00055775-A01), anti-Topoisomerase 1 (rabbit, 1:1000, Bethyl, cat# 302-590A), anti-KAP1 (rabbit, 1:2000, Abcam, cat# ab10484) and anti-phospho-KAP1 S824 (rabbit, 1:1000, Bethyl, cat# A300-767A), followed by appropriate horseradish peroxidase–conjugated secondary antibodies (1:2,000, GE Healthcare) and detected using ECL or ECL Prime chemiluminescence reagent (GE Healthcare).

Techniques: Immunofluorescence, Activity Assay, Phospho-proteomics, Quantitation Assay, Incubation

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: ATR Target Activation by the CHK1 Inhibitor AZD7762 in U2OS Cancer Cells (A) Western blot showing activation of ATR targets. U2OS cells were treated with the indicated concentrations for 30 and 60 min, lysed, and probed with anti-phospho (Serine 345) CHK1 and β-actin antibodies. (B) Induction of pre-apoptotic pan-nuclear γ-H2AX by ATR and CHK1 inhibitor in combination in cancer cells. U2OS cells were treated with the indicated drug concentrations for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody. Scale bar, 20 μm. (C) Quantitative data of γH2AX- (nine or more foci per cells) positive cells or pan-nuclear γH2AX signal after indicated treatments are shown (n = 3, mean ± SEM). (D) Western blot showing increased phosphorylation of H2AX after combination treatment. U2OS cells were treated with the indicated concentrations for 24 hr. At the end of incubation time, western blotting was performed using anti-phospho (Serine 139) H2AX, anti-phospho (Serine 345) CHK1, cleaved PARP, anti-phospho (Serine 10) H3, and β-actin antibodies. (E) Comet assay showing DNA damage induction by ATR and CHK1 inhibitor in combination. U2OS cells were treated with the indicated drug concentrations for 24 hr. At the end of incubation, cells were harvested and alkaline comet assay was performed. (F) Quantitative data of the tail moment are shown (n = 3, mean ± SEM, in each experiment ≥100 comets were measured). (G) Cancer-specific ssDNA formation by VE-821 and AZD7762, either alone or in combination. U2OS cells were treated with the indicated drug concentrations for 24 hr and pre-extracted using CSK buffer before fixation. Cells were stained with anti-RPA32 antibody; images were taken using a confocal microscope and were analyzed using ImageJ software. A mean intensity of ≥70 a.u. per cell was considered as positive. Quantitative data are presented as mean ± SEM from three independent experiments. (H) ssDNA formation in normal fibroblast VH-10 cells is shown. (I) Pre-apoptotic pan-nuclear γH2AX induction by combination treatment of ATR and CHK1 inhibitors in U2OS is mediated through the JNK pathway. U2OS cells were treated with the indicated drug concentrations for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody, and high-throughput microscopy was used to determine the percentage of γH2AX-positive cells (nine or more γH2AX foci per cell) or an average intensity of ≥2,000 a.u. for pan-nuclear γH2AX-positive cells (n = 2 with multiple wells, mean ± SEM). Statistical significance was determined using one-way ANOVA ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Incubation, Single Cell Gel Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Staining, Microscopy, Software, High Throughput Screening Assay

