Journal: Life Science Alliance

Article Title: SiR-DNA/SiR–Hoechst-induced chromosome entanglement generates severe anaphase bridges and DNA damage

doi: 10.26508/lsa.202302260

Figure Lengend Snippet: (A) Time-lapse series of anaphase in U2OS CENP-A-GFP/mCherry-α-tubulin cells treated with 100 nM SiR-DNA for 2 h. Scale bar 10 μm. (B) Quantification of percentage of severe chromatin bridges with centromeres marked by CENP-A found in central spindle in anaphase. P -values were calculated using t test. ** P ≤ 0.001. (C) Schematic illustration for correlative microscopy protocol. IF, immunofluorescence; ON, overnight. (D) Time-lapse series of control and SiR-DNA–treated U2OS H2B-GFP cells (left panel). After the live-cell imaging, cells were fixed and correlative immunofluorescence of DNA damage marker γ-H2AX was performed (right panel). Scale bars 10 μm. (E) Quantification of the number of γ-H2AX foci per nucleus upon SiR-DNA treatment. n.s., not significant, **** P < 0.0001.

Article Snippet: Cells were then incubated with γ-H2AX primary antibody (anti-γ-H2AX, pS139, rabbit polyclonal; Cell Signaling) diluted in PBS-TF at RT for 1 h and washed with PBS for 5 min.

Techniques: Microscopy, Immunofluorescence, Live Cell Imaging, Marker

Journal: Life Science Alliance

Article Title: SiR-DNA/SiR–Hoechst-induced chromosome entanglement generates severe anaphase bridges and DNA damage

doi: 10.26508/lsa.202302260

Figure Lengend Snippet: Summary of the quantifications, number of cells, and experiments.

Article Snippet: Cells were then incubated with γ-H2AX primary antibody (anti-γ-H2AX, pS139, rabbit polyclonal; Cell Signaling) diluted in PBS-TF at RT for 1 h and washed with PBS for 5 min.

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: A Targeted Thorium-227 Conjugate Demonstrates Efficacy in Preclinical Models of Acquired Drug Resistance and Combination Potential with Chemotherapeutics and Antiangiogenic Therapies

doi: 10.1158/1535-7163.MCT-22-0808

Figure Lengend Snippet: Biodistribution and mechanistic analyses on the effects of MSLN-TTC as monotherapy or in combination with chemotherapies in ST206B ovarian PDX tumors. Mechanistic analyses were conducted by IHC for necrosis, DNA damage, cell death and angiogenesis. A, Biodistribution of MSLN-TTC in tumor, blood, and selected organs 168 and 336 h after administration of MSLN-TTC in the human ovarian cancer PDX model ST206B. The mice were treated with MSLN-TTC (a single dose, 250 kBq/kg, i.v.) as monotherapy or in combination with the angiogenesis inhibitors regorafenib (30 mg/kg, QD, p.o.) or bevacizumab (5 mg/kg, Q5D, i.v.; n = 3/group). The values are presented as a percentage of remaining thorium-227 activity per injected gram of MSLN-TTC (ID%/g). B, Percentage of necrotic area as determined with Ki67 staining in ST206B tumor samples from mice treated with vehicle, non-radiolabeled MSLN antibody-chelate conjugate (a single dose, 0.43 mg/kg, i.v.), MSLN-TTC (a single dose, 250 kBq/kg, i.v.), docetaxel (7.5 mg/kg, Q7Dx9, i.v.), pegylated liposomal doxorubicin (5 mg/kg, Q7Dx9, i.v.), regorafenib (15 mg/kg, QDx63, p.o.), or bevacizumab (5 mg/kg, Q5Dx13, i.v.) as monotherapies or in combinations as indicated. Total antibody dose was 0.43 mg/kg. C, Nuclei positive for γH2AX in ST206B tumor samples from mice described in B . D, Ki67-positive nuclei in ST206B tumor samples from mice described in B . E, Number of CD31 and α-SMA positive vessels in ST206B tumor samples from mice described in B . Statistical analysis in A was performed using a linear model estimated with generalized least squares that included a common variance parameter for all study groups. Mean comparisons between the treatment and control groups were performed using the estimated model and corrected for family-wise error rate using Sidak method. No statistical significance was found. Statistical analysis in B – E was performed using ANOVA and Dunnett's test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 in comparison to vehicle.

Article Snippet: Western blot analysis was performed using primary antibodies specific for the DNA damage marker phospho-H2AX (γH2AX, #9718, Cell Signaling Technology) and cell death marker cleaved PARP (#9541, Cell Signaling Technology).

Techniques: Activity Assay, Injection, Staining, Comparison

Journal: bioRxiv

Article Title: Mitosis exit followed by death in interphase prevents the development of polyploid giant cancer cells

doi: 10.1101/2023.08.31.555795

Figure Lengend Snippet: a) Live-cell microscopy imaging of HCT116 cells treated with NOC and ST-401 for 24 h. NOC treatment results in comparable amounts of cell death in mitosis and interphase, while ST-401 treatment results in cells dying more frequently in interphase. Statistics (a). Data are shown as % of total cells from 3 independent experiments (5,500 cells for ST-401 and 7,200 cells for NOC). b-c) HCT116 cells treated for 3 days with the MTAs, harvested, and labeled with PI/annexin V to measure apoptosis and necrosis using flow cytometry. NOC dose-dependently and gradually increases apoptosis levels whereas ST-401 induces an all-or-nothing increase in apoptosis at 300 nM. Statistics (b-c): Data are expressed as mean of 3 independent experiments with >10,000 cells. Ordinary one-way ANOVA analysis. **P<0.01 significantly different from corresponding CTR. d-f) HCT116 cells stained with monodansylcadaverine (MDC) to detect autophagosomes in HCT116 cells treated with vehicle (CTR), NOC (100 nM) or ST-401 (100 nM) for 24 h. Fluorescence microscopy image of HCT116 cells (d). Flow cytometry histogram of MDC fluorescence (e). Quantification of MDC fluorescence by flow cytometry (f). NOC induces autophagy whereas ST-401 does not. Statistics (e-f): Data are expressed as mean ± s.e.m. of n=3 independent experiments. **P<0.01 significantly different from corresponding CTR (Two-way ANOVA followed by Dunnett’s). **P<0.01 significantly different from corresponding CTR. g-h) HCT116 cells were treated vehicle (CTR), NOC (100 nM) or ST-401 (100 nM) for 24 h, immunostained for P-γH2AX and P-P53, imaged by fluorescence microscopy and pixel intensity analyzed. NOC increased the levels of both P-H2AX (g) P-P53 (h) in the nucleus in a concentration-dependent manner, whereas ST-401 only induced such response at higher concentrations. Statistics (g-h): Data are shown as median with 95% CI for 3 independent experiments where 23,000 and 10,000 total cells were measured for ST-401 and NOC, respectively. Ordinary one-way ANOVA analysis. **P<0.01 significantly different from corresponding CTR.

Article Snippet: P-p53 and P-γH2AX were labeled using a mouse anti-P-p53 (1:250, Phospho-p53 (Ser15) (16G8), Cell Signaling Technology) or a rabbit anti-P-H2AX (1:250, Phospho-Histone γH2A.X (Ser139) (20E3), Cell Signaling Technology) applied overnight at 4 °C.

Techniques: Microscopy, Imaging, Labeling, Flow Cytometry, Staining, Fluorescence, Concentration Assay