Figure 4.

Methylene blue inhibits NLRP3 inflammasome formation in post-spinal cord injury (SCI) microglia. (A) Proportions of microglia and macrophages in spinal cords at indicated time points post SCI. Gr⁻CD45^lowCD11b⁺ microglia and Gr⁻CD45^hiCD11b⁺ macrophages were distinguished in spinal cord immune cells from 6 h to 7 days post SCI. These are representative dot plots of three independent experiments. Note that the leftmost dot plot showing the gating of Gr-negative cells was a sample collected 6 h post SCI. This is why only a small amount of Gr-positive cells were seen. Numbers in the plots are proportions of each gated population. (B–D) Expression of pro-IL-1β and mature IL-1β in microglia sorted from post-SCI spinal cords. Representative Western blot images are shown in (B), statistical analysis is shown in (C,D), respectively. N = 3 per group. (E) Representative images of Co-immunoprecipitation assay for the binding of ASC to NLRP3 in post-SCI microglia. IP: immunoprecipitation with the antibody against indicated protein. IB: detection of indicated protein. (F) Representative images of Co-immunoprecipitation assay for the binding of ASC to NLRC4 in post-SCI microglia. (G–J) Statistical analysis for the ASC bound to NLRP3 (G) or NLRC4 (I), and for the expression of NLRP3 (H) or NLRC4 (J). N = 5 per group. V: vehicle; M: methylene blue treatment. To analyze the relative expression of target proteins in (C,D), the band intensities of target proteins were normalized to corresponding band intensities of α-Tubulin, followed by calculating the target protein expression in other groups relative to “6 h (V)” group. For the quantification in (G–J), target proteins were normalized to α-Tubulin, followed by calculating the target protein expression in other groups relative to “Sham (V)” group. *p < 0.05 in comparison with the “6 h” vehicle group or sham group (if any). ^#p < 0.05; ^##p < 0.01 in comparison with the corresponding vehicle group at each time point.