Journal: Nucleic acids research

Article Title: The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress.

doi: 10.1093/nar/gku152

Figure Lengend Snippet: Figure 4. Analysis of DNA DSBs during recovery from UV irradiation in quiescent control and TK2 knock-down cells. LacZ control and TK2 knock-down cells were serum deprived for 10 days. After 10 J/M2 whole-cell UV irradiation and recovery at the indicated time, cells were (A) fixed for 53BP1 IF staining (Scale bar, 10 mm) and (B) analysed by neutral comet assay. Each cell containing 53BP1 foci 30 was counted and expressed as percentage. More than 100 cells were analysed for each 53BP1 foci experiment (n = 3) and 50 cells for each comet assay (n = 3). (C) Co-IF staining of 53BP1 and g-H2AX was performed at the indicated recovery time in TK2 knock-down cells (Scale bar, 10 mm). (D) At 12-h recovery, 5 mM NU7441 was added in the culture medium. Cells were fixed for 53BP1 IF staining at indicated time and analysed as described in (A) (Scale bar, 10 mm). RT-PCR of RNA extracted from indicated cells was shown. *P < 0.05, **P < 0.01, ***P < 0.001 based on Student’s t-test.

Article Snippet: Anti-human TS antibody (clone 4H4B1) was obtained from Zymed laboratories Inc. Anti-R1 (T16), anti-R2 (N18), anti-p53R2 (N16), antiPCNA (PC10), anti-53BP1 (H-300), horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit, donkey anti-goat antibodies and NU7441 (8-Dibenzo [b,d]thiophen-4-yl-2-morpholin-4-yl-4H-chromen-4-one) were from Santa Cruz.

Techniques: Irradiation, Control, Knockdown, Staining, Neutral Comet Assay, Single Cell Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: DNA-PKcs restricts Zika virus spreading and is required for effective antiviral response

doi: 10.3389/fimmu.2022.1042463

Figure Lengend Snippet: DNA-PKcs is critical for control of ZIKV infection. Virus replication measured by plaque assay, expressed as plaque-forming units per mL (PFU/mL), on (A) A549 WT and A549 PRKDC-/- or (B) RPE WT and RPE PRKDC-/- cells infected with ZIKV at indicated m.o.i. and time. RT-qPCR analysis to measure ZIKV RNA in (C) A549 WT and A549 PRKDC-/- or (D) RPE WT and RPE PRKDC-/- cells infected at indicated m.o.i. and time. (E) A549 WT and A549 PRKDC-/- or (F) RPE WT and RPE PRKDC-/- cells infected with 50 PFU of ZIKV at 48 hours in semi-solid medium, then ZIKV-E protein (green) was stained for immunofluorescence analysis, and the relative area of infection percentage was measured using the ImageJ software. The cell nuclei were stained with DAPI (blue). (G) Percentage of ZIKV-infected A549 WT and A549 PRKDC-/- cells at indicated m.o.i. and time, analyzed by flow cytometry. (H) Viability analysis by MTT assay of ZIKV-infected A549 WT and A549 PRKDC-/- cells relative to uninfected cells (mock) at indicated m.o.i. and time. (I) A549 WT and A549 PRKDC-/- were pretreated with NU7441 (0.5 and 1 µM) at 24 hours followed by ZIKV infection (m.o.i. 1) at 24 hours. We used two-way ANOVA with Sidak’s correction in (A–D, G, H) , and unpaired two-tailed Student’s t-test was used in (E, F) . * p<0.05, n = 3, error bars ± SEM.

Article Snippet: Cells were treated with 0.5 or 1 μM of NU7441 (BioGems, 5039598), 100 ng/mL human TNF (Peprotech, 300-01A), and 3 μM etoposide (Sigma, E1383).

Techniques: Control, Infection, Virus, Plaque Assay, Quantitative RT-PCR, Staining, Immunofluorescence, Software, Flow Cytometry, MTT Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: DNA-PKcs restricts Zika virus spreading and is required for effective antiviral response

doi: 10.3389/fimmu.2022.1042463

Figure Lengend Snippet: ZIKV infection does not induce DSB in A549 cells. A549 WT , A549 PRKDC-/- and 1 µM NU7441 pre-treated A549 WT infected with ZIKV (m.o.i. 1) at 24 hours. Stimulation with 3 µM etoposide for 12 hours was used as a DSB positive control. (A) Immunofluorescence to analyze γH2AX (green) in the ZIKV-infected cells (red, ZIKV-E protein). The cell nuclei were stained with DAPI (blue). (B) Percentage of γH2AX foci per cell showed in (A) . *Compared with WT cells; #Compared with mock. We used two-way ANOVA with Sidak’s correction. * or # p<0.05, n = 3, error bars ± SEM.

Article Snippet: Cells were treated with 0.5 or 1 μM of NU7441 (BioGems, 5039598), 100 ng/mL human TNF (Peprotech, 300-01A), and 3 μM etoposide (Sigma, E1383).

Techniques: Infection, Positive Control, Immunofluorescence, Staining

Journal: Journal of Radiation Research

Article Title: Expression of the DNA-dependent protein kinase catalytic subunit is associated with the radiosensitivity of human thyroid cancer cell lines

doi: 10.1093/jrr/rry097

Figure Lengend Snippet: Surviving fraction of non-treated and inhibitor-treated thyroid cancer cells. γ-ray sensitivity was measured using the colony-forming assay. (A) Non-treated control, (B) 5 μM NU7441, (C) 20 μM Wortmannin. The symbols used are: open square, filled square = FRO; open triangle (point down), filled triangle (point down) = WRO; open triangle (point up), filled triangle (point up) = TPC-1; open circle, filled circle = KTC-1; open diamond, filled diamond = KTC-2; filled star = primary thyrocyte. Open symbol indicates inhibitor-treated thyroid cancer cells; filled symbol indicates non-treated thyroid cancer cells. The γ-ray dose resulting in 10% survival (D 10 ) was estimated from the survival curves. The data is the average of two or three independent experiments.

Article Snippet: To inhibit DNA-PK activity, we used 5 μM NU7441 (a specific inhibitor of DNA-PK, AdooQ BioScience LLC, Irvine, CA, USA) [ , ] and 20 μM wortmannin (an inhibitor of PI-3 kinase) [ ].

Techniques: Control

Journal: Journal of Radiation Research

Article Title: Expression of the DNA-dependent protein kinase catalytic subunit is associated with the radiosensitivity of human thyroid cancer cell lines

doi: 10.1093/jrr/rry097

Figure Lengend Snippet: D 10 and plating efficiency of thyroid cancer cells treated with or without NU7441

Article Snippet: To inhibit DNA-PK activity, we used 5 μM NU7441 (a specific inhibitor of DNA-PK, AdooQ BioScience LLC, Irvine, CA, USA) [ , ] and 20 μM wortmannin (an inhibitor of PI-3 kinase) [ ].

Techniques: