Scriptaid

MTT assay was performed to determine the toxicity of the drugs on lung cancer cell lines. Cells seeded in 96-well plates, at a density of 1 × 10⁵ cells per well, were given the indicated drug treatment-Cisplatin (Sigma, USA) and Scriptaid (Sigma, USA) for 72 hrs. 100 μl of MTT solution (100 μg/ml) was added to each well and after its conversion to a soluble formazan, cell viability was measured by spectrophotometric absorbance at 490 nm. For clonogenic assay, cells were seeded at a density of 1000 cells per well in 6-well plates coated with poly-L-lysine (Sigma, USA). After giving the indicated treatment for 2 hrs, cells were grown in regular growth media for 2 weeks. The resulting colonies were fixed, stained with 0.5% crystal violet and counted using the ImageJ image analysis software.

At 3 dpf dye larvae were separated from unaffected sibling larvae based on phenotype. dye mutants and siblings were treated with either 1 µM Trichostatin A (TSA, Sigma) with or without 100–500 nM ANA-12, 6 µM Scriptaid (Sigma), 10 µM MC1568 (Tocris Biosciences), 10 µM MS275 (Cayman Chemicals), 10 µM 7,8-dihydroxyflavone hydrate (Sigma) or 0.1% dimethyl sulfoxide (DMSO) vehicle control in a final concentration of 0.1% v/v DMSO/embryo medium solution. Optimum drug concentrations for treatments were determined by prior treatment with 0.25–4 µM TSA, 2-10 µM Scriptaid, 5–20 µM MC1568 and 5-20 µM MS275, 2.5–10 µM 7,8-dihydroxyflavone hydrate, and assessment of defects in gross morphology and optokinetic response. For each treatment 12 larvae were transferred to 10 mL drug solution in a 60 × 15 mm petri dish, sealed in a container, and incubated under standard conditions stated above until 5 dpf.

We collected both oocytes and CCs from 8- to 10-week-old B6D2F1 female mice by superovulation. Superovulation was induced by sequentially injecting 7 IU of PMSG and 5 IU of hCG (San-Sheng Pharmaceutical, China) at an interval of 48 h. Then, cumulus–oocyte complexes were collected from oviducts 14 h after hCG injection and treated with hyaluronidase from bovine testes (Sigma, St. Louis, MO, United States) to obtain dissociated CCs and oocytes.
The oocytes were enucleated in a chamber containing oil-covered HCZB supplemented with 5 μg/ml of CB (Sigma) by Piezo-driven pipette (PrimeT 130 each) of an Olympus inverted microscope (Tokyo, Japan). The nuclei of donor CCs were transferred into enucleated oocytes by direct injection and activated through 5 h incubation in Ca²⁺-free CZB containing 1 mM SrCl₂ and 5 μg/ml CB. The reconstructed embryos were thoroughly washed and cultured in G1 medium under 5% CO₂ at 37°C.
For NT with the HDACi treatment, Scriptaid (Sigma, United States) was employed for a total of 10 h with a concentration of 5 nM by adding to the culture medium at the beginning of zygote activation.