Journal: Nature communications

Article Title: The chemotherapeutic CX-5461 primarily targets TOP2B and exhibits selective activity in high-risk neuroblastoma.

doi: 10.1038/s41467-021-26640-x

Figure Lengend Snippet: Fig. 3 CX-5461 causes DNA damage and cell death by apoptosis in neuroblastoma cells at sub-micromolar concentrations. a Apoptosis in CHP-134 cells. The cells were treated for 48 h. b Quantification of data in (a). Data represent the mean ± SD of three independent experiments. Significant P-values (left to right): 3.9 × 10−6, 7.2 × 10−7, and 4.8 × 10−7. c Quantification of flow cytometry data showing apoptosis up to 48 h in CHP-134 and IMR-5 (0.2 µM and 0.1 µM of CX-5461 respectively). CHP-134 cells were treated with 0.2 µM and IMR-5 cells with 0.1 µM of CX-5461. Data represent the mean ± SD of three independent experiments. Significant P-values (left to right): CHP-134, 2.2 × 10−4, 3.2 × 10−5, and 1.5 × 10−5; IMR-5, 1.8 × 10−4, 2.3 × 10−5, and 2.5 × 10−6. d Western blots for cleaved PARP (c-PARP). β-Actin was used as a loading control. Representative results of three biological replicates are shown. e Co-localization of 53BP1 and γ-H2AX. Cells were treated for 24 h. Representative results of three biological replicates are shown. Scale bar = 10 μm. See Fig. S3d for more time points. f Comet assay in CHP-134 cells. Tail length (left) and representative images (right) are shown. The red line indicates the mean value of tail length in each group. Scale bar = 5 µm. g Cell cycle analysis forCHP-134 (0.2 µM CX-5461) and IMR-5 (0.05 µM CX-5461) cells. Data represent the mean ± SD of three independent experiments. P-values vs 0 h: P1 = 4.2 × 10−5, P2 = 7.1 × 10−7, P3 = 6.3 × 10−5, P4 = 2.7 × 10−4, P5 = 6.3 × 10−3, P6 = 2.3 × 10−5, P7 = 1.8 × 10−4, and P8 = 0.044. h EdU incorporation assay to assess global nascent DNA transcription. Representative results of three biological replicates are shown. Blue, DAPI; green, EdU. Scale bar = 20 µm. i CX-5461 induced DNA damage (γ-H2AX, red) was enriched in cells with active DNA synthesis (positive for EdU (green) labeling). Representative results of three biological replicates are shown. Scale bar = 10 μm. Two-tailed t- test was used for (b, c, g). Source data for these panels are included in Source Data file.

Article Snippet: ProLong® Gold Antifade Reagent with DAPI (Cell Signaling, #8961) was used as coverslip mountant.

Techniques: Cytometry, Western Blot, Control, Single Cell Gel Electrophoresis, Cell Cycle Assay, DNA Synthesis, Labeling, Two Tailed Test

Journal: Communications Biology

Article Title: NUPR1 protects against hyperPARylation-dependent cell death

doi: 10.1038/s42003-022-03705-1

Figure Lengend Snippet: a OXPHOS metabolism, reflected by oxygen consumption rate (OCR) levels were measured in MiaPaCa-2 cells after 24 h treatments. b MiaPaCa-2 cells were treated with ZZW-115 (1.5 µM) and olaparib (25 µM) for 24 h, then, loaded with MitoTracker Deep-Red FM and, after fixation, stained with DAPI. Flow cytometry analysis were carried out using MitoProbe™ TMRM ( c ), MitoSOX™ Red ( d ), CellROX™ Orange Reagent ( e ) or Fluo-4-AM ( f ) for analysis of the mitochondrial membrane potential, mitochondrial superoxide levels, intracellular ROS levels and the cytosolic calcium concentration, respectively, after 24 h of incubation with the drugs. Data represent mean ± SEM. Two-way, Sidak was used, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.001. g PLA was performed in MiaPaCa-2 cells in the presence or in the absence of ZZW-115 together or not with 5-FU in the presence or absence of olaparib. Mouse anti-PAR and rabbit anti-Mitofusin2 antibodies were used. A representative experiment is shown ( n = 3). ImageJ was used to count the number of green dots. Mean ± SEM of foci/nucleus are included.

Article Snippet: Finally, samples were mounted using the Prolong Gold antifade reagent with DAPI (Thermon Fisher).

Techniques: Staining, Flow Cytometry, Membrane, Concentration Assay, Incubation

Journal: Journal of Innate Immunity

Article Title: Prognostic Value and Therapeutic Potential of TREM-1 in Streptococcus pyogenes- Induced Sepsis

doi: 10.1159/000348283

Figure Lengend Snippet: S. pyogenes induces expression of TREM-1 in BMDM. a Expression of trem-1 mRNA on BMDM after infection with S. pyogenes at MOI of 1:1, 10:1 and 100:1 bacteria per macrophage. BMDM were exposed to S. pyogenes for 1 h, washed and further incubated in the presence of gentamicin for 4 h. Total RNA was isolated followed by RT-PCR analysis of trem-1 and β-actin gene expression. b Quantitative expression of trem-1 mRNA on BMDM after infection with S. pyogenes at MOI of 1:1, 10:1 and 100:1 bacteria per macrophage measured by real-time PCR. c Fluorescence microscope photographs of TREM-1 in BMDM infected with S. pyogenes at MOI of 1:1 (cii), 10:1 (ciii) and 100:1 (civ) bacteria per macrophage. TREM-1 expression on uninfected macrophages is also shown (ci). TREM-1 appears in red and S. pyogenes in green. Macrophage nuclei are stained by DAPI (blue). Bar = 25 µm.

Article Snippet: Coverslips were washed with PBS, mounted on glass slides with Mowiol containing DAPI (Prolong Gold, Promega, Madison, Wisc., USA) and analyzed by fluorescence microscopy using an AxioVision microscope (Zeiss, Oberkochen, Germany).

Techniques: Expressing, Infection, Bacteria, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Real-time Polymerase Chain Reaction, Fluorescence, Microscopy, Staining