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 participates in DNA damage response. A, Schematics of the TRIM33 constructs showing relevant domains and the mutant constructs used. B, HeLa cells were treated with UV laser scissors and processed for IF using antibodies to γH2AX (green) and TRIM33 (red) at the times indicated. C, quantitation of TRIM33 dynamics at the site of UV laser-induced DNA breaks (mean ± S.D. of at least 20 cells). D, HeLa cells transfected with FLAG-WtTRIM33, TRIM33CA, TRIM33ΔPHD, TRIM33 PHD(AAA), or TRIM33ΔBromo were treated with UV laser scissors and, after 10 min, processed for IF using antibodies to FLAG and γH2AX. E, quantitation of TRIM33 constructs at UV laser-induced DNA breaks (mean ± S.D. of at least 20 cells).

Article Snippet: 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: Construct, Mutagenesis, Quantitation Assay, 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 knockdown results in DNA damage sensitivity. A, HeLa cells treated with control shRNA, TRIM33 shRNA1, TRIM33 shRNA2, and TRIM33 siRNA were exposed to increasing concentrations of bleomycin. Relative cell counts measured by MTS assay, normalized to no treatment, were performed on day 3 and were plotted. p < 0.005 for control versus TRIM33 shRNA or siRNA. Relative expression of TRIM33 and tubulin is shown. B, HeLa cells treated with control shRNA, TRIM33 shRNA, or TRIM33 shRNA cells complemented with WtTRIM33 were exposed to increasing concentrations of bleomycin, and relative cell counts were measured as above. The Western blot analysis shows levels of TRIM33 and tubulin. C, whole cell extracts from control and TRIM33 sh2-treated cells were processed for Western blotting using antibodies to the indicated proteins. D, TRIM33 localization to DNA damage is not dependent on ATM, ATR, or DNA-dependent protein kinase (DNApk). HeLa cells treated with vehicle or ATM inhibitor (ATMi) (KU-55933), GM18366 (ATR mutant), and M059J (DNApk−/−) cells were subject to laser scissor-induced DNA damage. Cells were fixed after 10 min and processed for IF using antibodies to γH2AX (green) and TRIM33 (red). E, quantitation of TRIM33 at sites of DNA damage is shown. Each data point is the mean ± S.D. of at least 20 cells.

Article Snippet: 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: shRNA, MTS Assay, Expressing, Western Blot, Mutagenesis, Quantitation Assay

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: 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 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: 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: 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: PLoS ONE

Article Title: A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids

doi: 10.1371/journal.pone.0154610

Figure Lengend Snippet: (A) Bar graph of the measurement of Ki67 positive cells, showing the fraction of cycling Calu-3 cells after static and dynamic suspension culture (*: p<0.05 vs static suspension). (B) Bar graph of the measurement of γH2AX positive cells, quantifying the double DNA strand breaks in Calu-3 cells harvested from static and dynamic suspension culture.

Article Snippet: Cell spots were stained by anti-Ki67 (Ki67, mouse monoclonal, DAKO, Italy) and anti-gamma histone H2AX (γH2AX, rabbit polyclonal, Bethyl Laboratories, TX, USA) antibodies and revealed by DAB (3,3’-diaminobenzidine) Peroxidase (HRP) Substrate Kit reaction (DAKO, Italy).

Techniques: Suspension

Journal: Oncology Letters

Article Title: Liver kinase B1 enhances chemoresistance to gemcitabine in breast cancer MDA-MB-231 cells

doi: 10.3892/ol.2014.2446

Figure Lengend Snippet: LKB1 overexpression alleviates gemcitabine-induced DNA damage possibly by affecting the ATM-CHK2 pathway. (A) Negative and positive examples of γH2AX foci in the immunofluorescence assay. (B) Positive rates of γH2AX foci in cells. Following gemcitabine treatment (1 μM; 24 h), three time points (0, 12 and 24 h) were selected to perform the immunofluorescence assay. Three fields were randomly selected for each coverslip and positive rates were calculated. LKB1 decreased positive rates of γH2AX foci. Each independent experiment was performed three times and data are presented as the mean ± SD. (C) γH2AX expression in normal conditions was analyzed by western blot analysis. LKB1 increased γH2AX expression. (D) Expression of DNA damage-associated proteins (p-ATR, -CHK1, -ATR, -CHK2 and γH2AX) 6 and 24 h following gemcitabine treatment (1 μM; 24 h), respectively. LKB1 affected the expression of p-ATR, p-CHK2 and γH2AX in the 24 h group. ( * P< 0.05, ** P<0.01 and *** P<0.001). LKB1, liver kinase B1; p, phosphorylated; WT, wild-type.

