Figure 2: The tetracycline COL-3 reduces the inflammatory response of microglial cells challenged with either LPS or αSa (A) (b) TEM images showing the presence of αS amyloid fibrils under shaken conditions, only (lower image). Scale bar: 1 μm. (B)Quantification of TNF-α levels in microglial cells exposed or not to LPS (10 ng/ml, 24-h) in the presence or not of COL-3 (1, 10 or 20 μM), DOX (50µM) or the reference antiinflammatory drug DEX (2.5 μM). Data are means ± SEM (n = 4), *p < 0.05 vs control.#p < 0.05 vs LPS. One-way ANOVA followed by Bonferroni post-hoc test. (C) Quantification of Iba-1 immunofluorescence density in microglial cultures exposed or not to to LPS (10 ng/ml, 24-h) in the presence or not of COL-3 (10 or 20 μM), DOX (50µM) or the reference glucocorticoid DEX (2.5 μM). Data are means ± SEM (n = 4), *p < 0.05 vs control.#p < 0.05 vs LPS. One-way ANOVA followed by Bonferroni post-hoc test. (D) Visualization of CD11b (green) and Iba-1 (red) immunofluorescence signals of LPS (10 ng/ml)-treated microglial cell cultures treated or not with COL-3 (20µM) or DOX (50 µM). Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 120 µm. (E) Quantification of TNF-α levels in microglial cells exposed or not to αSa (70 µg/ml, 24-h) in the presence or not of COL-3 (10 and 20 μM), DOX (50µM) or DEX (2.5 μM). Some cultures were also exposed αSm (70 µg/ml). Data are means ± SEM (n = 4), *p < 0.05 vs control. #p < 0.05 vs αSa. One-way ANOVA followed by Bonferroni post-hoc test.(F) Quantification of Iba-1 immunofluorescence signals in microglial cultures exposed or not to to α Sa (70 µg/ml, 24-h) in the presence or not of COL-3 (10 or 20 μM), DOX (50µM) or DEX (2.5 μM). Data are means ± SEM (n = 4), *p < 0.05 vs control.#p < 0.05 vs αSa. One-way ANOVA followed by Bonferroni post-hoc test. (G) Visualization of CD11b (green) and Iba-1 (red) immunofluorescent signals in αSa (70 µg/ml)-treated microglial cell cultures exposed or not to COL-3 (20µM) or DOX (50 µM). Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 120 µm.