Colcemid

Chromosome spread assays for Giemsa staining were prepared as previously described [30 (link)]. In brief, cells were treated with 100 ng/ml of colcemid (Sigma) for 2.5 h, and mitotic cells were collected by shaking-off. Cells were incubated in a hypotonic solution (DMEM: H₂O at a ratio of 2:3) for 5.5 min at room temperature and fixed with freshly prepared Carnoy’s solution (75% methanol, 25% acetic acid). Cells in Carnoy’s solution were dropped onto glass slides, stained with 5% Giemsa (Solarbio) and analyzed by bright field microscopy (BX51, Olympus). To perform chromosome spreads for immunofluorescence staining, HeLa cells grown on coverslips were treated with 100 ng/ml of colcemid (Sigma) for 2.5 h to enrich mitotic cells. After washing three times with PBS, the cells were incubated in 75 mM KCl hypotonic solution for 15 min at 37 °C and centrifuged for 5 min at 2000g to spread the chromosomes onto coverslips. The samples on coverslips were washed with PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH 6.9), fixed for 5 min with cold methanol (− 20 °C) and processed for immunofluorescence microscopy (LSM510, Zeiss).

For colcemid and Flavopiridol experiments, the following strains were used + ; worGal4, UAS-Cherry::Jupiter, Sqh::GFP; 
+ + ; worGal4, UAS-Cherry::Jupiter, Sqh::GFP; rodH4.8 + ; His2A::mRFP1; rod^H4.8 (this work)
Wild-type or rod^H4.8 mutant neuroblasts were incubated with colcemid (Sigma) in live imaging medium at a final concentration of 5 µg/mL, or with Flavopiridol hydrochloride (Sigma) at a final concentration of 5 µM. Live imaging was started without delay. Complete spindle depolymerization was seen ~ 30–60 min after colcemid addition.

Chromosome preparations were based on previous protocols with modifications for the pupal hindgut (Fox et al., 2010 (link); Gatti et al., 1994 (link)). For colcemid treatment, to enrich for metaphase cells, tissue was first incubated in colcemid (Sigma, St. Louis, MO) at 50 μg/ml for 30 min in PBS. For pre-mitotic chromosome spreads with Premature Chromosome Compaction, tissue was incubated in Calyculin A (Cell Signaling Technology, Danvers, MA) at 200 nM in PBS for 30 min, (Gotoh et al., 1995 (link); Miura and Blakely, 2011 (link)). FISH was performed as in Dej and Spradling, 1999 (link). BAC clone #BACN04H23 (Chromosome 3L, region 69C3-C8) from the PacMan collection (Venken et al., 2006 (link)) was labeled using the BioNick labeling system (Invitrogen, Carlsbad, CA). BAC probe signal was amplified through sequential labeling with Peroxidase-labeled Streptavidin followed by the TSA Peroxidase detection kit (Perkin Elmer, Waltham, MA). Imaging was performed on a Zeiss Axio Imager 2 with a 63x oil immersion lens.

Erwinia carotovora carotovora15 (Ecc15) was grown in LB medium at 29°C with shaking overnight, and harvested by centrifugation at 3000g at 4°C for 30 minutes. The pellet was then suspended in the residual LB, and bacterial concentration was adjusted to OD₆₀₀ = 200. Flies older than 3 days were first dry-starved in an empty tube for 2 hours, and then transferred into a classical fly food vial containing a filter paper that totally covers the food and was soaked with a solution consisting of 5% sucrose and Ecc15 at OD200 (1:1), or 6% DSS (average MW 40 kDa, sigma) treated flies were kept at 29°C until dissection. colcemid treatment was done as reported previously [34 (link)]. 200ug/ml colcemid (Sigma) was added to 5% sucrose to pre-treat the flies for 12 hours, and then an Ecc15 infection was performed in the presence of 200ug/ml colcemid.

Cell lines 4a-3A and Sua4.0 were cultured as described above in 250-ml flasks and were passaged at a 1:5 dilution to reach monolayer formation with a cell density of 50%–60%. Cells were treated for 1.5 h at 27 °C with 0.1 µg/ml colcemid (MilliporeSigma, St. Louis, MO, USA; catalog no. 10295892001) by adding 100 µl of 10 ug/ml colcemid to 10 ml of cell culture medium.

HeLa cells, post-transfection with the indicated siRNAs, were cultured in the presence of 100 μM BrdU for 42 h. Subsequently, cells were subjected to treatment with colcemid (0.2 μg/ml, Sigma) 4 h prior to harvesting through mitotic shake-off. After collection, cells were swollen in pre-warmed (37 °C) hypotonic solution (46.5 mM KCl, 8.5 mM NaCitrate) for 15 min, and fixed in ice-cold methanol/acetic acid (3:1). Metaphase cells was then spread onto glass slides and left to air-dry. After a 24 h interval, the slides were subjected to heating at 88 °C for 10 min in buffer (1.0 M NaH₂PO₄ [pH 8.0]), followed by rinsing in distilled water, staining with 5% Giemsa for 10 min, and a final rinse with water before allowing to air-dry.

Chromosome karyotype analysis of offspring mice was performed in the bone marrow mononuclear cells. The specific method is as follows. The mice were injected intraperitoneally with 0.04% colcemid (Sigma Aldrich, USA) at 0.1 ml/10 g. After 4 h of treatment, the mice were killed to harvest the femur. The marrow cavity was rinsed repeatedly with PBS to collect bone marrow cells. The samples were filtered through a 300-μm mesh filter to remove debris. Then, bone marrow cells were centrifuged at 1000 rpm for 10 min to obtain cellular pellets. The centrifuged cellular pellets were resuspended in 1.0 mL of chilled methanol-acetic acid fixative after low permeability treatment with 75 mmol/L potassium chloride. The cell suspension was dropped onto a pre-cooled slide and immediately used for chromosome preparation by trypsin-G-banding technology. The karyotype analysis of mouse bone marrow cells was carried out using the CV Cytovision karyotype analysis system (AI, UNK) and Cytovision 3.93 software (Leica Biosystems, Germany). Thirty metaphase mitotic phases were counted, and 5–10 chromosome karyotypes were analysed.