Article Snippet: The film was then incubated with blocking solution containing 5% bovine serum albumin [BSA; Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) in Tris-buffered saline with Tween 20 [Sangon Biotech (Shanghai) Co., Ltd.] at room temperature for 1 h. Subsequently, the film was immunoblotted with polyclonal rabbit anti-human LKB1 (Calbiochem, Dormstadt, Gemany), monoclonal mouse anti-human ribonucleotide reductase M1 (RRM1; Abcam, Hong Kong, China), monoclonal rabbit anti-human phosphorylated (p)-ATR, monoclonal rabbit anti-human p-CHK1, monoclonal rabbit anti-human p-ATR, monoclonal rabbit anti-human p-CHK2 and monoclonal mouse anti-human γH2AX (Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies, or monoclonal mouse anti-human GAPDH antibody (Sigma-Aldrich).

Techniques: Over Expression, Immunofluorescence, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: The role of TGFBI (βig-H3) in gastrointestinal tract tumorigenesis

doi: 10.1186/s12943-015-0335-z

Figure Lengend Snippet: TGFBI promotes liver cell survival and proliferation after irradiation. (A) Aml-12 liver cells overexpressed TGFBI after stable transfection with the TGFBI-expressing construct pCEP4-TGFBI. TGFBI mRNA levels of transfected Aml-12 liver cells were measured by RT-qPCR. β-actin mRNA levels served as internal controls, and data are expressed as ratios of TGFBI versus β-actin signals (left panel). The supernatants of these cells were quantified by ELISA in duplicate for TGFBI protein levels; regular culture medium was included as an additional control (right panel). Means ± SD of representative experiments are shown. (B) TGFBI promotes post-irradiation survival of liver cells carrying DNA damage. TGFBI-overexpressing Aml-12 and control cells were irradiated at 20 Gy and cultured for 20 h. They were then stained with Abs against γH2AX and caspase-3, and analyzed by flow cytometry. The percentages of caspase-3-positive cells among γH2AX-positive cells are presented (left), and the percentages of 3 independent experiments are summarized in the bar graph (right), with p -value indicated (2-tailed Student’s t test). (C) TGFBI promotes post-irradiation proliferation of liver cells. TGFBI-overexpressing Aml-12 and control cells after irradiation were cultured in complete DMEM/F12 medium containing 10% FCS for 40 h. 3 H-thymidine was added 16 h before the cells were harvested. Thymidine uptake of both irradiated and non-irradiated cells in a representative experiment is shown on the left. Post-irradiation proliferation indices of 3 independent experiments are summarized in a bar graph on the right with p -value indicated (2-tailed Student’s t test).

Article Snippet: They were first fixed and permeabilized using the eBioscience Fixation/Permeabilization Kit (eBioscience, San Diego, CA), and then stained with PE-conjugated rabbit anti-mouse phospho-histone H2AX (γH2AX) Ab and Alexa 488-conjugated rabbit anti-mouse cleaved caspase-3 Ab (Cell Signaling Technology).

Techniques: Irradiation, Stable Transfection, Expressing, Construct, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Flow Cytometry

Journal: Gene Therapy

Article Title: Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer

doi: 10.1038/gt.2016.5

Figure Lengend Snippet: Functional assays were used to measure viral NIS expression. ( a ) Representative confocal images of the effect of GLV-1h153 infection (VV), 2 Gy external beam radiotherapy (2 Gy RT), Combined GLV-1h153 infection and radioiodide treatment (VV+ 131 I), radioiodide treatment alone ( 131 I) and combined GLV-1h153 and 2 Gy external beam radiotherapy (2 Gy RT+VV) on DNA double-strand breaks measured by γH2Ax foci in WPMY (Top) and DU145 (Middle) and PC3 (Bottom) cells. Blue: DAPI, Green: Viral GFP, White: H2Ax foci. White arrows mark the non-infected ‘bystander' cells that have received DNA damage. ( b ) Quantification of confocal images examining the distribution of H2Ax foci among subsets of cells. Untreated control ( c ), radioiodide treated ( 131 I), 2 Gy external beam radiotherapy (RT), GLV-1h153 infection (VV) and combinations thereof. s.e.s are shown. Significance is the result of two-Way ANOVA with Sidak's multiple comparisons test, * P <0.05, ** P <0.001, *** P <0.0001. ( c ) Radioiodide uptake following GLV-1h153 infection at a range of multiplicities of infection (MOI), with and without the influence of external beam radiation (5 Gy) and potassium perchlorate (KClO 4 ). s.e.m.s are shown. Significance is the result of two-Way ANOVA with Bonferroni multiple comparisons test, * P <0.05, ** P <0.001, *** P <0.0001.

