h2b-cfp addgene  (Addgene inc)

Journal: BMC Biology

Article Title: Lineage tracing shows that cell size asymmetries predict the dorsoventral axis in the sea star embryo

doi: 10.1186/s12915-022-01359-3

Figure Lengend Snippet: The position of animal clones is not biased with respect to DV axis due to morphogenesis. Representative confocal image of a sea star larvae injected at the 8-cell stage ( A ). Oocytes were injected with H2B-CFP to mark nuclei, fertilized and raised until the 8-cell stage, when one animal blastomere was injected with DiI. Embryos were imaged in toto on a confocal microscope at three different developmental stages (26, 50 and 72 hpf). Orientation of the embryo is indicated in the upper left corner of each image and a schematic representation of the clone is provided in the upper right corner. B Quantification of the angles formed by the injected clone and the sagittal plane of the larva at 72 hpf. n = 55 embryos. Rayleigh test, ns, not significant. Scale bars: 50 μm

Article Snippet: To label individual blastomeres at the 2-, 4- or 8-cell stages, one blastomere of Patiria miniata and Patiriella regularis embryos was injected with either mRNAs coding for H2B-CFP (100 ng/μl), H2A-mCherry (400 ng/μl), Dextran Oregon Green 488 (1 mg/ml; Invitrogen, D7171) or DiI (Life Technologies, D282).

Techniques: Clone Assay, Injection, Microscopy

Journal: Molecular Biology of the Cell

Article Title: Mps1 regulates spindle morphology through MCRS1 to promote chromosome alignment

doi: 10.1091/mbc.E18-09-0546

Figure Lengend Snippet: MCRS1 S65 phosphorylation-deficient mutant exhibits a defect in chromosome segregation. (A) Time-lapse live-cell images. A549 cells expressing H2B-CFP were transfected with MCRS1 siRNA. Cells were then arrested at G2 by RO3306 and released into fresh medium for monitoring chromosome segregation. Time after nuclear envelope breakdown was counted by minutes. Misaligned chromosomes in MCRS1 siRNA cells were magnified in a rectangle. Bar: 5 μm. (B) Mitotic duration of MCRS1 depleted cells. Results from three independent experiments of panel A were combined and are shown together as mean ± SEM (total 120 mitotic cells). Two-tailed P value was calculated by unpaired Student’s t test. **** means P value is less than 0.0001, *** less than 0.001, and ** less than 0.01. NS stands for not significant. (C) Immunofluorescence of mitotic cells. HeLa Tet-on cells stably expressing MCRS1-GFP were fixed and processed for immunofluorescence using GFP antibody. DNA was stained by Hoechst 33342. Bar: 5 μm. (D) Stable cell line expressing MCRS1-GFP WT, S65A, or S65D. HeLa Tet-on cells were transfected with MCRS1 siRNA and processed for Western blot to examine MCRS1 protein level. (E) Mitotic duration of stable cell lines. Stable cell lines were transfected with siRNA and mitotic duration was counted by live-cell DIC microscope movie. Results from two independent experiments counting ∼40 cells were combined and are shown here as mean ± SEM. Two-tailed P value was calculated similarly as in panel B. (F) Abnormal chromosome alignment at metaphase. Stable cell lines were transfected with siRNA and treated with MG132 for 1 h. Metaphase cells were counted either normal or abnormal, based on DNA and mitotic spindle distribution. Results from three independent experiments counting ∼60 cells each were combined and are shown here as mean ± SEM. Two-tailed P value was calculated similarly as in panel B. Bar: 5 μm.

Article Snippet: Fluorescence time-lapse imaging of A549 cells expressing CFP-H2B was recorded every 10 min for a total duration of 24 h with a 10× objective in a Nikon Eclipse Ti-E microscope equipped with a temperature- and CO 2 -controlled stage incubation unit (Okolab).

Techniques: Mutagenesis, Expressing, Transfection, Two Tailed Test, Immunofluorescence, Stable Transfection, Staining, Western Blot, Microscopy