Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) HEK293 cells were treated with 0.5 µM doxorubicin (DOX) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32, and α-tubulin. ( B ) HEK293 cells were treated with 10 mM hydroxyurea (HU) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32, and α-tubulin. ( C ) HEK293 cells were treated with 10 nM camptothecin (CPT) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32, and α-tubulin. ( D ) HeLa cells were treated with 10 mM HU, and incubated as indicated. Cell lysates were collected and analyzed by immunoblotting for MASTL, phospho-H2AX Ser-139, RPA32, phospho-ATM/ATR substrates, and α-tubulin. ( E ) HeLa cells were treated with or without 0.5 µM DOX, 10 nM CPT, 10 mM HU, and 20 Gy ionizing radiation (IR) for 4 hr. Cell lysates were collected and analyzed by immunoblotting for MASTL, phospho-H2AX Ser-139, phospho-ATM/ATR substrates, and α-tubulin. ( F ) HeLa cells were treated with 0.5 μM DOX, and analyzed by immunoblotting for MASTL, α-tubulin, Aurora A, Aurora B, CDK1, cyclin B1, and phospho-ATM/ATR substrates. ( G ) SCC38 cells were incubated with or without HU and caffeine, as indicated, and analyzed by immunoblotting for MASTL and β-actin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Western Blot, Incubation

Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A–C ) HeLa cells expressing CFP-MASTL were treated without or with ionizing radiation (IR). Cells were analyzed by direct fluorescence for CFP-MASTL expression, and by immunofluorescence for the phosphorylation of ATM/ATR substrates. Quantifications are shown in panels B and C (>10 cells/time point). ( D, E ) HEK293 cells were treated without ( D ) or with ( E ) 10 mM hydroxyurea (HU) for 2 hr. These cells were then treated with cycloheximide (CHX, 20 μg/ml) at time 0 to block protein synthesis, and analyzed by immunoblotting for the protein stability of MASTL and α-tubulin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Expressing, Fluorescence, Immunofluorescence, Blocking Assay, Western Blot

Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) The sequence alignment of the conserved E6AP Ser-218 motif in human, mouse, and Xenopus . ( B ) A phospho-specific antibody recognizing E6AP Ser-218 was generated, as described in Materials and methods. WT or E6AP knockout (KO) HEK293 cells were treated without or with 10 mM hydroxyurea (HU) for 4 hr, and analyzed by immunoblotting for phospho-E6AP Ser-218, E6AP, and α-tubulin. ( C ) HeLa cells were treated without or with 0.5 μM doxorubicin (DOX) and 5 μM KU55933 (ATMi), as indicated, for 1 hr, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-E6AP Ser-218, and α-tubulin. ( D ) HeLa cells were transfected with HA-tagged WT, S218A, or S218D E6AP. Cell lysates were harvested for immunoprecipitation (IP) assays. The input, MASTL IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL and HA. ( E ) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX for 3 hr, and harvested for IP assays. The input, HA IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL, phospho-E6AP Ser-218, and HA. ( F ) E6AP KO HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX, incubated as indicated, and harvested for immunoblotting for MASTL and α-tubulin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Sequencing, Generated, Knock-Out, Western Blot, Transfection, Immunoprecipitation, Incubation

Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) WT or E6AP knockout HEK293 cells were treated without or with doxorubicin (DOX, 0.5 μM) for 4 hr. Cells were harvested and analyzed by immunoblotting for phospho-E6AP Ser-218 and α-tubulin. ( B ) HEK293 cells were treated without or with DOX (0.5 μM), or ATM inhibitor (KU55933, 10 μM), for 4 hr, and analyzed by immunoblotting. ( C ) HeLa cells were treated with hydroxyurea (HU, 10 mM) combined with ATM/ATR inhibitor (caffeine, 4 mM) or ATM inhibitor (KU55933, 10 μM), for 12 hr, as indicated. Cells were harvested and analyzed by immunoblotting.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Knock-Out, Western Blot

Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) E6AP knockout (KO) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in . Cells were treated with 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were then harvested at the indicated time points (after the removal of ETO) for immunofluorescence (IF) using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired two-tailed Student’s t test was used to determine the statistical significance (**p<0.01, n>500 cell numbers/measurement). ( B ) E6AP KO HeLa cells expressing HA-tagged WT or S218A E6AP, as in panel A, were treated with 2 mM hydroxyurea (HU) for 18 hr. Cells were then released in fresh medium, and incubated as indicated, for recovery. Cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS). ( C ) WT or S218A E6AP was expressed in E6AP KO HEK293 cells. Cells were treated without or with 0.1 μM ETO for 18 hr, released in fresh medium for recovery, and incubated as indicated. Cells were analyzed by immunoblotting for phospho-cyclin-dependent kinase (CDK) substrates and histone H3. ( D ) WT or S218A E6AP was expressed in E6AP KO HEK293 cells, as in panel C. Cells were treated without or with 1 μM CPT for 90 min, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, and α-tubulin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Knock-Out, Transfection, Immunofluorescence, Activation Assay, Two Tailed Test, Expressing, Incubation, Fluorescence, FACS, Western Blot

Journal: Cells

Article Title: Investigation of the Antifungal and Anticancer Effects of the Novel Synthesized Thiazolidinedione by Ion-Conductance Microscopy

doi: 10.3390/cells12121666

Figure Lengend Snippet: SICM topography ( 1 ) and a Young’s modulus map ( 2 ) of control cells ( A , D ); cells treated with fluconazole ( B , E ); cells treated with drug L-173 ( C , F ), where PC3 is in ( A – C ) and HEK 293 in ( D – F ). Histogram of PC3 and HEK 293 cell stiffness with the addition of antifungals, SE and (*) p < 0.01 (one-way ANOVA) (“ns” is not significant) ( G ).

Article Snippet: Human prostate adenocarcinoma PC3 and human embryonic kidney HEK 293 cell lines (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (Gibco, Grand Island, NE, USA) containing 1% GlutaMAX (Gibco, USA), 1% Penicillin-Streptomycin (Gibco, USA), 10% fetal bovine serum (Gibco, USA) and maintained in a humidified incubator at 5% CO 2 and 37 °C.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Characterization and Biological Activities of Melanin from the Medicinal Fungi Ophiocordyceps sinensis

doi: 10.3390/ijms241210282

Figure Lengend Snippet: Antioxidant activity of O. sinensis melanin. ( A ) ABTS and DPPH radical scavenging activities. ( B ) The scavenging effect of O. sinensis melanin on intracellular ROS; green fluorescence indicates DCFH-DA labeled ROS. Bar = 20 μm. ( C ) Quantification and statistical analysis of the scavenging effect of O. sinensis melanin on intracellular ROS. ( D ) Preincubation with O. sinensis melanin reduces the apoptosis induced by H 2 O 2 in HEK293 cells. Different lowercase letters indicate significant differences between groups ( p < 0.05).

Article Snippet: Human hepatocellular carcinoma (HepG2) cell line (HB-8065, ATCC), mouse leukemia cells of monocyte-macrophage (RAW264.7) cell line (TIB-71, ATCC), dendritic (DC2.4) cell line (HTX2245, ATCC), human colon cancer (SW620) cell line (CCL-227, ATCC), and human embryonic kidney 293 (HEK293) cell line (CRL-1573, ATCC) were purchased from the American Type Culture Collection and grown in complete growth medium supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C with 5% CO 2 .

Techniques: Antioxidant Activity Assay, Fluorescence, Labeling

Journal: Journal of medical virology

Article Title: Cryptic resolution sites in the vector plasmid lead to the heterogeneities in the rAAV vectors

doi: 10.1002/jmv.28433

Figure Lengend Snippet: Single ITR plasmids reduce rAAV vector yield. Cotransfection of pH22, pFA6, and pssAAV-CB-EGFP with pCI, pCI-ITR-CBD, pCI-ITR or pRB21-ITR at the ratio of 1:1:0.1:0.9 into HEK 293 cells. AAV vector yield was quantified by qPCR 72-h post transfection. Significant differences in vector yield of the other groups versus pCI group denoted by a star flag. ***p < 0.001. qPCR, quantitative polymerase chain reaction.

Article Snippet: Cell lines Human embryonic kidney (HEK) 293 cell line was purchased from ATCC.

Techniques: Plasmid Preparation, Cotransfection, Transfection, Real-time Polymerase Chain Reaction