Characterization of telomeric repeats in metaphase chromosomes and interphase nuclei of Syrian Hamster Fibroblasts

Abstract

BackgroundRodents have been reported to contain large arrays of interstitial telomeric sequences (TTAGGG)n (ITS) located in pericentromeric heterochromatin. The relative sizes of telomeric sequences at the ends of chromosomes (TS) and ITS in Syrian hamster (Mesocricetus auratus) cells have not been evaluated yet, as well as their structural organization in interphase nuclei.ResultsFISH signal distribution analysis was performed on DAPI-banded metaphase chromosomes of Syrian hamster fibroblasts, and relative lengths of telomere signals were estimated. Besides well-distinguished FISH signals from ITS located on chromosomes ##2, 4, 14, 20 and X that we reported earlier, low-intensity FISH signals were visualized with different frequency of detection on all other metacentric chromosomes excluding chromosome #21. The analysis of 3D-distribution of TS in interphase nuclei demonstrated that some TS foci formed clearly distinguished associations (2–3 foci in a cluster) in the nuclei of cells subjected to FISH or transfected with the plasmid expressing telomeric protein TRF1 fused with GFP. In G0 and G1/early S-phase, the average total number of GFP-TRF1 foci per nucleus was less than that of PNA FISH foci in the corresponding cell cycle phases suggesting that TRF1 overexpression might contribute to the fusion of neighboring telomeres. The mean total number of GFP-TRF1 and FISH foci per nucleus was increased during the transition from G0 to G1/early S-phase that might be the consequence of duplication of some TS.ConclusionsThe relative lengths of TS in Syrian hamster cells were found to be moderately variable. All but one metacentric chromosomes contain ITS in pericentromeric heterochromatin indicating that significant rearrangements of ancestral genome occurred in evolution. Visualization of GFP-TRF1 fibrils that formed bridges between distinct telomeric foci allowed suggesting that telomere associations observed in interphase cells are reversible. The data obtained in the study provide the further insight in the structure and dynamics of telomeric sequences in somatic mammalian cells.

Laminin differentially regulates the stemness of type I and type II pericytes

“…3d and e ) PKO pericytes without affecting that of WT pericytes, indicating a reversible role of laminin in the proliferation of both type I and type II pericytes. In addition to Edu, an S-phase-specific marker [ 33 ], we also examined proliferation using phospho-Histone H3 (pH3), an M-phase-specific marker [ 34 – 36 ]. Similar results were observed; specifically, a higher percentage of pH3 + cells was found in PKO pericytes (both type I and type II), and exogenous laminin substantially reduced their proliferation to baseline levels (Additional file 1 : Figure S2), again suggesting that laminin inhibits the proliferation of both type I and type II pericytes in vitro.…”

Aloperine Protects Mice against Bleomycin-induced Pulmonary Fibrosis by Attenuating Fibroblast Proliferation and Differentiation

“…Primary lung fibroblast proliferation assay was performed by EdU (5-ethynyl-2′-deoxyuridine) staining according to protocols as reported 31 . Briefly, the mouse lung fibroblasts were treated with recombinant PDGF-BB (20 ng/ml) + acetic acid,PDGF-BB (20 ng/ml) + aloperine (0.3125 mM), or PBS + acetic acid for 48 h. Next, the cells were washed with PBS followed by incubation in serum-free DMEM containing 10 μmol/L EdU (RiboBio, China) for 2 h. Cells were fixed, and then underwent Apollo staining and DNA staining, according to the manufacturer’s instructions.…”

Variations in brain defects result from cellular mosaicism in the activation of heat shock signalling

“…Following hybridization, the samples were washed with wash buffer for 30 min at 37 °C, counterstained with DAPI (1:2,000), mounted with VECTASHIELD (VECTOR laboratories), and imaged using a Zeiss LSM510 confocal microscope. For the combination of smFISH analysis and Ki67 staining, which was used to distinguish between G1-S and G2 phases of the cell cycle 47 48 the cells were incubated with anti-Ki67 (1:10, Clone B56, BD Biosciences, 550609) in addition to smFISH probes (human HSP70 , 1:100). The data were analysed using semi-automated software for counting mRNA particles (StarSearch; http://rajlab.seas.upenn.edu/StarSearch/launch.html ).…”