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Cells were fed three times a week with medium containing compound Ia.
At 80% confluency, cells were washed with PBS and supplemented with fresh medium containing compound or the topoisomerase I-inhibitor, camptothecin, at 0, 0.3, 0.3, 1, 3, 10, 30, 100, 300 µM.
The medium was replaced with serum-free medium containing compound under high glucose conditions for 24 h.
On the day of measurement, change medium containing compound into PBS with phosphodiesterase inhibitors, and incubate 2 hr and then the procedures were performed following manufacture's instruction.
Culture medium was then replaced with 100 μL of fresh medium containing compound (serially diluted), and this was further incubated for 24 h.
Microinjection of embryos with XDsh mRNA and incubation in medium containing compound 3289 8625 increased the prevalence of the non-canonical Wnt signaling associated CE phenotype.
Similar(54)
The culture medium containing compounds was removed, and the cells were fixed in 4% paraformaldehyde for 10 min.
The culture medium containing compounds was removed, and the cells were fixed in 4% paraformaldehyde for 10 min. The cells were washed twice with PBS, and were consequently stained with 0.5 mL of Hoechst 33258 staining for 5 min. The stained nuclei were washed twice with PBS, and were consequently observed under fluorescence microscope at 350 nm excitation and 460 nm emissions.
For each strain, the tolerance was evaluated as relative growth (optical density (OD) value, %) by comparing the yeast growth in the medium containing inhibitory compound(s) with that in medium lacking these compound(s).
For experiments using ML171, chloroquine or MG132, the compound was continued during the stimulation, whereas for other treatments, medium containing the compound was replaced by fresh medium according to the experimental requirements.
Next, a fresh medium containing tested compound was added.
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