Click it edu kit

HS01 cells were seeded at 250,000 cells/well in 6-well CellBIND (Corning) plates, grown to ~80% confluency, and treated with CUDC907 for 24 h. EdU (10 μM; Click-iT EdU kit; Molecular Probes, ThermoFisher) was added during the last 3 h. Cells were harvested with 0.05% trypsin, stained with violet live/dead stain (ThermoFisher), and permeabilized. DNA labeling with FxCycle stain (ThermoFisher) was conducted according to manufacturer’s protocol. Cell populations were identified by flow cytometry on CytoFlex (Beckman Coulter) and analyzed by CytExpert software (Beckman Coulter).

OMV-associated DNA was labeled using the Click-iT EdU kit (Molecular Probes). Briefly, P. aeruginosa bacteria were cultured in tryptic soy broth (TSB; Bacto, BD) containing 1.2 mM of EdU (5-ethynyl-2-deoxuridine), a nucleoside analogue of thymidine, for 4 h on an orbital shaker (225 rpm) at 37 °C. The EdU-labeled OMVs were isolated as described above and added to epithelial cells in 8-well chambers (Ibidi). After the required incubation, cells were washed twice in PBS before fixing in 4% paraformaldehyde (PFA) and permeabilization in 3% Triton-X 100. Click-iT EdU labeling involves a two-step process. The first step involves incubation with 12.5 μM biotin azide (Molecular Probes), during which the azide moiety binds specifically to the alkyne backbone of the EdU molecule in the presence of a copper catalyst. The second step involves incubation with 5 μg/ml streptavidin-conjugated 568 Alexa Fluor^® (Molecular Probes) to enable fluorescent detection of the biotin-azide bound to the EdU.

JC-1, Newport Green™ DCF diacetate, DQ-Green BSA and Click-iT EdU Kit were acquired from Molecular Probes, Inc. (Eugene, OR, USA). Zinc pyrithione, acridine orange, cyclosporin A, monodansylcadaverine (MDC), N-acetyl cysteine (NAC), 3-methyladenine (3-MA), chloroquine (CQ), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), horseradish peroxidase, Triton-X, dithiotreitol (DTT), bisBenzimide H 33342 trihydrochloride (Hoechst 33342) and 4’,6-Diamidino-2-Phenylindole (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). WST-1 was purchased from Roche Diagnostics (Manheim, Germany). Caspase-3 inhibitor z-devd-fmk was from ICN Biomedicals Inc. (Irvine, CA, USA). Necrostatin-1 was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Primary and secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were of the highest analytical grade.

To calculate cell cycle and S phase duration, HH10 embryos were exposed to EdU for defined periods (1 or 4 h), fixed and processed to detect EdU-labelled DNA using Click-iT EdU kit (Molecular Probes) following manufacturer's instructions. The percentage of EdU-positive cells was determined by counting EdU+ cells/all nuclei (DAPI labelled) in five sections through each region in five embryos for each exposure time. Values obtained were then used in the following simultaneous equations, after (Storey, 1989 ): where T=total cell-cycle time and S=S phase length. T was calculated by subtracting equations:
S phase length was determined by substituting the calculated value for T into one of the original equations. Mitotic index was determined by counting phospho-H3-positive cells/all nuclei (DAPI labelled) in five sections through each region in five embryos for each exposure time. Statistical comparisons were made using Student's t-test (Fig. 5B).

HS01 cells were seeded at 125,000 cells/well in a 6-well CellBIND Corning plate and al-lowed to attach overnight. Cells were then treated with 0.001% DMSO, 2.5 µM pictilisib, 150 nM PF-3748309, or a combination of the two drugs for 24 h. EdU (10 μM) (Click-iT EdU kit; Molecular Probes, ThermoFisher) was added during the last 3 h. Cells were collected with 0.05% trypsin, stained with violet live/dead stain (ThermoFisher Scientific), and permeabilized. DNA labeling with FxCycle stain (ThermoFisher Scientific) was performed according to manufacturer’s instructions. Cell populations were identified by flow cytometry on CytoFlex (Beckman Coulter, Brea, CA, USA) and analyzed by CytExpert software (Beckman Coulter). Experiment was performed in three replicates.