Figure 3.
TLRs contribute to Notch target gene activation by IKK- and p38-dependent pathways
(A) Human macrophages were treated with 10 μg/ml of Jagged1-Fc, cocultured with OP9-DL1 cells, or stimulated with 10 ng/ml of LPS for 3 h. mRNA was measured by real time PCR. Similar results were obtained when cells were treated with plate-bound Jagged1-Fc. Results are shown as means + SD of triplicate determinants and are representative of three independent experiments.
(B) RAW264.7 cells were cotransfected with a Hes1 reporter gene construct and an expression plasmid encoding NICD1 or a control empty vector. One day post transfection, cells were stimulated with 1 μg/ml of LPS for 6 h. Results were shown as normalized firefly luciferase activity relative to internal control and expressed as mean + SD from duplicate transfections. One representative experiment out of four performed is shown.
(C) Human macrophages were pretreated with DMSO vehicle control, 10 μM of Bay11-7082 (Bay11), or 10 μM of SB203580 for 30 min and stimulated with 10 ng/ml of LPS for 3 h. mRNA was measured using real time PCR. Results are shown as means + SD of triplicate determinants and are representative of seven independent experiments.
(D and E) Primary human macrophages were transfected with control or IKKβ-specific siRNAs (D) or MEKK3-specific siRNAs (E) using an Amaxa nucleofector. 4 d after transfection, cells were stimulated with LPS (10 ng/ml) for 3 h, and mRNA was measured using real time PCR. Data are shown as means + SD of triplicate determinants and are representative of three independent experiments.
(F) Bone marrow derived macrophages from control or Mapk14-deficient mice were stimulated with 10 ng/ml of LPS for the indicated time periods. mRNA was measured using real time PCR. Unstimulated controls within each genotype were set as 1. Data shown as means + SD of triplicate determinants and one experiment representative of two is shown.
(G) ChIP was perform with LPS-treated primary human macrophages using anti-phospho-serine 10 histone H3 (pSer10-H3) or rabbit IgG as a control. Immunoprecipitated DNA was analyzed by semiquantitative PCR with Hes1 promoter-specific primers.
(H) ChIP was done as in (G) with 30 min preincubation of inhibitors and 2 h of Pam3Cys stimulation. Data in (G) and (H) are representative of three independent experiments.