Inhibitory effect of SiRNA TNFR on TNF-α-induced genes in HMEC endothelial cells.

a<p>Results are expressed as ratio (in percentage) of the mRNA content in siRNA TNFR-transfected cells stimulated with TNF-α (40 ng/ml) for 4 h to the mRNA content in unstimulated cells. After transfection with siTNFR1 or siTNFR2 RNAs, TNFR1 and TNFR2 expression were strongly inhibited (45 % and 77 %, respectively). Results are expressed as the mean of two different independent experiments.</p

List of the 55 selected genes tested.

<p>List of the 55 selected genes tested.</p

Inhibitory effects of Bay 11–7082 and IKK2 inhibitor V on TNF-α-induced genes in HMEC endothelial cells.

<p>Cells were pre-treated with pharmacological inhibitors of NF-κB, Bay 11–7082 10 µM or IKK2 inhibitor V (1 and 10 µM), for 30 min before incubation with 40 ng/ml TNF-α for 4 h. mRNA levels were normalized to unstimulated cells which values were set to 1.</p

Characteristics of the 48 ERα-positive and the 48 ERα-negative breast tumors.

a<p>Log-rank test. NS: not significant.</p>b<p>Scarff Bloom Richardson classification.</p

RelA knock-down by RNAi blocks TNFα-induced gene expression in HMEC cells.

<p>(A) Nuclear extracts from HMEC cells treated with TNF-α for the indicated periods of time were analyzed by EMSA using a ³²P-labeled HIV-LTR tandem κB oligonucleotide as a probe. (B) For supershift, nuclear extracts from HMEC cells treated with TNFα for 4 hours were incubated with the indicated antibodies before incubation with the labeled probe. Complex I: RelA/p50. (C) Nuclear extracts from HMEC cells stably transduced with a lentivirus encoding a shRNA targeting either RelA or a scrambled control, and treated by TNFα for the indicated periods of time were analyzed by EMSA as described in (A).</p

Kinetics of selected NFkB gene expression in TNF-α treated HMEC.

a<p>Ratio of the mRNA content in cells stimulated with TNF-α (40 ng/ml) for 2, 8, 15, 30 min or 1, 4, 24 and 72 h to the mRNA content in the control cell.</p

Relationship between expression of <i>TNF</i> and expression of the 19 others identified putative TNF-inducible genes in 48 REα negative breast tumors and 48 REα positive breast tumors.

<p><b>^a</b>Spearman correlation coefficient. <b>^b</b>P value, Spearman rank correlation test.</p><p><b>NS</b>, not significant.</p

Effect of ShRNA RelA on TNF-α-induced genes in HMEC endothelial cells.

<p>For each gene and each sample, mRNA levels were normalized such that the value of the Control shRNALuc sample was 1.The inhibition by ShRNA RelA is expressed as pourcentage of the difference between control shRNA Luc vs shRNA RelA in cells stimulated with TNF-α (40 ng/ml) for 4 h. Genes were classified by alphabetical order.</p

mRNA expression of CCL2/MCP1, IL8 and IL1A in TNF-α-stimulated and unstimulated HUVEC and HMEC cells.

<p>For each gene, mRNA levels were normalized such that the value for the “basal mRNA level” (smallest amount of target gene mRNA quantifiable, Ct = 35) was 1.</p

Western blot analyses of IL-1β, ICAM, RelA, RelB, NF-κB1/p50 and NF-κB2/p52 proteins in TNF-α stimulated HMEC.

<p>HMEC cells were incubated for 4 h with 4–40 ng/ml TNF-α. Whole cell lysates were prepared and subjected to Western blotting using anti-ICAM, anti-IL-1β anti-RelA, anti-RelB, anti-NF-κB1/p50 or anti-NF-κB2/p52 antibodies, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021589#s2" target="_blank"><i>Materials and Methods</i></a>. As loading controls, total proteins were also analyzed with anti-GAPDH. Each lane contains 50 µg of cellular protein. Lanes 1–4 correspond to control endothelial cells (lane 1), endothelial cells stimulated with 40 ng/ml (lanes 2 and 3) or 4 ng/ml (lane 4) TNF-α. Results were similar in two independent experiments.</p