Representative figures for apoptosis of a MN-free HeLa CENP B-GFP H2B-mCherry cell. apoptosis of a cell in mitosis. Arrows point to the initial cell nucleus, its pyknosis and Karyorrhexis. 12935_2019_917_MOESM3_ESM.docx (127K) GUID:?8A70D64C-5A5C-4C34-A354-2E4BCFB0C768 Additional file 4: Table S1. Colcemid and actinomycin D induced K? MNi and K+MNi in HeLa CENP B-GFP H2B-mCherry cells. 12935_2019_917_MOESM4_ESM.docx (15K) GUID:?AF082AB9-1F78-462F-AAE8-D0BC1590F4A3 Additional file 5: Figure S3. DNA degradation was not obvious in Hela Cells containing mainly K? MNs and K+MNs. Cells were exposed to actinomycin D and colcemid and for 24?h, and DNA were isolated from each treatment for gel electrophoresis as described in Methods section. (1) 100?bp DNA ladder marker (Takara Corp.); (2) Control; (3) Cells treated with 150?ng/mL actinomycin D; (4) Cells treated with 15?ng/mL actinomycin D; (5) Cells treated with 25?ng/mL colcemid. Results suggested that there was no DNA degradation TPT-260 in control cells. DNA degradation was obvious in the high concentration of actinomycin D treatment (150?ng/mL), while slight DNA degradation occurred in the colcemid and low concentration actinomycin D (15?ng/mL) treatment cells. 12935_2019_917_MOESM5_ESM.docx (38K) GUID:?20DB4E2A-4AAA-4691-BCA2-AF916C8619DE Data Availability StatementNot applicable. Abstract Background Micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability. However, the roles of kinetochore of MN in mitosis have not been completely addressed. Methods The HeLa CENP B-GFP H2B-mCherry cells are applied to address these questions via the long-term live-cell imaging. In the cells, the kinetochore-positive micronucleus (K+MN) contained CENP B-GFP, while the kinetochore-negative micronucleus (K?MN) did not. Results K?MN-bearing cells produced much more chromosome fragments than did MN-free cells. Most of the chromosome fragments eventually merged into K?MNi. K+MN-bearing cells yielded more kinetochore-positive lagging chromosomes (K+LCs) and K+MNi than MN-free cells did. The results suggested the differences in the fates of K+MNi and K?MNi in mitosis. The cycle of K?MN??Chromosome fragment??K?MN may occur in generations of K?MN-bearing cells, while part of K+MNi might reincorporate into the main nucleus. The K+MN-bearing cells prolonged significantly duration of mitosis compared with MN-free cells. The presence of micronuclei, regardless of K? MN and K+MN, enhanced apoptosis cell death. And K+MN-bearing cells were inclined to apoptosis more than K?MN-bearing cells. The results suggested differences in fates between K? MN-bearing and TPT-260 K+MN-bearing cells. Conclusions Kinetochore determined the fates of micronuclei. Kinetochore in micronuclei indirectly prolonged the duration of mitosis. Kinetochore enhanced cytotoxicity of micronuclei. Our data are direct evidences showing the roles of kinetochore of micronucleus in mitosis of HeLa cells. Electronic supplementary material The online version of this article (10.1186/s12935-019-0917-8) contains supplementary material, which is available to authorized users. Keywords: Micronucleus, Kinetochore, Lagging chromosome, Chromosome fragment, Mitosis, Live cell imaging Background The micronucleus (MN) test determines chromosomal level DNA damage and is widely used to biomonitor humans exposed to clastogens and aneugens [1, 2]. Elevated frequencies of MNi are also found in patients with cancer and other diseases [3, 4]. MNi are formed from an entire chromosome or from a chromosomal fragment. The kinetochore is an essential structure composed of a number of conserved protein complexes on the centromere in eukaryotes. It serves as a bridge between the spindle microtubules and chromosomes and regulates chromosome segregation [5, 6]. Based on the presence of kinetochores, MNi are further classified into TPT-260 K+MNi and K?MNi. In fixed cells, kinetochores in MNi can be Rabbit Polyclonal to ADCK3 detected by immunofluorescent staining using anti-kinetochore antibodies from the serum of scleroderma (CREST syndrome) patients. Aneugenic agents mainly induce K+MNi in human cells, while clastogenic agents enhance K?MNi. The classification increases the specificity of the MN test [7C11]. In live cells, kinetochores in MNi were identified in a dual-colour fluorescent cell line, HeLa CENP B-GFP H2B-mCherry cells [12]. In these cells, chromosomes and kinetochores were labelled by H2B-mCherry and CENP B-GFP, respectively. MNi were marked by H2B-mCherry. K+MNi were identified by CENP B-GFP, while K?MNi did not have the GFP signal. The differences in the origins of K+MNi and K?MNi were investigated using this construction [12]. However, the roles of kinetochore of micronucleus in mitosis of HeLa cells have not been completely addressed. Dynamic MN formation was analysed in several types of living cells [13C15]. The MN-bearing cells frequently produced daughter cells with MNi through chromosome lagging during cell division [16]. MNi were partly reincorporated into daughter nuclei after mitosis [17]. If this is the case, there should be significant differences.
