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. 2019 Jan 10;9:3118. doi: 10.3389/fimmu.2018.03118

Figure 2.

Figure 2

Myo1F is required to stimulate a pro-inflammatory phenotype in macrophages. (A) iNOS expression was analyzed by RT-PCR in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. ***p = 0.0005. (B) iNOS and pro-IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M0, M1, or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. (C) Expression of CD80 and CD86 was analyzed by flow cytometry in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype and expressed as Mean Florescence Intensity. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. *p = 0.05, **p = 0.01. (D) NLRP3, Caspase 1, proIL-1β, and IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. Densitometric analyses obtained from those results are shown as graphs. **p = 0.01. (E) Secretion of IL-1β and IL-6 in supernatants of WT and Myo1F−/− derived BMM was performed by ELISA assay. LPS and IFN-γ/LPS stimulation was carried out for 5 h. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. *p = 0.05, **p = 0.01, ***p = 0.0005. (F) Western blotting for NLRP3, Caspase 1 and IL-1β in J774 cells overexpressing Myo1F or GFP under homeostatic conditions. GAPDH was used as loading control. n = 3. Densitometric analysis obtained from IL-1β is shown as graph. **p = 0.01. (G) Secretion of IL-1β in supernatants of J774 cells overexpressing Myo1F or GFP was performed by ELISA assay. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. *p = 0.05. (H) Quantification of IL-1β release was analyzed in colonic explants of WT and Myo1F deficient mice stimulated with IFN-γ/LPS. Inflammatory stimulus was administered for 5 h. IL-1β was quantified by ELISA. Graphs are derived from three independent experiments. n = 6. Results are given as mean values ± SEM. *p = 0.05.