Cytochalasin B

SCNT was performed as previously described [23] (link), [25] (link). After culturing for 38 h to 42 h, oocytes with expanded cumulus cells were briefly treated with 0.1% (w/v) hyaluronidase and denuded of cumulus cells using a finely drawn glass capillary pipette. Oocytes extruding the first polar body with uniform cytoplasm were cultured in NCSU23 medium supplemented with 0.1 µg/mL demecolcine, 0.05 M sucrose, and 4 mg/mL bovine serum albumin (BSA) for 0.5 h to 1 h. The oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette (approximately 20 µm in diameter) in Tyrode’s lactate medium supplemented with 10 µM hydroxyethyl piperazineethanesulfonic acid (HEPES), 0.3% (w/v) polyvinylpyrrolidone, and 10% FBS in the presence of 0.1 µg/mL demecolcine and 5 µg/mL cytochalasin B. Any protrusion observed on the surface of an oocyte was removed along with the polar body. Fetal, newborn, and adult fibroblasts of the fourth to ninth passages were used as nuclear donors after cell cycle synchronization by 0.5% FBS serum starvation for 48 h. A single donor cell was inserted into the perivitelline space of an enucleated oocyte.
Donor cells were fused with the recipient cytoplasts with a single direct current pulse of 200 V/mm for 20 µs using an embryonic cell fusion system (ET3, Fujihira Industry Co. Ltd., Tokyo, Japan) in fusion medium [0.25 M d-sorbic alcohol, 0.05 mM Mg(C₂H₃O₂)₂, 20 mg/mL BSA, and 0.5 mM HEPES (free acid)]. The reconstructed embryos were cultured for 2 h in PZM-3 and then activated with a single pulse of 150 V/mm for 100 µs in an activation medium containing 0.25 M d-sorbic alcohol, 0.01 mM Ca(C₂H₃O₂)₂, 0.05 mM Mg(C₂H₃O₂)₂, and 0.1 mg/mL BSA. The reconstructed embryos were equilibrated in PZM-3 supplemented with 5 µg/mL cytochalasin B for 2 h at 38.5°C in humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ (APM-30D, ASTEC, Japan).

Laconic and FLIIP were excited at 870 nm wavelength and images were acquired at 5.94 Hz with a 256x256-pixel resolution. ACSF containing 20 mM oxamate was used in the trans-acceleration imaging protocol. ACSF containing 5 $\mathrm{\mu }$ M AR-C155858 (BioTechne Tocris) was used in the lactate transport inhibition imaging protocol. ACSF containing 20 $\mathrm{\mu }$ M cytochalasin B (BioTechne Tocris, 5474) was used in the glucose transport inhibition imaging protocol. ACSF containing 0 mM glucose was used in the aglycemia imaging protocol.
Whole-frame image analysis was carried out using custom-made code for MATLAB 2017b (MathWorks) and ImageJ (National Institutes of Health). For each experiment, images were aligned using a 2D convolution engine to account for xy drift in time. To optimize signal to noise ratio, time smoothing of 11 frames for Laconic and 5 frames for FLIIP was applied, as well as automatic thresholding using the Li’s minimum cross entropy method [86 (link)]. Ratiometric data were visualized with Prism software (GraphPad). Mann-Whitney nonparametric test was used to compare $ro{d}^{\Delta Vhl}$ and ctrl mice. Kuskal-Wallis nonparametric test and Dunnett’s multiple comparison test were used to compare PRs, GCL and INL cells. Because AAVs were injected subretinally and biosensor expression was CMV driven, both rod and cone PRs may have been transduced. However, given that rods outnumber cone PRs by a factor of 30 in the mouse retina, all imaging data represent primarily rods.

The cellular models consisted
of chromosomally stable human colon cancer DLD1 diploid (2N) and tetraploid
(4N) cells, along with tetraploid centrosome -amplified (4NCA) DLD1
cells and DLD1 HSET knockout (KO) cells. DLD1 2N and 4N cells were
generated and characterized as previously described.^{27 (link),41 ,42 (link)} In order to generate 4NCA cells,
we used dihydro-cytochalasin B (DCB) to transiently block cytokinesis
and induce tetraploidisation and centrosome amplification in DLD1
cells. Centrosome amplification in 4NCA cells is transient and therefore
they were generated from 2N cells for each run of the assay.^{27 (link)} The DLD1 HSET KO cells were knockout clones
generated by CRISPR and validated by sequencing to have a homozygous
deletion in the KIFC1 gene.

The experiments were conducted according to the guideline OECD 487 [14 ] with few modifications [15 (link)].
For the mutagenic effect evaluation, the CHO-K1 cells (2 × 10⁵ cells well⁻¹) in 6-well plates were exposed to rutin; hydrolyzed rutins (4, 8, and 12 h reaction times) and quercetin (2.5 µg ml⁻¹, in duplicate) for 4 h. Untreated cells, as the negative control, DMSO-treated (0.25%) as the solvent control and MMS-treated (25 µg ml⁻¹) as the micronuclei inductor control cells were prepared. After cell washing (phosphate buffer, pH = 7.0, 2 mL well⁻¹), cytochalasin B solution (3 µg ml⁻¹, 2 mL well⁻¹) was added. After 20 h, cells were detached, fixed with sodium citrate solution (1% w/v), followed by methanol:acetic acid 3:1 (v/v) (three times) and dropped into slides (3 slides/cell suspension). All slides were maintained at 65 °C in a wet atmosphere for 3 min before being stained with 5% Giemsa solution (Dinâmica^®) for 20 min, washed with distilled water and dried at room temperature.
For the anti-mutagenic assay, the CHO-K1 cells (2 × 10⁵ cells well⁻¹) in 6-well plates were exposed for 4 h to rutin, hydrolyzed rutin (8 h reaction time), quercetin (2.5 µg ml⁻¹, in duplicate) and MMS (25 µg ml⁻¹). After 4 h exposure, cells were washed (phosphate buffer, pH = 7.0, 2 mL well⁻¹), exposed to cytochalasin B solution (3 µg ml⁻¹, 2 mL well⁻¹) for 20 h and the slides were prepared as explained for mutagenic evaluation.
For each experiment, at least 2000 binucleated (BN) cells, besides mononucleated (MoN) and multinucleated cells (MuN), were counted (1000 binucleated cells/replicate) under 400× magnification using a light microscope (Leica, SME model). In each cell population, the micronuclei frequency (CBMN, Equation (1)) was calculated as:
where MN was the number of binucleated cells with 1, 2 or 3 micronuclei and BN was total number of binucleated cells.
For the evaluation of the cytotoxic effects, two parameters, named the Cytokinesis-Block Proliferation Index (CBPI, Equation (2)) and Replication Index (RI, Equation (3)), were calculated as: