Journal: Cancer Research

Article Title: NEIL3 Prevents Senescence in Hepatocellular Carcinoma by Repairing Oxidative Lesions at Telomeres during Mitosis

doi: 10.1158/0008-5472.CAN-20-1028

Figure Lengend Snippet: NEIL3 recruits APE1 to TIFs during mitosis. A, Images of NEIL3 foci (red) and telomere (TTAGGG; green) colocalization by anti-NEIL3 IF and telo-FISH in HEP3B cells. Cells were treated with DMSO, 24-hour vincristine (VCR; 20 nmol/L), 24-hour vincristine (20 nmol/L) + 24-hour Reversine (REV; 0.5 μmol/L) or 24-hour RO-3306 (10 μmol/L). Scale bar, 5 μm. B, Quantification of NEIL3 foci–positive cells (top left), TIFs (top right), NEIL3 on telomere (bottom left), and NEIL3-positive TIFs (bottom right) in HEP3B cells. Means ± SEM; at least 200 cells were analyzed. C, Images of NEIL3 foci (red), telomere (TTAGGG, green), and γH2AX foci (cyan) colocalization by anti-NEIL3, PNA telC probe, and anti-γH2AX IF in HEP3B metaphase spread. Cells were treated with 24-hour vincristine (20 nmol/L) or synchronized in 1-hour colcemid (20 ng/mL) with 30 min KBrO3 (5 mmol/L) or DMSO. White arrowheads, NEIL3/telomere/γH2AX colocalization. Scale bar, 5 μm. D, Quantification of relative TIFs (left), NEIL3 on telomere (middle), or NEIL3-positive TIFs (right) in HEP3B metaphase spread. Means ± SEM; at least 20 metaphase cells were analyzed in each group. E, Quantification of APE1 foci on telomere (left) and APE1/NEIL3 colocalization (right) in HEP3B metaphase spread. Cells were treated with 24-hour vincristine (20 nmol/L) or synchronized in 1-hour colcemid (20 ng/mL) with 30-minute KBrO3 (5 mmol/L) or DMSO. Means ± SEM; at least 20 metaphase cells were analyzed in each group. F, Quantification of APE1 on telomere (left) and APE1-positive TIFs (right) after NEIL1, NEIL2, or NEIL3 silencing in HEP3B metaphase spread. Means ± SEM; at least 20 metaphase cells were analyzed in each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Inhibitors used are as follows: vincristine (Sigma), Colcemid (Gibco), RO-3306 (CDK1 inhibitor, MedChemExpress), Reversine (GSK923295, Axon MedChem), APE1 inhibitor (Sigma, CAS 6960–45–8), PARP inhibitor (olaparib, SelleckChem, 763113–22–0).

Techniques:

Journal: Cancer Research

Article Title: NEIL3 Prevents Senescence in Hepatocellular Carcinoma by Repairing Oxidative Lesions at Telomeres during Mitosis

doi: 10.1158/0008-5472.CAN-20-1028

Figure Lengend Snippet: NEIL3 repairs telomere damage through base excision repair in mitotic cells. A, Schematic of mitotic telomere damage repair assay. Cells were treated with 10 μmol/L RO-3306 for 16 hours to synchronize cells in G 2 phase. No damage was induced in group 0 and oxidative damage was induced in groups A–C . Group A represents no repair, group B 1 hour of repair, and group C 1 hour of repair in the presence of inhibitors. DNA repair at telomeres were analyzed by META-FISH. B, Images of APE1 foci (red), telomere PNA probe (TTAGGG, green), and γH2AX (cyan) by anti-APE1, anti-γH2AX IF, and telo-FISH in HEP3B metaphase spread. Cells were treated as indicated in A with 10 μmol/L APE1 inhibitor or 10 μmol/L PARP inhibitor (olaparib). White arrowheads, APE1/telomere/γH2AX colocalization. Scale bar, 5 μm. C, Quantification of TIFs (left) and APE1-positive TIFs (right) in HEP3B metaphase spread described in B . Means ± SEM; at least 20 metaphase cells were analyzed in each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. D, Quantification of TIFs (top) and APE1-positive TIFs (bottom) in HEP3B metaphase spread described in A . Cells were transfected with NT siRNA and siRNA targeting PolB or NEIL3 for 72 hours. Means ± SEM; at least 20 metaphase cells were analyzed in each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, Telomere qPCR of NEIL1 digestion in HEP3B cells transfected with NEIL3 siRNA s2, s4, or nontargeting control (siCtrl) for 72 hours. After transfection, cells were synchronized in G 1 with 2 mmol/L thymidine, followed by synchronization in G 2 with RO-3306. Cells were released into mitosis and treated with 2.5 mmol/L KBrO 3 for 30 minutes, followed by recovery for 60 minutes. Controls are shown with no treatment (untreated) and with no recovery period (no repair). Values were normalized to untreated siCtrl and represent the mean ± SEM from three experiments. ns, nonsignificant; **, P < 0.01, one-way ANOVA analysis.

Article Snippet: Inhibitors used are as follows: vincristine (Sigma), Colcemid (Gibco), RO-3306 (CDK1 inhibitor, MedChemExpress), Reversine (GSK923295, Axon MedChem), APE1 inhibitor (Sigma, CAS 6960–45–8), PARP inhibitor (olaparib, SelleckChem, 763113–22–0).

Techniques: Transfection, Control

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Relative levels of intercellular and intracellular cyclins. (A) A representative example of the immunoblotting analysis showing the relative levels of mitotic cyclins in HeLa and RPE1 cells. Different concentrations of plasmids expressing AID-cyclin A, B1, or B2 were transfected into HeLa cells to serve as standards. Lysates from asynchronous (Asy) and G 2 HeLa and RPE1 cells were loaded. The samples were analyzed with antibodies against mAID or individual cyclins. (B) RPE1 cells contain relatively lower concentrations of B-type cyclins compared to HeLa cells. HeLa and RPE1 cells from asynchronous (Asy) and RO3306-treated (G 2 ) populations were analyzed with flow cytometry or immunoblotting. (C) Comparison of the expression of mitotic cyclins in HeLa and RPE1 cells. The relative expression of cyclin A, B1, and B2 in asynchronous (Asy) and G 2 lysates was determined using the respective AID-cyclin standards. The expression level of each cyclin was normalized to that cyclin in asynchronous HeLa cells. Mean of two independent experiments. (D) B-type cyclins are more abundant than cyclin A. The relative expression of cyclin A, B1, and B2 in asynchronous (Asy) and G 2 lysates was determined using the respective AID-cyclin standards. The relative levels between each AID-cyclin were first determined using an antibody against AID. The expression level of endogenous cyclins was normalized to that of cyclin B1 in asynchronous HeLa (upper panel) or RPE1 (lower panel). Mean of two independent experiments.

Article Snippet: Unless specified, cells were treated with the following reagents at the indicated final concentrations: AZ3146 (Selleck Chemicals; 2 μM), blasticidin (2.5 μg/ml; Thermo Fisher Scientific), doxycycline (Dox) (2 μg/ml; Sigma-Aldrich), hygromycin B (0.25 mg/ml; Thermo Fisher Scientific), IAA (50 μg/ml; Sigma-Aldrich), NOC (100 ng/ml; Sigma-Aldrich), puromycin (0.3 μg/ml; Sigma-Aldrich), RO3306 (10 μM for HeLa cells and 5 μM for RPE1 cells; Santa Cruz Biotechnology), and thymidine (2 mM; Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Transfection, Flow Cytometry, Comparison