Figure 2

Ndst1 silencing results in increased NFκB activation in response to CpG and interferon-β expression in DC2.4 cells as well as augmented interferon-β protein production in primary Ndst1 mutant DCs stimulated by the TLR7 ligand R848. (A) CpG-dependent NFκB activation is significantly augmented by Ndst1 silencing of DC2.4 cells, designated si(Ndst1), compared to that of si(Control) DC2.4 cells (i.e., scrambled RNA treated controls). Specifically, the level of phosphorylated NFκB subunit p65, as measured by western blotting, was indexed to the corresponding total p65 value; and the graphed values for each condition (at baseline and at 1 h post stimulation) are shown normalized to that of un-stimulated si(Control) cells in the graph (mean of n = 3 experiments, *P = 0.03 and **P < 0.01 for indicated differences in means; paired T-test). (B) A representative Western blot from one NF-κB activation experiment is shown to the right of the graph in (A), illustrating bands for phospho-p65 and total p65 collected from DC2.4 cells stimulated with 1 μM CpG for 15 min and 1 h (with data for baseline and 1 h stimulation plotted in the graph); and stimulation in the absence or presence of Ndst1 silencing denoted by “–” or “ + ” above each lane (full length blot shown in Supplemental Fig. S8). (C) Interferon-β quantitative PCR results for basal/unstimulated DC2.4 cells as well as cells stimulated with CpG. Among unstimulated cells: Ndst1 silenced DC2.4 cells demonstrate a significant increase in interferon-β expression (IFN-β) relative to that of control cells. Percent IFN-β expression was normalized to GAPDH expression in the respective cells, and responses for each condition are normalized to the response by resting si(Control) DC2.4 cells. Graph shows mean +/− SD from triplicate runs; **P < 0.01 for difference in means for si(Control) versus si(Ndst1) cells. Stimulation with CpG showed increased IFN-β expression responses by both control and Ndst1-silenced cells (to 1 μM CpG). While expression responses to 18 h CpG exposure were significant compared to baseline for both si(Control) and si(Ndst1) cells (***P < 0.001 or **P < 0.01 for respective differences in means relative to baseline), they reached essentially equivalent expression levels by the 18 h exposure time point (right bars on Fig. 2C graph). (D) IFN-β protein production into the culture medium was examined by ELISA assay in primary Ndst1f/f CD11cCre + mutant or Ndst1f/f CD11cCre− control bone marrow DCs in response to either (i) presence versus absence of 1 μM CpG stimulation overnight (high dose/ maximum stimulation from prior studies); or (ii) presence (up to 10 μg/ml) versus absence of overnight stimulation with the TLR7 agonist R848. While we were able to measure only minimal IFN-β protein responses upon CpG stimulation, with no significant differences compared to basal/resting cells (graph portion at left), primary DCs responded with significant IFN-β production in response to R848 at the 1.0 or 10 μg/ml range, with markedly greater responses associated with DC Ndst1 mutation (right portion of graph; ***P < 0.001 and **P < 0.01 for differences in indicated mean values).