FIGURE 2.

Nos2, caspase-11 or CD38 expression is preferentially induced by the LPS/IFNγ treatment; DNA-microarray and protein expression analysis. N9 cells were treated without (control) or with LPS (100 ng/ml) or IFNγ (100 units/ml) or LPS/IFNγ (LPS (100 ng/ml) and IFNγ (100 units/ml). RNA samples and protein extracts were prepared after 6 and 15 h respectively. The RNA was subjected to gene expression profiling and the results obtained were analyzed as described in Materials and Methods. The protein extracts were analyzed by immunoblotting (A) for the expression of Nos2, and caspase-11, as described in Materials and Methods. The immunoblotting results shown are from a representative experiment for each protein (one of 3–5 independent experiments). Immunoblots obtained in at least three independent experiments (including the one shown here) were scanned and the intensities of the different proteins were assessed by densitometric analysis. Data are presented as mean values of the intensities obtained for each protein after its normalization to β-tubulin. The data presented are expressed as the ratios of the mean intensities of caspase-11 and Nos2 in LPS-treated, IFNγ-treated, or control cells to those of LPS/IFNγ-treated cells (defined as 1 A.U). Values shown in the histograms are means ± SEM (bars) (n = 3–5). The gene-expression results (obtained from the DNA-microarray analysis) are shown (in parentheses) above each of the corresponding immunoblot results and are expressed for each gene in the same manner as described for the corresponding proteins. For determination of CD38 expression (B), N9 cells were treated as described above for 15 h and then were harvested, stained with PE-conjugated anti-CD38 Abs (PE-CD38), and analyzed by flow cytometry as described in Materials and Methods. The results of a representative experiment for each treatment (one of at least three independent experiments) are shown in the right panels. The empty histogram represents the background staining using an isotype-matched control Ab; the filled area represents CD38 staining. The mean fluorescence intensities (MFIs) for the different treatments of the (at least) three experiments were calculated and their mean values determined. The left panels present the quantification of the results of three independent experiments (including the one shown here), expressed as CD38 expression values (as determined by the MFIs) in the LPS-, IFNγ-, or LPS/IFNγ-treated cells relative to CD38 expression values in the control (defined as 1 A.U.). Values shown are means ± SEM (bars) (n = 3–4). The gene-expression results (obtained from the DNA-microarray analysis) are shown (in brackets) above each of the corresponding flow cytometry results.