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

Figure 3.

Figure 3

Cycling hypoxia increases the nuclear abundance of p65, the abundance of p65 phosphorylated on serine 536, and the transcriptional activity of NF-κB, induced by TNFα. (A–B) EAhy926 cells were exposed to N, chH, or cyH with or without TNFα (0.1 ng/ml or 1 ng/ml) for 6 hours. (A) Representative immunoblots and the relative quantification bar graph represent p65 nuclear abundance normalized by NUP98 (n= 3, mean ± 1 SD). (B) Representative immunoblots and the relative quantification bar graph represent the total abundance of p65 phosphorylated on serine 536 normalized for α-tubulin (n= 3, mean ± 1 SD). (C) EAhy926 cells were transiently co-transfected with the pNFκB-Fluc reporter plasmid encoding firefly luciferase and the pRL-TK normalization plasmid encoding renilla luciferase. Twenty-four hours posttransfection, cells were exposed to N, chH, or cyH with or without TNFα (0.1 ng/ml or 1 ng/ml) for 6 hours. Luciferase activities were measured 4 hours after the incubation using dual luciferase reporter assay. Results are expressed as mean of the ratio between firefly and renilla luciferase activity ± 1 SD (n= 3). (A–C) 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, Δ TNFα 1 ng/ml versus corresponding TNFα 0.1 ng/ml. *, $, #, Δ, P < .05; **, $$, ##, ΔΔ, P < .01; ***, $$$, ###, ΔΔΔ, P < .001.