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. 2015 Jan 23;17(1):66–78. doi: 10.1016/j.neo.2014.11.003

Figure 2.

Figure 2

Cycling hypoxia increases IL-6 and IL-8 secretion induced by TNFα and stabilizes TNFα-induced ICAM-1 expression along reoxygenation time. (A–E) EAhy926 cells and HUVEC were exposed to N, chH, or cyH with or without TNFα (0.1 ng/ml) for 6 hours. (A–B) Conditioned media were recovered directly after the incubation (6h+0hR) or after 16-hour reoxygenation (6h+16hR) and assayed to determine IL-8 and IL-6 concentration by ELISA (n= 3, mean ± 1 SD). Statistical analysis was performed by two-way ANOVA and Holm-Sidak test as post hoc test. * chH/cyH versus corresponding N, $ cyH versus corresponding chH, # TNFα versus corresponding control. *, $, #, P < .05; **, $$, ##, P < .01; ***, $$$, ###, P < .001. (C) EAhy926 cells were fixed directly after the incubation (6h+0hR) or after 4-hour (6h+4hR) or 16-hour (6h+16hR) reoxygenation and were immunostained for ICAM-1 (green). Nuclei were labeled with TOPRO-3 (blue). (D–E) After 16-hour reoxygenation, total abundance of ICAM-1 in EAhy926 cells (D) and HUVEC (E) was detected by Western blotting (n= 1). α-Tubulin was used as loading control. ICAM-1 fluorescence intensity was quantified and was normalized for α-tubulin.