Journal: Oncotarget

Article Title: DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response

doi:

Figure Lengend Snippet: (A and B) BMH-21-caused nucleolar stress is independent of ATM pathway activation. A375 cells were pretreated with ATM inhibitor (ATMi) KU55933 (10 μM) for 30 min as indicated, followed by treatment with BMH-21 (1 μM) or IR (2 Gy) and incubation for 3 h. Cells were stained for (A) S1891-phosphorylated ATM (PATM, green ) or (B) NCL ( green ) and counterstained for DNA ( blue ). (C) Parent DLD and DLD cells with ATR-knock in mutation (DLD Seckel cells) were treated with BMH-21 for 6 h followed by staining for NPM ( green ). Merged images with DNA ( blue ) are shown. (D) Inhibition of DDR pathways does not affect BMH-21-mediated RPA194 degradation. A375 cells were pretreated for 30 min with the following: KU55933 (10 μM), caffeine (2 mM), wortmannin (10 μM), NU7441 (5 μM) followed by addition of BMH-21 (1 μM) and incubation for 2 h. Cells were stained for RPA194 ( green ), UBF ( red ) and counterstained for DNA ( blue ) Arrowheads , nucleolar caps. (E) A375 cells were pretreated with KU55933 (10 μM) for 1 h as indicated, followed by IR (2 Gy) and incubation for the indicated times. BMH-21 (1 μM) was added for the final 3 h as indicated. Cell lysates were analyzed by western blotting for RPA194 and GAPDH was used as a loading control. (F) A375 cells were pretreated with NU7441 (10 μM) for 1 h as indicated, followed by addition of ActD (50 ng/ml) or BMH-21 (1 μM) and incubation for 3 h. Cell lysates were analyzed by western blotting for RPA194, NCL and GAPDH was used as a loading control. Scale bars, 10 μm.

Article Snippet: Other reagents were KU55933 and caffeine (Calbiochem), ActD, camptothecin, wortmannin (Sigma) and NU7441 (Santa Cruz Biotechnology).

Techniques: Activation Assay, Incubation, Staining, Knock-In, Mutagenesis, Inhibition, Western Blot, Control

Journal: Oncotarget

Article Title: DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response

doi:

Figure Lengend Snippet: U2OS cells were treated with LI-216 (10 μM) for 3 h in the presence or absence of NU7441 (10 μM). Cells were fixed and stained for (A) PATM, (B) γH2AX, (C) PKAP1, (D) PDNA-PK and counterstained for DNA. Scale bars, 10 μm.

Article Snippet: Other reagents were KU55933 and caffeine (Calbiochem), ActD, camptothecin, wortmannin (Sigma) and NU7441 (Santa Cruz Biotechnology).

Techniques: Staining

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