Smz645

Zebrafish embryos were obtained by mating wild-type female and male zebrafish. The embryos were then cultured in embryo medium containing PTU until they reached 24 h post-fertilization (hpf). After the 24 h culture period, the embryos were treated with varying concentrations of CHNQD-00824, ranging from 0 to 15 μM, for an additional 24 h. Following the treatment, the embryos were stained with acridine orange (AO) at a concentration of 2 μg/mL for 30 min. The stained embryos were then washed three times with embryo culture medium to remove any excess dye. The results were visualized and photographed using a fluorescence microscope (SMZ645, Nikon, Tokyo, Japan). The Image-pro-plus software (version 10.0.5, Media Cybernetics Inc., Rockville, Maryland) was used to analyze the images and quantify the results obtained from the stained embryos.

Electrical conductivity (σ) was measured using the modified four‐point probe method. By applying an AC voltage (±1 V, 1000 Hz), voltage (V) and current (I) were recorded using a probe station (MST4000A, MSTECH, Korea). The thickness (T), length (L), and width (W) were measured using an optical microscope (SMZ645, Nikon, Japan). The electrical conductivity (σ) was calculated using the equation: σ = (L × I)/(W × T × V).
The electrical impedance was characterized by using a potentiostat (SP‐200, Bio‐Logic, USA). The hydrogel samples were loaded between two platinum electrodes with a 10 mm × 10 mm area and ≈200‐µm thickness. The two electrodes were connected to the potentiostat, and EIS measurement was conducted. To characterize the electrical resistance of the hydrogel, the output voltage and current across the hydrogel were measured in real‐time using an electrometer (Keithley 6514, Tektronix, USA).

Human SCNT pluripotent cell lines, CHA-SCNT-hPSC-17 [10 (link)] and hPSC-18 ([10 (link)], Korea Stem Cell Registry code hES12019001), and embryonic stem cell line, CHA-hESC-15 ([25 (link)], Korea Stem Cell Registry code hES12010028), were cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) in DMEM/F12 medium supplemented with 20% KnockOut Serum Replacement (KO-SR), 1% non-essential amino acids (NEAA), 0.1 mM β-mercaptoethanol (β-ME), and 4 ng/mL hrbFGF (ES culture media; all of them from Invitrogen). Human PSCs were mechanically passaged every 5 days under a stereomicroscope (SMZ 645, Nikon, Tokyo, Japan).

At day 7 of the bioassay, the number of laid egg masses per treatment was recorded. Masses were carefully removed from the containers and were placed individually in plaques of 6-well plates (10 ml) containing 8 ml of each water sample (L1, L2, L3 or TW). Broken masses were discarded. Bioassay was carried out under controlled conditions of temperature (23 ± 1°C) and photoperiod 12:12 h (Light:Dark). Total egg masses (46) were examined daily under a stereoscopic microscope (Nikon SMZ645). The number of eggs per mass, embryonated eggs per mass, morphological abnormalities and arrested eggs were registered. The hatching success was determined by counting the number of hatched juveniles per egg mass. The percentage of hatching was calculated on the total number of eggs and on the total number of embryonated eggs.

To measure the hydrostatic pressure of the blastocoel cavity, a 900 A micropressure system (World Precision Instruments, SYS-900A), which has a resolution of 13 Pa, was used in accordance with previous work^{77 (link)}. In brief, 0.5–1 μm microneedles (World Precision Instruments, TIP05TW1F, TIP1TW1) were filled with 1 M KCl solution and attached to the microelectrode holder (World Precision Instruments, MEH6SF) and connected to 900 A system. The microelectrode was calibrated using a calibration chamber (World Precision Instruments, CAL900A). The microelectrode was positioned onto a micromanipulator and the reference electrode was mounted in a fixed position inside the media under LeicaMZ6 or a Nikon SMZ645 dissecting microscope. Embryos were fixed in position using plasticine to ensure embryos were stable during pressure measurement. The microelectrode was inserted into the blastocyst at a depth above the ectoderm thickness of 100 µm, around 300 µm (precisely measured using a micromanipulator) and then maintained in place for ~10 s. The pressure was calculated as the mean pressure of 10 s. Data points with decreased pressure within the 10 s of probe insertion failed to stabilize and were discarded due to repature of the ectoderm.