Journal: The Journal of Cell Biology

Article Title: BLM helicase regulates DNA repair by counteracting RAD51 loading at DNA double-strand break sites

doi: 10.1083/jcb.201703144

Figure Lengend Snippet: Ablation of Blm rescues genomic instability, T cell development, and poly (ADP-ribose) polymerase inhibitor sensitivity in Brca1 Δ11/Δ11 cells. (A) Metaphase spreads from primary mouse B lymphocyte cells stained with DAPI and Cy3-labeled telomeric probe. The arrows point to chromatid breaks, closed arrowheads point to chromosome breaks, and open arrowheads point to radial chromosomes. Bars, 10 µm. (B) Quantification of genomic instability in metaphase spreads after 2 µM overnight treatment with the poly (ADP-ribose) polymerase inhibitor olaparib. CSB, chromosome breaks; CTB, chromatid breaks. (C) Flow cytometry data from primary T lymphocyte cells from mice of indicated genotypes stained with CD4 and CD8 antibodies. (D) Quantification of CD4 − CD8 − double-negative T cells. (E) Clonogenic survival assay after BLM knockdown in WT and BRCA1 Δ11/Δ11 cells with no treatment (NT) and chronic treatment with 100 nM Olaparib (OLA), a poly (ADP-ribose) polymerase inhibitor. (F) Quantification of clonogenic survival assay after shBLM in WT and BRCA1 Δ11/Δ11 MEFs. Graphs represent mean ± SD of three independent experiments.

Article Snippet: Telomere DNA FISH analysis was performed by first preparing the telomeric Cy-3 PNA probe (Cy3-00-CCCTAACCCTAACCCTAA, F1002; PNA Bio Inc.).

Techniques: Staining, Labeling, Flow Cytometry, Clonogenic Cell Survival Assay, Knockdown

Journal: bioRxiv

Article Title: Modulation of fracture healing by the transient accumulation of senescent cells

doi: 10.1101/2021.05.18.444618

Figure Lengend Snippet: (A) Femoral fractures (Fx) were induced in male C57BL/6 mice with contralateral (Contra), intact control femora. Callus and controls were analyzed 4, 8, 14 and 28 days after fracture (n=4 per time point). (B) p21 Cip1 and p16 Ink4a mRNA expression levels during fracture healing were determined at each time point using qRT-PCR. p21 Cip1 was strongly induced 8-14 days and p16 Ink4a 14 days post fracture. (C) Senescence-associated distension of satellites (SADS) were detected using immune-fluorescent in situ hybridization (FISH) Top: representative image of the contralateral side. Below: fractured side with four SADS. (D) The total number of SADS as well as percentage of SADS-positive cells (≥4 SADS/cell) at all time points was increased in the callus compared to the contralateral control femur. (E) Telomere-associated foci (TAF) were determined using FISH to detect telomeres (red) and immunofluorescent staining for γH2A.X (green). TAF (yellow, see arrowheads) were defined as sites of γH2A.X-associated DNA damage co-localized with telomeres (n=70 cells were analyzed per bone). (F) The highest percentage of TAF were detected on day 14 of fracture healing. The number of TAF-positive senescent cells (≥3 TAF/cell) was increased in fractured bones throughout the entire healing period. Mean ± SEM. One-way ANOVA or Student’s t -test for pairwise comparisons; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Sections were dried and denatured for 10 min at 80°C in hybridization buffer (0.1M Tris, pH 7.2, 25mM MgCl 2 , 70% formamide [Sigma-Aldrich, Saint Louis, MO], 5% blocking reagent [Roche] with 1.0 μg/mL of Cy-3-labeled telomere-specific [CCCTAA] peptide nucleic acid [PNA] probe [TelC-Cy3, Panagene Inc., Korea; Cat. #F1002]).

Techniques: Expressing, Quantitative RT-PCR, In Situ Hybridization, Staining