Journal: Biomedical research (Tokyo, Japan)

Article Title: TBC1D8B, a GTPase-activating protein, is a novel apoptosis inducer.

doi: 10.2220/biomedres.42.95

Figure Lengend Snippet: Fig. 4 Effect of the N-terminal TBC1D8B overexpression on caspase 3 and PARP cleavage. A. Estimation of the number of suspended and adherent cells in culture. The closed bar represents the cells in which pcDNA3.1 plasmid DNA was in- troduced. The hatched column represents cells in which the N-terminal TBC1D8B was overexpressed. B. Collected sus- pended cells as a pellet from the culture medium. C. The effect of the N-terminal TBC1D8B overexpression in cultured HCT116 cells on caspase 3 cleavage. Uncleaved and cleaved caspase 3 status is shown in the top and bottom panel, re- spectively. Note that the images are of the same caspase 3 blotting, but the exposure period of the top panel was much shorter than that of the bottom panel. D. The effect of the N-terminal TBC1D8B overexpression in the cultured HCT116 cells on the PARP cleavage. Uncleaved and cleaved PARP statuses are shown in the top and bottom panels, respectively. Note that the exposure period of the top panel (10 seconds) was much shorter than that of the bottom panel (10 minutes exposure). Red * represents cleaved PARP in the bottom panel.

Article Snippet: Anti-caspase 3 (9662), anti-phospho-eIF2α (eukaryotic initiation factor 2) (Ser51) (3597), anti-eIF2α (5234), anti-α-tubulin (2144), and poly ADP-ribose polymerase (PARP) (9542) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, Plasmid Preparation, Cell Culture

Journal: Science advances

Article Title: Flap endonuclease 1 repairs DNA-protein cross-links via ADP-ribosylation-dependent mechanisms.

doi: 10.1126/sciadv.ads2919

Figure Lengend Snippet: Fig. 6. PARP1 induces FEN1-dependent repair of nonenzymatic and enzymatic DPCs. (A) The modified RADAR assay in HEK293 cells treated with the indicated DNA damaging agents: FA, 400 μM for 2 hours; hydroxyurea (HU), 1 mM for 2 hours; mitomycin C (MMC), 10 μM for 2 hours; cisplatin, 10 μM for 2 hours; ultraviolet (UV), 20 J/ m2; infrared (IR), 20 Gy. Cells were exposed to 10 μM PARGi for 1 hour before cotreatments. (B) The modified RADAR assay in HEK293 cells. PARGi was used at 10 μM for 1-hour pretreatment and then 1-hour cotreatment with 10 μM ETOP. (C) HEK293 cells were transfected with empty vector or FEN1-FLAG overexpression plasmid, followed by the indicated treatments: FA, 400 μM for 2 hours; FEN1i, 10 μM, 1-hour pretreatment +2-hour cotreatment with FA; PARPi, 10 μM, 1-hour pretreatment +2-hour cotreat- ment with FA. Cells were subjected to the modified RADAR assay for the detection of total DPCs. (D) The modified RADAR assay in HEK293 cells transfected with or without FEN1-FLAG expression plasmid for 48 hours before the indicated treatments. PARPi and FEN1i were used at 10 μM for 1-hour pretreatment, followed by cotreatment with ETOP at 10 μM for 1 hour. (E) GFP–proliferating cell nuclear antigen (PCNA) expression plasmid-transfected U2OS cells were treated as indicated: FA, 400 μM for 1 hour; APH, 1 μM, 30-min pretreatment +1-hour cotreatment with FA; PARPi, 10 μM, 1-hour pretreatment +1-hour cotreatment with FA). Cells were then subjected to PLA assay to measure TOP1 and FEN1 interaction. Scale bar, 10 μm. (F) U2OS cells were treated as indicated: ETOP, 10 μM for 1 hour; PARPi, 10 μM, 1-hour pretreatment +1-hour cotreatment with ETOP). PLA assay was used to measure TOP2α and FEN1. Scale bar, 10 μm.

Article Snippet: The following antibodies were used: anti- PAR, mouse monoclonal, Trevigen, 4335- MC- 100; anti- PAR (10H), mouse monoclonal, Enzo Life Sciences, ALX- 804- 220; anti- TOP1, mouse monoclonal, BD Biosciences, 556597; anti–double- stranded DNA (dsDNA), mouse monoclonal, Abcam, ab27156; anti- FLAG, mouse monoclonal, SigmaAldrich, F1804; anti- FLAG, rabbit polyclonal, Sigma- Aldrich, F7425; anti- FEN1, rabbit polyclonal, Cell Signaling Technology, 2746; antiXPF, rabbit monoclonal, 13465; anti- XPG, mouse monoclonal, Santa Cruz Biotechnology, 13563; anti- APE1, rabbit monoclonal, Cell Signaling Technology, 10519; anti- EXO1, rabbit polyclonal, Abcam, 95068, anti- DNA2, rabbit polyclonal, Abcam, 96488; anti- MRE11, mouse monoclonal, GeneTex, 70212; anti- TDP1, rabbit polyclonal, Bethyl Laboratories, A301- 618A; anti- TDP2, mouse monoclonal, Santa Cruz Biotechnology, 377280; anti- PARP1 (F2), mouse monoclonal, Santa Cruz Biotechnology, sc- 8007; anti- PARG, rabbit monoclonal, Cell Signaling Technology, 66564; and anti- γH2AX, rabbit polyclonal, Cell Signaling Technology, 66564; anti- BrdU (5- bromo- 2′- deoxyuridine), mouse monoclonal, ab8152.