Article Snippet: Permeabilised with 0.2% Triton X-100 in PBS for 20 min. H2aX foci were detected by a 1:200 dilution of rabbit anti-γH2aX primary antibody (Cell signaling) incubated at 4 °C overnight and Alexafluor-546 conjugated secondary antibody (Life Technologies) diluted 1:1000 at room temperature for 90 min. DNA was stained with 1:10 000 dilution of DAPI (Life technologies).

Techniques: Functional Assay, Expressing, Infection, Control

Journal: Gene Therapy

Article Title: Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer

doi: 10.1038/gt.2016.5

Figure Lengend Snippet: Mechanisms of cell death in cells treated with GLV-1h153 and radiation. ( a ) Caspase-3/7 activity 48 h post treatment. s.e.m.s are shown. Significance is the result of two-way ANOVA with Bonferroni multiple comparisons test, * P <0.05, *** P <0.0001. ( b ) Activation of apoptosis by cleavage of caspase-3 and phosphorylation of JNK in response to external beam radiation and GLV-1h153 infection at MOIs of 0, 0.01, 0.1 and 1. ( c ) Induction of apoptosis in response to GLV-1h153 infection (MOI 0.1), external beam radiotherapy (2 Gy), 131 I (5 μCi) and combinations thereof, measured by γH2Ax expression, caspase-3 cleavage and PARP-1 cleavage.

Article Snippet: Permeabilised with 0.2% Triton X-100 in PBS for 20 min. H2aX foci were detected by a 1:200 dilution of rabbit anti-γH2aX primary antibody (Cell signaling) incubated at 4 °C overnight and Alexafluor-546 conjugated secondary antibody (Life Technologies) diluted 1:1000 at room temperature for 90 min. DNA was stained with 1:10 000 dilution of DAPI (Life technologies).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Infection, Expressing

Journal: Gene Therapy

Article Title: Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer

doi: 10.1038/gt.2016.5

Figure Lengend Snippet: Efficacy of GLV-1h153 in TRAMP models. ( a ) Confocal images of H2Ax foci resulting from DNA double-strand breaks in TRAMP cells treated with GLV-1h153 and 131 I. Blue: DAPI, Green: Viral GFP, White: γH2Ax foci. White arrows mark the non-infected ‘bystander' cells that have received DNA damage. ( b ) TRAMP cells treated with GLV-1h153 and external beam radiation. Reduction of proliferative capability was measured by MTT assay at 48 h post infection. s.e.m.s are shown. Significance is the result of two-way ANOVA with Bonferroni multiple comparisons test, * P <0.05, ** P <0.001, *** P <0.0001. ( c ) Long-term survival of TRAMP mice bearing spontaneous prostate tumors and treated intravenously with 5 × 10 7 PFU GLV-1h153 (VV-NIS) and 1 mCi 131 I (VV-NIS+ 131 I) ( n =4). Kaplan–Meier plot significance is the result of log-rank (Mantel Cox) test. ( d ) Representative examples of the histology of the above TRAMP mouse prostates, showing H&E staining and IHC for KI67 and probasin at the time of death.

Article Snippet: Permeabilised with 0.2% Triton X-100 in PBS for 20 min. H2aX foci were detected by a 1:200 dilution of rabbit anti-γH2aX primary antibody (Cell signaling) incubated at 4 °C overnight and Alexafluor-546 conjugated secondary antibody (Life Technologies) diluted 1:1000 at room temperature for 90 min. DNA was stained with 1:10 000 dilution of DAPI (Life technologies).

Techniques: Infection, MTT Assay, Staining