Karyomax

Karyotypic analysis was contracted out to Nihon Gene Research Laboratories (Sendai, Japan). To assess diploidy, 50 cells at metaphase were examined. Metaphase spreads were prepared from cells treated with 100 ng/mL of Colcemid (KaryoMax, Gibco; Thermo Fisher Scientific, MA, USA) for 6 h. The cells were fixed with methanol: glacial acetic acid (2:5) three times and placed onto glass slides. Giemsa banding was applied to metaphase chromosomes. A minimum of 10 metaphase spreads was analyzed for each sample and karyotyped using a chromosome imaging analyzer system (Applied Spectral Imaging, CA, USA).

Fixed cell suspensions were prepared for FISH. Colcemid (Gibco® KaryoMAX®) was added to the culture medium to a final concentration of 0.1 μg/mL (1 in 100) and the flask incubated at 37 °C for 1 h. Cells were spun and supernatant discarded. Ten millilitres prewarmed hypotonic solution was added (for human: 1:1 1% (w/v) sodium citrate: 0.56% (w/v) (0.075 M) KCl and for mouse: 0.56% (w/v) (0.075 M) KCl only) and incubated at 37 °C for 12 min. Cells were pelleted, the supernatant discarded and the cells washed with and then stored (at –20 °C) in fresh 3:1 methanol: acetic acid fix. Bacterial artificial chromosomes (BACs) were obtained from BACPAC Resource Center (BPRC) at the Children’s Hospital Oakland Research Institute. Clones were grown and DNA extracted according to BPRC protocols. BAC DNA was labelled using ARES™ Alexa Fluor® Labelling Kits (Alexa Fluor® 488 and Alexa Fluor® 594) according to the manufacturer’s protocol. FISH was performed on fixed cell suspensions according to standard methods [40 (link), 41 (link)].

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Iowa State University. Metaphase chromosome preparations were obtained from C. picta and T. scripta cell cultures established previously using our standard protocols [12 (link),21 (link),26 (link)]. Briefly, turtle tissues were digested with collagenase (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) and cultured using a medium composed of 1:1 RPMI 1640:Leibowitz media (GIBCO) supplemented with 15% fetal bovine serum (One Shot, GIBCO), 2 mM L-glutamine (GIBCO), and 1% antibiotic–antimycotic solution (GIBCO). Cell cultures were incubated at 30 °C without CO₂ supplementation, and 10 µg/mL colcemid (KaryoMAX^®, GIBCO) was added four hours prior to harvesting. Metaphase chromosomes were harvested after hypotonic exposure and fixed in 3:1 methanol:acetic acid. Cell suspensions were dropped onto glass slides and air-dried. The slides were digested with pepsin and fixed with formaldehyde. High quality metaphase chromosomes were imaged and used for FISH. Because the subspecies of the slider turtle individual used for cytogenetics was not confirmed, we will use T. scripta or TSC to refer to cytogenetic data related to this individual, and T. s. elegans or TSE to refer to data from the recently published genome assembly [13 (link)].

For metaphase spreads of splenocytes^{70 (link)}, freshly harvested spleens were minced and filtered through a 40 μm cell strainer into warm PBS. Cells were spun at 1000 × g for 5 min, resuspended in warm DMEM supplemented with 10% FBS, 100 U/mL penicillin,100 U/mL streptomycin. Cells were then treated with colcemid (KaryoMAX, GIBCO) at 0.1 μg/ml for 1 h at 37 °C^{74 (link)}. Cells were swollen in prewarmed 75 mM KCl for 30 min at 37 °C, then carefully fixed in methanol: acetic acid (3:1) and kept at −20 °C. Metaphase spreads were prepared by dropping cells in the fixative onto Superfrost glass slides (Fisher Scientific) at 25 °C and 60% of humidity. After air dry, metaphase images were captured and analyzed using a 20X Nikon objective (PL APO, 1.4 NA). At least 10 metaphases from each tissue were scored for chromosomal aberration.

Exponentially growing cells transfected with ILK-RNAi-lentivirus and negative control lentivirus were suspended in complete growth medium and seeded in 6-well plates at 200 cells per well. The plates were maintained at 37°C in a humidified incubator with 5% CO₂ for 2 weeks. The visible colonies were subsequently recorded under an inverted fluorescence microscope (MicroPublisher 3.3RTV; Olympus). Following fixation in paraformaldehyde, the colonies were subjected to Giemsa (Karyomax; Gibco, Grand Island, NY, USA) staining for 10 min followed by acquisition of images with an Olympus C5050 digital camera attached to an Olympus CKX1 inverted microscope (Olympus).