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Cells were then incubated for 5 h or the indicated time with 5% CO2 at 37 °C before analysis.

At first, 1 × 10 POBs per well in 24-well culture plates were cultured and differentiated in α-MEM medium containing 50  μM ascorbic acid, 10 mM β-glycerophosphate, 100  μM indomethacin and 100 nM Dex for indicated time with regular medium changed.

Stimulation was performed for the indicated time with 1.0 mM freshly prepared CaCl2.

HeLa cells transfected with Bmi1 siRNAs were treated for the indicated time with VM26.

In the cases of 24 hpf and 48 hpf inhV withdrawal, media was replaced at the indicated time with E3 media and PTU only.

Shortly, cells were washed once with PBS after treatment for the indicated time with the BioPORTER preparations (4 h unless stated otherwise).

Mungbean seedlings at 4 DAI were exposed to 4 °C and Fv/Fm ratios were measured at indicated times with a portable Pulse Amplitude Modulated (PAM) fluorometer (PAM 2100, Walz, Germany) after 30 min of dark adaptation.

The resulting phosphorylation eIF2α-NTD was incubated with affinity-purified phosphatase in 20 mM Tris HCL pH 7.4, 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.8 mM ATP at 30°C for indicates times with shaking.

For TGF-β time course experiments, NMuMG cells and HMEC were treated with 2 ng/ml TGF-β (rhTGF-β1 240-B, R&D systems) for indicated time points with TGF-β replenishment and medium change every 2 days.

For cytokine analysis B-1P, B-1S and\or B-2S (1−2.5×105) cells were cultured in triplicate for indicated time points with various stimulants.

Four animals in each group were assessed for biomarkers at each indicated time point, with survival assessed in eight mice in the Sham group and 22 each in the HS and HSXBJ groups.