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: Combination of the ATR Inhibitor VE-821 and the CHK1 Inhibitor AZD7762 Synergistically Kills Cancer Cells (A) Clonogenic survival of U2OS, VH-10, and MCF-7 cells. The 500 (U2OS and MCF-7) or 1,000 (VH-10) cells were seeded in 10-cm 2 dishes, and, after 5-hr incubation, the inhibitors were added directly to the media. After 72-hr incubation, drug-containing media were replaced with fresh media and cells were kept for another 5–8 days before colonies were stained with methylene blue. Quantitative data: n = 3, mean ± SEM. (B) Parental and cMYC-transformed cells were treated with the indicated doses for 72 hr. At the end of the incubation period, resazurin was added and cell viability was measured. Quantitative data: n = 3, mean ± SEM. (C) BJ-hTERT, BJ-hTERT SV40, and BJ SV40 RAS cells were treated with the indicated doses for 72 hr. At the end of the incubation period, resazurin was added and cell viability was measured. Quantitative data: n = 3, mean ± SEM. (D) CHK1 functionally compromised cells are sensitive to ATR inhibitor. Clonogenic survival of DLD-1, DLD-1 CHK1 S317A/− , DLD-1 CHK1 +/− , and DLD-1 ATR S/S after ATR inhibitor VE-821 treatment is shown. A similar protocol was used as for U2OS and VH-10 cells. Quantitative data: n = 3, mean ± SEM. (E) Therapeutic efficacy of combined inhibition of ATR and CHK1 in mouse tumor models. Therapeutic efficacy of VX-970 and AZD7762 in H460 lung cancer xenografted mice is shown. BALB/c nude mice bearing H460 xenograft were divided in four groups (five animals in each group) with a tumor volume of ∼130 mm 3 in each group. The first control group of animals was treated with vehicle (orally and intraperitoneally). The second group of animals was treated with 25 mg/kg body weight of CHK1 inhibitor AZD7762 (intraperitoneal route). The third group of animals was treated with 60 mg/kg body weight of ATR inhibitor VE-822 (oral administration), and the fourth group received a combination of both CHK1 and ATR inhibitors. Vehicle and drugs were administered on days 0–3, 10–12, and 18–20 irrespective of no mice survival in each group. Tumor volume was measured with calipers and is shown here as mean ± SEM. Statistical significance was determined using two-way ANOVA with repeated measurement ( ∗ p < 0.05 and ∗∗ p < 0.01). (F) Kaplan-Meier survival curve of H460-xenografted mice. When tumor size reached 1,000 mm 3 , the animal was sacrificed.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Incubation, Staining, Transformation Assay, Drug discovery, Inhibition, Control

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: ATR and CHK1 Inhibitors, Alone or in Combination, Decrease Replication Fork Speed Only in Cancer Cells (A) Treatment regimen is shown. U2OS and VH-10 cells were treated for 60 min with the indicated drug concentrations and sequentially labeled with 5-chlorodeoxyuridine (CldU) and 5-iododeoxyuridine (IdU) for 30/20 min each in the presence of the inhibitors. DNA fibers were stained and replication speed was measured by IdU labeling. (B and C) Representative images show stained replication fork tracts for each treatment group. (D) Quantitative data of replication fork speed (kb/min), mean ± SEM, and p values were analyzed with one-way ANOVA for each condition and cell line. (E and F) Average distribution of replication fork rates. A minimum of 450 forks per condition were analyzed from at least three independent repeats.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Labeling, Staining

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: Combination Treatment of VE-821 and AZD7762 Results in S Phase Arrest in U2OS Cells (A) U2OS cells were treated with the indicated drug concentrations for 24 hr and propidium iodide (PI) staining was carried out to measure cell-cycle profile using flow cytometry. (B) Quantitative data were obtained using Modfit software. (C) ATR and CHK1 inhibitors in combination decrease EdU incorporation in U2OS cells. U2OS cells were treated for 24 hr with the indicated doses. Images were taken with a confocal microscope and analyzed using ImageJ software. A mean intensity of ≥80 a.u. per cell was considered as EdU-positive cells. Quantitative data: n = 3, mean ± SEM. (D) No significant decrease in EdU incorporation in normal fibroblast VH-10 cells treated with the ATR and CHK1 inhibitors either alone or in combination. VH-10 cells were treated for 24 hr with the indicated doses. Quantitative data: n = 3, mean ± SEM. Statistical significance was determined using one-way ANOVA ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Staining, Flow Cytometry, Software, Microscopy

