Exploring New Inflammatory Biomarkers and Pathways during LPS-Induced M1 Polarization

Figure 5

M1 polarized N9 microglia evidence activation of TLR4/TLR2/NF-κB signaling pathway and of inflammasome complex. Protein expression of glycosylated toll-like receptor 4 (TLR4) (a) and TLR2 (b) was evaluated by Western blot analysis. Translocation of nuclear factor kappa B (NF-κB) to the nucleus is represented by considering the Western blot analysis of nuclear expression (c). To observe NF-κB cellular localization, immunocytochemistry against anti-NF-κB was performed as indicated in Materials and Methods and representative results are shown (d) and presented as the percentage of cells with nuclear staining versus total number of cells (e). NOD-like receptor family, pyrin domain containing 3 (Nlrp3), interleukin- (IL-) 1beta, and IL-18 expression were measured by qRT-PCR ((f), (g), and (h), resp.). Detection of caspase-1 activation was achieved by a colorimetric assay, as described in Materials and Methods (i). Results are mean ± s.e.m. from six (a, c), five (f, g, h), four (b), or three (e, i) independent experiments; (c) and (i) were performed in duplicate. Comparisons between lipopolysaccharide- (LPS-) treated N9 cells and nontreated cells (control) were made via two-tailed Student’s -test for all the parameters, except for (c, e), where unpaired -test with Welch’s correction was applied. , , and versus nontreated cells (control). Scale bar represents 20 µm.

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