Techniques: Modification, Transfection, Plasmid Preparation, Over Expression, Expressing

Journal: Science advances

Article Title: Flap endonuclease 1 repairs DNA-protein cross-links via ADP-ribosylation-dependent mechanisms.

doi: 10.1126/sciadv.ads2919

Figure Lengend Snippet: Fig. 7. ADP-ribosylation of FEN1 at glutamic acid 285 residue localizes FEN1 to DPC sites for repair. (A) Experimental setup for PAR antibody-IP-MS in HEK293 cells. PSM, peptide spectrum match. (B) Representative in vitro PARylation assay with recombinant FEN1 protein, recombinant PARP1 protein, NAD+, and activated DNA. Following 20-min incubation at room temperature, the samples were subjected to Western blotting using anti-PAR and anti-FEN1 antibodies. Anti-FEN1 antibody failed to detect PARylated FEN1 likely because PAR polymers blocked the epitope. (C) Representative activity assay testing unmodified and PARylated recombinant FEN1 toward unmodified and C6-modified DNA substrates for the indicated times. 32P labeled DNA products following the activity assay were visualized by PAGE electrophoresis. (D) Structure of FEN1 with DNA substrate, SM3+ and K+ (PDB: 3q8l) highlighting glutamic acid residue 285 (E285), an ADP-ribosylation site identified by ELTA-MS. (E) GFP-PCNA expressing U2OS cells transfected with FEN1-FLAG WT or FEN1-FLAG E285Q expression plasmid were treated with 400 μM FA for 1 hour. Cells were then subjected to PLA assay to measure TOP1 and FEN1-FLAG interaction using their TOP1 and FLAG antibodies. Scale bar, 10 μm. (F) U2OS cells transfected with FEN1-FLAG WT or FEN1-FLAG E285Q ex- pression plasmid were treated with 10 μM ETOP for 1 hour. Cells were then subjected to PLA assay to measure TOP2α and FEN1-FLAG interaction using TOP2α and FLAG antibodies. Scale bar, 10 μm. (G) HEK293 cells were transfected with empty vector or FEN1-FLAG overexpression plasmid, followed by treatment with 400 μM FA for 2 hours. Cells were subjected to the modified RADAR assay for detection of total DPCs by Coomassie stain. (H) HEK293 cells were transfected with empty vector or indicated FEN1- FLAG overexpression plasmid, followed by treatment with 10 μM ETOP for 30 min. Cells were subjected to the RADAR assay for detection of enzymatic TOP2α-DPCs and DNA using their antibodies.

Article Snippet: The following antibodies were used: anti- PAR, mouse monoclonal, Trevigen, 4335- MC- 100; anti- PAR (10H), mouse monoclonal, Enzo Life Sciences, ALX- 804- 220; anti- TOP1, mouse monoclonal, BD Biosciences, 556597; anti–double- stranded DNA (dsDNA), mouse monoclonal, Abcam, ab27156; anti- FLAG, mouse monoclonal, SigmaAldrich, F1804; anti- FLAG, rabbit polyclonal, Sigma- Aldrich, F7425; anti- FEN1, rabbit polyclonal, Cell Signaling Technology, 2746; antiXPF, rabbit monoclonal, 13465; anti- XPG, mouse monoclonal, Santa Cruz Biotechnology, 13563; anti- APE1, rabbit monoclonal, Cell Signaling Technology, 10519; anti- EXO1, rabbit polyclonal, Abcam, 95068, anti- DNA2, rabbit polyclonal, Abcam, 96488; anti- MRE11, mouse monoclonal, GeneTex, 70212; anti- TDP1, rabbit polyclonal, Bethyl Laboratories, A301- 618A; anti- TDP2, mouse monoclonal, Santa Cruz Biotechnology, 377280; anti- PARP1 (F2), mouse monoclonal, Santa Cruz Biotechnology, sc- 8007; anti- PARG, rabbit monoclonal, Cell Signaling Technology, 66564; and anti- γH2AX, rabbit polyclonal, Cell Signaling Technology, 66564; anti- BrdU (5- bromo- 2′- deoxyuridine), mouse monoclonal, ab8152.