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: Synergistic Cytotoxic Effect in U2OS Cancer Cells by Combination Treatment of AZD7762/VE-821 Is Mainly Due to CDK-Mediated Excess Origin Firing (A) U2OS cells were pretreated with the indicated concentrations of the CDK inhibitor Roscovitine for 1 hr prior to the addition of VE-821 and AZD7762 for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody and anti-53BP1, and DNA was counterstained with ToPro. (B) Quantitative data of pan-nuclear γH2AX are shown (mean ± SEM from two independent experiments). (C) Treatment regimen for DNA fiber assay in U2OS cells is shown. (D) CDK inhibitors Roscovitine and PHA-767491 enhance the replication fork speed of U2OS cells treated with VE-821 and AZD7762 in combination. U2OS cells were pre-treated with Roscovitine or PHA-767491 for 1 hr prior to the addition of VE-821 and AZD7762. Representative images show stained replication fork tracts for each treatment group. (E) Average distribution of replication fork rates. A minimum of 450 forks per condition were analyzed from at least three independent repeats. (F) Roscovitine abolishes the synergistic cytotoxic effect of combination treatment of AZD7762 and VE-821 in U2OS cancer cells. U2OS cells were individually treated with VE-821, AZD7762, or the combination with our without Roscovitine for 24 hr, followed by recovery for another 48 hr. Cell viability was measured by using resazurin at 72 hr. (G) Model for ATR/CHK1 synthetic lethality. CHK1 is activated by replication stress both by ATR-dependent and -independent pathways ( Yang et al., 2008 ) to suppress replication stress in cancer, promoting restart and survival. CHK1 inhibitors increase oncogene-activated CDK activity and origin firing, leading to replication stress and accumulation of stalled replication forks, requiring ATR activity to prevent replication collapse. Red arrows indicate primary route in the presence of both ATR and CHK1 inhibitors.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Staining, Activity Assay

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: HU-Induced Replication Stress in Combination with VE-821 and AZD7762 Causes Fragmented Nuclei and the Early Onset of Apoptosis Only in U2OS Cells (A) U2OS cells were treated for 24 hr with the indicated drug concentrations and stained with anti-cleaved caspase 3 and β-actin antibodies. Representative confocal images are shown. (B) Quantitative data of fragmented nuclei and cleaved caspase-3 positive cells presented as mean ± SEM from three independent experiments. (C) Western blot showing apoptosis in U2OS cells treated with ATR and CHK1 inhibitors alone or in combination. U2OS cells were treated with the indicated concentrations for 24 hr; lysed; protein extracted; and western blotting was performed with anti-Cleaved PARP, anti-phospho (Serine 10) Histone H3, and anti-β-actin antibodies. (D) HU-induced replication stress does not cause fragmentation of nuclei or apoptosis in combination with VE-821 and AZD7762 in VH-10 normal fibroblast cells in 24 hr. Etoposide treatment (4 μM) was used to induce apoptosis as a positive control. Scale bar represents 20 μM.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Staining, Western Blot, Positive Control

Journal: Frontiers in Pharmacology

Article Title: USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

doi: 10.3389/fphar.2017.00274

Figure Lengend Snippet: USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. (A) Comet assay was performed to assess DNA damage after 0.33 μM cisplatin treatment for 6 h in A549-WT, A549-USP22, and A549/CDDP cells (scale bar 20 μM). (B,C) H2A interacts with USP22 in A549-WT cells. A549-WT cells lysates were subjected to immunoprecipitation (IP) with control IgG, anti-USP22 (B) , and anti-H2A (C) antibodies. The immunoprecipitates were then blotted with the indicated antibodies. (D) H2A binding to USP22, γH2AX, and the ubiquitination of H2A (Lys119) was detected by IP and western blot after 0.33 μM cisplatin treatment for 48 h in A549-WT, A549/CDDP and A549-USP22 cells. (E) Western blot analysis of the proteins of USP22, γH2AX and Ubi-H2A after various concentrations of cisplatin treatment for 48 h. Every experiment was conducted at least three times, and the average is shown (mean ± SD). ∗ P < 0.05, ∗∗ P < 0.01, significant.

Article Snippet: Membranes were blocked in a buffer (TBS: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 5% bovine serum albumin and 0.1% Tween-20, followed by incubation with the primary antibodys USP22 (ab4812, 1:2000, Abcam, Cambridge, UK), Sirt1 (2496, 1:2000, CST, United States), H2AX (2595, 1:1000, CST, United States), γ-H2AX (Ser 139)(9718, 1:1000, CST, United States), Ubi-H2A (Lys119) (8240, 1:1000, CST, United States), Ku70 (10723-1-AP, 1:1000, Proteintech, United States), Bax (50599-2-lg, 1:1000, Proteintech, United States), cytochrome C (10993-1-AP, 1:1000, Proteintech, United States) or GAPDH (10494-1-AP, 1:3000, Proteintech, United States).

Techniques: Phospho-proteomics, Single Cell Gel Electrophoresis, Immunoprecipitation, Control, Binding Assay, Ubiquitin Proteomics, Western Blot