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 (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 1. Isolation of ALC1 from 1q21 amplicon. (A) Amplification of different regions along chromosome 1q in 60 HCC cases was detected by FISH. The number of cases amplified is indicated above each bar. (B) FISH analysis showed that the amplified microdissected DNA probe was specifically hybridized to normal chromosome 1q21 (red signals). This microdissected DNA was used for cDNA selection from an HCC case with 1q21 amplification. (C) ALC1 was mapped to 1q21 by FISH with a BAC clone containing ALC1 (indicated by arrows). (D) A representative exam- ple of ALC1 gene amplification detected in H-4 cells by FISH with the BAC probe (red signals). A BAC probe from 1p32 (green signals) was used as a control. (E) ALC1 was cloned to pEGFP vector. The exogenously expressed ALC1-EGFP fusion protein (green color) was sublocalized in the nucleus. (F) Compared to CHD1, the predicted protein structure of ALC1 also has SNF2_N and HELICc domains.

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Isolation, Amplification, Selection, Control, Clone Assay, Plasmid Preparation

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 2. Overexpression of ALC1 in primary HCCs and HCC cell lines. (A) A representative example of ALC1 amplification in HCC TMA detected by FISH with the BAC clone contain- ing ALC1. (B) A 98-kD protein was detected by anti-ALC1 antibody. (C) Positive nuclear staining of ALC1 was frequently detected in primary HCC (left) but not in its matched adjacent nontumor tissue specimen (right) by IHC. (D) The IHC result in TMA was verified on a larger tissue section containing HCC tissue (upper part) and surrounding nontumor liver tissue (lower part). RNA expression of ALC1 was tested in (E) primary HCC cases and(F) HCC cell lines by north- ern blot analysis. In primary HCC, ALC1 expression was compared be- tween tumors (T) and their matched nontumor liver tissues (N).

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Over Expression, Staining, RNA Expression, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 3. Oncogenic ability of ALC1. (A) Expression of ALC1 in ALC1- transfected LO2 and QGY-7703 cells detected by northern blot hybrid- ization. Blank vector–transfected cells were used as controls. (B) Rates of colony formation in soft agar detected in ALC1-transfected and blank vector–transfected LO2 and QGY-7703 cells (**P 0.05). (C,D) Rep- resentative examples of tumors formed in nude mice following injection of ALC1-expressing LO2 cells (left) and QGY-7703 cells (right). ALC1- expressing cells and mock cells were injected into the right and left dorsal flanks, respectively. (E) Flow cytometry histogram showing that overex- pression of ALC1 in ALC1-expressing QGY-7703 cells could promote G1/S phase transition compared to vector-transfected QGY-7703 cells.

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Expressing, Transfection, Northern Blot, Plasmid Preparation, Injection, Flow Cytometry, Sublimation

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 4. Silencing ALC1 expression by siRNA. (A) Two siRNAs (ALC1-si1 and ALC1-si2) could efficiently reduce the expression of ALC1 in H2-M cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. (B) Colony formation ability in soft agar was decreased significantly in siRNA-treated H2-M cells (**P 0.05). (C) Flow cytometry analysis showed that ALC1-si1 could inhibit the cell cycle at the G1/S checkpoint. The percentage of cells in the S phase was decreased from 35% to 23.7%. (D) Western blot analyses indicated that p53 and p21Waf1/Cip1 were down-regulated, whereas cyclin E and Cdk2 were up-regulated in ALC1-transfected QGY-7703 cells in comparison with vector-transfected QGY-7703 cells. -Actin was used as a loading control. (E) Western blot results were quantified by densitometry, and data are presented as mean standard error (n 3). Fold values were first normalized with actin and then compared with vector-transfected QGY-7703 cells.

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Expressing, Control, Flow Cytometry, Western Blot, Transfection, Comparison, Plasmid Preparation

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 5. The inhibition role of ALC1 in apoptosis. (A) Representative figures of TUNEL staining images. After cells were treated with STS for 4 hours, more apoptotic cells (bright white) were detected in vector- transfected QGY-7703 cells in comparison with ALC1-transfected 7703 cells. (B) Detection of the apoptotic index between ALC1-transfected and vector-transfected QGY-7703 cells before and after STS treatment (**P 0.05). The data showed that ALC1-transfected QGY-7703 cells could resist STS-induced apoptosis in comparison with QGY-7703 only. (C) Expressions of caspase 3 and Bax were compared between ALC1- transfected and vector-transfected QGY-7703 cells before and after STS treatment by western blot analyses. -Actin was used as a loading control. (D) Protein levels of caspase 3 and Bax were quantified by densitometry, and data are shown as mean standard error (n 3).

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Inhibition, TUNEL Assay, Staining, Plasmid Preparation, Transfection, Comparison, Western Blot, Control