Techniques: Residue, Protein-Protein interactions, In Vitro, Recombinant, Incubation, Western Blot, Activity Assay, Modification, Labeling, Electrophoresis, Expressing, Transfection, Plasmid Preparation, Over Expression, Staining

Journal: Science advances

Article Title: Flap endonuclease 1 repairs DNA-protein cross-links via ADP-ribosylation-dependent mechanisms.

doi: 10.1126/sciadv.ads2919

Figure Lengend Snippet: Fig. 8. Working models depict the role of PARylation in nonenzymatic and enzymatic DPC repair by FEN1. (A) DPCs formed within the 5′-flap of Okazaki fragments during strand displacement are sensed by PARP1 leading to PARylation, which activates FEN1 for repair following dePARylation of the DPCs by PARG. Concurrent damaged bases adjacent to non-end DPCs (in the front of the fork and on the leading strand behind the fork) are converted into DPC-harboring 5′-flaps by the BER proteins with SSBs that can be detected by PARP1 for PARylation to signal FEN1. FEN1 enriched near Okazaki fragments are in PARylated by PARP1 for their translocation to the front of fork and to the leading strand behind the fork to cleave DPC-harboring 5′-flaps following dePARylation by PARG. (B) TOP2α are homodimeric enzymes that can act in front of the replication fork to relax accumulated positive supercoils. The homodimers cut both strands of DNA and pass another DNA molecule through the break. The 5′ end of each break is covalently linked to the tyrosine in the active center of each of the two subunits of the protein (TOP2αcc). In this configuration, the two sides of the nicked DNA are held together by the strong protein-protein interactions between the two subunits of TOP2, allowing the nicks to be faithfully resealed in situ. These transient enzyme-DNA intermediate can be trapped by their inhibitors such as ETOP and DOX. The binding of the inhibitor to the interface between one of the subunits and DNA leads to the formation of a single-stranded 5′ TOP2-DPC, which can be converted to a 5′ flap structure via posttranslational modifications, conformational changes, or polymerase activities. PARP1 senses the TOP2-linked SSB and PARylates the DPC to signal FEN1. FEN1 at Okazaki fragments at the rear of the fork is also PARylated by PARP1, which drives FEN1 to the DPC site for cleavage upon dePARylation by PARG.

Article Snippet: The following antibodies were used: anti- PAR, mouse monoclonal, Trevigen, 4335- MC- 100; anti- PAR (10H), mouse monoclonal, Enzo Life Sciences, ALX- 804- 220; anti- TOP1, mouse monoclonal, BD Biosciences, 556597; anti–double- stranded DNA (dsDNA), mouse monoclonal, Abcam, ab27156; anti- FLAG, mouse monoclonal, SigmaAldrich, F1804; anti- FLAG, rabbit polyclonal, Sigma- Aldrich, F7425; anti- FEN1, rabbit polyclonal, Cell Signaling Technology, 2746; antiXPF, rabbit monoclonal, 13465; anti- XPG, mouse monoclonal, Santa Cruz Biotechnology, 13563; anti- APE1, rabbit monoclonal, Cell Signaling Technology, 10519; anti- EXO1, rabbit polyclonal, Abcam, 95068, anti- DNA2, rabbit polyclonal, Abcam, 96488; anti- MRE11, mouse monoclonal, GeneTex, 70212; anti- TDP1, rabbit polyclonal, Bethyl Laboratories, A301- 618A; anti- TDP2, mouse monoclonal, Santa Cruz Biotechnology, 377280; anti- PARP1 (F2), mouse monoclonal, Santa Cruz Biotechnology, sc- 8007; anti- PARG, rabbit monoclonal, Cell Signaling Technology, 66564; and anti- γH2AX, rabbit polyclonal, Cell Signaling Technology, 66564; anti- BrdU (5- bromo- 2′- deoxyuridine), mouse monoclonal, ab8152.

Techniques: Translocation Assay, Protein-Protein interactions, In Situ, Binding Assay

Journal: Cancers

Article Title: SFN Enhanced the Radiosensitivity of Cervical Cancer Cells via Activating LATS2 and Blocking Rad51/MDC1 Recruitment to DNA Damage Site

doi: 10.3390/cancers14081872

Figure Lengend Snippet: LATS2 suppresses cervical cancer cell proliferation and promotes apoptosis. ( A ) Proliferation of SiHa and C33A with LATS2-overexpressing cells and their respective controls was assessed by DAPI staining and nuclear counting using CellProfiler. ( B ) Cell apoptosis was evaluated by flow cytometric analysis. SiHa and C33A were treated with SFN (20 and 15 μM, respectively) for 48 h, or transfected with LATS2 overexpression plasmid (ex-LATS2) or control vector. The data shown are from a single representative experiment performed in triplicate. ( C ) Western blot analysis of the levels of cleaved caspase-3 and cleaved PARP. Cropped blots were showed in Western blotting. The samples derived from the same experiment and those blots were processed in parallel. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For the antibodies, mouse monoclonal anti-cleaved PARP, rabbit monoclonal anti-cleaved caspase-3, and rabbit monoclonal anti-γ-H2AX monoclonal antibodies (7631) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Staining, Transfection, Over Expression, Plasmid Preparation, Control, Western Blot, Derivative Assay